Winkler light-dark dissolved O2 bottle

• Page authors: Anabel von Jackowski
• Responsible curator: Anabel von Jackowski

Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)

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

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. .

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

1. 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.
2. 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.
3. 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!!
4. 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.
5. 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.
6. 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

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 ($V$) of the bottle in milliliters.

$V=\frac{\text{Weight B}-\text{Weight A}}{\text{Density of Water at }{T}^{\circ }\text{C}}$

Sample Collection

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
10. 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.
11. 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

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.
13. 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.
14. Titration - Step 1
    Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
15. 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.
16. 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.
17. 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.
18. 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 $10\text{ ml}$ of $5\times 10^{-3}{\text{ N KH(IO}}_3{)}_2$, which corresponds to $5\text{ ml}$ of $0.02{\text{ M Na}}_2{\text{S}}_2{\text{O}}_3$.

$\text{factor}=\frac{5}{V}$

where $V$ = volume of thiosulphate used $[\text{ml}]$

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

${\text{O}}_2[\text{ml}]=0.112\times a\times \text{factor}$

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

Where

$a$ = consumption of thiosulphate $[\text{ml}]$

$b$ = volume of the sampling bottle $[\text{ml}]$

$2$ = volume of reagents added $[\text{ml}]$ (Attention: This volume may vary depending on the protocol)

Note: A sample calculation sheet is linked here: File: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) or a size-fraction Winkler method.

Community Respiration

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)
Arctic Ocean	Fram Strait	July 2007	6.2 ± 0.87	Regaudie-de-Gioux and Duarte (2010)
Arctic Ocean	Greenland Current	July 2007	2.1 ± 1.02	Regaudie-de-Gioux and Duarte (2010)
Pacific Ocean	Station ALOHA	May 2001 - May 2002	0-1.5	Williams et al., (2004)
Global Ocean	Global	-	3.3 ± 0.15	Robinson & Williams et al., (2005)

Bacterial Respiration

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
Arctic Ocean	Kara Sea	Aug-Sep 2001	0.48 ± 0.57	Meon and Amon (2004)

More CR and BR rates are available via the "A global dataset of marine pelagic microbial respiration" database