Responses of Freshwater Calcifiers to Carbon-Dioxide-Induced Acidification

Abstract

Increased anthropogenic carbon dioxide (CO₂) in the atmosphere can enter surface waters and depress pH. In marine systems, this phenomenon, termed ocean acidification (OA), can modify a variety of physiological, ecological, and chemical processes. Shell-forming organisms are particularly sensitive to this chemical shift, though responses vary amongst taxa. Although analogous chemical changes occur in freshwater systems via absorption of CO₂ into lakes, rivers, and streams, effects on freshwater calcifiers have received far less attention, despite the ecological importance of these organisms to freshwater systems. We exposed four common and widespread species of freshwater calcifiers to a range of pCO₂ conditions to determine how CO₂-induced reductions in freshwater pH impact calcium carbonate shell formation. We incubated the signal crayfish, Pacifastacus leniusculus, the Asian clam, Corbicula fluminea, the montane pea clam, Pisidium sp., and the eastern pearlshell mussel, Margaritifera margaritifera, under low pCO₂ conditions (pCO₂ = 616 ± 151 µatm; pH = 7.91 ± 0.11), under moderately elevated pCO₂ conditions (pCO₂ = 1026 ± 239 uatm; pH = 7.67 ± 0.10), and under extremely elevated pCO₂ conditions (pCO₂ = 2380 ± 693 uatm; pH = 7.32 ± 0.12). Three of these species exhibited a negative linear response to increasing pCO₂ (decreasing pH), while the fourth, the pea clam, exhibited a parabolic response. Additional experiments revealed that feeding rates of the crayfish decreased under the highest pCO₂ treatment, potentially contributing to or driving the negative calcification response of the crayfish to elevated pCO₂ by depriving them of energy needed for biocalcification. These results highlight the potential for freshwater taxa to be deleteriously impacted by increased atmospheric pCO₂, the variable nature of these responses, and the need for further study of this process in freshwater systems.

1. Introduction

Increased partial pressure of atmospheric carbon dioxide (pCO₂) drives large scale alterations to environmental systems. In particular, this additional CO₂ can enter surface waters and perturb the aquatic carbonate system, lowering pH, carbonate ion concentration ([CO₃²⁻]), and the saturation state of water with respect to calcium carbonate (CaCO₃). In marine systems, this process, termed ocean acidification, can impair tissue and shell growth and alter the behavior of many marine species, yielding ecological changes across a range of spatial, temporal, and trophic scales [1].

Calcifying organisms, those producing calcium carbonate shells or skeletons, are especially sensitive to CO₂-induced changes in carbonate system chemistry, although the drivers of this sensitivity can be complex [2,3,4]. In many taxa, calcium carbonate material is produced more slowly under increased pCO₂ [1,5], with shell and skeletal material produced under these conditions tending to be weaker [6,7]. Some species, however, grow faster under increased pCO₂ or can maintain or enhance the quality of their shell or skeletal material [5,8]. In many cases, the effects of pCO₂ operate indirectly, often via pathways related to pH, bicarbonate, or carbonate ions [3,9]. Indeed, the diversity of responses to increased pCO₂ complicates the process of generalizing how calcifying taxa will respond to CO₂-induced acidification in the future [4].

Although there have been many studies examining effects of CO₂-induced acidification on marine species, far fewer have considered responses of freshwater species. Freshwater typically has lower alkalinity, or chemical buffering capacity, which makes the CaCO₃ saturation state of freshwater less than that of seawater for equivalent pCO₂ conditions, while also rendering the carbonate chemistry of freshwater systems more sensitive to variations in atmospheric pCO₂ than that of marine systems [10,11]. Freshwater species, for example, will experience larger shifts in pH and lower absolute CaCO₃ saturation states than their marine analogs for a given increase in anthropogenic CO₂. The lower alkalinity of freshwater systems also makes the carbonate chemistry of these systems more vulnerable to diurnal and seasonal cycles in photosynthesis and respiration than marine systems [10]. It is, therefore, possible that freshwater calcifiers will be more sensitive than marine calcifiers to equivalent shifts in atmospheric pCO₂. Alternatively, the naturally higher variability in carbonate chemistry and lower absolute saturation state of freshwater systems could select for freshwater calcifying taxa that are relatively resilient to CO₂-induced perturbations to carbonate system chemistry.

Although past research has examined responses of freshwater calcifying species to elevated aqueous CO₂, most of this earlier work was conducted with the aim of understanding physiological acid–base dynamics within the organisms themselves, rather than understanding the impacts of CO₂-induced acidification on shell and skeletal formation. Thus, much of this prior work employed unrealistically high pCO₂ conditions that exceed projected end-of-century CO₂ partial pressures by tens of thousands of microatmospheres (µatm), resulting in unrealistic pH reductions in the order of 2 units [12]. Furthermore, among the modest number of studies where pCO₂ treatments reflected realistic future scenarios, prior work has focused on shifts in primary productivity by phytoplankton and changes in food quality [10] as driving calcifier performance, rather than on the direct effects of CO₂-induced changes in freshwater carbonate chemistry. Therefore, to determine the effect of increased atmospheric pCO₂ on calcifying invertebrates in the context of increasing anthropogenic pCO₂, we reared four species of freshwater calcifiers under three atmospheric pCO₂ conditions that bracket the range of conditions expected to occur over the next two centuries given the range of future emissions scenarios [13].

2. Materials and Methods

This project was conducted at the University of California, Davis, Tahoe Environmental Research Center (Tahoe City Field Station) and Lot #4 of Alpine Meadows, CA, USA between December 2020 and August 2021. The freshwater employed in the flow-through CO₂-induced acidification experiments was sourced from Burton Spring, a tributary of Lake Tahoe. The carbonate chemistry of the treatments was controlled by maintaining constant pH in three 800 L sumps with a pH-stat system (American Marine Inc. Pinpoint pH Controller) that reduced pH by dosing pure compressed CO₂ with a solenoid-valve controlled gas regulator (FZone Pro Series CO₂ Regulator), or increased pH by dosing ambient air through an air pump (Simply Deluxe Electromagnetic Air Pump). The compressed CO₂ and compressed air were sparged into the sumps and treatments tanks with flexible, microporous bubbling tubes that were designed to expedite equilibration between aqueous and gas phases. Spring water (filtered to 10 µm) entered the sumps at a rate of 1 L min⁻¹ to minimize accumulation of metabolic byproducts and prevent depletion of alkalinity and calcium ions through the calcification process. The sumps and experimental tanks were covered with plastic sheeting and plastic lids, respectively, to prevent room air from equilibrating with the water in the sumps and the experimental treatments. The water within each of the three sump systems was recirculated through 12–40 L tanks (3 sumps × 12 tanks = 36 tanks total), with the recirculating water passing through an activated carbon filter (changed biweekly) before entering the tanks and through 10 L of loose activated carbon media (changed monthly) before re-entering the sump (Figure 1). Temperature varied naturally with the seasons but was maintained below 22 °C with chillers and the addition of frozen spring water.

Water chemistry was measured three times per week with a Thermo Scientific Orion Star A329 Multiparameter Meter. Oxygen (Orion RDO Optical Dissolved Oxygen Probe) and pH sensors (Atlas Scientific Spear-tip pH) were calibrated daily in air-equilibrated water and with NIST-traceable NBS buffers (pH 7 and 10), respectively. The conductivity probe (Duraprobe) was calibrated every other week with Oakton 84 and 1413 µS cm⁻¹ standards. Discrete 200 mL water samples were collected from the three sumps during each of these sampling events and frozen until they could be analyzed for alkalinity with a Metrohm 855 Robotic Titrosampler.

In addition to these higher frequency measurements, discrete 500 mL water samples were collected weekly from each of the 36 tanks and analyzed at the Tahoe Environmental Research Center (Incline Village Field Station) for dissolved inorganic carbon (DIC) on a Lachat IL 500 TOC instrument. The ‘seacarb’ package in R was used to calculate total alkalinity (TA) from these lower frequency DIC and pH measurements using constants from Waters et al. [14]. This information was used to define the linear relationship between specific conductance and TA for water employed in this experiment (Figure 2). We then used this relationship to estimate TA from the higher frequency measurement of water-specific conductance (described above). The calculated TA was 96 ± 0.3% (mean ± se, n = 149) of the measured alkalinity described above. Given this good agreement between calculated and measured alkalinity, we then used these higher frequency measurements of pH and conductivity-estimated alkalinity, along with measured temperature and total dissolved solids, to calculate carbonate system parameters for each tank throughout the duration of the experiment. The saturation state of calcite (Ω_calcite) was calculated assuming that the calcium concentration was half the alkalinity as calcium concentrations in the Lake Tahoe area tend to be between 0.5 and 1 times the alkalinity [15]. Small deviations in Ca²⁺ concentration from this relationship will not materially impact the calculated saturation states or the interpretation of the results.

The species investigated in this experiment were collected from the field and acclimated to laboratory conditions for at least 5 days prior to starting the experiments. Signal crayfish, Pacifastacus leniusculus, were collected from Pomin Park in Tahoe City, CA, USA, in late April 2021. Two cohorts of Asian clams, Corbicula fluminea, were collected from Marla Bay and Lakeside near Stateline, NV, on 15 April 2021 and 24 May 2021.

Two cohorts of pea clams, Pisidium sp., were collected from a spring feeding into the Truckee River near Polaris, CA, on 13 May 2021 and 19 June 2021. Pearlshell mussels, Margaritifera margaritifera, were collected from a lake near Jupiter, Florida, in early May 2021. Bivalves were held in three of the 40 L tanks per CO₂ treatment while crayfish were held in a different set of three tanks (9 tanks total per species). Remaining tanks (6 per pCO₂ treatment) were used for respiration and feeding trials described below. Bivalves were fed a commercially available Shellfish Diet twice daily at a concentration of 5 mL per 40 L of tank water. Crayfish were fed 1 × 1 cm dehydrated algae sheets and raw shrimp ad libitum every other day, with uneaten food removed at the time of feeding. All tanks were cleaned of accumulated solid waste three or four times per week.

Organisms remained within the tanks for the duration of the experiment and net calcification rates were calculated as the fractional change in shell mass determined from buoyant weights at the beginning and end of the experiment, normalized to a 30-day interval. Shell mass was calculated from buoyant weight measurements, where the density of the water was determined daily by the air and buoyant weights of glass bead standards (measured daily) with densities of either 2.55 or 2.23 g cm⁻³ [16]. Daily buoyant and dry weights of four half shells of Corbicula fluminea and two half shells of Margaritifera margaritifera revealed that these species had shell densities of 2.81 and 2.71 g cm⁻³, respectively and these values were used for calculating shell mass from buoyant weight [16]. A shell density of 2.71 g cm⁻³ was used for calculating the crayfish shell mass from their buoyant weight. Because crayfish transport calcium carbonate between their external shell and internal gastrolith during the molting process [17], crayfish that, at the conclusion of the incubation, had recently molted and had not yet remineralized their shell (i.e., the shell was soft) were excluded from further analysis. Due to their small size, net calcification rate of pea clams was calculated as the fractional change in wet mass between the beginning and end of the experiment normalized to a 30-day interval, measured with a Sartorius 2120T microbalance after removing excess water from the shell with lint-free wipes and allowing the clams to air dry for precisely 15 min prior to weighing. Net calcification rates were analyzed with a maximum likelihood routine fitting various functional models identified previously [5], including linear, parabolic, and threshold (exponential) relationships with average pCO₂ during the incubation period using individuals as replicates. The statistically significant model with the lowest AIC was selected as the best one to describe the relationship between pCO₂ and net calcification rate.

At the conclusion of the growth experiments, additional experiments were conducted to test whether CO₂-induced changes in carbonate chemistry impaired crayfish feeding. A new batch of live crayfish prey (pea clams) was collected and acclimated to the pCO₂ treatments for three days. Five pea clams of comparable size were placed in each of six tanks per pCO₂ treatment with a single crayfish per tank. The number of pea clams consumed after each 25 min trial was recorded. The respiration rates of the crayfish were also obtained to determine whether pCO₂-induced changes in water chemistry altered metabolic rates and, therefore, feeding requirements. Before the feeding trials, we placed each crayfish in a sealed incubation vessel and measured the change in oxygen concentration during the incubation time with the Orion RDO dissolved oxygen probe. Respiration and feeding trials both took place at night in the dark, as this is when the crayfish are most active.

3. Results

The freshwater calcifying organisms in this study were exposed to pCO₂ conditions approximately double and quadruple those of average present-day conditions. Mean daily pCO₂ ± SD (calculated from pH, total alkalinity, TDS, and temperature) across all tanks in the control, intermediate, and high treatments were 616 ± 151 µatm, 1026 ± 239, and 2380 ± 693 (Figure 3d,

Table 1. Average treatment conditions during the experiment. Measurements are the mean (standard deviation) of 12 tanks per treatment over 35 sampling days.

Parameter	Units	Low pCO2	Intermediate pCO2	High pCO2
Temperature	°C	17.9 (2.6)	17.9 (2.5)	17.6 (2.5)
Specific conductance	µS cm−1	113 (11)	111 (10)	113 (11)
Dissolved oxygen	µmol L−1	232 (13)	230 (13)	227 (9)
pH (NBS scale)		7.91 (0.11)	7.67 (0.10)	7.32 (0.12)
Total alkalinity	µmol kg−1	1098 (116)	1078 (102)	1093 (110)
pCO2	µatm	616 (151)	1026 (239)	2380 (693)
[HCO3−]	µmol kg−1	1088 (114)	1072 (102)	1090 (110)
[CO32−]	µmol kg−1	4.7 (1.4)	2.7 (0.7)	1.2 (0.4)
Ωaragonite		0.50 (0.16)	0.28 (0.08)	0.13 (0.04)

, n = 35 sampling days), respectively, corresponding to mean daily pH ± SD of 7.91 ± 0.11, 7.67 ± 0.10, and 7.32 ± 0.12 (Figure 3b, Table 1, n = 35 sampling days). Daily mean alkalinity decreased throughout the course of the experiment from 1272 to 927 µmol kg⁻¹ (Figure 3c), probably due to increased contribution of snowmelt relative to groundwater through the spring–summer transition. This trend was accompanied by an increase in daily mean temperature from 12.2 to 21.4 °C (Figure 3a). The relationship between specific conductance (SC, µS cm⁻¹) and total alkalinity (TA, µmol kg⁻¹) was TA = −75.46 + 10.38 ∗ SC (R² = 0.96, F₁₃₁₄ = 6937, p < 0.001).

Net calcification rates of the four species as a function of pCO₂ followed two general response types (

Table 2. Net calcification rates (fractional change in buoyant or wet weight normalized to a 30-day growth interval) in each treatment for each species during the experiment. Values reported are mean (standard error), sample size.

Species	Low pCO2	Intermediate pCO2	High pCO2
P. leniusculus	−0.007 (0.003), 10	−0.009 (0.002), 15	−0.019 (0.003), 8
C. fluminea	−0.018 (0.004), 33	−0.019 (0.004), 31	−0.032 (0.004), 31
M. margaritifera	−0.087 (0.006), 25	−0.082 (0.008), 19	−0.108 (0.008), 18
Pisidium sp.	0.003 (0.003), 29	0.013 (0.003), 35	0.007 (0.003), 21

and

Table 3. Parameter estimates for tested models describing the relationship between net calcification rate and pCO₂. Linear models took the form net calcification rate = b0 + b1 ∗ pCO₂. Parabolic models took the form net calcification rate = b0 + b1 ∗ pCO₂ + b2 ∗ (pCO₂)². Exponential models took the form net calcification rate = b0 + b1 ∗ e^b2∗pCO₂. Bold rows indicate statistically significant model (α = 0.05) with the lowest AIC value for a given species.

Species	Model	b0	b1	b2	R2	RMSE	F-Statistic	p-Value	AIC
P. leniusculus	linear	−1.79 × 10−3	−5.42 × 10−6		0.257	0.008	10.71 (1, 31)	0.0026	−217.18
P. leniusculus	exponential	−1.10 × 10−4	−4.71 × 10−3	4.41 × 10−4	0.260	0.008	5.29 (2, 30)	0.0104	−215.33
P. leniusculus	parabolic	−5.84 × 10−3	−4.32 × 10−7	−1.19 × 10−9	0.260	0.008	5.27 (2, 30)	0.0105	−215.32
C. fluminea	linear	−1.35 × 10−2	−5.76 × 10−6		0.052	0.024	5.13 (1, 93)	0.0258	−433.29
C. fluminea	exponential	1.32 × 10−2	−2.80 × 10−2	1.49 × 10−4	0.050	0.024	2.4 (2, 92)	0.0963	−431.04
C. fluminea	parabolic	2.53 × 10−3	−2.80 × 10−5	5.64 × 10−9	0.067	0.024	3.3 (2, 92)	0.0410	−432.78
M. margaritifera	linear	−7.38 × 10−2	−1.15 × 10−5		0.099	0.032	6.6 (1, 60)	0.0126	−243.26
M. margaritifera	exponential	−4.67 × 10−2	−3.66 × 10−2	8.29 × 10−5	0.044	0.033	0.52 (2, 59)	0.5973	−229.62
M. margaritifera	parabolic	−7.86 × 10−2	−4.40 × 10−6	−1.90 × 10−9	0.100	0.032	3.27 (2, 59)	0.0447	−241.29
Pisidium sp.	linear	8.06 × 10−3	−5.23 × 10−8		0.000	0.017	0.001 (1, 83)	0.9815	−442.70
Pisidium sp.	exponential	−7.37 × 10−3	1.54 × 10−2	−3.09 × 10−6	0.000	0.017	0.0002 (2, 82)	0.9998	−440.70
Pisidium sp.	parabolic	−1.68 × 10−2	3.37 × 10−5	−8.68 × 10−9	0.076	0.017	3.393 (2, 82)	0.0383	−447.46

). Net calcification rates of Pacifastacus leniusculus (Figure 4a), Corbicula fluminea (Figure 4b), and Margaritifera margaritifera (Figure 4c) exhibited a linear decline in net calcification with increasing pCO₂ (decreasing pH). Net calcification rate of Pisidium sp. exhibited a parabolic relationship with pCO₂, in which maximum rates of net calcification were observed in the intermediate pCO₂ treatment (Figure 4d).

Metabolism and food consumption of the crayfish, Pacifasticus leniusculus, exhibited more complex responses to altered pCO₂ treatments. Respiration rates averaged 14.7 µmol g⁻¹ h⁻¹ (n = 43 crayfish, 13 in low and high pCO₂ treatments, respectively, 17 in the intermediate pCO₂ treatment), but did not vary with pCO₂ (Figure 5a, F_1,41 = 0.281, p = 0.60). Feeding rates, however, were impacted by pCO₂ (Figure 5b, ANOVA, F_2,15 = 4.47, p = 0.030), such that feeding rates in the highest pCO₂ trials were significantly less than in the intermediate pCO₂ trials (post-hoc Tukey, p = 0.043), and with nearly significantly less than in the lowest pCO₂ trials (post-hoc Tukey, p = 0.060).

4. Discussion

Elevated pCO₂, resulting in decreased pH, caused a reduction in net calcification rates in three species of freshwater calicifiers, the Asian clam Corbicula fluminea, the pearlshell mussel Margaritifera margaritifera, and the crayfish Pacifastacus leniusculus. In the pea clam, Pisidium sp., however, net calcification rate was highest under intermediate pCO₂. The linearly negative response of the Asian clam, pearlshell mussel, and crayfish suggests that these species will be negatively impacted by CO₂-induced acidification of freshwater systems over the coming decades. However, optimization of calcification under moderate acidification exhibited by the pea clam suggests that this species may be more resilient to CO₂-induced acidification predicted for the near future, but this resilience may diminish at higher pCO₂ conditions predicted for the next century or two. Although the high inter-specimen variability in growth rates of the pea clam may argue for selection of a simpler model (i.e., linear), the large number of individuals employed in the pea clam experiment (85) supports adoption of the more complex parabolic model.

There are various mechanisms that can lead to the observed functional relationships between net calcification rate and pCO₂. Because we did not manipulate calcium ion concentrations, shell formation here is likely the culmination of two main processes the uptake of inorganic carbon, primarily in the form of bicarbonate ion and, to a lesser extent, aqueous CO₂, and the efflux of protons (H⁺) from the site of calcification that effectively converts bicarbonate ion to carbonate ion available for calcification [18,19]. The linear reduction in net calcification rate with decreased pH (increased pCO₂) exhibited by the Asian clam, pearlshell mussel, and crayfish suggests that these species are limited in their ability to control carbonate chemistry at the site of calcification in support of calcification, such as by removing H⁺ from their calcifying fluid.

This linear negative calcification response to elevated pCO₂ exhibited by the crayfish is somewhat surprising given that various species of marine decapod crustacea are reported to calcify faster under elevated pCO₂ [5,20], suggesting that freshwater crustacea utilize dissolved inorganic carbon in shell formation differently than marine crustacea and/or that other processes within their physiological repertoire are more sensitive to pH (see discussion below). The parabolic relationship between net calcification rate and pH exhibited by the pea clam suggests that it may be able to utilize the additional dissolved inorganic carbon (DIC) resulting from increased pCO₂ by, for example, converting it to carbonate ions by removing protons from the site of calcification. Under the highest pCO₂ condition, it is possible that the benefits of this process are overwhelmed by other processes that are more deleteriously impacted by the low pH conditions, such as the dissolution of pre-formed, exposed shell in water that is relatively undersaturated with respect to the clam’s aragonite [21] shell mineral. This trend may be further reinforced by nonlinearities imposed by scaling differences between surface- vs. volume-related processes. For example, because Pisidium sp. broods its larvae [22], calcification occurs throughout the clam’s body cavity as its offspring build their shells. This contrasts the calcification processes of M. margaritifera and C. fluminea, which do not brood their calcifying offspring. Dissolution, however, acts only on shell surfaces exposed to water with a low saturation state, meaning that the brooding larval Pisidium sp. are potentially shielded from this process, which may confer some resilience to calcification of the whole pea clam (i.e., both adults and brooded larvae) under intermediate pCO₂ conditions, potentially resulting in their observed parabolic responses to elevated pCO₂.

It is also possible that decreased water pH impacts rates of shell formation indirectly, potentially through physiological processes beyond biocalcification. For the crayfish, P. leniusculus, we observed that feeding rates were reduced at the lowest pH despite no pH-dependent variation in respiration rates. We did not test for specific mechanisms, but similar reductions in feeding rates, arising from modified behavior, have been observed for marine decapods exposed to increased pCO₂ [23]. Alternatively, lowered pH has been shown to cause a biomechanical weakening of the exoskeleton in some species of marine crustacea [24], and it is possible that this could impair their ability to handle and consume prey. Regardless of the cause, these reduced feeding rates under CO₂-induced reductions in water pH would yield less food and energy for shell production by the crayfish, potentially contributing to or driving their linear decline in net calcification rate with increasing pCO₂. Although the feeding rates of the other species were not quantified in the present study, obtaining these types of measurements in future experiments should provide valuable insight into the mechanisms responsible for the observed reductions in net calcification rate of freshwater calcifiers under conditions of elevated pCO₂.

Anthropogenic increases in atmospheric pCO₂ and the resulting decrease in freshwater pH has the potential to negatively impact freshwater aquatic ecosystems by altering the net calcification rate and behavior of calcifying invertebrates [1]. Differential sensitivities to CO₂-induced changes in water chemistry can further shape populations, communities, and ecosystems [25]. Given the ecological importance of freshwater calcifiers in lakes, ponds, rivers, and streams [26,27,28], further research is merited to determine the range of responses that such species exhibit to elevated atmospheric pCO₂, and the mechanisms driving these diverse responses. A better understanding of how freshwater species perform under acidification can help inform strategies for managing freshwater systems amidst the various threats that they face, including species invasion, eutrophication, sedimentation, warming, and CO₂-induced acidification.

5. Conclusions

• CO₂-induced acidification (doubling, quadrupling of ambient pCO₂ causing pH decline of between 0.23 and 0.57 units) caused negative linear calcification responses in three species of freshwater calcifiers and a more complex parabolic calcification response in one species (pea clam). Increased pCO₂ decreased the feeding rates of the signal crayfish on its natural pea clam prey but had no significant impact on crayfish respiration rate (i.e., constant metabolism), potentially driving this species’ negative calcification response to acidification by depriving it of energy required for its molt-mediated calcification.
• Although increased pCO₂ can impair calcification rates of freshwater organisms, variation in these effects amongst species, combined with impacts on predator–prey dynamics, could yield complex ecological consequences for freshwater systems.
• Results highlight the importance of further elucidating the understudied effects of CO₂-induced acidification within freshwater systems.