OceanWiki - User contributions [en]

Radiolabeled tracer method

2026-05-15T18:38:46Z

Anabelvonjackowski:

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1. For example, nmol Leucine L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

Leucine incorporation can be overestimated see Giering & Events (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://aslopubs.onlinelibrary.wiley.com/doi/10.1002/lno.12032</ref>

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

{| class="wikitable sortable" style="text-align: center;"

|+ Summary of published LeuCFemp (in kg C [mol Leu]-1). Based on 54 publications and 296 published values. From Giering & Evans (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://aslopubs.onlinelibrary.wiley.com/doi/10.1002/lno.12032</ref>
.
! Hydrographic setting !! Min !! First Qu. !! Median !! Third Qu. !! Max !! n
|-
| Coast and shelf || 0.21 || 0.98 || 1.35 || 2.47 || 36.40 || 160
|-

| Open ocean || 0.02 || 0.25 || 0.56 || 1.29 || 19.20 || 105
|-

| Mesopelagic || 0.13 || 0.33 || 0.54 || 0.63 || 2.38 || 15
|-

| All marine || 0.02 || 0.52 || 1.14 || 2.00 || 36.40 || 280
|-

| Freshwater || 0.18 || 0.88 || 1.15 || 2.41 || 8.60 || 16
|-

| Sediment || 0.21 || 0.24 || 0.82 || 0.89 || 1.45 || 16
|-

| All environments || 0.02 || 0.53 || 1.14 || 2.03 || 36.40 || 296
|}

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:35:45Z

Anabelvonjackowski:

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1. For example, nmol Leucine L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

Leucine incorporation can be overestimated see Giering & Events (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://aslopubs.onlinelibrary.wiley.com/doi/10.1002/lno.12032</ref>

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

{| class="wikitable sortable" style="text-align: center;"

|+ Summary of published LeuCFemp (in kg C [mol Leu]-1). Based on 54 publications and 296 published values. From Giering & Evans (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://aslopubs.onlinelibrary.wiley.com/doi/10.1002/lno.12032</ref>
.
! Hydrographic setting !! Min !! First Qu. !! Median !! Third Qu. !! Max !! n
|-
| Coast and shelf || 0.21 || 0.98 || 1.35 || 2.47 || 36.40 || 160
|-

| Open ocean || 0.02 || 0.25 || 0.56 || 1.29 || 19.20 || 105
|-

| Mesopelagic || 0.13 || 0.33 || 0.54 || 0.63 || 2.38 || 15
|-

| All marine || 0.02 || 0.52 || 1.14 || 2.00 || 36.40 || 280
|-

| Freshwater || 0.18 || 0.88 || 1.15 || 2.41 || 8.60 || 16
|-

| Sediment || 0.21 || 0.24 || 0.82 || 0.89 || 1.45 || 16
|-

| All environments || 0.02 || 0.53 || 1.14 || 2.03 || 36.40 || 296
|}

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:35:09Z

Anabelvonjackowski:

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1. For example, nmol Leucine L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

Leucine incorporation can be overestimated see Giering & Events (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://aslopubs.onlinelibrary.wiley.com/doi/10.1002/lno.12032>

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

{| class="wikitable sortable" style="text-align: center;"

|+ Summary of published LeuCFemp (in kg C [mol Leu]-1). Based on 54 publications and 296 published values. From Giering & Evans (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://aslopubs.onlinelibrary.wiley.com/doi/10.1002/lno.12032>
.
! Hydrographic setting !! Min !! First Qu. !! Median !! Third Qu. !! Max !! n
|-
| Coast and shelf || 0.21 || 0.98 || 1.35 || 2.47 || 36.40 || 160
|-

| Open ocean || 0.02 || 0.25 || 0.56 || 1.29 || 19.20 || 105
|-

| Mesopelagic || 0.13 || 0.33 || 0.54 || 0.63 || 2.38 || 15
|-

| All marine || 0.02 || 0.52 || 1.14 || 2.00 || 36.40 || 280
|-

| Freshwater || 0.18 || 0.88 || 1.15 || 2.41 || 8.60 || 16
|-

| Sediment || 0.21 || 0.24 || 0.82 || 0.89 || 1.45 || 16
|-

| All environments || 0.02 || 0.53 || 1.14 || 2.03 || 36.40 || 296
|}

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:34:11Z

Anabelvonjackowski: /* Limitations */

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1. For example, nmol Leucine L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

Leucine incorporation can be overestimated see Giering & Events (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://aslopubs.onlinelibrary.wiley.com/doi/10.1002/lno.12032>

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

{| class="wikitable sortable" style="text-align: center;"

|+ Summary of published LeuCFemp (in kg C [mol Leu]-1). Based on 54 publications and 296 published values. From Giering & Evans (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://doi.org</ref>
.
! Hydrographic setting !! Min !! First Qu. !! Median !! Third Qu. !! Max !! n
|-
| Coast and shelf || 0.21 || 0.98 || 1.35 || 2.47 || 36.40 || 160
|-

| Open ocean || 0.02 || 0.25 || 0.56 || 1.29 || 19.20 || 105
|-

| Mesopelagic || 0.13 || 0.33 || 0.54 || 0.63 || 2.38 || 15
|-

| All marine || 0.02 || 0.52 || 1.14 || 2.00 || 36.40 || 280
|-

| Freshwater || 0.18 || 0.88 || 1.15 || 2.41 || 8.60 || 16
|-

| Sediment || 0.21 || 0.24 || 0.82 || 0.89 || 1.45 || 16
|-

| All environments || 0.02 || 0.53 || 1.14 || 2.03 || 36.40 || 296
|}

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:33:13Z

Anabelvonjackowski: /* Units & currency */

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1. For example, nmol Leucine L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

Leucine incorporation can be overestimated see Giering & Events (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://doi.org</ref>

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

{| class="wikitable sortable" style="text-align: center;"

|+ Summary of published LeuCFemp (in kg C [mol Leu]-1). Based on 54 publications and 296 published values. From Giering & Evans (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://doi.org</ref>
.
! Hydrographic setting !! Min !! First Qu. !! Median !! Third Qu. !! Max !! n
|-
| Coast and shelf || 0.21 || 0.98 || 1.35 || 2.47 || 36.40 || 160
|-

| Open ocean || 0.02 || 0.25 || 0.56 || 1.29 || 19.20 || 105
|-

| Mesopelagic || 0.13 || 0.33 || 0.54 || 0.63 || 2.38 || 15
|-

| All marine || 0.02 || 0.52 || 1.14 || 2.00 || 36.40 || 280
|-

| Freshwater || 0.18 || 0.88 || 1.15 || 2.41 || 8.60 || 16
|-

| Sediment || 0.21 || 0.24 || 0.82 || 0.89 || 1.45 || 16
|-

| All environments || 0.02 || 0.53 || 1.14 || 2.03 || 36.40 || 296
|}

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:32:46Z

Anabelvonjackowski: /* Limitations */

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1. For example nmol Leucine L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

Leucine incorporation can be overestimated see Giering & Events (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://doi.org</ref>

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

{| class="wikitable sortable" style="text-align: center;"

|+ Summary of published LeuCFemp (in kg C [mol Leu]-1). Based on 54 publications and 296 published values. From Giering & Evans (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://doi.org</ref>
.
! Hydrographic setting !! Min !! First Qu. !! Median !! Third Qu. !! Max !! n
|-
| Coast and shelf || 0.21 || 0.98 || 1.35 || 2.47 || 36.40 || 160
|-

| Open ocean || 0.02 || 0.25 || 0.56 || 1.29 || 19.20 || 105
|-

| Mesopelagic || 0.13 || 0.33 || 0.54 || 0.63 || 2.38 || 15
|-

| All marine || 0.02 || 0.52 || 1.14 || 2.00 || 36.40 || 280
|-

| Freshwater || 0.18 || 0.88 || 1.15 || 2.41 || 8.60 || 16
|-

| Sediment || 0.21 || 0.24 || 0.82 || 0.89 || 1.45 || 16
|-

| All environments || 0.02 || 0.53 || 1.14 || 2.03 || 36.40 || 296
|}

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:32:09Z

Anabelvonjackowski: /* Common calculations/conversions */

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1. For example nmol Leucine L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

Leucine incorporation can be overestimated see Giering & Events (2022) doi: 10.1002/lno.12032

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

{| class="wikitable sortable" style="text-align: center;"

|+ Summary of published LeuCFemp (in kg C [mol Leu]-1). Based on 54 publications and 296 published values. From Giering & Evans (2022)<ref name="Giering2022">Giering, S. L. C., & Evans, C. (2022). Overestimation of prokaryotic production by leucine incorporation—and how to avoid it. ''Limnology and Oceanography'', 67(3), 726–738. https://doi.org</ref>
.
! Hydrographic setting !! Min !! First Qu. !! Median !! Third Qu. !! Max !! n
|-
| Coast and shelf || 0.21 || 0.98 || 1.35 || 2.47 || 36.40 || 160
|-

| Open ocean || 0.02 || 0.25 || 0.56 || 1.29 || 19.20 || 105
|-

| Mesopelagic || 0.13 || 0.33 || 0.54 || 0.63 || 2.38 || 15
|-

| All marine || 0.02 || 0.52 || 1.14 || 2.00 || 36.40 || 280
|-

| Freshwater || 0.18 || 0.88 || 1.15 || 2.41 || 8.60 || 16
|-

| Sediment || 0.21 || 0.24 || 0.82 || 0.89 || 1.45 || 16
|-

| All environments || 0.02 || 0.53 || 1.14 || 2.03 || 36.40 || 296
|}

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:29:54Z

Anabelvonjackowski: /* Limitations */

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1. For example nmol Leucine L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

Leucine incorporation can be overestimated see Giering & Events (2022) doi: 10.1002/lno.12032

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:28:15Z

Anabelvonjackowski: /* Units & currency */

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1. For example nmol Leucine L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:25:39Z

Anabelvonjackowski: /* Classic examples */

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Publication examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:25:27Z

Anabelvonjackowski: /* Classic examples */

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Classic examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

== References ==

[[Category:Main Pages|Model types]]

Radiolabeled tracer method

2026-05-15T18:23:53Z

Anabelvonjackowski:

{{BreadcrumbsSecondaryProduction}}

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]], [[User:Hagi BucknWise|Hagen Buck-Wiese]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Bacterial production
|-
| '''Approach:''' radiolabeled tracer incorporation
|-
| '''Context:''' incubation, lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' hours
|-
| '''Units:''' mol incorporated tracer L-1 h-1
|-
| '''Community captured:''' bulk, size-fractionated
|-
| '''Co-measurements:''' cell abundance
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate<ref name="Kirchman2001">Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. ''Methods in Microbiology'', 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8</ref>.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

=== Classic examples ===
* Kirchman (2001) ''Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments'' <ref name="Kirchman2001" />
Recent applications ===
* Kirchman et al. (2009) ''Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean'' <ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep-Sea Research Part II'', 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

=== Scale of measurement ===

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

=== Data generated ===

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.

=== Units & currency ===

Units are mol incorporated tracer L-1 h-1.

=== Sample size ===

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

=== Repositories & databases ===

== Limitations ==

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

== Step-by-Step Protocol ==

# '''Dilute Leucine Stock:''' Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
#* ''Example:'' 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
#* ''Note:'' A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
#* ''High Density Note:'' For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
# '''Label Tubes:''' Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
# '''Set Incubator:''' Set the incubation chamber to the desired target temperature and place the incubation racks inside.
# '''Pre-chill Equipment and Reagents:'''
#* Pre-chill the centrifuge to 4 °C.
#* Store one tube rack at 4 °C.
#* Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
#* ''Batch Limit Note:'' Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
# '''Aliquot Water Samples:''' Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
# '''Add Leucine Substrate:''' Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
# '''Add Sample:''' Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
# '''Prepare Killed Controls (Blanks):''' Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
# '''Incubation:''' Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
# '''Pre-cooling Transfer:''' Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
# '''Terminate Reaction:''' Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). '''Do not add TCA to the already killed controls!''' Record the exact termination timestamp.
# '''First Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
# '''Aspirate Supernatant:''' Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
# '''First Wash Step:''' Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
# '''Second Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
# '''Aspirate Supernatant:''' Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
# '''Second Wash Step:''' Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
# '''Third Centrifugation:''' Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
# '''Final Aspiration:''' Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
# '''Drying the Pellet:''' Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
# '''Add Scintillation Cocktail:''' Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
# '''Load and Measure:''' Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
#* ''Note on Timing:'' For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
#* ''Expected Yield:'' The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

=== Common calculations/conversions ===
* Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
* Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).

== References ==

[[Category:Main Pages|Model types]]

Winkler light-dark dissolved O2 bottle

2026-05-15T17:57:29Z

Anabelvonjackowski: /* Method Overview */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

The Baltic Marine Environment Protection Commission – Helsinki Commission (HELCOM) has published a sampling guideline see here: https://helcom.fi/wp-content/uploads/2019/08/Guidelines-for-sampling-and-determination-of-dissolved-oxygen.pdf

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|-
| Global Ocean || Global || - || 3.3 ± 0.15 || Robinson & Williams et al., (2005) <ref name="Robinson2005" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

More CR and BR rates are available via the "A global dataset of marine pelagic microbial respiration" database <ref name="Robinson2026" />

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009a). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., & Amon, R. (2004). Heterotrophic bacterial activity and fluxes of dissolved free amino acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. ''Aquatic Microbial Ecology'', 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C. (2008). Heterotrophic Bacterial Respiration. In: Kirchman, D. L. (Ed.), ''Microbial Ecology of the Oceans''. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Robinson2005">Robinson, C., & Williams, P. J. l. B. (2005). Respiration and its measurement in surface marine waters. In: del Giorgio, P. A., & Williams, P. J. l. B. (Eds.), ''Respiration in Aquatic Ecosystems''. Oxford University Press, Oxford, pp. 147–180 </ref>

<ref name="Robinson2026">Robinson, C., Seguro, I., Dall'Olmo, G., Moncoiffe, G., Aranguren-Gassis, M., Aristegui, J., Azzaro, M., Baek, Y. J., Baltar, F., Cohn, M. R., Eissler, Y., Evans, C., Fennel, K., Fernandez-Urruzola, I., Ferron, S., Fukuda, H., Garcia-Martin, E. E., Gifford, S., Goddard-Dwyer, M., Hernandez-Hernandez, N., Herndl, Y., Hyun, J. H., Kim, B., Kirchman, D., Kitidis, Vassilis., LaBrie, R., Lefevre, D., Lonborg, C., Maranger, R., Martinez-Garcia, S., Montero, M. F., Mourino-Carballido, B., Nagata, T., Osma, N., Panton, A., Regaudie de Gioux, A., Reinthaler, T., Serret, P., Sulpis, O., Uchimiya, Mario., Wang, B., Wang, Q., & Yokokawa, T. (2026). A global dataset of marine pelagic microbial respiration. NERC EDS British Oceanographic Data Centre NOC. https://www.bodc.ac.uk/data/published_data_library/catalogue/10.5285/4b2a5ac6-b6db-c98e-e063-7086abc040c6 </ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., & Duarte, C. M. (2010). Plankton metabolism in the Greenland Sea during the polar summer of 2007. ''Polar Biology'', 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1</ref>

<ref name="Williams2004">Williams, P. J. M., & Karl, D. M. (2004). Net community production and metabolic balance at the oligotrophic ocean site, station ALOHA. ''Deep Sea Research Part I: Oceanographic Research Papers'', 51(11), 1563–1578. https://doi.org/10.1016/j.dsr.2004.07.001</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:57:13Z

Anabelvonjackowski: /* Method Overview */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

The Baltic Marine Environment Protection Commission – Helsinki Commission (HELCOM) has published a sampling guideline see here https://helcom.fi/wp-content/uploads/2019/08/Guidelines-for-sampling-and-determination-of-dissolved-oxygen.pdf

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|-
| Global Ocean || Global || - || 3.3 ± 0.15 || Robinson & Williams et al., (2005) <ref name="Robinson2005" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

More CR and BR rates are available via the "A global dataset of marine pelagic microbial respiration" database <ref name="Robinson2026" />

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009a). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., & Amon, R. (2004). Heterotrophic bacterial activity and fluxes of dissolved free amino acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. ''Aquatic Microbial Ecology'', 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C. (2008). Heterotrophic Bacterial Respiration. In: Kirchman, D. L. (Ed.), ''Microbial Ecology of the Oceans''. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Robinson2005">Robinson, C., & Williams, P. J. l. B. (2005). Respiration and its measurement in surface marine waters. In: del Giorgio, P. A., & Williams, P. J. l. B. (Eds.), ''Respiration in Aquatic Ecosystems''. Oxford University Press, Oxford, pp. 147–180 </ref>

<ref name="Robinson2026">Robinson, C., Seguro, I., Dall'Olmo, G., Moncoiffe, G., Aranguren-Gassis, M., Aristegui, J., Azzaro, M., Baek, Y. J., Baltar, F., Cohn, M. R., Eissler, Y., Evans, C., Fennel, K., Fernandez-Urruzola, I., Ferron, S., Fukuda, H., Garcia-Martin, E. E., Gifford, S., Goddard-Dwyer, M., Hernandez-Hernandez, N., Herndl, Y., Hyun, J. H., Kim, B., Kirchman, D., Kitidis, Vassilis., LaBrie, R., Lefevre, D., Lonborg, C., Maranger, R., Martinez-Garcia, S., Montero, M. F., Mourino-Carballido, B., Nagata, T., Osma, N., Panton, A., Regaudie de Gioux, A., Reinthaler, T., Serret, P., Sulpis, O., Uchimiya, Mario., Wang, B., Wang, Q., & Yokokawa, T. (2026). A global dataset of marine pelagic microbial respiration. NERC EDS British Oceanographic Data Centre NOC. https://www.bodc.ac.uk/data/published_data_library/catalogue/10.5285/4b2a5ac6-b6db-c98e-e063-7086abc040c6 </ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., & Duarte, C. M. (2010). Plankton metabolism in the Greenland Sea during the polar summer of 2007. ''Polar Biology'', 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1</ref>

<ref name="Williams2004">Williams, P. J. M., & Karl, D. M. (2004). Net community production and metabolic balance at the oligotrophic ocean site, station ALOHA. ''Deep Sea Research Part I: Oceanographic Research Papers'', 51(11), 1563–1578. https://doi.org/10.1016/j.dsr.2004.07.001</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:55:39Z

Anabelvonjackowski: /* References */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|-
| Global Ocean || Global || - || 3.3 ± 0.15 || Robinson & Williams et al., (2005) <ref name="Robinson2005" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

More CR and BR rates are available via the "A global dataset of marine pelagic microbial respiration" database <ref name="Robinson2026" />

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009a). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., & Amon, R. (2004). Heterotrophic bacterial activity and fluxes of dissolved free amino acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. ''Aquatic Microbial Ecology'', 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C. (2008). Heterotrophic Bacterial Respiration. In: Kirchman, D. L. (Ed.), ''Microbial Ecology of the Oceans''. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Robinson2005">Robinson, C., & Williams, P. J. l. B. (2005). Respiration and its measurement in surface marine waters. In: del Giorgio, P. A., & Williams, P. J. l. B. (Eds.), ''Respiration in Aquatic Ecosystems''. Oxford University Press, Oxford, pp. 147–180 </ref>

<ref name="Robinson2026">Robinson, C., Seguro, I., Dall'Olmo, G., Moncoiffe, G., Aranguren-Gassis, M., Aristegui, J., Azzaro, M., Baek, Y. J., Baltar, F., Cohn, M. R., Eissler, Y., Evans, C., Fennel, K., Fernandez-Urruzola, I., Ferron, S., Fukuda, H., Garcia-Martin, E. E., Gifford, S., Goddard-Dwyer, M., Hernandez-Hernandez, N., Herndl, Y., Hyun, J. H., Kim, B., Kirchman, D., Kitidis, Vassilis., LaBrie, R., Lefevre, D., Lonborg, C., Maranger, R., Martinez-Garcia, S., Montero, M. F., Mourino-Carballido, B., Nagata, T., Osma, N., Panton, A., Regaudie de Gioux, A., Reinthaler, T., Serret, P., Sulpis, O., Uchimiya, Mario., Wang, B., Wang, Q., & Yokokawa, T. (2026). A global dataset of marine pelagic microbial respiration. NERC EDS British Oceanographic Data Centre NOC. https://www.bodc.ac.uk/data/published_data_library/catalogue/10.5285/4b2a5ac6-b6db-c98e-e063-7086abc040c6 </ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., & Duarte, C. M. (2010). Plankton metabolism in the Greenland Sea during the polar summer of 2007. ''Polar Biology'', 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1</ref>

<ref name="Williams2004">Williams, P. J. M., & Karl, D. M. (2004). Net community production and metabolic balance at the oligotrophic ocean site, station ALOHA. ''Deep Sea Research Part I: Oceanographic Research Papers'', 51(11), 1563–1578. https://doi.org/10.1016/j.dsr.2004.07.001</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:54:45Z

Anabelvonjackowski: /* References */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|-
| Global Ocean || Global || - || 3.3 ± 0.15 || Robinson & Williams et al., (2005) <ref name="Robinson2005" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

More CR and BR rates are available via the "A global dataset of marine pelagic microbial respiration" database <ref name="Robinson2026" />

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009a). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., & Amon, R. (2004). Heterotrophic bacterial activity and fluxes of dissolved free amino acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. ''Aquatic Microbial Ecology'', 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C. (2008). Heterotrophic Bacterial Respiration. In: Kirchman, D. L. (Ed.), ''Microbial Ecology of the Oceans''. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Robinson2005">Robinson, C., & Williams, P. J. l. B. (2005). Respiration and its measurement in surface marine waters. In: del Giorgio, P. A., & Williams, P. J. l. B. (Eds.), ''Respiration in Aquatic Ecosystems''. Oxford University Press, Oxford, pp. 147–180. https://doi.org</ref>

<ref name="Robinson2026">Robinson, C., Seguro, I., Dall'Olmo, G., Moncoiffe, G., Aranguren-Gassis, M., Aristegui, J., Azzaro, M., Baek, Y. J., Baltar, F., Cohn, M. R., Eissler, Y., Evans, C., Fennel, K., Fernandez-Urruzola, I., Ferron, S., Fukuda, H., Garcia-Martin, E. E., Gifford, S., Goddard-Dwyer, M., Hernandez-Hernandez, N., Herndl, Y., Hyun, J. H., Kim, B., Kirchman, D., Kitidis, Vassilis., LaBrie, R., Lefevre, D., Lonborg, C., Maranger, R., Martinez-Garcia, S., Montero, M. F., Mourino-Carballido, B., Nagata, T., Osma, N., Panton, A., Regaudie de Gioux, A., Reinthaler, T., Serret, P., Sulpis, O., Uchimiya, Mario., Wang, B., Wang, Q., & Yokokawa, T. (2026). A global dataset of marine pelagic microbial respiration. NERC EDS British Oceanographic Data Centre NOC. https://www.bodc.ac.uk/data/published_data_library/catalogue/10.5285/4b2a5ac6-b6db-c98e-e063-7086abc040c6 </ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., & Duarte, C. M. (2010). Plankton metabolism in the Greenland Sea during the polar summer of 2007. ''Polar Biology'', 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1</ref>

<ref name="Williams2004">Williams, P. J. M., & Karl, D. M. (2004). Net community production and metabolic balance at the oligotrophic ocean site, station ALOHA. ''Deep Sea Research Part I: Oceanographic Research Papers'', 51(11), 1563–1578. https://doi.org/10.1016/j.dsr.2004.07.001</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:53:50Z

Anabelvonjackowski: /* References */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|-
| Global Ocean || Global || - || 3.3 ± 0.15 || Robinson & Williams et al., (2005) <ref name="Robinson2005" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

More CR and BR rates are available via the "A global dataset of marine pelagic microbial respiration" database <ref name="Robinson2026" />

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009a). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., & Amon, R. (2004). Heterotrophic bacterial activity and fluxes of dissolved free amino acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. ''Aquatic Microbial Ecology'', 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C. (2008). Heterotrophic Bacterial Respiration. In: Kirchman, D. L. (Ed.), ''Microbial Ecology of the Oceans''. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Robinson2005">Robinson, C., & Williams, P. J. l. B. (2005). Respiration and its measurement in surface marine waters. In: del Giorgio, P. A., & Williams, P. J. l. B. (Eds.), ''Respiration in Aquatic Ecosystems''. Oxford University Press, Oxford, pp. 147–180. https://doi.org</ref>

<ref name="Robinson2026">Robinson, C., Seguro, I., Dall'Olmo, G., Moncoiffe, G., Aranguren-Gassis, M., Aristegui, J., Azzaro, M., Baek, Y. J., Baltar, F., Cohn, M. R., Eissler, Y., Evans, C., Fennel, K., Fernandez-Urruzola, I., Ferron, S., Fukuda, H., Garcia-Martin, E. E., Gifford, S., Goddard-Dwyer, M., Hernandez-Hernandez, N., Herndl, Y., Hyun, J. H., Kim, B., Kirchman, D., Kitidis, Vassilis., LaBrie, R., Lefevre, D., Lonborg, C., Maranger, R., Martinez-Garcia, S., Montero, M. F., Mourino-Carballido, B., Nagata, T., Osma, N., Panton, A., Regaudie de Gioux, A., Reinthaler, T., Serret, P., Sulpis, O., Uchimiya, Mario., Wang, B., Wang, Q., & Yokokawa, T. (2026). A global dataset of marine pelagic microbial respiration. NERC EDS British Oceanographic Data Centre NOC. https://doi.org</ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., & Duarte, C. M. (2010). Plankton metabolism in the Greenland Sea during the polar summer of 2007. ''Polar Biology'', 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1</ref>

<ref name="Williams2004">Williams, P. J. M., & Karl, D. M. (2004). Net community production and metabolic balance at the oligotrophic ocean site, station ALOHA. ''Deep Sea Research Part I: Oceanographic Research Papers'', 51(11), 1563–1578. https://doi.org/10.1016/j.dsr.2004.07.001</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:52:31Z

Anabelvonjackowski: /* Bacterial Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|-
| Global Ocean || Global || - || 3.3 ± 0.15 || Robinson & Williams et al., (2005) <ref name="Robinson2005" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

More CR and BR rates are available via the "A global dataset of marine pelagic microbial respiration" database <ref name="Robinson2026" />

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135.https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2005">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Robinson2008">Carol Robinson and Peter J. le B. Williams (2005) Respiration and its measurement in surface marine waters. doi: 10.1093/acprof:oso/9780198527084.003.0009 In book: Respiration in Aquatic Ecosystems </ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1 </ref>

<ref name="Williams2004">Peter J. le B. Williams, Paul J. Morris, David M. Karl, Net community production and metabolic balance at the oligotrophic ocean site, station ALOHA, Deep Sea Research Part I: Oceanographic Research Papers,Volume 51, Issue 11,2004,https://doi.org/10.1016/j.dsr.2004.07.001 </ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:41:09Z

Anabelvonjackowski: /* References */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|-
| Global Ocean || Global || - || 3.3 ± 0.15 || Robinson & Williams et al., (2005) <ref name="Robinson2005" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135.https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2005">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Robinson2008">Carol Robinson and Peter J. le B. Williams (2005) Respiration and its measurement in surface marine waters. doi: 10.1093/acprof:oso/9780198527084.003.0009 In book: Respiration in Aquatic Ecosystems </ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1 </ref>

<ref name="Williams2004">Peter J. le B. Williams, Paul J. Morris, David M. Karl, Net community production and metabolic balance at the oligotrophic ocean site, station ALOHA, Deep Sea Research Part I: Oceanographic Research Papers,Volume 51, Issue 11,2004,https://doi.org/10.1016/j.dsr.2004.07.001 </ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:39:18Z

Anabelvonjackowski: /* Community Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|-
| Global Ocean || Global || - || 3.3 ± 0.15 || Robinson & Williams et al., (2005) <ref name="Robinson2005" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135.https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1 </ref>

<ref name="Williams2004">Peter J. le B. Williams, Paul J. Morris, David M. Karl, Net community production and metabolic balance at the oligotrophic ocean site, station ALOHA, Deep Sea Research Part I: Oceanographic Research Papers,Volume 51, Issue 11,2004,https://doi.org/10.1016/j.dsr.2004.07.001 </ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:32:38Z

Anabelvonjackowski: /* References */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135.https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1 </ref>

<ref name="Williams2004">Peter J. le B. Williams, Paul J. Morris, David M. Karl, Net community production and metabolic balance at the oligotrophic ocean site, station ALOHA, Deep Sea Research Part I: Oceanographic Research Papers,Volume 51, Issue 11,2004,https://doi.org/10.1016/j.dsr.2004.07.001 </ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:31:30Z

Anabelvonjackowski: /* Community Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-
| Pacific Ocean || Station ALOHA || May 2001 - May 2002 || 0-1.5 || Williams et al., (2004) <ref name="Williams2004" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135.https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1 </ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:12:30Z

Anabelvonjackowski: /* Community Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1O2 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135.https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1 </ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:11:26Z

Anabelvonjackowski: /* Community Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="RegaudiedeGioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135.https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1 </ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:11:12Z

Anabelvonjackowski: /* References */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135.https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="RegaudiedeGioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-1 </ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:10:44Z

Anabelvonjackowski: /* References */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135.https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Regaudie-de-Gioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-
1</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:10:20Z

Anabelvonjackowski: /* References */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Regaudie-de-Gioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-
1</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:07:51Z

Anabelvonjackowski: /* Bacterial Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Nguyen2012">Nguyen, D., Maranger, R., Tremblay, J., Gosselin, M., 2012. Respiration and bacterial carbon
dynamics in the Amundsen Gulf, western Canadian Arctic. J. Geophys. Res. 117, 2011JC007343. https://doi.org/10.1029/2011JC007343</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Regaudie-de-Gioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-
1</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:07:28Z

Anabelvonjackowski: /* Community Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || May-August 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date (Season.yyyy) !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Nguyen2012">Nguyen, D., Maranger, R., Tremblay, J., Gosselin, M., 2012. Respiration and bacterial carbon
dynamics in the Amundsen Gulf, western Canadian Arctic. J. Geophys. Res. 117, 2011JC007343. https://doi.org/10.1029/2011JC007343</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Regaudie-de-Gioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-
1</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:07:07Z

Anabelvonjackowski: /* Bacterial Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || Summer 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date (Season.yyyy) !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || July-August 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Aug-Sep 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Nguyen2012">Nguyen, D., Maranger, R., Tremblay, J., Gosselin, M., 2012. Respiration and bacterial carbon
dynamics in the Amundsen Gulf, western Canadian Arctic. J. Geophys. Res. 117, 2011JC007343. https://doi.org/10.1029/2011JC007343</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Regaudie-de-Gioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-
1</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:06:45Z

Anabelvonjackowski: /* Community Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || Summer 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || July 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Greenland Current || July 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date (Season.yyyy) !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || Summer 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Summer 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Nguyen2012">Nguyen, D., Maranger, R., Tremblay, J., Gosselin, M., 2012. Respiration and bacterial carbon
dynamics in the Amundsen Gulf, western Canadian Arctic. J. Geophys. Res. 117, 2011JC007343. https://doi.org/10.1029/2011JC007343</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Regaudie-de-Gioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-
1</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T17:05:57Z

Anabelvonjackowski: /* Community Respiration */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || Summer 2004 || 4.4 ± 0.034 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || Summer 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Greenland Current || Summer 2007 || 2.1 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date (Season.yyyy) !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || Summer 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Summer 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Nguyen2012">Nguyen, D., Maranger, R., Tremblay, J., Gosselin, M., 2012. Respiration and bacterial carbon
dynamics in the Amundsen Gulf, western Canadian Arctic. J. Geophys. Res. 117, 2011JC007343. https://doi.org/10.1029/2011JC007343</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Regaudie-de-Gioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-
1</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T16:51:43Z

Anabelvonjackowski: /* Respiration by Ocean Region */

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date (Season.yyyy) !! CR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Chukchi Sea || Summer 2004 || 0.5 ± 0.49 || Kirchman et al. (2009) <ref name="Kirchman2009" />
|-

| Arctic Ocean || Fram Strait || Summer 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Greenland Current || Summer 2007 || 1.2 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
|-

| Arctic Ocean || Beaufort Sea || Summer 2009 || 8.1 ± 3.6 || Nguyen et al., 2012 <ref name="Nguyen2012" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"
! Ocean !! Marginal Sea (if available) !! Sampling Date (Season.yyyy) !! BR (µmol L-1 d-1) !! Reference

|-
| Arctic Ocean || Beaufort Sea || Summer 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
|-

| Arctic Ocean || Kara Sea || Summer 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Nguyen2012">Nguyen, D., Maranger, R., Tremblay, J., Gosselin, M., 2012. Respiration and bacterial carbon
dynamics in the Amundsen Gulf, western Canadian Arctic. J. Geophys. Res. 117, 2011JC007343. https://doi.org/10.1029/2011JC007343</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Regaudie-de-Gioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-
1</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T16:51:10Z

Anabelvonjackowski:

* [[Page authors|Page authors]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]
* [[Responsible curator|Responsible curator]]: [[User:Anabelvonjackowski|Anabel von Jackowski]]

__TOC__

<div style="display: flex; flex-direction: row; align-items: flex-start; justify-content: flex-end; gap: 15px; margin-bottom: 1em;">


<div style="flex-shrink: 0;">
[[File:VonJackowski titration.jpg|thumb|200px|Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)]]
</div>


<div class="model-box" style="margin: 0;">
{| class="model-ib" style="margin: 0;"
! Oxygen Concentration

|-
| '''Approach:''' Winkler titration (iodometric)
|-

| '''Context:''' discrete measurement
|-
| '''Spatial scale:''' mL

|-
| '''Temporal scale:''' ''in situ''
|-

| '''Units:''' mmol O2 L-1
|-
| '''Community captured:''' all

|-
| '''Co-measurements:''' temperature, salinity, depth
|}
</div>

</div>
<div style="clear:both"></div>

== Method Overview ==

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. <ref name = "Winkler1888">Winkler, L. W. (1888). *Die Bestimmung des im Wasser gelösten Sauerstoffes*. Berichte der Deutschen Chemischen Gesellschaft, 21(2), 2843–2854. https://doi.org/10.1002/cber.188802102122</ref>.

== Step-by-Step Protocol ==

=== Preparing Chemicals ===
# '''Winkler 1 (Manganese(II) chloride)''' The reagent is stable for an extended period but should be stored protected from light.
#* '''Option A)''' Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
#* '''Option B)''' Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
# '''Winkler 2 (Alkaline iodide solution)''' 
#* '''Option A)''' Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
#* '''Option B)''' Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
# '''Sulfuric Acid 50%''' Carefully dilute 98% sulfuric acid with MQ water '''ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!'''
# '''Sodium Thiosulfate Solution (0.02 M)''' Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
# '''Starch Solution''' Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
# '''Standard Solution''' Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

=== Calibrate Bottles ===
# <li value="8">'''Gravimetric Calibration'''
:* ''Note: This can also be done after sampling but is needed to calculate oxygen concentration.''

: '''a.''' Weigh the Empty Bottle: Weigh the clean, dry Winkler bottle and its stopper together (Weight A, in grams).

: '''b.''' Weigh the Full Bottle: Fill the bottle completely with distilled water at a known temperature, insert the stopper to ensure no air bubbles are trapped, and dry the outside of the bottle. Weigh the full bottle (Weight B, in grams).

: '''c.''' Calculate Volume: Calculate the exact volume (<math>V</math>) of the bottle in milliliters.

:<math>V = \frac{\text{Weight B} - \text{Weight A}}{\text{Density of Water at } T^\circ\text{C}}</math>

=== Sample Collection ===
# <li value="9">'''Sample Preparation'''
#* Place Winkler 1 and 2 solutions close to where you are taking your sample
#* Write down the number of bottle you will fill with water
#* Have the bottle you will use and a tube ready for sampling
# '''Sample for oxygen''' Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.</li>
# '''Fix Oxygen''' Immediately add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to '''maximum''' 12 hours.
#* Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
#* Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2
#* Tipp: Dispensers may be useful to add Winkler solutions

=== Analysis via Titration ===
# <li value="12">'''Dissolving the Precipitate''' Add 50% sulfuric acid, avoid disturbing of the precipitate, and close the bottle again. Gently swirl until the precipitate dissolves.
#* Note: Some protocols say a few drops of H2SO4, I use 2 mL for 100 mL. </li>
# '''Transfer Sample options'''
#* '''Option A)''' Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
#* '''Option B)''' Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
#* '''Option C)''' No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
# '''Titration - Step 1''' Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
# '''Titration - Step 2''' Add starch solution (iodine indicator), which turns the pale-yellow into a deep blue-black color. Again protocols vary from 3-5 drops, but I use 1 mL for 100 mL.
# '''Titration - Step 3''' Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
# '''Disposal''' Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.
# '''Standardization of the thiosulphate solution''' The thiosulphate solution needs to be calibrated daily. Use the same volume of MQ as your sample and add 2 ml sulphuric acid and, while stirring, add 1 ml Winkler 1 and 1 ml Winkler 2 solution. Then, add 1 ml of 0,833∙10-2 M iodate standard solution and titrate with thiosulphate solution (0.02 M Na2S2O3) as described above. This standardization is used to calculate the "Factor Sodium thiosulphate" of the thiosulphate solution. You should have 3-5 replicates and the standard deviation should be +/- 0.02ml.

=== Calculation of O2 Concentration ===
The factor for sodium thiosulphate is determined by standardizing with <math>10\text{ ml}</math> of <math>5 \times 10^{-3}\text{ N KH(IO}_3)_2</math>, which corresponds to <math>5\text{ ml}</math> of <math>0.02\text{ M Na}_2\text{S}_2\text{O}_3</math>.

:<math>\text{factor} = \frac{5}{V}</math>

:where <math>V</math> = volume of thiosulphate used <math>[\text{ml}]</math>

<math>1\text{ ml}</math> of <math>0.02\text{ M}</math> thiosulphate solution contains <math>20\ \mu\text{mol}</math> of thiosulphate. This titrates <math>5\ \mu\text{mol}</math> of oxygen (<math>= 0.16\text{ mg O}_2 = 0.112\text{ ml O}_2</math>).

:<math>\text{O}_2\ [\text{ml}] = 0.112 \times a \times \text{factor}</math>

:<math>\text{O}_2\ [\text{ml/l}] = \frac{a \times \text{factor} \times 112}{b - 2}</math>

; Where:
: <math>a</math> = consumption of thiosulphate <math>[\text{ml}]</math>
: <math>b</math> = volume of the sampling bottle <math>[\text{ml}]</math>
: <math>2</math> = volume of reagents added <math>[\text{ml}]</math> ('''Attention:''' This volume may vary depending on the protocol)

::: ''Note: A sample calculation sheet is linked here: [[File:Sample-Oxygen Calculation-Winkler.xls|Sample-Oxygen Calculation-Winkler.xls]]''

== Respiration by Ocean Region ==
Oxygen can be fixed at different timepoints to give community respiration. Bacterial respiration can be calculated using BR = 0.45 x CR0.93 (Robinson, 2008)<ref name="Robinson2008" /> or a size-fraction Winkler method.

=== Community Respiration ===
{| class="wikitable sortable" style="text-align: center;"

|+
! Ocean !! Marginal Sea (if available) !! Sampling Date (Season.yyyy) !! CR (µmol L-1 d-1) !! Reference
|-
| Arctic Ocean || Chukchi Sea || Summer 2004 || 0.5 ± 0.49 || Kirchman et al. (2009) <ref name="Kirchman2009" />
| Arctic Ocean || Fram Strait || Summer 2007 || 6.2 ± 0.87 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
| Arctic Ocean || Greenland Current || Summer 2007 || 1.2 ± 1.02 || Regaudie-de-Gioux and Duarte (2010) <ref name="Regaudie-de-Gioux2010" />
| Arctic Ocean || Beaufort Sea || Summer 2009 || 8.1 ± 3.6 || Nguyen et al., 2012 <ref name="Nguyen2012" />
|}

=== Bacterial Respiration ===
{| class="wikitable sortable" style="text-align: center;"

|+
! Ocean !! Marginal Sea (if available) !! Sampling Date (Season.yyyy) !! BR (µmol L-1 d-1) !! Reference
|-
| Arctic Ocean || Beaufort Sea || Summer 2009 || 1.2 ± 0.94 || Ortega-Retuerta et al., 2012 <ref name="Ortega-Retuerta2012" />
| Arctic Ocean || Kara Sea || Summer 2001 || 0.48 ± 0.57 || Meon and Amon (2004) <ref name="Meon2004" />
|}

== References ==
<references>
<ref name="Kirchman2009">Kirchman, D.L., Hill, V., Cottrell, M.T., Gradinger, R., Malmstrom, R.R., Parker, A., 2009a. Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. ''Deep Sea Research Part II: Topical Studies in Oceanography'', 56(15), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018</ref>

<ref name="Meon2004">Meon, B., Amon, R., 2004. Heterotrophic bacterial activity and fluxes of dissolved free amino
acids and glucose in the Arctic rivers Ob, Yenisei and the adjacent Kara Sea. Aquat. Microb. Ecol. 37, 121–135. https://doi.org/10.3354/ame037121</ref>

<ref name="Nguyen2012">Nguyen, D., Maranger, R., Tremblay, J., Gosselin, M., 2012. Respiration and bacterial carbon
dynamics in the Amundsen Gulf, western Canadian Arctic. J. Geophys. Res. 117, 2011JC007343. https://doi.org/10.1029/2011JC007343</ref>

<ref name="Ortega-Retuerta2012">Ortega-Retuerta, E., Jeffrey, W. H., Babin, M., Bélanger, S., Benner, R., Marie, D., Matsuoka, A., Raimbault, P., & Joux, F. (2012). Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance, production and respiration on the Beaufort Sea margin. ''Biogeosciences'', 9(9), 3679–3692. https://doi.org/10.5194/bg-9-3679-2012</ref>

<ref name="Robinson2008">Robinson, C., 2008. Heterotrophic Bacterial Respiration, in: Kirchman, D.L. (Ed.), Microbial
Ecology of the Oceans. Wiley-Blackwell, New Jersey, pp. 299–334.</ref>

<ref name="Regaudie-de-Gioux2010">Regaudie-de-Gioux, A., Duarte, C.M., 2010. Plankton metabolism in the Greenland Sea during the polar summer of 2007. Polar Biol 33, 1651–1660. https://doi.org/10.1007/s00300-010-0792-
1</ref>

</references>

Winkler light-dark dissolved O2 bottle

2026-05-15T16:10:29Z

Anabelvonjackowski: /* Calculation of O2 Concentration */

File:Sample-Oxygen Calculation-Winkler.xls

2026-05-15T16:09:46Z

Anabelvonjackowski:

Winkler light-dark dissolved O2 bottle

2026-05-15T16:08:01Z

Anabelvonjackowski: /* Calculation of O_2 Concentration */

Winkler light-dark dissolved O2 bottle

2026-05-15T16:07:38Z

Anabelvonjackowski: /* Calculation of O2 Concentration */

Winkler light-dark dissolved O2 bottle

2026-05-15T16:07:10Z

Anabelvonjackowski: /* Calibrate Bottles */

Winkler light-dark dissolved O2 bottle

2026-05-15T16:06:16Z

Anabelvonjackowski: /* Prepare Bottles */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:56:14Z

Anabelvonjackowski: /* Calculation of O2 Concentration */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:53:27Z

Anabelvonjackowski: /* Calculation of O2 Concentration */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:53:16Z

Anabelvonjackowski: /* Calculation of O2 Concentration */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:52:54Z

Anabelvonjackowski: /* Calculation of O2 Concentration */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:45:54Z

Anabelvonjackowski: /* Analysis via titration */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:41:07Z

Anabelvonjackowski: /* Analysis via titration */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:40:17Z

Anabelvonjackowski: /* Analysis via titration */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:38:00Z

Anabelvonjackowski: /* Analysis via titration */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:37:25Z

Anabelvonjackowski: /* Analysis via titration */

Winkler light-dark dissolved O2 bottle

2026-05-15T15:35:47Z

Anabelvonjackowski: /* Sample Collection */