Study site and sampling

Surface water was collected from Lake Svartkulp (59.9761313° N, 10.7363544° E; southeast Norway) north of Oslo, Norway, on 4 September 2019. A 5 L plastic bottle was gently pushed into the water and progressively tilted to let the water flow into the bottles without bubbling. The bottle aperture was covered with a 90 µm plankton net to avoid sampling large particles. This procedure was repeated five times to yield a total water volume of 25 L. The 5 L water bottles were immediately brought back to the lab. Upon arrival at the laboratory, after temperature equilibration, water from the 5 L bottles was slowly poured, to limit gas exchange with the ambient air, into a 25 L tank to provide a single bulk sample to start the incubation experiment. Filtration, e.g. with 0.45 or 0.2 µm filters, was avoided to minimize changes in dissolved gas concentrations (e.g. Magen et al., 2014). The mixed water sample (25 L) was sub-sampled (0.5 L) for the determination of alkalinity (127 µmol L⁻¹), pH (6.73), ammonium (3 µg N L⁻¹), nitrate (5 µg N L⁻¹), total N (230 µg N L⁻¹), phosphate (1 µg P L⁻¹), total P (9 µg P L⁻¹) and total organic carbon (TOC; 8.9 mg C L⁻¹), all analysed by standard methods at the accredited Norwegian Institute for Water Research (NIVA) lab (see Table S1 in the Supplement). The in situ temperature of the lake water was measured with a handheld thermometer and was 18.5 °C. Note that particulate organic carbon is a negligible fraction of TOC in Norwegian lake waters, representing on average less than 3 % (de Wit et al., 2023).

Lake Svartkulp was selected for this experiment because it is representative of low-ionic-strength Northern Hemisphere lakes, typically found in granitic bedrock regions in northeast America and Scandinavia. It is a typical low-productivity, heterotrophic, slightly acidic to neutral, moderately humic lake. Similar lakes are found in southern Norway (de Wit et al., 2023), large parts of Sweden (Valinia et al., 2014), Finland, Atlantic Canada (Houle et al., 2022), Ontario, Quebec and northeast USA (Skjelkvåle and de Wit, 2011; Weyhenmeyer et al., 2019).

Laboratory incubation experiment with different preservatives and storage times

The experimental design involved incubating 72 borosilicate glass bottles (120 mL) filled with lake water from our 25 L bulk sample subjected to four different treatments: addition of 240 µL of a preservative solution of (i) HgCl₂, (ii) CuCl₂ or (iii) AgNO₃ or addition of 240 µL of (iv) Milli-Q water. The bottles amended with Milli-Q water are hereafter referred to as “unfixed”. The 72 bottles were divided into three groups which were incubated cold (+4 °C) and dark for 24 h, 3 weeks or 3 months, respectively, before being processed for dissolved gas analysis by gas chromatography. These incubation times were selected to represent situations where samples are processed directly upon return to the laboratory (24 h) or after medium-term (3 weeks) to long-term (3 months) storage. At each time point and for each treatment, a group of six bottles were further processed for dissolved gas analysis. Concentrations of O₂, N₂, N₂O, CO₂ and CH₄ were determined by gas chromatography (see below) using the headspace technique following Yang et al. (2015). The pH was not measured at the end of the storage period.

In detail, within 3 h of lake water sampling, the 120 mL bottles were gently filled with water from the mixed sample (25 L). Each 120 mL bottle was slowly lowered into the water and progressively tilted to let the water flow into the bottle without bubbling. The bottle was then capped under water with a gastight butyl rubber stopper after ensuring that there were no air bubbles in the bottle. The bottles were randomized prior to preservative or Milli-Q treatment. The preservative or Milli-Q amendment was pushed in each bottle with a syringe and needle through the rubber septum. To avoid overpressure, another needle was placed through the septum at the same time, at least 2 cm above the other needle, to allow an equivalent volume of clean water to be released.

Stock solutions of HgCl₂, CuCl₂ and AgNO₃ were prepared according to Table 1 using high-accuracy chemical equipment (e.g. high-accuracy scale, volumetric flasks). The Ag (silver nitrate EMSURE^® ACS; Merck KGaA, Germany) Cu (copper(II) chloride dihydrate; Merck Life Science ApS, Norway) and Hg (mercury(II) chloride; undetermined) salts were dissolved in Milli-Q ultrapure water (> 18 MΩ cm). For measurement of CO₂ in seawater samples, the standard method involves poisoning the samples by adding a saturated HgCl₂ solution in a volume equal to 0.05 %–0.02 % of the total volume (Dickson et al., 2007). We used this as a starting point and added 0.02 % saturated HgCl₂ solution to 18 bottles (240 µL of HgCl₂ 10× diluted saturated solution), resulting in a sample concentration of 14 µg HgCl₂ mL⁻¹ (51.6 µM; Table 1). Based on estimated toxicity relative to Hg (Deheyn et al., 2004; Halmi et al., 2019), the silver and copper salts were added in molar concentrations equal to 2 and 3 times the molar concentration of HgCl₂, respectively (Table 1), although this varies between species of microorganisms and environmental matrices (Hassen et al., 1998; Rai et al., 1981).

Table 1Stock and sample concentrations of HgCl₂, CuCl₂ and AgNO₃.

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Additional 24 h incubation experiment with different preservatives for pH measurements

Since pH was not measured at the end of the first incubation experiment, we performed an additional experiment to document any potential rapid (within 24 h) impacts of preservative on pH. A total of 48 borosilicate glass bottles (120 mL) filled with lake water were subjected to the same four different treatments as the first experiment described above: HgCl₂, CuCl₂, AgNO₃ or Milli-Q water amendments. To this end, a 20 L water tank was filled with surface water from Lake Svartkulp on 14 December 2023. The water tank was immediately returned to the laboratory and left for 24 h to equilibrate to the room temperature. On 15 December, 120 mL bottles were gently filled with water from the bulk 20 L sample, as described above. The bottles were randomized prior to preservative or Milli-Q treatment performed as described above. The bottles were then incubated at room temperature for 2 or 24 h. The pH was measured in the initial unamended lake water, in 24 bottles opened after 2 h incubation, and in 24 bottles opened after 24 h incubation. The pH measurements were performed with a WTW Multi 3620 pH meter calibrated using a two-point calibration at pH 4 and 7. All pH measures were corrected for temperature. The water temperature of the water samples during pH measurements ranged between 19.1 and 21.2 °C.

Study site and sampling

Water samples were collected from Lake Lundebyvannet located southeast of Oslo (59.54911° N, 11.47843° E; southeast Norway). Two sets of samples were taken from 1, 1.5, 2 and 2.5 m depth using a water sampler once or twice a week between April 2020 and January 2021 for the determination of (i) dissolved CO₂ by gas chromatography (GC) analysis following fixation with HgCl₂ and (ii) DIC analysis with a TOC analyser. Samples for GC analysis were filled into 120 mL glass bottles (as described above for the 72 incubation bottles), which were sealed with rubber septa under water without air bubbles. Samples for GC analysis were preserved in the field by adding a half-saturated (at 20 °C) solution of HgCl₂ (150 µL) through the rubber seal of each bottle using a syringe, as described above the 72 incubation bottles, resulting in a concentration of 161 µM similar to previous studies (Clayer et al., 2021; Hessen et al., 2017; Yang et al., 2015). Samples for DIC analysis were filled without bubbles in 100 mL Winkler glass bottles that were sealed airtight directly after sampling. These samples were not fixed in any way and were analysed by a TOC analyser within 2 h. Note that estimation of dissolved CO₂ concentrations from pH and DIC is the least uncertain method of indirect CO₂ concentration with estimated relative error of 6 % or less (Golub et al., 2017). Lake water temperature and pH were measured in situ using HOBO pH data loggers placed at 1, 1.5, 2 and 2.5 m (Elit, Gjerdrum, Norway).

Lake Lundebyvannet has a surface area of 0.4 km² and a maximum depth of 5.5 m. It often experiences large blooms of Gonyostomum semen over the summer between May and September (Hagman et al., 2015; Rohrlack et al., 2020). The lake water is characterized by high and fluctuating concentrations of humic substances (with DOC concentrations ranging from 8 to 28 mg C L⁻¹), ammonium (5 to 100 µg N L⁻¹), nitrate (20 to 700 µg N L⁻¹), total N (average of 612 µg N L⁻¹), phosphate (2 to 4 µg P L⁻¹), total P (average of 28 µg P L⁻¹; Rohrlack et al., 2020; Hagman et al., 2015), a fluctuating pH (from 5.5 to 7.3), weak ionic strength with alkalinity ranging between 30 and 150 µmol L⁻¹, and electric conductivity varying from 40 to 70 µS cm⁻¹. For more details, see Rohrlack et al. (2020).

Lake Lundebyvannet was selected for this experiment because it is representative of productive, low-ionic-strength Northern Hemisphere lakes typically found in the southern part of granitic bedrock regions in northeast America and Scandinavia.

Gas chromatography

Headspace was prepared by gently backfilling sample bottles with 20–30 mL helium (He; 99.9999 %) into the closed bottle while removing a corresponding volume of water. Care was taken to control the headspace pressure within 5 % of ambient air, and a slight He overpressure was released before equilibration. The bottles were shaken horizontally at 150 rpm for 1 h to equilibrate gases between the sample and headspace. The temperature during shaking was recorded by a data logger. Immediately after shaking, the bottles were placed in an autosampler (GC-Pal, CTC, Switzerland) coupled to a GC instrument with He back-flushing (model 7890A, Agilent, Santa Clara, CA, USA). Headspace gas was sampled (approx. 2 mL) by a hypodermic needle connected to a peristaltic pump (Gilson MINIPULS 3), which connected the autosampler with the 250 µL heated sampling loop of the GC instrument.

The GC instrument was equipped with a 20 m wide-bore (0.53 mm) PoraPLOT Q column for separation of CH₄, CO₂ and N₂O and a 60 m wide-bore Molsieve 5 Å PLOT column for separation of O₂ and N₂, both operated at 38 °C and with He as the carrier gas. N₂O and CH₄ were measured with an electron capture detector run at 375 °C with $\mathrm{Ar}/{\mathrm{CH}}_{\mathrm{4}}$ ($\mathrm{80}/\mathrm{20}$) as makeup gas and a flame ionization detector, respectively. CO₂, O₂ and N₂ were measured with a thermal conductivity detector (TCD). Certified standards of CO₂, N₂O and CH₄ in He were used for calibration (AGA, Germany), whereas air was used for calibrating O₂ and N₂. The analytical error for all gases was lower than 2 %. For the Lake Lundebyvannet time series, CO₂ was separated from other gases using the 20 m wide-bore (0.53 mm) PoraPLOT Q column, while the other gases were not measured.

The results from gas chromatography give the relative concentration of dissolved gases (in ppm) in the headspace in equilibrium with the water. For the lab experiment with Svartkulp samples (Sect. 2.1), the concentration of dissolved gases in the water at equilibrium with the headspace was calculated from the temperature-corrected Henry constant in water using Carroll et al. (1991) for CO₂, Weiss and Price (1980) for N₂O, Yamamoto et al. (1976) for CH₄, Millero (2002) for O₂, and Hamme and Emerson (2004) for N₂. For the Lake Lundebyvannet time series (Sect. 2.2), the concentration of CO₂ in the water samples was determined using temperature-dependent Henry's law constants given by Wilhelm et al. (1977). The quantities of gases in the headspace and water were summed to find the concentrations and partial pressures of dissolved gases from the water collected in the field as follows:

where [gas] is the gas aqueous concentration, p_gas is the gas partial pressure, H is the Henry constant, V_water is the volume of water sample during headspace equilibration, V_headspace is the headspace gas volume during equilibration, R is the gas constant and T is the temperature during headspace equilibration (recorded during shaking). The calculations were similar to those of Yang et al. (2015).

DIC analyses

DIC analysis was performed for the Lake Lundebyvannet time series using a Shimadzu TOC-V CPN (Oslo, Norway) instrument equipped with a non-dispersive infrared (NDIR) detector with O₂ as a carrier gas at a flow rate of 100 mL min⁻¹. Two to three replicate measurements were run per sample. The system was calibrated using a freshly prepared solution containing different concentrations of NaHCO₃ and Na₂CO₃, and standards were measured in between each sixth sample. CO₂ concentrations in water samples ([CO₂]) were calculated on the bases of temperature, pH and DIC concentrations as follows (Rohrlack et al., 2020):

where [H⁺] is the proton concentration (10^−pH), C_T is the dissolved inorganic carbon concentration and Z is given by

Here K₁ and K₂ are the first and second carbonic acid dissociation constants adjusted for temperature (pK₁=6.41 and pK₂=10.33 at 25 °C; Stumm and Morgan, 1996).

pCO₂ and saturation deficit

Lake Lundebyvannet CO₂ concentrations provided by GC and DIC analyses were converted to pCO₂ (in µatm) as follows:

where K_H is the Henry constant for CO₂ adjusted for the in situ water temperature (Stumm and Morgan, 1996) and P_atm is the atmospheric pressure in bar approximated by

Here altitude is the altitude above sea level of Lake Lundebyvannet (158 m). Finally, the CO₂ saturation deficit (${\text{Sat}}_{{\mathrm{CO}}_{\mathrm{2}}}$ in µatm) was given by

where [CO₂]_air is the pCO₂ in the air (416 µatm for 2020 in southern Norway retrieved from the EBAS database; NILU, 2022; Tørseth et al., 2012). ${\text{Sat}}_{{\mathrm{CO}}_{\mathrm{2}}}$ gives the direction of CO₂ flux at the water–atmosphere interface, and its product with gas transfer velocity determines the CO₂ flux at the water–atmosphere interface, i.e. whether lake ecosystems are sinks (${\text{Sat}}_{{\mathrm{CO}}_{\mathrm{2}}}<\mathrm{0}$) or sources (${\text{Sat}}_{{\mathrm{CO}}_{\mathrm{2}}}>\mathrm{0}$) of atmospheric CO₂.

Statistical analyses

The effect of storage time and treatment on five dissolved gases (O₂, N₂, CO₂, CH₄, N₂O) from the Lake Svartkulp samples was tested with a two-way ANOVA at an alpha level adapted using the Bonferroni correction for multiple testing; i.e. $\mathit{\alpha }=\mathrm{0.05}/\mathrm{5}=\mathrm{0.01}$. To evaluate the impact of Hg fixation on Lake Lundebyvannet samples, [CO₂] values determined by headspace equilibration and GC analysis of HgCl₂-fixed samples were compared with those calculated from DIC measurements of unfixed samples with a paired t test.

A regression analysis was performed to describe the overestimation of CO₂ concentrations caused by HgCl₂ fixation in Lake Lundebyvannet samples as a function of pH. The total CO₂ concentration in the HgCl₂-fixed samples ($[{\mathrm{CO}}_{\mathrm{2}}{]}_{{\mathrm{HgCl}}_{\mathrm{2}}}$) can be expressed as

where [CO₂]_i is the initial CO₂ concentration prior to HgCl₂ fixation, i.e. CO₂ concentration in the unfixed samples, and [CO₂]_ex is the excess CO₂ concentration caused by a decrease in pH following HgCl₂ fixation. The relative CO₂ overestimation (E in %) is given by

The impact of pH (or [H⁺]) on E was mathematically described by running a regression analysis using MATLAB^®. The fminsearch MATLAB function from the Optimization Toolbox was used to find the minimum sum of squared residuals (SSR) for functions of the form of $E=A/\left[{\mathrm{H}}^{+}\right]$ or $E=A\times {\mathrm{10}}^{-B\times \text{pH}}$. For each optimal solution, the root-mean-square error (RMSE) and coefficient of determination (R²) were calculated against observed values of E, i.e. values of E determined empirically from observed [CO₂]_i and [CO₂]_ex.

Chemical speciation, saturation index calculations and prediction of CO₂ overestimation

The speciation of solutes and saturation index (SI) values of selected minerals were calculated with the program PHREEQC developed by the USGS (Parkhurst and Appelo, 2013), neglecting the effect of dissolved organic matter. This was used to assess the impact of the addition of preservative on pH and shifting the carbonate equilibrium as well as dissolved inorganic carbon losses due to carbonate mineral precipitation. PHREEQC is commonly used to calculate the speciation of inorganic carbon and the SI of carbonate minerals and to help estimate the fate of inorganic carbon in carbon-cycling studies (Atekwana et al., 2016; Clayer et al., 2016; Klaus, 2023). For each PHREEQC simulation, two files, the database (with input reactions) and input files, respectively, were used to define the thermodynamic model and the type of calculations to perform. The database of MINTEQA2 (i.e. minteq.dat; Allison et al., 1991) was used to describe the chemical system because it includes, inter alia, reactions and constants for Ag, Cu and Hg complexation with Cl, NO₃ and carbonates.

Three PHREEQC simulations were run representing the addition of each preservative solution to sample water from Lake Svartkulp. The input files described the composition of two aqueous solutions: (i) the preservative solution assumed to contain only the preservative (i.e. HgCl₂ solution) and (ii) sample water from Lake Svartkulp with observed major element concentrations (pH, Al, Ca, Cl, Cu, Fe, Mg, Mn, N as nitrate, K, Na, S as sulfate, Zn; Table S1) and Hg and Ag natural concentration assumed to be 10⁻⁵ mg L⁻¹. The output file provided the activities of the various solutes in the preserved samples, i.e. simulating the mixing of 120 mL of lake water with 240 µL of the AgNO₃, CuCl₂ and HgCl₂ preservative solutions, as described in Sect. 2.1. This procedure allows the estimation of the pH of the preserved samples as well as the SI for various mineral phases. The SI is calculated by PHREEQC comparing the chemical activities of the dissolved ions of a mineral (ion activity product, IAP) with their solubility product (K_s). When SI>1, precipitation is thermodynamically favourable. Note however that PHREEQC does not give information about precipitation kinetics.

Similarly, PHREEQC was used to estimate the decrease in pH caused by adding 150 µL of a half-saturated HgCl₂ solution to Lake Lundebyvannet samples prior to GC analyses, as described in Sect. 2.2. In the absence of data on the chemical composition of Lake Lundebyvannet, we assumed that it had the same composition as Lake Svartkulp water samples. This assumption is supported by the fact that waters from both lakes have circumneutral pH, low ionic strength (poor buffering capacity) and high DOC concentration and would therefore behave similarly in the presence of acids. Briefly, for each 0.1 pH value between a pH of 5.4 and 7.3, the carbonate alkalinity was first adjusted by increasing HCO₃ concentrations in the input files for PHREEQC to confirm that the water was at equilibrium at the given pH value. Then, the effect of adding 150 µL of a half-saturated HgCl₂ solution was simulated as described above for Lake Svartkulp. Knowing the new equilibrated pH, after addition of HgCl₂, the overestimation of CO₂ concentration in Hg-fixed samples relative to unfixed samples (E, described in Eq. 8 above) can be predicted as described below.

Adapting Eq. (2), we obtain

and

where [H⁺]_i is the proton concentration measured in the initial water samples prior to HgCl₂ fixation and $[{\mathrm{H}}^{+}{]}_{{\mathrm{HgCl}}_{\mathrm{2}}}$ is the proton concentration estimated by PHREEQC following HgCl₂ fixation, and a similar definition applies for Z_i and $Z_{{\mathrm{HgCl}}_{\mathrm{2}}}$ from Eq. (3). Combining Eqs. (7), (9) and (10), we obtain

Hence

Alternatively, E can also simply be predicted based on the carbonic acid dissociation:

At equilibrium, we have

When pH is decreased upon addition of HgCl₂, a fraction (α) of the initial bicarbonate concentration $[{{\mathrm{HCO}}_{\mathrm{3}}}^{-}{]}_{\mathrm{i}}$ is turned into CO₂. This fraction, expressed as [CO₂]_ex in Eq. (7) above, can be estimated with Eq. (13) as follows:

Introducing the expression of [CO₂]_ex from Eq. (14) into Eq. (8) yields

When the decrease in pH, or acidification, is greater than the buffering capacity of the water, α=1. The value of α cannot exceed 1 because the amount of CO₂ produced by a decrease in pH cannot exceed the amount of ${{\mathrm{HCO}}_{\mathrm{3}}}^{-}$ initially present. In all the other cases, we have α<1. For both predictions of E, i.e. with Eqs. (12) and (15), the root-mean-square error (RMSE) and coefficient of determination (R²) were calculated.

Finally, additional sources of CO₂ overestimation were investigated by analysing the residuals of the model described by Eq. (12), i.e. the difference between E predicted with Eq. (12) and E determined empirically with Eq. (8). Briefly, residuals were plotted against pH and in situ temperature. Residuals were separated into two groups based on the empirical value of $[{{\mathrm{HCO}}_{\mathrm{3}}}^{-}{]}_{\mathrm{i}}-[{\mathrm{CO}}_{\mathrm{2}}{]}_{\text{ex}}$; i.e. the first group had values of $[{{\mathrm{HCO}}_{\mathrm{3}}}^{-}{]}_{\mathrm{i}}-[{\mathrm{CO}}_{\mathrm{2}}{]}_{\text{ex}}\ge a$, while the second group had values of $[{{\mathrm{HCO}}_{\mathrm{3}}}^{-}{]}_{\mathrm{i}}-[{\mathrm{CO}}_{\mathrm{2}}{]}_{\text{ex}}\le -a$, where different values for a were used: 20, 10 or 5 µM. The justification for separating residuals into two groups is that (i) the first group represents samples for which bicarbonate alkalinity in the original sample is, as expected, higher than CO₂ overestimation after HgCl₂ fixation, while (ii) the second group represents samples for which bicarbonate alkalinity is not sufficient to explain CO₂ overestimation after HgCl₂ fixation.

CO₂ diffusion fluxes from Lake Lundebyvannet

The diffusive flux of CO₂ ($F_{{\mathrm{CO}}_{\mathrm{2}}}$ in $\mathrm{mol}{\mathrm{m}}^{-\mathrm{2}}{\mathrm{d}}^{-\mathrm{1}}$) from Lake Lundebyvannet surface water was estimated according to

where $k_{{\mathrm{CO}}_{\mathrm{2}}}$ is the CO₂ transfer velocity (m d⁻¹), [CO₂] is the surface water CO₂ concentration (µM), 1000 is a factor to ensure consistency in the units, and [CO₂]_eq is the theoretical water CO₂ concentration (µM) in equilibrium with atmospheric CO₂ concentration calculated with Eq. (3) and a pCO₂ of 416 µatm (see above).

The CO₂ transfer velocity ($k_{{\mathrm{CO}}_{\mathrm{2}}}$) was estimated as follows (Vachon and Prairie, 2013):

where k₆₀₀ is the gas transfer velocity (m d⁻¹) estimated from empirical wind-based models and ${\mathit{\text{Sc}}}_{{\mathrm{CO}}_{\mathrm{2}}}$ is the CO₂ Schmidt number for in situ water temperature (unitless; Wanninkhof, 2014). We used n values of 0.5 or $\mathrm{2}/\mathrm{3}$ when wind speed was below or above 3.7 m s⁻¹, respectively (Guérin et al., 2007). Empirical k₆₀₀ models included those from Cole and Caraco (1998; $k_{\mathrm{600}}=\mathrm{2.07}+\mathrm{0.215}{U}_{\mathrm{10}}^{\mathrm{1.7}}$), Vachon and Prairie (2013; $k_{\mathrm{600}}=\mathrm{2.51}+\mathrm{1.48}{U}_{\mathrm{10}}+\mathrm{0.39}{U}_{\mathrm{10}}{\log }_{\mathrm{10}}\text{LA}$), and Crusius and Wanninkhof (2003; power model: $k_{\mathrm{600}}=\mathrm{0.228}{U}_{\mathrm{10}}^{\mathrm{2.2}}+\mathrm{0.168}$ in cm h⁻¹). U₁₀ and LA refer to mean wind speed at 10 m in m s⁻¹ and lake area in km², respectively. Sub-hourly U₁₀ data for 2020 were retrieved from a weather station of the Norwegian Meteorological Institute located 1.5 km west of Lake Lundebyvannet (station name: E18 Melleby; ID: SN 3480; 59.546° N, 11.4535° E) using the Frost application programming interface (Frost API, 2022). Daily, monthly and yearly (only covering the ice-free season: April–November) $F_{{\mathrm{CO}}_{\mathrm{2}}}$ was estimated using Eq. (12). Daily [CO₂] was interpolated from weekly data using a modified Akima spline (makima spline in MATLAB^® based on Akima, 1974). This interpolation method is known to avoid excessive local undulations.