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1
Biological response of eelgrass epifauna, Taylor’s Sea hare
(Phyllaplysia taylori) and eelgrass isopod (Idotea resecata), to elevated
ocean alkalinity
Kristin Jones1, Lenaïg G. Hemery1, Nicholas D. Ward1-2, Peter J. Regier1, Mallory Ringham3, Matthew
D. Eisaman4-5
5
1 Coastal Sciences Division, Pacific Northwest National Laboratory,1529 W Sequim Bay Rd, Sequim, WA, USA
2 School of Oceanography, University of Washington, Seattle, WA, USA
3 Ebb Carbon, Inc., San Carlos, CA, USA
4 Department of Earth & Planetary Sciences, Yale University, New Haven, CT, USA
5 Yale Center for Natural Carbon Capture, Yale University, New Haven, CT, USA 10
Correspondence to: Kristin Jones (Kristin.Jones@pnnl.gov), Lenaig Hemery (Lenaïg.Hemery@pnnl.gov)
Abstract. Marine carbon dioxide removal (mCDR) approaches are under development to mitigate the effects of climate change
with potential co-benefits of local reduction of ocean acidification impacts. One such method is ocean alkalinity enhancement
(OAE). A specific OAE method that avoids issues of solid dissolution kinetics and the release of impurities into the ocean is 15
the generation of aqueous alkalinity via electrochemistry to enhance the alkalinity of the surrounding water and extract acid
from seawater. While electrochemical acid extraction is a promising method for increasing the carbon dioxide sequestration
potential of the ocean, the biological effects of this method are relatively unknown. This study aims to address this knowledge
gap by testing the effects of increased pH and alkalinity, delivered in the form of aqueous base, on two ecologically important
eelgrass epifauna in the U.S. Pacific Northwest, Taylor’s sea hare (Phyllaplysia taylori) and eelgrass isopod (Idotea resecata), 20
across pH treatments ranging from 7.8 to 9.3. Four-day experiments were conducted in closed bottles to allow measurements
of the evolution of carbonate species throughout the experiment with water refreshed twice daily to maintain elevated pH. Sea
hares experienced mortality in all pH treatments, ranging from 40% mortality at pH 7.8 to 100% mortality at pH 9.3. Isopods
experienced lower mortality rates in all treatment groups, which did not significantly increase with higher pH treatments.
Different invertebrate species will likely have different responses to increased pH and alkalinity, depending on their 25
physiological vulnerabilities. Investigation of the potential vulnerabilities of local marine species will help inform the decision-
making process regarding mCDR planning and permitting.
1 Introduction
Among the many other impacts of climate change, increased levels of atmospheric carbon dioxide (CO2) drive global decreases
in ocean pH and calcium carbonate (CaCO3) saturation states (Doney et al., 2009; Doney et al., 2020) known as ocean 30
acidification (OA), posing a significant threat to marine organisms and ecosystems (Doney et al., 2020). These increased CO2
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levels are primarily caused by human sources, such as fossil fuel combustion, cement production, and land use changes (Doney,
et al., 2009; Orr et al., 2005). OA can lead to various detrimental effects on marine life, including decreases in survival, growth,
calcification, development, and abundance, particularly for slow moving or sessile animals (Kroeker et al., 2013). Marine
invertebrates can experience physiological effects such as oxidative stress, decreased immunity, decreased growth and 35
development, and lower reproductive success (Shi and Li, 2023). OA can be particularly harmful to organisms in early life
stages, affecting fertilization, larval development, dispersal, and settlement (Ross et al., 2011). Moreover, OA can negatively
impact food web dynamics and ecosystem processes (Fabry et al., 2008).
Natural carbon sinks on land and in the ocean help to reduce atmospheric CO2 but are not keeping pace with increasing
anthropogenic emissions, prompting research efforts to explore methods of enhancing the ocean’s natural carbon sink through 40
marine carbon dioxide removal (mCDR) (National Academies of Sciences, Engineering, and Medicine, 2022). One method of
mCDR is an ocean alkalinity enhancement (OAE) approach that electrochemically processes salt (e.g., sodium chloride NaCl)
to generate aqueous acid (hydrochloric acid HCl), which is removed from the system, and base (sodium hydroxide NaOH),
which is mixed with the seawater stream and returned to the ocean, thus enhancing the alkalinity of the surrounding water (de
Lannoy et al., 2018; Eisaman et al., 2018; Eisaman et al., 2023; Lu et al., 2022; Ringham et al., 2024; Tyka et al., 2022; Wang 45
et al., 2023). The increased surface alkalinity drives additional ocean uptake of atmospheric CO2, which is ultimately stored
in seawater as dissolved bicarbonate (Cross et al., 2023, Eisaman et al., 2023, Ringham et al., 2024). While OAE could be a
promising avenue for reducing atmospheric CO2, the biological response of marine organisms and impact on ecosystems of
locally increasing pH and alkalinity remains largely unknown.
Changes in ocean pH can have implications for marine life and the health of marine ecosystems. pH shifts can affect physiology 50
of aquatic organisms by disrupting acid-base regulation essential for cellular function, can inhibit fixation and respiration of
CO2, and reduce nutrient uptake (Tresguerres et al. 2020, Tresquerres et al. 2023). Multicellular marine organisms rely on
intracellular and extracellular pH gradients and modulation for metabolic processes. This is regulated through an acid-base
balance, which can be disrupted if environmental conditions, such as pH or CO2, are altered (National Research Council,
2010). Many organisms have the ability to control their internal pH to an extent, but some may be able to acclimate better than 55
others at the cost of high metabolic demand (Portner et al. 2000). Previous studies have explored the impact of pH changes,
particularly in the context of OA, on a variety of marine organisms, but only a few have studied the impacts of increasing pH.
A method used in aquaculture to reduce biofouling involves increasing local alkalinity with the addition of calcium hydroxide
(Comeau et al. 2017). In this study, bivalves were quickly exposed to a 12.7 pH solution and exhibited short-term behavioral
stress, but did not show any mortality, likely due to the quick dispersal of the alkaline solution. The bivalves were then exposed 60
to weakly elevated pH (9.2) consistently for three days, in which they experienced prolonged closure of their valves, indicating
an “avoidance behavior,” however the behavior ceased when treatment was completed, and no mortality was observed
(Comeau et al. 2017). Another study investigated the effects of increased ocean alkalinity on red calcifying algae. The algae
experienced a 60% increase in carbonate production when alkalinity was increased from 2694 μEq L-1 to 3454 μEq L-1
(resulting in a pH increase from 7.97 to 8.2), but these alkalinity and pH increases had no significant negative impacts on 65
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primary productivity, respiration or photophysiology (Gore et al., 2019). In a controlled laboratory experiment, European green
crabs were exposed to calcium hydroxide to determine biological effects of increased alkalinity. Calcium hydroxide was added
in two concentrations (0.28 mmol L-1 and 0.54 mmol L-1), to raise the pHNBS (8.0 - 8.7). The green crabs experienced
physiological disruption in acid-base regulation, respiratory alkalosis and hyperkalemia (Cripps et al., 2013). Few studies have
investigated biological response to enhanced ocean alkalinity, but this research is needed to move this field forward, following 70
best practices for collaborative OAE research (Oschilies et al. 2023).
In the Pacific Northwest region of the U.S. and Canada, eelgrass is critical to many nearshore ecosystems, playing key roles
such as providing habitat for other species and acting as a food source, directly and indirectly to support food webs (Thayer &
Phillips, 1977). In addition, eelgrass ecosystems provide a variety of supporting services, such as refuge, nursery habitat,
foraging areas, and habitat areas for reproduction, as well as regulating services, such as shoreline protection, sediment 75
stability, water quality improvement, and climate change regulation (Sherman & DeBruyckere, 2018). Pacific salmon, both a
culturally and commercially important species, rely on valuable eelgrass habitat. This ecosystem provides foraging
opportunities for juvenile salmon that promote growth and survival during their critical early life stage (Kennedy et al., 2018).
However, Pacific Northwest eelgrass ecosystems are at risk from a variety of threats, including invasive species, anthropogenic
contaminants, and global shifts in temperature and sea level rise (Sherman & DeBruyckere, 2018). Although manipulation 80
experiments have shown that acidic conditions may actually alleviate stress and promote eelgrass productivity (Zayas-Santiago
et al., 2020; Zimmerman et al., 2017), the synergistic impacts on eelgrass epifauna under either acidic or alkaline treatments
remains unknown.
Eelgrass isopods (Idotea resecata) typically range from Alaska, U.S. to California, U.S. and are found in eelgrass ecosystems.
They feed on eelgrass blades and kelp and play a significant role in food webs as a prey source for many fish species, including 85
Pacific salmon (Bridges, 1973, Ricketts and Calvin, 1952; Welton and Miller, 1980). Taylor’s sea hares (Phyllaplysia taylori)
typically range from British Columbia, Canada to California, U.S. and spend their lives on the blades of eelgrass, feeding on
epiphytic diatoms (Beeman, 1963). Sea hares are herbivores and use their green coloration and vertical stripes as camouflage
from predation among the eelgrass blades (Bridges, 1973). In the eelgrass ecosystem, both isopods and sea hares graze on the
epiphytic algae, which block the eelgrass from the sun and limit photosynthesis. This grazing reduces the epiphyte load on the 90
eelgrass blades, allowing for continued photosynthesis (Lewis & Boyer, 2014). Studies examining eelgrass mesograzer species
sensitivity to environmental changes, such as pH, salinity, and temperature, found that shifts in environmental conditions are
likely to affect their feeding on epiphytes, and this can lead to indirect effects on the growth and productivity of the eelgrass
ecosystem (Hughes et al., 2017; Tanner et al., 2019).
Marine epifauna local to the Pacific Northwest experience substantial natural variability in pH over daily to seasonal 95
timescales. Over the course of the year, pH in Puget Sound surface layer waters can vary by more than one pH unit with even
greater variability at the numerous river outlets (e.g., 6.5 to 8.5) around the region (Bianucci et al., 2018; Fassbender et al.,
2018). This variability is driven primarily by tides, diel productivity patterns, river discharge, and seasonal weather variability.
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The objective of this study was to determine the biological responses of eelgrass epifauna (Taylor’s sea hares and eelgrass
isopods) to increased pH and alkalinity levels to inform future mCDR field trials and identify knowledge gaps pertaining to 100
laboratory and field trials in the context of OAE. We investigated both the biological and chemical responses to the addition
of NaOH to seawater to determine safe bounds of operation for OAE interventions on specific species. We contextualized the
carbonate speciation conditions organisms were exposed to throughout these experiments through measurements of pH and
partial pressure of CO2 (pCO2) and investigated animal mortality and behavioral trends. We hypothesized that prolonged
exposure to pH and alkalinity outside the bounds of typical coastal variability would cause widespread mortality of the studied 105
organisms.
2 Methods
2.1 Laboratory experiment
The experiments were conducted at the Pacific Northwest National Laboratory marine laboratory in Sequim, WA, U.S.
Eelgrass has been cultivated in outdoor mesocosm tanks on the dock at this facility for over 20 years, supplied with unfiltered 110
seawater from the mouth of Sequim Bay. pH measured from the unfiltered seawater at the facility was shown to display large
variability throughout the day/year (Myers et al., in prep; Ward, personal communication). Adult Taylor’s sea hares and
eelgrass isopods were collected from these eelgrass ecosystem tanks in July to September, 2023. Three batches of 120 sea
hares were collected from late July to early August 2023 and three batches of 120 isopods were collected from late August to
late September 2023, each batch undertaking a week of acclimation before being used for the experiments. Sea hares and 115
isopods were gently collected by hand or with nets from the outdoor eelgrass tanks and transferred to three acclimation tanks
in the on-site wet laboratory. Acclimation tanks were filled with about 2.5 cm of sediment collected from one of the outdoor
eelgrass tanks, and had continuously flowing raw seawater from Sequim Bay to provide both flow and nutrients to the
organisms. The flow rate (not measured) was fast enough to allow for the water to rapidly refresh and maintain a temperature
as close to the natural environment as possible. Animals were provided daily with eelgrass blades and diatom masses from the 120
outdoor tanks as habitat and food sources in the acclimation tanks. The animals acclimated for one week during which time
they were checked daily. Any mortality was noted, and the deceased animals were removed from the tank. At the end of
acclimation, 100 sea hares and 100 isopods were randomly collected from the surviving organisms to enter the experiments.
After acclimation, animals were not fed for the remainder of the experiment, based on the standards for acute toxicity tests
with macroinvertebrates (ASTM, 2000). 125
To start the experiment and for each water change, 7 L of unfiltered seawater were gently poured into an 8-L bucket and the
pHNBS (NBS scale), salinity, temperature, and dissolved oxygen (DO) of the seawater were measured using a YSI ProDSS
probe that was calibrated daily. The control group pH was the pH of Sequim Bay water collected via the facility’s seawater
intake, located near the seafloor at about 10 m depth, at the time of water change (generally around 7.8 +/- 0.3 due to natural
variability). Temperature and salinity readings, along with water volume, were used to calculate the amount of NaOH needed 130
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to reach the desired pH for each treatment group, using a CO2SYS Excel macro (Pierrot et al., 2021). The low treatment group
had a target pH of 8.3, the medium treatment group had a target pH of 8.8, and the high treatment group had a target pH of
9.3. Commercial aqueous NaOH (0.5 M, Honeywell Chemicals 352576X1L) was used to replicate the concentration of NaOH
derived from an electrodialysis method for creating acid and base from seawater (Eisaman et al. 2023; Ringham et al., 2024).
After mixing in NaOH to the seawater, the pH level was measured using the YSI probe and adjusted on a drop-by-drop basis 135
until within 0.1 of the target pH. Preparation of pH treatment was always completed in a separate container from the organisms
to avoid exposing animals to incorrect pH levels.
Once the seawater pH was adjusted to a target value, pH, salinity, temperature, and DO were recorded (Table 1). A gas sample
(process described in sect. 2.2) was taken for pCO2 analysis, and the water was carefully transferred to fill to the top six 1-L
glass jars without creating bubbles. For each of the sea hares and isopods experiments, five animals were randomly chosen 140
from the acclimation tanks and placed into five of the six jars per pH treatment. Lids were placed on the jars, which were then
placed randomly on a laboratory water table (Fig. 1) to account for potential variances in environmental parameters (e.g., light,
air temperature). Lids were not perforated to limit air exchange and abiotic alteration of pH (i.e., atmospheric equilibration,
which would decrease the intended pH treatment and increase pCO2 as CO2 diffused into the seawater in response to the NaOH
addition). For each pH treatment, a sixth jar was filled with controlled seawater or treated seawater, but without organisms, as 145
a chemical control.
Figure 1. Experimental design of laboratory water table, including the 1-liter glass jars (colored circles) used in the experiment and
the acclimation tanks (white rectangles). The white circles indicate control jars with pH ≈ 7.8, yellow circles indicate low treatment
150
with pH ≈ 8.3, orange circles indicate medium treatment with pH ≈ 8.8, and red circles indicate high treatment with pH ≈ 9.3. The
circles with “C” indicate the chemical control jars in which only treated, or control seawater was added without the presence of sea
hares or isopods. The circle with “D” indicates the drain on the water table.
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155
This water change process was repeated twice a day to ensure proper oxygenation and pH treatment in the jars and to remove
any excrement or deceased organisms. For each water change, used water from all the jars within a treatment group was
carefully pooled into a single bucket, and pH, salinity, temperature, and DO within this bucket were measured and a gas sample
was taken for pCO2 analysis. Water quality measurements and a gas sample were also taken from the chemical control from
each treatment group. Water was refilled as described above. Organisms were checked for mortality, any casualties were 160
removed from the jars, and any unusual behavior, such as reproduction or cannibalism among the animals, was also noted.
Between water changes, a standpipe was inserted into the drain of the water table to create a water bath of about 10 cm to keep
the jars at a cooler temperature akin to the natural seawater in Sequim Bay. The temperature of the water from Sequim Bay
ranged from 10.9℃ to 15.3℃ depending on the time of day and month of year (Table 1). The experiment was conducted over
four days and was repeated three times for both sea hares and isopods, with a new batch of animals each time. 165
Table 1. Range in pH, salinity, dissolved oxygen, and temperature of ambient seawater before NaOH treatment was added.
2.2 pCO2 sampling and carbonate speciation calculations
To collect gas samples from the treated seawater, a water sample was collected in a 300-mL bottle, poured gently to avoid 170
extraneous bubbles, and poured to overflow to eliminate headspace. 60 mL of nitrogen (N2) were injected with a syringe into
the bottle; simultaneously, 60 mL of water were removed from the bottle with another syringe. This created a headspace of N2
at the top of the bottle. After vigorously shaking the bottle for one minute to distribute the N2 throughout the water sample, the
gas sample was extracted using a unique plastic syringe for each sample. A gas analyzer, Picarro G2508 Cavity Ring‐Down
Spectrometer with a flow limiter installed on the inlet to reduce gas flow rates, was used to measure the partial pressure of CO2 175
present in the gas sample (e.g., Regier et al., 2023).
Calculation of carbonate speciation from measured pH and pCO2 was not attempted considering these are correlated parameters
and the least accurate for making such calculations. However, the goal of these measurements was not to precisely quantify
Round 1
Round 2
Round 3
Sea hares
pH
Salinity
(ppt)
DO
(mg/L)
Temp
pH
Salinity
(ppt)
DO
(mg/L)
Temp
pH
Salinity
(ppt)
DO
(mg/L)
Temp
Minimum
7.70
29.87
7.07
12.90
7.72
29.89
7.24
12.40
7.59
30.55
6.23
12.00
Maximum
8.09
30.51
9.93
15.30
8.09
31.01
10.00
14.40
8.00
31.07
8.94
14.50
Average
7.84
30.16
8.31
13.74
7.89
30.28
8.70
13.45
7.73
30.90
6.99
12.56
Isopods
pH
Salinity
(ppt)
DO
(mg/L)
Temp
pH
Salinity
(ppt)
DO
(mg/L)
Temp
pH
Salinity
(ppt)
DO
(mg/L)
Temp
Minimum
7.65
31.94
6.27
12.30
7.62
32.19
5.80
12.00
7.59
32.57
5.52
10.90
Maximum
8.02
32.50
9.25
14.70
7.85
32.48
7.74
13.40
7.84
32.99
7.94
12.10
Average
7.81
32.24
7.22
13.04
7.71
32.29
6.56
12.57
7.67
32.78
6.34
11.38
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total alkalinity and DIC dynamics, but rather to provide basic context for the biological responses. Directly measuring DIC
and total alkalinity was not practical given the amount of replication, treatments, and water refreshes that were performed. 180
2.3 Data analysis
All data analyses were conducted in R Statistical Software version 4.3.1 (R Core Team, 2023). Data exploration, visualization,
and analysis were completed using the following libraries: ggplot2, tidyverse, dplyr, ggpubr, readxl, lmtest, nlme, rstatix and
car (Fox and Weisberg, 2019; Kassambara, 2022; Kassambara, 2023; Pinheiro et al. 2023; Wickham, 2016; Wickham et al.
2019; Wickham et al. 2023; Wickham and Bryan, 2023; Zeilis and Hothorn, 2002). One-way analysis of variance (ANOVA) 185
tests were applied to mortality data from the four different treatment groups to determine whether differences between groups
were statistically significant. Data from each round were pooled into one data file for statistical analysis. When p-values
indicated a significant difference (p < 0.05), Tukey tests were applied to identify between which groups these differences
appeared. Multiple linear regression models were used in conjunction with the boxplots to determine whether pH was the
driving factor behind mortality, as opposed to abnormal salinity, low DO levels, or temperature variation. 190
Chemical toxicity tests use a Lethal Concentration 50 (LC50) value to measure the toxicity of a substance and its concentration
that results in 50% mortality of a test subject (Government of Canada, 2024). While LC50 tests generally measure the
concentration of a particular substance, we were more interested in looking at the overall effect the pH values had on the
organisms’ mortality, as opposed to the actual amount of NaOH added to achieve said pH. Therefore, for each round of sea
hare and isopod experiments, the number of days at which 50% mortality occurred for each pH treatment was used as an LC50 195
value.
3 Results
3.1 Change in pH and pCO2
Changes in pH and pCO2 were evaluated for geochemical context for the biological experiments, but were not a major focus
of this study. In the chemical control jars for the sea hare experiment, i.e., with no organisms present, the pCO2 in the pH 7.8 200
control group (i.e., without addition of NaOH) was consistently higher both before and after the water changes than any of the
other pH treatment groups (Fig. 2 top). This is consistent with the expected decrease in the pCO2 resulting from the addition
of NaOH to seawater. With organisms present in the pH 7.8 control group (i.e., without the addition of NaOH), there was a
large increase in pCO2 after the water changes. However, as pH increased, this change in pCO2 appeared to decrease, even
sometimes experiencing a decrease in pCO2 in the medium (pH 8.8) and high (pH 9.3) treatment groups (Fig. 2 bottom). 205
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210
215
220
225
230
235
240
Figure 2. pCO2 (ppm) measured in sea hare jars, before and after twice daily water changes for both jars without organisms (top), 245
and jars with organisms (bottom). Closed symbol = start of a segment using refreshed water, open symbol = end of a segment before
a water change.
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Similarly to the sea hare experiment, in the isopod experiment the chemical control jars without organisms had higher pCO2
levels in the control group (i.e., pH 7.8) both before and after the water changes (Fig. 3 top). With organisms present, control
group pCO2 mostly increased after the water change (79% of the time), but no clear pattern was discernible within the treatment 250
groups; no treatment group had consistently lower pCO2 than the rest and all treatment groups had variable pCO2 levels before
and after the water changes (Fig. 3 bottom).
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260
265
270
275
280
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285
290
295
300
305
Figure 3. pCO2 (ppm) measured in isopod jars, before and after twice daily water changes for both jars without organisms (top),
and jars with organisms (bottom). Closed symbol = start of a segment using refreshed water, open symbol = end of a segment before
a water change.
310
3.2 Animal mortality
In all three rounds of the experiment, sea hares experienced 100% mortality at pH 9.3: on the last day of experiment in the first
round, after 3 days in the second round, and at 3.5 days in the third and last round. The other treatment groups never saw 100%
mortality. Mortality was lowest (28%) for the control group, and mortality decreased as the pH lowered from 9.3 to 8.8, 8.3,
and 7.8 (or control group) (Fig. 4 top). 315
In all rounds, the ANOVA test showed significant differences between the four treatment groups (ANOVA, p < 0.001). Further
analysis with a Tukey test (Table 2) showed that the mortality in the high treatment group was significantly larger than
mortality in control, low, and medium treatment groups in the first round (TukeyHSD, p < 0.001, p < 0.001, p = 0.02
respectively) and third round (TukeyHSD, p < 0.001, p = 0.003, p = 0.02 respectively). In the second round, mortality in the
high treatment group was significantly larger than mortality in control and low treatment groups (TukeyHSD, p <0.001) but 320
not compared to the medium treatment. However, unlike the other rounds, mortality in the medium treatment group was
significantly higher than that of the control treatment group (TukeyHSD, p = 0.019).
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Isopod mortality showed similarity between treatment groups, and no clear patterns were present in any of the three rounds of
experiments (Fig. 4 bottom). Mortality was observed in every treatment group in round 1 and round 2 of the experiments,
however no mortality was seen in the low treatment group in round 3. Maximum mortality never reached above 50% for any 325
treatment group in any round of the experiment. There were no significant differences in mortality between any of the treatment
groups in round 1 and 2 (ANOVA, p = 0.104, p = 0.086). In round 3, the mortality in the low treatment group was significantly
less than that of the medium and high treatment group (TukeyHSD, p < 0.001, p = 0.02; Table 2). The medium treatment group
also had significantly higher mortality than the control group (TukeyHSD, p = 0.008).
330
335
340
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Figure 4. Mortality of sea hares (top) and isopods (bottom) is shown over the four-day period for each of the three rounds of the
experiment and for each treatment group. Yellow circles indicate pH ≈ 7.8, blue squares indicate pH ≈ 8.3, orange triangles indicate
345
pH ≈ 8.8, and purple diamonds indicate pH ≈ 9.3.
Table 2. Tukey HSD matrices for sea hare and isopod mortality comparisons between treatments. Stars (*) indicate a significant p-
value of p < 0.05.
Sea Hare Mortality
Isopod Mortality
Round 1
Round 1
Low
Medium
High
Low
Medium
High
Control
p = 0.98
p = 0.15
p < 0.001*
Control
p = 0.33
p = 0.99
p = 0.18
Low
p = 0.27
p < 0.001*
Low
p = 0.43
p = 0.98
Medium
p = 0.02*
Medium
p = 0.25
Round 2
Round 2
Control
p = 0.96
p = 0.02*
p < 0.001*
Control
p = 0.90
p = 0.99
p = 0.27
Low
p = 0.06
p < 0.001*
Low
p = 0.94
p = 0.07
Medium
p = 0.22
Medium
p = 0.22
Round 3
Round 3
Control
p = 0.76
p = 0.30
p < 0.001*
Control
p = 0.62
p = 0.008*
p = 0.25
Low
p = 0.85
p = 0.003*
Low
p < 0.001*
p = 0.02*
Medium
p < 0.001*
Medium
p = 0.42
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350
Averaged over the course of all three rounds, a clear trend of increasing mortality correlating with increasing pH can be
observed for sea hares (Fig. 5 top). The low treatment group displayed the most variation in mortality over the three rounds,
and the high treatment group showed the least variation in mortality due to each round resulting in 100% mortality. The average
mortality was not significantly higher in the low treatment group than the control group but was significantly higher for both
medium and high treatment groups (Fig. 5 top). 355
While there appears to be a slight increasing trend in the average mortality of isopods with each treatment group, the overlap
between treatment groups is considerable, and shows high levels of variation in results between the three rounds of
experiments. Average isopod mortality was not significantly different from the control group to any of the treatment levels
(Fig. 5 bottom).
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365
370
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375
Figure 5. Mortality of sea hares (top) and isopods (bottom) in each treatment group averaged over the three rounds of the
experiment. T-test p-values are presented, ns indicates non-significance, ** indicates a significant p-value < 0.05.
Multiple linear regression models for sea hare mortality data were created to determine if mortality was based only on changes
in pH, or if other environmental variables could have contributed, such as DO, salinity, and temperature. Fitting these models 380
and using the drop 1 function, pH was the only variable to have significant p-value in any of the models (Table 3). The same
models were made for the isopods; however, upon analysis none of the four variables had any significant effect on mortality
(no p-value < 0.05) in any of the models, so the data was not presented.
Table 3. Multiple linear regression models for sea hare mortality p-value comparison using drop 1 function. Stars (*) indicates a 385
significant p-value of p < 0.05.
Model
Variables
p value
death~ph + do + temp + salinity
pH
0.03693*
DO 0.47927
Temperature 0.91881
Salinity
0.55388
death ~ ph + do + salinity pH 0.03604*
DO 0.445
Salinity
0.45183
death ~ ph + do
pH
0.02908*
DO 0.78374
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3.3 Animal Lethal Concentration (LC50) 390
On average, 50% mortality (LC50) of the sea hares was observed after the first water refresh (between day 0.5 and day 1) in
the high treatment group (pH 9.3) and after 1.5 days (between day 1 and day 2.5) in the medium treatment group (pH 8.8) (Fig.
6 top). In the low treatment group (pH 8.3), LC50 was reached in only one of the three rounds of experiment, at day 3, and
never reached in the control group (pH 7.8) (Fig. 6 top).
For the isopods, LC50 was never reached in any treatment group, in any round of the experiment (Fig. 6 bottom). While some 395
mortality was observed in every treatment group, isopod moulting and reproduction was observed throughout the experiment.
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Figure 6. LC50 plots for sea hare (top) and isopod (bottom) mortality at each treatment group. 50% mortality is indicated by the 400
dashed line.
4 Discussion
4.1 Biological response
The two eelgrass epifauna invertebrates investigated in this study, isopods and sea hares, responded differently to seawater
treated with NaOH. In increasingly alkaline environments with increasing pH, sea hares exhibited higher mortality in a shorter 405
amount of time than isopods, with over 50% mortality occurring in pH 8.3 within two days and reaching 100% mortality after
one day in pH 9.3. Isopods did not reach 50% mortality in any pH treatment.
In addition to showing higher survivorship, isopods exhibited growth and reproductive behaviors during the experiments. The
process of molting for eelgrass isopods promotes growth, as well as provides the animals with an opportunity to reproduce
(Kuris et al., 2007; Sadro, 2001). The isopods in the experiment exhibited molting in the control group and all treatment groups 410
in all three rounds of the experiment, indicating that growth and reproduction still occurred in highly alkaline waters. Because
the variation in mortality between control and treatment groups was small, we suggest that the mortality observed was likely
not due to variations in pH. During the experiment, isopods were observed cannibalizing each other in control and all treatment
groups. It is possible that the mortality we observed during the experiment could have been due to lack of food rather than
changes in pH. A previous study showed Idotea resecata as being the fastest daily consumers of eelgrass among six common 415
mesograzer species (Best and Stachowicz, 2012), suggesting they may not handle four days of fasting well. A previous study
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investigating resilience to ocean acidification found opposing results, in that sea hare mortality was low while isopod mortality
was high, but isopod mortality was likely not due to pH (Hughes et al., 2017). However, there is limited information on sea
hare and eelgrass isopod biology, and this warrants further studies.
Few previous studies have investigated the effect of increased alkalinity and pH on various marine organisms. Cripps et al. 420
(2013) investigated the response of European green crabs to increased alkalinity (through the introduction of calcium
hydroxide) and found that female crabs were more susceptible to increases in pH affecting their physiology, though no
mortality in any control or treatment group was observed. Shellfish species (blue mussel, eastern oyster, and bay scallop) were
assessed for their behavioral response to increased alkalinity (through the introduction of calcium hydroxide). All three species
exhibited behavioral stress, but responses were short lived, and recovery occurred after treatment was halted (Comeau et al., 425
2017).
Results from these previous studies and the present experiments indicate that different species will likely have different
responses to increased pH, depending on their physiological vulnerabilities. Investigation of the potential vulnerabilities of
local marine species can help inform decision making in the mCDR planning process regarding the placement of highly
alkaline seawater outflow pipes in nearshore environments. This will also allow scientists to perform targeted monitoring on 430
specific species that might be more sensitive during this process within the mixing zone of mCDR projects.
Additionally, the dependence of mortality on the duration of exposure is critical to the practical application of these results. In
practice, the maximum pH resulting from an OAE intervention will be observed at the outfall or point of dispersal. The pH
will decrease rapidly with distance through the mixing zone until the alkaline plume has diluted enough to where it will be
indistinguishable from natural variation in the open environment (Ho et al., 2023; Wang et al., 2023). If the species are mobile 435
over an area larger than the mixing zone radius, they will only experience the mixing zone maximum pH for some period of
time. However, immobile species, or species with small mobility ranges such as sea hares and isopods, located in the mixing
zone will experience it continuously. Understanding the potential exposure of OAE projects on marine organisms will likely
involve a combination of near-field dilution modeling of the release of alkalinity into seawater and in-situ sensing for pH
changes within the mixing zone. Studies investigating the impact of increasing alkalinity and pH on specific species should 440
take into account the natural chemical variations experienced before an OAE perturbation, the range of chemical changes
during an OAE perturbation based on the expected dilution of alkalinity and pH in time and space at varying distances from
an outfall, and the potential for acclimatization to increased alkalinity and pH as OAE projects scale up from pilot experiments.
4.2 Limitations and Future Recommendations
To avoid air exchange causing the pH to drop below the target level, we needed to secure our experiment jars with airtight 445
lids. This also prevented any animals from escaping the experiment. While this kept the pH within 0.1 of our target pH, it did
cause some lowering of dissolved oxygen levels between water changes (on average, sea hares: -2.67 mg/L, isopods: - 0.57
mg/L). This was mitigated by refreshing the water twice daily, however in an ideal experiment, we would have a flow-through
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system. A flow-through system would have a continuous flow of the treated water so the organisms would have ample oxygen
and the water would not need to be manually refreshed, reducing the need for physically handling the organisms. Decreasing 450
handling of organisms during the experiment might reduce unnecessary stress on sensitive species. Due to funding and
wastewater safety considerations, a flow-through system was not feasible at the time of this study.
It is also worth noting that during the initial treatment of seawater with NaOH, precipitation was visually observed but rapidly
dissipated upon mixing, indicating that this was likely Mg(OH)2 (Ringham et al., 2024). No additional precipitation was
observed after incubation or before refreshing water throughout the experiments, even for the high pH treatment (9.3), 455
suggesting that these experiments did not surpass thresholds for runaway CaCO3 precipitation, in which more alkalinity would
have been removed by precipitation than was initially added by the alkalinity treatment (Moras et al., 2022; Hartmann et al.,
2023; Suitner et al., 2023). Determination of precipitation thresholds is a major area of research for OAE because CaCO3
precipitation can reverse the intended effect of OAE by removing alkalinity from the surface ocean and releasing CO2 to the
water column. In addition, the increased turbidity resulting from precipitation may impact photosynthesis and predator-prey 460
interactions in the natural environment. Understanding connections between changes in carbonate chemistry and biological
activity is crucial for characterizing potential interactions between OAE and the biota in the receiving environment.
Experiments like those presented here will provide important baseline information for permitting OAE, particularly in coastal
waters where shallow well-mixed waters interact strongly with benthic biological communities that host organisms like sea
hares and isopods that are critical to marine food webs. 465
5. Conclusion
Understanding the biological implications of mCDR methods is an important consideration as this field expands. This current
study focused on the species level effects of locally important species in the Pacific Northwest. Future research should focus
on additional commercially, culturally, or ecologically important species. This work should be scaled up to understand the
effects of alkalinity enhancement on an ecosystem level using mesocosm studies, allowing for a more realistic depiction of a 470
natural ecosystem while still keeping the treatment confined and allow for both benthic and pelagic species or ecosystem
studies (Riebesell et al. 2023). Future research could leverage lessons learned from laboratory experiments and mesocosm
studies to eventually scale up to small scale field studies in sites under consideration for enhanced alkalinity approaches
(Cyronak et al. 2023).
Data Availability 475
Raw data will be made available by the authors.
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Author Contributions
Kristin Jones: Investigation, Formal analysis, Visualization, Writing - original draft preparation, Writing - review & editing
Lenaïg G. Hemery: Conceptualization, Methodology, Supervision, Writing - original draft preparation, Writing - review &
editing 480
Nicholas D. Ward: Funding acquisition, Methodology, Project administration, Writing - original draft preparation, Writing -
review & editing
Peter J. Regier: Formal analysis, Visualization, Writing - original draft preparation, Writing - review & editing
Mallory C. Ringham: Writing - original draft preparation, Writing - review & editing
Matthew D. Eisaman - Writing - original draft preparation, Writing - review & editing 485
Competing Interests
Matthew D. Eisaman is Co-Founder and Chief Scientific Advisor at Ebb Carbon, Inc.
Acknowledgments
This study was led by Pacific Northwest National Laboratory which is operated for the U.S. Department of Energy by Battelle
Memorial Institute under contract DE-AC05-76RL01830. Funding was provided by a multi-agency award funded by the 490
NOAA National Oceanographic Partnership Program, U.S. Department of Energy Water Power and Technology Office
(WPTO), and Climate Works as part of the Electrochemical Acid Sequestration to Ease Ocean Acidification (EASE-OA)
project, led by Brendan Carter (UW-PMEL), who we thank for his assistance throughout. The WPTO funded portion of the
EASE-OA project was a sub-task of the Oceans for Climate AOP led by Chinmayee Subban. We thank Ebb Carbon team
members and especially Tyson Minck for helping us understand the electrochemical OAE process and for assisting with the 495
experimental setup. The team also acknowledges former PNNL interns Kira Burch and Grace Weber, and PNNL researcher
Tristen Meyers for their help running the experiments, and PNNL staff Jakob Bueche for assisting with the experimental setup
and Ioana Bociu and Corey O’Donnell for assisting with permitting and compliance.
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