# Sampling in low oxygen aquatic environments: The deviation from anoxic conditions

Emilio Garcia-Robledo, Aurélien Paulmier, Sergey M. Borisov, Niels Peter
Revsbech

► To cite this version:

Emilio Garcia-Robledo, Aurélien Paulmier, Sergey M. Borisov, Niels Peter Revsbech. Sampling in low oxygen aquatic environments: The deviation from anoxic conditions. Limnology and Oceanography: Methods, 2021, 10.1002/lom3.10457. hal-04720525

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# Sampling in low oxygen aquatic environments: The deviation from anoxic conditions

Emilio Garcia-Robledo ID,<fnref n="1" />* Aurelien Paulmier ID,<fnref n="2" /> Sergey M. Borisov ID,<fnref n="3" /> Niels Peter Revsbech ID<fnref n="4" />

## Abstract

Studies of the impact of hypoxic or anoxic environments on both climate and ecosystems rely on a detailed characterization of the oxygen (O₂) distribution along the water column. The former trivial separation between oxic and anoxic conditions is now often redefined as a blurry concentration range in which both aerobic and anaerobic processes might coexist, both in situ and during experimental incubations. The O₂ concentrations during such incubations have often been assumed to be equal to in situ levels, but the concentration was rarely measured. In order to evaluate the actual oxygen concentration in samples collected from low-oxygen environments, a series of measurements were performed on samples collected in the Pacific oxygen minimum zones. Our results show a significant deviation from in situ anoxic conditions in samples collected by Niskin bottles where leakage from the bottle material resulted in O₂ concentrations of up to 1 µM. Subsequent sampling further increased the O₂ contamination. Sampling and analysis by Winkler method resulted in variable apparent concentrations of 2–4 µM. Two common procedures to avoid atmospheric contamination were also tested: allowing gentle overflow and keeping the sampling bottle submersed in a portion of the sampled water. Both procedures resulted in similar O₂ contamination with values of 0.5–1.5 µM when bottles were immediately closed and measurements performed with optical sensors, and 3–4 µM apparent concentration when analyzed by the Winkler method. Winkler titration is thus not suited for analysis of low-O₂ samples. It can be concluded that incubation under anoxic conditions requires deoxygenation after conventional sampling.

The characterization of low oxygen and anoxic environments has received increasing attention during the past few decades. The increased number of hypoxic events detected in the coastal areas and the expansion of the oceanic oxygen minimum zones (OMZs) (Horak et al. 2016; Stramma et al. 2008) place ocean deoxygenation as one of the most relevant problems caused by global warming (Breitburg et al. 2018; Sampaio et al. 2021). The distribution and extension of low oxygen environments has not only a direct impact in the abundance, diversity and distribution of macroorganisms (Breitburg et al. 2009; Vaquer-Sunyer and Duarte 2008), but also affect the composition and activity of the microbial community.

At the boundaries of the OMZs and other anoxic water masses, variations in the oxygen concentration of a few micromolar may cause large differences in microbial metabolism, pathways and rates. In the Eastern Tropical South Pacific

(ETSP) OMZ, aerobic processes such as ammonium and nitrite oxidation were active at nanomolar O₂ concentrations, and 3–5 µmol O₂ L⁻<fnref n="1" /> sufficed to reach maximum rates (Bristow et al. 2016). On the other side, anaerobic processes in the same OMZ such as denitrification and anammox were inhibited at nanomolar oxygen concentrations, reaching complete inhibition at concentrations below 1–2 µmol O₂ L⁻<fnref n="1" /> (Dalsgaard et al. 2014), while others have found substantial anammox and denitrification rates at considerably higher O₂ concentrations (Bristow 2018; Kalvelage et al. 2011). The water layers in the OMZs where both aerobic and anaerobic processes might coexist (< 5 µmol O₂ L⁻<fnref n="1" />) are often tens of meters in the upper oxycline and up to hundreds of meters in the lower oxycline (Tiano et al. 2014a). Estimates of global impacts of OMZs commonly assume an O₂ threshold when anaerobic processes are upscaled (Paulmier and Ruiz-Pino 2009), but due to the resolution of the historical oxygen concentration measurements, values of 5–20 µmol L⁻<fnref n="1" /> have often been assumed (Codispoti et al. 2001). Recent advances in sensor technology now makes it possible to measure O₂ in

<footnote n="1">¹Departamento de Biologia, Universidad de Cadiz, Cadiz, Spain</footnote>

<footnote n="2">²Laboratoire d'Etudes en Géophysique et Océanographie Spatiales (LEGOS), Université de Toulouse, Centre National de la Recherche Scientifique, Centre National d'Etudes Spatiales, Institut de Recherche pour le Développement, Toulouse, France</footnote>

<footnote n="3">³Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Graz, Austria</footnote>

<footnote n="4">⁴Department of Biology, Aarhus University Centre for Water Technology, Aarhus University, Aarhus, Denmark</footnote>

<footnote>*Correspondence: emilio.garcia@uca.es</footnote>
situ at few nanomolar detection limit and resolution (Larsen et al. 2016; Revsbech et al. 2011), and the same sensor technologies now show that low O₂ samples for laboratory incubations are O₂ contaminated during sampling and analysis. Although the oxygen contamination during the ex situ incubation of OMZs samples were previously quantified (De Brabandere et al. 2012; Lehner et al. 2015), the initial oxygen contamination produced by the sampling procedure remains poorly characterized.

Besides OMZs, other stratified aquatic environments such as freshwater lakes, fjords, and marine basins can also develop anoxic bottom waters. Unlike open ocean OMZs, euxinia is commonly present in these environments, showing certain accumulation of free sulfide (van Vliet et al. 2020). The potential O₂ contamination from the sampling procedure might be considered negligible under these conditions as the reduced compounds can consume the oxygen (Ingvorsen and Jorgensen 1979) and the O₂ contamination during sampling could pass unnoticed. However, the presence of small amounts of oxygen significantly increase some microbial processes such as methane and sulfide oxidation (Jørgensen et al. 1991; Milucka et al. 2015; Steinle et al. 2017) while the generation of oxidized sulfur compounds (elemental sulfur, thiosulfate, sulfite...) might enhance reducing microbial processes (Hayes et al. 2006). As a consequence, the measured rates of microbial processes in euxinic environments might be altered by the uncharacterized intrusion of oxygen during sampling.

In order to explore the oxygen contamination produced during sampling in anoxic environments, we performed a series of measurements in the Eastern Tropical North and South Pacific (ETNP and ETSP) OMZs as well as in the laboratory. By the use of STOX sensors (Revsbech et al. 2011) and high-sensitivity optodes (Lehner et al. 2015), we quantified the increase in measured oxygen concentration introduced by (1) the collection of the sample using standard Niskin bottles; (2) the transfer of the water sample from the Niskin bottle to gas-tight containers under various conditions (under N₂ atmosphere, with gentle overflow and in submerged containers); and (3) a standard Winkler O₂ determination procedure.

## Material and procedures

### Cruises locations, sampling, and in situ data collection

Data and samples were collected during three oceano-graphic cruises in the ETNP and ETSP OMZs. Samplings in the ETSP were performed during the activities of research dedicated to the minimum of oxygen in the Eastern Pacific (AMOP) cruise (February 2014) on board the R/V L'Atalante (IFREMER, France) off Peru. Another two samplings were performed in the ETNP off the Mexican coast: OC1705, May 2017, on board of the R/V Oceanus (Oregon State University) and SR1805, May 2018, on board of the R/V Sally Ride (Scripps Institution of Oceanography). During the three

cruises, ships were equipped with a Seabird SBE CTD system and a SBE-43 oxygen sensor. The SBE-43 oxygen sensors were calibrated before the cruises by the manufacturer (Seabird). Seawater samples were collected using Niskin bottles mounted on a CTD rosette with volumes of 12 L for R/V L'Atalante, 10 L for R/V Oceanus, and 20 L for R/V Sally Ride.

High-resolution oxygen profiles were carried out using custom-made STOX sensors mounted on an in situ STOX electronics unit (UNISENSE A/S, Denmark) that was connected to the Seabird CTD as described previously (Revsbech et al. 2011; Tiano et al. 2014a). STOX sensor signals were analyzed and calibrated as described previously (Tiano et al. 2014a). Based on the information obtained by the STOX sensors, different depths inside the anoxic core were selected for sampling. Up-cast STOX data were used to define the in situ oxygen concentration of samples collected by the Niskin bottles. In situ STOX sensors were not available during the AMOP cruise and depths were selected based on the information of the SBE-43 sensor.

### Oxygen in collected samples: Concentration inside the Niskin bottle and Winkler determination

The oxygen concentrations in the Niskin bottles were measured during the AMOP cruise (Table 1). Samples from the anoxic core at depths between 150 and 400 m were collected in 12-L Niskin bottles. Once the rosette arrived on board, the bottles were dismounted from the rosette and oxygen concentrations were measured using STOX sensors. For that purpose, a short (ca. 10 cm) piece of Tygon tubing (6 mm i.d.) was connected to the Niskin bottle and water flow was simultaneously directed toward two STOX sensors by the use of a glass Y-piece. Sensors tips were inserted inside the glass of the Y-piece to avoid oxygen contamination from the atmosphere. After 2 min of flow, a minimum of three STOX cycles (1 min each) were recorded.

Immediately after STOX readings, samples for Winkler oxygen determination were collected from the same bottle. From each Niskin bottle, three 160-mL Winkler flasks were filled, trying to minimize contamination by extensive initial rising and gentle overflow. Winkler reagents were added and flasks were closed and kept in darkness for 24 h prior to analysis (Carpenter 1965). The potential contamination caused by the replacement of the headspace in the Niskin bottle with air was minimized by collecting less than 50% of the total Niskin volume. Some contamination was thus possible, but the high reproducibility of the Winkler replicates in the sequentially collected samples suggests no significant impact. Dissolved oxygen titration was performed on board applying an autotitrator, a 20-mL burette and potentiometric end-point determination (reproducibility of 0.9% ± 0.1%) (Granéli and Granéli 1991).

### Sample collection and oxygen measurements: Overflow vs. submerged bottles

Samples were collected during cruise SR1805 off Mexico (May 2018) from the anoxic layers at 65 m at the coastal
<table><caption>**Table 1.** Oxygen concentration (in µmol O₂ L⁻¹) measured in the same Niskin bottle using STOX sensors and Winkler titration during the AMOP cruise in the ETSP off Peru. All samples were collected in the core of the OMZ in water layers apparently anoxic. All data are means ± standard deviation (SD), determined from the corresponding triplicates.</caption><thead><tr><th>Station_Cast</th><th>Date</th><th>Depth (m)</th><th>T (°C)</th><th>STOX</th><th>Winkler</th></tr></thead><tbody><tr><td>am11_01</td><td>04 Feb 2014</td><td>400</td><td>8.95</td><td>0.83 ± 0.14</td><td>4.07 ± 0.02</td></tr><tr><td>am11_14</td><td>06 Feb 2014</td><td>400</td><td>8.83</td><td>0.67 ± 0.07</td><td>3.21 ± 0.08</td></tr><tr><td>am13_03</td><td>07 Feb 2014</td><td>300</td><td>10.43</td><td>0.96 ± 0.07</td><td>2.56 ± 0.11</td></tr><tr><td>am25_02</td><td>14 Feb 2014</td><td>282</td><td>12.54</td><td>0.6 ± 0.08</td><td>2.85 ± 0.05</td></tr><tr><td>am25_02</td><td>14 Feb 2014</td><td>200</td><td>11.86</td><td>0.56 ± 0.24</td><td>3.27 ± 0.02</td></tr><tr><td>am25_11</td><td>16 Feb 2014</td><td>288</td><td>13.74</td><td>0.45 ± 0.06</td><td>2.87 ± 0.02</td></tr><tr><td>am25_11</td><td>16 Feb 2014</td><td>150</td><td>13.29</td><td>0.25 ± 0.06</td><td>3.99 ± 0.01</td></tr><tr><td>am25_11</td><td>16 Feb 2014</td><td>80</td><td>11.92</td><td>0.46 ± 0.07</td><td>4.47 ± 0.04</td></tr><tr><td>am28_02</td><td>17 Feb 2014</td><td>300</td><td>13.45</td><td>0.47 ± 0.12</td><td>3.82 ± 0.02</td></tr><tr><td>am28_02</td><td>17 Feb 2014</td><td>153</td><td>12.71</td><td>0.39 ± 0.05</td><td>3.2 ± 0.06</td></tr><tr><td>am28_02</td><td>17 Feb 2014</td><td>100</td><td>10.63</td><td>0.66 ± 0.09</td><td>3.23 ± 0.04</td></tr><tr><td>am28_16</td><td>19 Feb 2014</td><td>300</td><td>12.80</td><td>0.49 ± 0.09</td><td>5.02 ± 0.02</td></tr><tr><td>am28_16</td><td>19 Feb 2014</td><td>200</td><td>12.29</td><td>0.58 ± 0.18</td><td>6.43 ± 1.42</td></tr><tr><td>am28_16</td><td>19 Feb 2014</td><td>150</td><td>10.85</td><td>0.47 ± 0.14</td><td>4.12 ± 0.04</td></tr></tbody></table>

station PS3. A piece of 50-cm Tygon tubing piece was connected to the Niskin bottle upon arrival on deck and water was transferred into six Winkler bottles (65 mL) by two standard procedures:

1. Gentle overflow: tubing was inserted inside the bottles and water was left to overflow for several volumes before closing the bottles with a solid glass stopper.

2. Submerged bottles: one beaker (0.5 L) was quickly filled up with water from one Niskin while Winkler bottles were kept submerged. Water flowing from another Niskin bottle collected at the same depth was used to overflow the submerged Winkler bottles by several volumes before closing with a solid glass stopper while submerged.

Winkler bottles were equipped with O₂ optode sensing spots and oxygen concentration was measured after 10 min by luminescence measuring oxygen sensor (LUMOS) technology (Lehner et al. 2015).

In order to exclude the oxygen contamination from Niskin bottle or chemical interferences, the same experiment was repeated in the laboratory using demineralized water. Water was deoxygenated by gentle bubbling with N₂ (g) for 20–30 min in a 5-L glass bottle. Winkler bottles (65 mL) were filled up with the artificial anoxic water following the same two procedures described above. Oxygen concentrations in the collected samples were measured in the Winkler bottles with LUMOS optodes (Lehner et al. 2015) or trace oxygen optodes (PyroScience GmbH). Samples for the determination of oxygen concentration by Winkler titration were also collected in 270-mL glass bottles, following the procedures described above of gentle overflow and submerged vessels. Immediately after collection, Winkler reagents were added to

the bottles exposed to air (filled by overflow) or in submerged conditions using micropipettes and closed with glass stoppers. The bottles were kept submerged in water and analyzed after 24 h following the procedures described by Labasque et al. (2004).

## Results and discussion

### Sample collection in anoxic layers: Oxygen contamination from Niskin bottles

The standard procedure for sample collection in oceanography is by use of Niskin bottles, commonly attached to a rosette for multiple sample collection and controlled by the CTD unit. These bottles, normally made of polyvinyl chloride (PVC), are closed by the action of an elastic mechanism localized inside the bottle, commonly made of silicone or rubber tubing and more recently by polytetrafluoroethylene-coated (commercially known as Teflon) or epoxy-coated springs. These polymers have certain oxygen permeability and solubility (Robb 1968) and the often long resting periods on deck between casts ensure equilibration of these materials with the atmosphere. After closing of the bottle, the O₂ dissolved in the polymers is equilibrating with the O₂ concentration in the enclosed water. Sampling of anoxic water masses induces the largest oxygen gradient between the polymers and the confined water mass and oxygen thus leaks into the confined water sample.

We performed STOX measurements of O₂ contamination in the Niskin bottles during two cruises to the Pacific OMZs (AMOP and OC1705). In both cases and with two different approaches, O₂ contamination values in water samples collected at anoxic water depths were similar. Oxygen concentrations measured in a direct flow from the Niskin bottle ranged from 0.25 to 0.96 µM, being on average 0.56 ± 0.18 µM
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Oxygen concentration measured in the same Niskin bottle using STOX sensors and Winkler titration during the AMOP cruise at the ETSP off Peru. All samples were collected in apparently anoxic water layers. Data measured *in situ* with the SBE43 oxygen sensor mounted on the CTD are shown for comparison. Bar graph in the right panel shows mean ± standard deviation (SD) for each sample.</figcaption></figure>

(mean ± SD, *n* = 14, Fig. 1; Table 1). Similarly, oxygen concentration in water samples collected from the Niskin bottles into glass bottles under a N₂ (g) atmosphere ranged from 0.39 to 2.08 µM, being on average 0.81 ± 0.15 µM (mean ± SD, *n* = 4, Table 2). In spite of following the same procedure during several days, in one of the samplings (07 May 2017, Table 2) oxygen contamination values in the glass bottles were much higher, ranging from 0.87 to 4.45 µM, being on average 2.24 ± 1.41 µM (mean ± SD, *n* = 5).

The amount of oxygen dissolved in the polymers might depend of the history of the Niskin bottle before the sample confinement and thus it will be highly variable and unpredictable. Oxygen contamination values in the Niskin bottles showed a significant and negative correlation with temperature (*p* < 0.01, Spearman's rank correlation), with higher oxygen contamination values at the lowest in situ temperatures. In addition, colder waters are usually located at greater depths increasing the time between the confinement

of the water sample and the collection of samples on deck. The O₂ contamination thus also exhibited a positive correlation with depth, but the correlation was not significant (*p* = 0.055, Spearman's rank correlation).

Besides these factors, other unpredictable events might significantly increase the contamination inside the bottles, such as the presence of air bubbles inside the bottle. Bubbles can be trapped inside the Niskin bottles, being imprisoned or attached in the closing mechanism, lids, walls, and so on. After the closing of the bottle, the oxygen in the bubble can dissolve in the sampled water, increasing the oxygen concentration. For instance, the high oxygen contamination measured in one of the samplings (07 May 2017, Table 2), could be caused by the presence of one single air bubble with a diameter of 6 mm at 100 m, increasing the oxygen concentration by 1.2 µM. Although a 6-mm diameter bubble might be unlikely, smaller bubbles might be more frequent and they also equilibrate faster, providing an unpredictable contamination of the sampled water.

The average contamination in the Niskin bottles of 0.56 ± 0.18 µM is similar to the accuracy of the Winkler method when analyzing air-equilibrated samples (Carpenter 1965; Labasque et al. 2004), and it might be unnoticeable when measurements in oxygenated water masses are performed. Contrary, for the anoxic water masses of the OMZ core or other euxinic environments, the increase in O₂ concentration might significantly change chemical and microbial transformations in the water samples. Some of the most relevant microbial processes have been shown to be highly sensitive to low O₂ concentrations, and processes like denitrification and anammox may be partly or totally inhibited (Dalsgaard et al. 2014) at submicromolar O₂ levels. On the other side, aerobic processes, such as ammonium and nitrite oxidation (Bristow et al. 2016), methane oxidation (Milucka et al. 2015; Steinle et al. 2017), or aerobic respiration (Garcia-Robledo et al. 2016; Gong et al. 2016), are highly efficient at low oxygen concentrations, with Km values below or similar to the O₂ contaminations measured in this study. The unavoidable oxygen contamination of the collected samples thus most likely result in wrong interpretations or estimations of microbial metabolism in the anoxic core of the OMZs if the

<table><caption>**Table 2.** Oxygen concentration (in µmol O₂ L⁻¹) measured in samples collected from Niskin bottles in full glass bottles using LUMOS sensors during the OC1705 cruise in the ETNP off Mexico. All samples were collected in the core of the OMZ in apparently anoxic water layers. Vigorous N₂ flow was applied inside the glass sampling bottles during the filling procedure to prevent air contact and oxygen contamination. Values are means ± SD, determined from the *N* replicates.</caption><thead><tr><td>Date</td><td>St.</td><td>Lon. (°E)</td><td>Lat. (°N)</td><td>Depth (m)</td><td>T (°C)</td><td>[O₂]</td><td>N</td></tr></thead><tbody><tr><td>06 May 2017</td><td>6</td><td>255.099</td><td>18.900</td><td>100</td><td>13.94</td><td>0.94 ± 0.30</td><td>7</td></tr><tr><td>07 May 2017</td><td>6</td><td>255.099</td><td>18.900</td><td>100</td><td>14.02</td><td>2.24 ± 1.41</td><td>5</td></tr><tr><td>10 May 2017</td><td>8</td><td>255.103</td><td>18.198</td><td>110</td><td>13.78</td><td>0.81 ± 0.58</td><td>7</td></tr><tr><td>11 May 2017</td><td>8</td><td>255.103</td><td>18.198</td><td>105</td><td>14.03</td><td>0.67 ± 0.32</td><td>7</td></tr></tbody></table>
collected samples are investigated without prior deoxygenation. Even with prolonged N₂ or He bubbling of water in a large container, and transfer with procedures optimized to minimize atmospheric contamination it was, however, very difficult to get final O₂ concentrations inside the custom-made all-glass bottles of less than 100 nM (Tiano et al. 2014b). To obtain samples much below this O₂ concentration it is apparently necessary to bubble with strictly O₂-free gas like helium directly within the glass container (Lehner et al. 2015), which is only possible with a gas headspace. By this approach, it is possible to reach O₂ concentrations below a detection limit of 1 nM (Sun et al. 2021).

Using an intensive bubbling deoxygenation procedure, other gases are also removed along with oxygen. Some gases of general interest in OMZs, such as methane or nitrous oxide, are added subsequently for transformation studies (Bourbonnais et al. 2021; Thamdrup et al. 2019). CO₂ is also partially removed, which can modify the final pH of the sample. However, CO₂ (g) concentration in seawater is relatively low as compared to the bicarbonate pool in the pH range of OMZ seawater (Hernandez-Ayon et al. 2019) and the change in both total CO₂ content (carbonate + bicarbonate + carbon dioxide) and pH could be considered negligible for most studies. We measured a drop of 0.1–0.2 pH units after 30 min of vigorous bubbling of coastal seawater with N₂ (g) in laboratory tests (data not shown). However, longer deoxygenation procedures could change these values, so specific validations are required if the main objective of the study is on pH-sensitive compounds.

The oxygen contamination might be removed by processing the sample after collection. However, it should be noted that this procedure does not guarantee the conservation of reduced compounds (sulfide, iodine, and so on), commonly present in anoxic waters. The oxygen contamination (up to 3–4 µM according to our measurements) might oxidize some of the reduced compounds initially present in the hypoxic/ anoxic water and modify the chemical composition of the sample. Oxidized compounds such as elemental sulfur, thiosulfate, sulfite, or iodate are the substrate for different microbial processes (Hayes et al. 2006; Moriyasu et al. 2020; van Vliet et al. 2020) and the generation of such compounds during the sampling might result in the overestimation of microbial processes. Although this impact was not studied in detail in the present study, sampling in euxinic environments should consider the indirect effects of oxygen contamination by interpretation of the results.

## Sample collection in anoxic layers: Overestimation by Winkler measurements

Measurement by the Winkler method is the gold standard for the determination of oxygen concentrations in the ocean. Besides their use for monitoring the oxygen distribution, they are also used for the calibration of sensors mounted on CTDs and other autonomous platforms such as moorings, buoys, and so on. The method is relatively simple and provides high

accuracy and precision in seawater samples (Carpenter 1965). However, by all modifications of the method (Carpenter 1965; Helm et al. 2012; Labasque et al. 2004; Langdon 2010), the determined O₂ concentrations may deviate strongly from actual values in low O₂ samples, and in addition come contamination problems by sampling.

As discussed before, collecting low-O₂ samples by Niskin bottles increases the oxygen concentration of the bottled water with 0.5–1 µmol L⁻¹. During the AMOP cruise, oxygen concentrations were measured in samples from the same bottles by Winkler, and values ranged from 2.56 to 6.43 µmol L⁻¹, with an average of 3.79 ± 1.03 µmol L⁻¹ (mean ± SD, *n* = 14, Table 1; Fig. 1). The titrations thus resulted in apparent O₂ concentrations that were 1.60–5.85 µmol L⁻¹ higher than the actual values, with an average increase of 3.23 ± 1.08 µmol L⁻¹ (mean ± SD, *n* = 14).

Measurement by Winkler in low O₂ waters has two important sources or error: Gas exchange during the manipulation of the samples/addition of reagents (excluding the oxygen added with reagents, taken into account by calculations), and interference from chemical compounds in the sample (Broenkow and Cline 1969; Langdon 2010; Wong and Li 2009). Some reducing compounds such as sulfide decrease the oxygen concentration measured (Broenkow and Cline 1969). However, most OMZs are characterized by lack of sulfide at relevant concentrations (Cutter et al. 2018; Schunck et al. 2013). On the other hand, nitrite and iodate increase the apparent oxygen concentration measured (Broenkow and Cline 1969; Wong 2012; Wong and Li 2009) and might be found at relevant concentrations in OMZs.

Iodate concentration in the oceans is in the range of 0.35–0.45 µmol L⁻¹, and might eventually increase to concentrations of 0.75 µmol L⁻¹ in coastal areas or in layers with recent interaction with the sediment (Evans et al. 2020; Moriyasu et al. 2020; Wong and Li 2009). The interference corresponds to 1.5 mol of O₂ per mole of iodate (Wong and Li 2009). Consequently, the overestimation range in the open oxic ocean environment might be between 0.52 and 0.63 µmol O₂ L⁻¹. In the anoxic layers of the OMZ, iodate is biologically consumed and samples collected in anoxic waters of the ETNP OMZ and Arabian Sea OMZ have iodate concentrations below 0.2 µmol L⁻¹ (Evans et al. 2020; Farrenkopf and Luther III 2002) that could increase the apparent oxygen concentration measured by up to 0.3 µmol O₂ L⁻¹. Iodate could thus only contribute by 5–18% to the apparent increase in oxygen concentration measured with Winkler method.

One mole of nitrite gives an interference equivalent to 0.25–0.5 mol of oxygen (Wong 2012), and as NO₂⁻ in the anoxic core may be present at concentrations up to 7 µmol L⁻¹ (Peters et al. 2016), nitrite could potentially cause all the overestimation by our Winkler titrations (Broenkow and Cline 1969). We therefore conducted a series of laboratory determinations with nitrite, iodate and O₂ free demineralized water (Fig. 2). The Winkler determinations were in this case
<figure><img src="image_3.png" /><figcaption>**Fig 2.** ETNP OMZ (optodes): Oxygen concentration in samples collected at anoxic water in the ETNP during the OC1705 cruise. Samples were collected directly from the Niskin bottle to glass Winkler bottles (60–65 mL total volume) allowing gentle overflow before the closing of the bottle with glass stoppers, or filling the bottles submerged in a beaker containing the sampled water to avoid atmospheric contamination (n = 3). Oxygen optode spots were preinstalled inside the bottles and oxygen was measured with LUMOS technology. Laboratory (optodes): The same procedure was repeated in laboratory, deoxygenating 5 L of distilled water by gentle N₂ (g) bubbling for 35–40 min and transferring the anoxic water by siphoning to the same Winkler bottles as used during the OC1705 cruise. The experiment was repeated to get *n* = 12. Laboratory (Winkler): Similar procedure was performed in the laboratory, collecting samples in 270-mL bottles and measuring O₂ concentration with Winkler determination as described in Labasque et al. (2004; *n* = 7). Bar graphs are means ± SE from the *n* replicates.</figcaption></figure>

done with both bottles filled the usual way with Tygon tubing at the bottom of the bottles and allowing gentle overflow, and with submerged bottles. Under the well-controlled laboratory conditions, the determined O₂ concentrations were extremely similar to the O₂ concentrations measured by Winkler in Niskin bottle water (Table 1), indicating that nitrite and iodate were not the major factors causing the overestimation by Winkler observed with anoxic core samples. There was no statistically significant difference between the two types of bottle filling and the average determined concentration was 3.03 ± 0.36 µM which is very close to the average O₂ concentration measured in water from Niskin bottles by Winkler (3.81 ± 0.28 µM).

To avoid the uncertainty introduced by Winkler on the difference between usual filling procedure and submerged bottles, the comparison was also conducted with Winkler bottles containing optode dots. Here, the field sampling directly from the Niskin bottle showed less contamination by submerged bottles than by bottles filled under air (ETNP OMZ, Fig. 2). The picture was not so clear when the experiment was repeated in the laboratory with a higher number of replicates

(Laboratory, Fig. 2). It is thus not clear whether the filling of bottles while submerged constitutes a real advantage. The O₂ concentration in the water bath evidently affect the effectiveness of the submerged procedure, and the number of volume changes by flushing have an affect by both procedures.

## Recommendations

O₂ contamination of anoxic or near-anoxic water by collection in PVC Niskin bottles and subsequent transfer into glass containers is unavoidable and O₂ is introduced at concentrations of 0.5–1.0 µM. Determination of the in situ O₂ concentration from such water samples is thus not possible, and by even very careful use of the Winkler technique, more O₂ is introduced so that in situ anoxic water appeared to contain 2–4 µM O₂. Determination of in situ O₂ concentrations must therefore be conducted by equipment that can actually measure in situ and Winkler data at low concentrations (< 30 µM) should not be used for calibration of in situ sensors to avoid significant errors (> 10%). Water sampling from anoxic environments introduces O₂ in the samples and deoxygenation of samples are mandatory if truly anoxic conditions are required. Deoxygenation of water samples by bubbling with O₂-free gas should preferably be conducted in all-glass containers in which the experiments are to be conducted, as a laboratory transfer of water leads to unpredictable O₂ contamination.

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## Acknowledgments

We would like to thank Lars B. Pedersen at Aarhus University for the construction of STOX sensors. We are grateful to the cruise leaders Bess B. Ward and Frank Stewart for the invitation to participate in OMZ cruises. We also thank the captains and crews of the R/Vs L'Atalante, New Horizon, Oceanus and Sally Ride. We additionally thank A. Franco-Garcia, M. Giraud, J. Ledesma, F. Baurand, D. Lefèvre, B. Dewitte, C. Maes, V. Garçon and the PACOP platform (Toulouse) for operational and experimental support during the AMOP cruise. This work was funded by the Poul Due Jensen Foundation and co-financed by the 2014-2020 ERDF Operational Programme and by the Department of Economy, Knowledge, Business and University of the Regional Government of Andalusia (to EGR, project reference FEDER-UCA18-107225).

## Conflict of Interest

None declared.

Submitted 15 February 2021

Revised 06 August 2021

Accepted 11 August 2021

Associate editor: Gregory Cutter