A novel methodology to characterize the potential impacts of electrochemical ocean alkalinity enhancement on juvenile coho salmon (Oncorhynchus kisutch)

Ocean alkalinity enhancement (OAE) includes a branch of marine carbon dioxide removal (mCDR) methods that add alkalinity to the surface ocean, leveraging the ocean's vast natural ability to capture and store atmospheric CO₂. The impact of OAE on marine ecosystems will depend on the type and delivery of alkaline feedstock to the ocean, which typically results in elevated pH and total alkalinity and decreased pCO₂ in the near-field of an OAE application. These signals will decrease in space and time away from the point of alkaline addition until are no longer measurable against the background of natural variability in the marine environment. It is important to evaluate potential impacts of OAE on marine ecosystems within the context of realistic OAE deployments. This study highlights the use of an effluent dilution model to describe the measurable extent of the release of electrochemically-generated aqueous alkalinity from Ebb Carbon's research pilot in Port Angeles, WA. We describe a novel laboratory method to simulate the potential exposure of juvenile coho salmon to the pilot's alkaline discharge, representing exposure to OAE field conditions as salmon swim through the pilot's mixing zone. Salmon were exposed to an electrochemically generated alkalinity-enhanced seawater solution pulsed into a test chamber at a dilution factor predicted approximately 3 m from the alkaline outfall. The alkalinity-enhanced seawater was held for 30 s, 1 min, and 5 mins, then was slowly flushed with ambient seawater. The alkaline solution, initially at pH_NBS 10.0, was released into seawater at pH_NBS 7.6, resulting in peak pH of the mixed solution of 8.04-8.09, with an increase in total alkalinity of ~60 μmol/kg. The results of the study indicated no impact on juvenile coho salmon behavior, survival, or physical effects on gills, eyes, or external body tissues, relative to control tests. The experimental design, developed for performance by a commercial toxicology laboratory and supported by standard mixing analyses, allows for rapid repetition with species of interest near OAE deployments.

1 Introduction

Efforts to constrain global warming to avoid the worst impacts of climate change require active removal of atmospheric CO₂ on the order of 5-15 gigatons per year by 2,100 on top of drastic emissions reductions (IPCC, 2021; Rogelj et al., 2018). The enormous carbon storage capacity of the ocean has brought increasing attention to marine carbon dioxide removal (mCDR) methods (National Academies of Sciences Engineering and Medicine, 2021; Cross et al., 2023). Ocean Alkalinity Enhancement (OAE) is notable for its high durability of carbon storage and ability to scale (Oschlies et al., 2023; Renforth and Henderson, 2017). OAE methods aim to increase alkalinity in the surface ocean by the direct addition of alkaline materials or removal of acid from seawater. The addition of alkalinity shifts inorganic carbon speciation toward bicarbonate (${\text{HCO}}_3^{-}$) and carbonate (${\text{CO}}_3^{2-}$), resulting in a disequilibrium in pCO₂ across the air-sea boundary. This allows for additional uptake of atmospheric CO₂ into seawater or reduced outgassing of CO₂ to the atmosphere. Atmospheric CO₂ is stored in seawater in the form of primarily ${\text{HCO}}_3^{-}$.

One OAE method uses electrochemistry to convert salt (NaCl) from seawater or brine into aqueous base (NaOH) and acid (HCl) streams (Eisaman et al., 2023; Eisaman, 2024). The acid is removed from the system and the combined base and remaining seawater or brine streams are returned to the surface ocean. There, the added alkalinity results in an immediate increase in pH and total alkalinity (TA) and decrease in pCO₂, the magnitude of which decreases as the alkaline plume dilutes away from the point source. Over the timeline of air-sea gas exchange (on order of weeks to months (He and Tyka, 2023; Wang et al., 2023), the re-equilibration of CO₂ across the air-sea boundary results in carbon storage as an increase in dissolved or total inorganic carbon (DIC or TIC).

Because electrochemical OAE produces aqueous alkalinity from seawater, it has potential advantages for scaling including limitation of trace metal impurities relative to mineral alkalinity sources, and simplified distribution and near-field dilution modeling in the ocean. At the current scale of OAE field pilots, alkalinity is released into the surface ocean through coastal outfalls, which may take the form of pipes, diffusers, or channels. As OAE scales, integration with other coastal facilities such as desalination may impact the way in which aqueous alkalinity is introduced to the ocean.

While the impacts of ocean acidification (i.e., decreased pH and increased pCO₂) on marine organisms have been long studied, there is limited information on ecological responses to OAE (Bach et al., 2019). OAE may impact marine ecosystems in both: (1) the near-field and short-term of a deployment (i.e., experiencing increased pH and TA and decreased pCO₂), and (2) through the increase in DIC resulting from scaled OAE and mCDR over time. Marine ecosystems may also impact OAE: biological processes such as biotic calcification have the potential to decrease the efficacy of OAE within both near-field and scaled contexts. A particular focus in ecological response studies centers on impacts to ecologically, culturally, and economically important species present near OAE deployments, often including fish. There are few direct studies to date on OAE impacts to fish (e.g., Goldenberg et al., 2024; Marx et al., 2025; Sloterdijk et al., 2025).

To understand the impact of OAE pilots and field trials on the marine environment, we must: (1) understand how alkalinity is introduced and how it dilutes in the surface ocean, and (2) identify the species and habitats that may be impacted by altered seawater chemistry. Within the United States, coastal outfalls are regulated under the Clean Water Act Section 402 by the Environmental Protection Agency (Murthy et al., 2025). National Pollutant Discharge Elimination System (NPDES) permits are required for the discharge of any pollutants (defined broadly including any type of industrial, municipal, and agricultural waste) through a point source (defined broadly as any discernable, confined, and discrete conveyance, including pipes, ditches, channels, and containers) into US waters [noting that mCDR activities beyond the US baseline from which the territorial sea is measured are regulated under the Marine Protection, Research, and Sanctuaries Act (MPRSA)]. NPDES permits are issued by the EPA or authorized States in coordination with appropriate Tribal, federal, state, and local entities. The permitting process includes consideration of related acts (Endangered Species Act, Magnuson Stevens Fishery Conservation and Management Act, Coastal Zone Management Act), opportunities for public review and comment, and project assessment to ensure permitted activities do not harm water quality or human health (EPA-833-K-10-001).

One regulatory concept that can be applied by NPDES permits is that of a mixing zone. A mixing zone is defined as a limited volume of water in which initial dilution of an effluent occurs, within which certain water quality criteria may exceed water quality standards. If no mixing zone is permitted, then water quality criteria must be reached at the point of discharge at the ‘end of the pipe.' To permit a mixing zone, a steady-state water quality model is used to characterize the dilution of an effluent into receiving water, which is then used to determine both the size of the permitted mixing zone and allowable water quality exceedances for each pollutant of concern. In the context of OAE, pH can be regulated by these mechanisms.

Herein we focus on the first NPDES-permitted mCDR project, Ebb Carbon's Project Macoma. Project Macoma is a temporary electrochemical OAE pilot operating in Port Angeles Harbor, WA, US (Industrial NPDES permit number WA0991051) (Department of Ecology State of Washington, 2024) (Figure 1). In broad strokes, the pilot is designed to pump seawater from Port Angeles Harbor to a land-based electrochemical treatment facility where it is processed and de-acidified. The alkalinity-enhanced seawater is discharged back to Port Angeles Harbor from a barge-based outfall. The Project Macoma pilot is conducted to demonstrate the electrochemical OAE technology's efficacy in carbon removal, while evaluating potential ecological impacts and co-benefits associated with the process. The Project was granted a mixing zone authorization that specifies allowed water quality exceedances and monitoring requirements within an acute mixing zone, a circle of radius 20.7 ft or 6.3 m measured from the center of each discharge port, and a chronic mixing zone, a circle of radius 207 ft or 63.1 m, measured from the center of each discharge port, each extending from the bottom to the top of the water column. Routinely discharged effluent has a maximum pH_NBS of 9.8 set by the NPDES permit. The concentration of pollutants at the edge of the acute and chronic zones must meet Acute and Chronic Aquatic Life Criteria, respectively. As an example, pH_NBS must fall within the range of 7.0-8.5 with a human-caused variation within this range of less than 0.5 units at the edge of the chronic mixing zone boundary. The requirements were determined based on WA Department of Ecology water quality standards and the NPDES permit application process (Washington State Department of Ecology, 2002, 2016, 2018; US Environmental Protection Agency, 2010). This includes modeling of the alkaline effluent into Port Angeles Harbor performed by a professional engineering firm using Visual Plumes (https://www.epa.gov/hydrowq/visual-plumes) for dynamics and chemistry modeling using commercial OLI Systems software (DeBoer and Oza, 2024). While further discussion of these analyses is out of the scope of this study, the results are publicly available and were significant in providing context for the biological methods to follow, as discussed in Section 2.1.

Figure 1

Figure 1. Location of Project Macoma (a, b), utilizing a barge-based outfall diffuser to release alkalinity-enhanced seawater to Port Angeles Harbor (c, d). The borders of the acute mixing zone (20.7 ft or 6.3 m) and chronic mixing zones (207 ft or 63.1 m) are represented on panel (d).

Port Angeles Harbor is one of the largest natural deep water harbors on the west coast of the US, with near-shore depths within the Project mixing zone ranging from 5-15 m depth, sloping to >50 m along the natural arm of Ediz Hook. Ambient conditions within the harbor include average seawater temperatures of 10.0 °C (October through April) and 11.4 °C (May-September), with an average surface salinity of 30.8. The WA Department of Ecology reports an average pH 7.8 and dissolved oxygen of 7.3 mg/L in this region. Few direct historical observations for seawater carbonate chemistry are available within the harbor, particularly toward the western end. Ebb Carbon has continuously collected water quality, seawater carbonate chemistry, and current data at the location of Project Macoma since October 2024, which is described in the Supplementary material.

Port Angeles, a city of about 20,000 in Clallam County on the northern edge of the Olympic Peninsula, has a rich history of Native American habitation dating back over 2,700 years. In the nineteenth and twentieth centuries, logging and farming were primary industries, and Port Angeles Harbor was once dominated by large sawmills, plywood manufacturing, pulp, and paper production facilities, as well as marine shipping/transportation, boat building and refurbishing, petroleum bulk fuel facilities, marinas, and commercial fishing industries (NOAA, 2024). These industries discharged heavy metals, dioxins, PCBs, and petrochemicals into the harbor. The WA Department of Ecology is conducting eight environmental cleanup projects in and around Port Angeles (including at the planned site of Project Macoma) as part of the Puget Sound Initiative.

Ebb Carbon conducted extensive rights holder and stakeholder engagement to inform the design of Project Macoma and to hear directly from community members about how the project might affect their community. Among those most potentially impacted by Project Macoma's operations are local Indian Tribes, who serve as co-managers of Washington's natural resources and hold federally recognized treaty rights to the region's lands and waters. Through early and ongoing consultation with local Indian Tribes, a key area of interest emerged: understanding the potential impact of the alkaline-enhanced seawater on juvenile salmon. Port Angeles Harbor provides vital habitat for a wide range of species—including shellfish, fish, birds, and marine mammals—that rely on this shoreline ecosystem. Salmon are especially important, serving a vital ecological function and carrying deep cultural significance for local Tribes.

Ebb Carbon collaborated with the Lower Elwha Klallam Tribe (LEKT), Anchor QEA, and EcoAnalysts, a Spheros Environmental Company, to evaluate potential impacts of temporary alkaline exposure on juvenile coho salmon within simulated mixing conditions predicted for Project Macoma. We developed a novel laboratory methodology– a “pulse test”—intended to simulate the brief exposure fish might experience while swimming past the pilot project's alkaline-enhanced seawater discharge, approximately 3 m from the outfall and within the acute mixing zone. LEKT contributed juvenile coho salmon (Oncorhynchus kisutch) from their 2023 hatchery brood year for use in the testing.

The design criteria for this study included:

1. Appropriate simulation of alkaline discharge representing dilution ratios predicted to occur at ~3-6.3 m from the outfall, conservatively chosen within the acute mixing zone.

2. A range of exposure times conservatively representing durations predicted for juvenile salmon to swim through this portion of the mixing zone.

3. Experimental design suitable for performance by a commercial toxicology laboratory.

Further, the study was designed to answer the following questions:

1. How does the alkaline discharge interact with seawater upon release?

2. Are there acute effects on juvenile coho salmon in the form of mortality or physical damage (i.e., gills, eyes, and skin) during or after exposure to the alkaline discharge?

3. Are behavioral differences observed during or after exposure to the alkaline discharge?

We note that the study is not intended to assess impacts of chronic exposure to alkaline discharge, interactions with marine food webs, or potential impacts of OAE at large. This study was designed and conducted well in advance of any alkaline release from Project Macoma (e.g., December 2024 vs. August 2025). While Ebb Carbon staff were involved in the design of the study, the laboratory experiments, analysis, and interpretation were conducted independently by EcoAnalysts in December 2024. The report was summarized by Anchor QEA into a memo submitted to the WA Department of Ecology and LEKT (Galaska and Soccorsy, 2025b), and Ebb Carbon has since led this description of the experimental design, results, and context with Project Macoma and prior studies for the purpose of peer review.

2 Methods

2.1 Overview

A laboratory study was designed to expose fish to an alkaline discharge for a brief period, simulating their exposure within the alkalinity plume approximately 3-6.3 m from the outfall of Project Macoma, within the acute mixing zone. Juvenile salmon were acclimated to ambient seawater in test chambers as described in the following sections. They were exposed to a pulse of undiluted alkaline discharge (at pH_NBS 10.0) introduced into the test chamber, where it mixed into filtered seawater, resulting in the dilution ratio expected at 3-6.3 m from the outfall as predicted by a mixing analysis. The fish were continuously exposed to this alkalinity-enhanced seawater solution for full exposure durations of 30 s, 1 min, or 5 min, followed by decreasing exposure during slow flushing with ambient seawater, to represent potential exposure times of a juvenile salmon swimming through the mixing zone, as described in Section 2.4. Studies were conducted by EcoAnalysts at their Port Gamble Environmental Laboratory in Port Gamble, Washington, US (Galaska and Soccorsy, 2025a).

The mixing analysis for routine operations at Project Macoma is summarized in the Supplementary material (DeBoer and Oza, 2024). The dilution model was validated using the release of a dye tracer from the Project's outfall diffuser, paired with current velocity data collected with a Nortek Eco Acoustic Doppler Current Profiler (ADCP) placed within the Project's mixing zone (see Supplementary material). These analyses indicate that the routine pH 9.8 alkaline discharge from this OAE pilot will rapidly dilute into Port Angeles Harbor, resulting in a dilution ratio of seawater to alkaline discharge that ranges from ~70-123 to 1 at ~3 m from the center of the outfall, depending on conservative assumptions used for ambient currents in the receiving water (0.02 and 0.05 m/s, respectively,) and ambient pH (assuming ambient pH_NBS 7.8 but including an analysis of sensitivity to ambient pH ranging from 7.5-8.5). These dilution factors correspond to a predicted pH_NBS at this location in the alkaline effluent plume of ~8.30-8.07, respectively, dependent on initial ambient seawater conditions and assuming continuous release of effluent at the maximum permitted value of pH_NBS 9.8. The predictions indicate that the temperature and salinity of the plume return to ambient conditions at the targeted location in the acute mixing zone. We note here and in the Supplementary material that these dilution predictions are conservative both within the modeling methods and parameterization and in the Project's operations. As of November 26, 2025, the Project has never released effluent at the maximum pH_NBS of 9.8 in order to remain safely within bounds of the permit during research activities. For these reasons, the modeled release of effluent at the permit limit of pH_NBS 9.8 over represents the maximum change in pH expected in situ during routine operations.

Based on these considerations and equipment availability within the laboratory that conducted these tests, this study focused on monitoring pH during the alkaline exposures. We note that other impacts on seawater chemistry, including increased total alkalinity and decreased pCO₂, have the potential to impact marine organisms. However, as discussed in Section 4 below, pH changes are likely the most important factor that may impact the health of salmon passing through an OAE field site, through alteration of H⁺ and carbonate ion concentrations. This study was not designed to interrogate the mechanisms for physiological impacts of increased pH on juvenile salmon in depth, but instead to test the realistic physical and behavioral responses of mobile juvenile salmon passing through the acute mixing zone at Project Macoma.

2.2 Coho smolting

Approximately 250 hatchery-reared juvenile coho salmon (Oncorhynchus kisutch) were provided by the Lower Elwha Klallam Tribe. These were hatched on January 8, 2024, began feeding on February 17, 2024, and were reared in freshwater. The fish averaged 8.45 g upon arrival at EcoAnalysts Port Gamble Laboratory on November 15, 2024. The culture was slowly acclimated from freshwater to marine conditions to ensure the fish were conditioned for testing parameters that mimic environmental conditions in Port Angeles Harbor. Water quality measurements were taken in the culture tanks daily prior to partial exchanges with seawater that increased the overall salinity by no more than 2 ± 1 ppt per day. The culture was fully acclimated to marine conditions (30 ± 2 ppt) after 15 days. Fish were received and held at 6 ± 2 °C for approximately 1.5 weeks during the initial smolting process. Temperatures were slowly increased to approximately 9 °C after smolting was complete and experimental testing was set to begin, which occurred on December 3-6, 2024. Netting was placed over the culture tubs to prevent fish from escaping. The culture was fed 2-3 times daily at 3.6 g per feeding (1% their total weight) using food provided by the Lower Elwha Klallam Tribe. The fish were withheld food beginning at 24 h prior to test initiation and no food was provided during the tests.

2.3 Test chamber design

Testing was conducted in four 167 L cylindrical polycarbonate drums filled with ~146 L of sand-filtered seawater sourced from the northern Hood Canal at Port Gamble, WA, ~70 km east of Project Macoma. The size of the test chamber, water volume, and maximum number of organisms per chamber was determined following loading rate recommendations in USEPA OPPTS guidance (i.e., 0.8 g fish per liter; USEPA 1996). Four replicate chambers containing 10 fish each were tested in each of three exposure conditions described in Section 2.4 below. A separate control exposure was also performed where the alkaline solution was replaced with sand filtered seawater. Each replicate test chamber used a 5 L glass separatory funnel to gravity-feed the alkaline sample beneath the surface of the seawater. The discharge end was designed to simulate Project Macoma's outfall design, which is angled up and away from the seafloor to promote mixing (Figure 2). Ebb Carbon provided the electrochemically-generated NaOH and seawater alkaline solutions that were created following the Project Macoma routine discharge scenario. This alkaline sample had a pH_NBS of 10.0 ± 0.1, TA ~ 2400 μmol/kg, and salinity 40 ppt ± 2 ppt. Flow-through piping within each chamber allowed for seawater to be refreshed through the bottom of the tank and for excess seawater to flow out of a screened port at the top. HOBO pH loggers (MX2501) were placed at the surface and bottom of one test chamber to track pH time-series at a frequency of 30 s to 1 min with a pH resolution of 0.01 and ± 0.10 accuracy. For each test, temperature, salinity, pH, and dissolved oxygen were measured in the alkaline sample, in one test chamber prior to the alkaline discharge, and in one test chamber at the end of a 3 h flushing period post alkaline discharge. A video camera (Logitech webcam model HD 1080p) was placed above each test chamber to monitor fish behavior throughout the test, beginning before the alkaline discharge and ending after the removal of fish for physical measurements. Video was recorded using Bandicam software. For video clarity, test chambers were not aerated during testing.

Figure 2

Figure 2. Schematic of test chamber with alkaline discharge and flow-through seawater lines (a); photograph from experimental setup (b).

All laboratory instruments were calibrated daily or on their recommended schedules. Water quality measurements were conducted with an Orion Star meter (Model A329) with pH, ISE, conductivity/salinity, and dissolved oxygen (DO) probes. Orion Star water quality measurements had a precision of ± 1 ppt, ± 0.05 pH, 0.1 mg/L DO, and ± 0.10 °C. Salinity probes underwent a 2-point calibration weekly using 1413 μS/cm and 12.9 mS/cm standard solutions with an additional check standard of 35 ppt to ensure salinity measurements were within ± 1 ppt of calibration. Temperature was measured from the salinity probe, which is calibrated annually against a NIST-certified (National Institute of Standards and Technology) thermometer to confirm temperature accuracy. DO probes underwent daily auto calibration with a calibration sleeve and deionized water saturated sponge. For measurement of pH, pH probes underwent a 2-point calibration daily using 7.00 and 10.01 pH buffer solutions with an additional check standard of 8.00 to ensure pH measurements were within ± 0.05. The pH probe was calibrated at the test temperature of 12 ± 2 °C. The HOBO pH data loggers were calibrated the day of or day prior to each experiment using a 2 point calibration with 7.00 and 10.01 pH buffer solutions and an additional check standard of 8.00 to ensure pH measurements were within ± 0.05. HOBO loggers were calibrated at the test temperature of 12 ± 2 °C.

2.4 Test procedure

The salmon were randomly divided into one control group and four test groups designated for different alkalinity exposure periods. Due to space restrictions, the control and three exposure experiments were tested independently of one another over a period of four consecutive days. All employed the same organism batch, alkaline sample, and test conditions. Each individual fish was only used for one experiment.

For each of the control and exposure experiments, 10 fish were loaded into each of four test chambers the day prior to testing and flow through seawater was turned on to allow the fish time to acclimate to test conditions. Each test chamber was dosed with 3.8 L of alkaline sample at an initial pH_NBS of 10.0 ± 0.1 via a control valve on the separatory funnel, representing an estimated increase in TA by ~2400 μmol/kg. This brought the final volume of the alkalinity-enhanced seawater of each test chamber to ~150 L (or 2.5% concentration of the alkaline discharge, with a TA increase of ~60 μmol/kg), representative of the dilution modeled within the acute mixing zone during mixing analyses. The alkaline sample dosing period took ~ 1.5 min for the sample to be fully discharged and mixed into the test chamber. The exposure period began immediately following the complete discharge of the alkaline solution. This duration was initially verified visually and via pH measurements using a dyed alkalinity solution without the presence of test organisms, and is further described in the Supplementary material.

Following the initial alkaline discharge period, each test chamber was held at static conditions to expose the fish to the discharge sample. The salmon were exposed to the alkalinity-enhanced conditions for 30 sec, 1 min, and 5 min. These conditions were determined based on the typical swimming speed of a juvenile coho salmon (0.3-0.5 m/s; Glova and McInerney, 1977; Griffiths and Alderdice, 1972; Taylor and McPhail, 1985), such that a salmon could pass through the 6.3 m acute mixing zone within 14-21 s and through the entire 63.1 m chronic mixing zone within 2-4 min. Without further assumptions on time spent in the mixing zone (i.e., while feeding or hiding from predators) these exposure periods would conservatively reflect or exceed the estimated time that juvenile salmon would spend within the mixing zone of Project Macoma's outfall.

Following the given exposure period, each test chamber was flushed with seawater at a rate of ~11.4 L per min to slowly dilute and flush out the added alkalinity. This flushing period continued for 3 h. During this period, fish were still exposed to elevated pH and alkalinity conditions, albeit at increasing dilution over time.

All 4 test chambers were used for simultaneous replicates at each exposure condition. Biological measurements, as described in Section 2.5 below, were conducted on fish within three of the test chambers at the end of the 3 h flushing period for each exposure condition. Fish were removed from the fourth chamber immediately after the discharge period for inspection for physical impacts, without the 3 h flushing period.

The control was conducted similarly to that of the longest exposure experiment, but in this case, filtered seawater was discharged into the test chamber in place of the alkaline sample. The control was held for 5 mins following the seawater discharge, then flushed for 3 h as in other test cases.

2.5 Biological observations

Fish from all replicates in all test chambers and exposure experiments were inspected for physical conditions, survival rates, and behavioral changes during and after exposure using video analysis and physical measurements. Each fish was measured in length and weight. Bodies, gills, and eyes were inspected for physical abnormalities. Still photographs were captured for all fish from the fourth chamber, for which fish were inspected immediately after the discharge period without a flushing period. Inspection of both fish behavior and physical condition was unblinded.

Each test subject was examined for the following characteristics (following best practices including EPA 823-B-00-007 and WA Department of Ecology WQ-R-95-80): gills were examined for pale, darkened or reddened gills associated with anemia or hypermia, frayed lamellae, and unusual mucus production. Eyes were examined for cloudiness or opacity, sunken or popped features, deformities, and hemorrhage. External skin tissues were examined for lesions, nodules, cysts, spots, abnormal pigmentation, hemorrhage, visible parasites, and skin conditions.

Each test chamber video was analyzed to note behavioral changes during and after the alkaline sample discharge. Observations included: notation if fish attempted to avoid the discharge outlet to avoid contact with the alkaline sample; multiple fish clustering within the test chamber; flashing or scratching on the walls or piping within the chamber to relieve itching; erratic swimming; gulping, and gill flushing. Video observations were notated continually throughout the sample discharge and exposure period, then at intervals of 30 s, 1 min, 5 mins, 30 mins, 1 h, 2 h, and at the end of the test at 3 h post-exposure during the flushing period. Observations are provided in the Supplementary material.

3 Results

Water quality within the test chambers measured within recommended limits following USEPA OPPTS (US Environmental Protection Agency, 1996) test procedures for temperature, salinity, dissolved oxygen, and pH during the control experiment. A summary of water quality parameters and impacts to test subjects for the control and exposure experiments is available in Table 1. Time-series of pH data for the control and exposure experiments are available in Figure 3. Detailed results for each replicate test chamber are available in the Supplementary material.

Table 1

Table 1. Summary of experimental observations for water quality ranges and impacts on fish health and behavior during alkaline exposures.

Figure 3

Figure 3. Time series of pH data collected in the control (a) and exposure 30 s (b); 1 min (c); 5 min (d) experiments from surface and bottom pH loggers. The shaded error bars represent the ~0.05 precision of the pH loggers, where the logger at the top of the test chamber is indicated in blue, the logger at the bottom of the chamber is indicated in gray, and the darker blue bar identifies the overlap in measurements between the two loggers. The vertical green (control, a) and gold (exposure experiments, b-d) bars indicate the ~1.5 mins during which the seawater control or alkaline sample was drained into each test chamber beginning at t = 0 mins, followed by a given exposure holding period represented by the vertical magenta (control, a) or red (exposure, b-d) bars. Within each experiment, three test chambers were slowly flushed with ambient seawater during the remaining 180 mins time period, followed by physical analysis of each fish. Within the fourth test chamber of each experiment, fish were collected and analyzed immediately following the discharge period.

The test chamber seawater before the control experiment had a temperature of 11.2 ± 2 °C, salinity of 30 ± 2, dissolved oxygen of 6.9 mg/L, and pH of 7.62 ± 0.05. The filtered seawater discharged into the test chambers in place of an alkaline sample measured identically to that of the starting seawater. The test chamber seawater at the end of the 3 h flushing period measured 11.2 ± 2 C, 31 ± 2 ppt, and dissolved oxygen 7.2 mg/L. The pH HOBO data loggers measured consistently throughout the experiment, with a range of 7.62-7.63 (top) and 7.66-7.67 (bottom), within the ± 0.05 tolerance of the instruments.

The alkaline solution was similar between exposure experiments, initially measuring pH_NBS 10.0 ± 0.05 and salinity 38 ± 2 ppt prior to discharge into the test chambers. Water quality within the test chambers measured within recommended limits following USEPA OPPTS (US Environmental Protection Agency, 1996) test procedures for temperature, salinity, dissolved oxygen, and pH for all exposure experiments, but initial conditions varied slightly as exposure experiments were conducted on separate days using fresh seawater (Table 1).

The 30 s exposure experiment had an initial temperature of 11.2 ± 2 °C, salinity 30 ± 2, dissolved oxygen 7.0 mg/L, and pH 7.63 ± 0.05. p^H increased due to the alkalinity addition, reaching maximum values of 7.92 ± 0.05 (top) and 8.04 ± 0.05 (bottom), respectively, (Figure 3). The fish were exposed to alkalinity for the full 30 s experimental duration, plus an extended period of exposure to increasingly diluted alkaline discharge as the chambers were slowly flushed. At the end of the 3 h flushing period, test chamber seawater measured 11.2 ± 2 °C, 31 ± 2 ppt, dissolved oxygen 7.0 mg/L, and pH 7.65 (top) and 7.72 (bottom).

The 1 min exposure experiment had an initial temperature of 10.8 ± 2 °C, salinity 30 ± 2, dissolved oxygen 6.9 mg/L, and pH 7.70 ± 0.05. pH increased due to the alkalinity addition, reaching maximum values of 7.98 ± 0.05 (top) and 8.05 ± 0.05 (bottom), respectively, (Figure 3). The fish were exposed to alkalinity for the full 1 min experimental duration, plus an extended period of exposure to increasingly diluted alkaline discharge as the chambers were slowly flushed, but in general the test chambers were better homogenized and more rapidly returned to baseline initial seawater conditions than in the 30 s exposure experiment. At the end of the 3 h flushing period, test chamber seawater measured pH 7.70 (top) and 7.71 (bottom).

The 5 mins exposure experiment had an initial temperature of 11.3 ± 2 °C, salinity 31 ± 2, dissolved oxygen 7.7 mg/L, and pH 7.70 ± 0.05. pH increased due to the alkalinity addition, reaching maximum values of 8.02 ± 0.05 (top) and 8.09 ± 0.05 (bottom), respectively, (Figure 3). The fish were exposed to alkalinity for the full 5 mins experimental duration, plus an extended period of exposure to increasingly diluted alkaline discharge as the chambers were slowly flushed, but in general the test chambers were better homogenized and more rapidly returned to baseline initial seawater conditions. At the end of the 3 h flushing period, test chamber seawater measured 11.0 ± 2 °C, 31 ± 2 ppt, dissolved oxygen 7.1 mg/L, and pH 7.73 (top) and 7.75 (bottom).

There were no mortalities in any of the control or timed exposure experiments. During the control experiment, one fish exhibited an injured operculum and another appeared to be slightly bloated with a darker complexion. No other anomalies were noted in the gills, eyes, or external body tissues of control subjects. During the 30 s exposure experiments, two fish exhibited a slightly darker complexion. No other anomalies were noted in the gills, eyes, or external body tissues of the test subjects during this set of experiments. No anomalies were noted in the gills, eyes, or external body tissues of the test subjects in the 1 min and 5 min exposure experiments (Figure 4).

Figure 4

Figure 4. Representative photographs of test subjects from control (a), 30 s (b), 1 min (c), and 5 min (d) alkaline exposures.

All fish were alert and active through the duration of each test in each chamber of the control and all timed exposure experiments. All fish attempted to evade capture for physical observations at the end of each test. At least one fish was observed swimming directly below the visible brine plume of the alkaline sample outlet during alkalinity discharge without noticeable avoidance or stress behaviors in two test chambers in the control, 30 s, and 1 min exposures, and in all test chambers of the 5 min alkaline exposure. There were no instances of visible stress behaviors in the way of gulping, flashing, scratching, gill flushing, or erratic swimming.

4 Discussion

4.1 Representation of mixing zone via alkaline discharge design

The dilution factors predicted at ~3 m from the Project Macoma outfall correspond to an estimated pH_NBS of ~8.07-8.30, based on the conservatively assumed ambient current used in the mixing analysis and assuming continuous release of an effluent with pH_NBS 9.8 into ambient seawater of pH_NBS 7.8. The lower end of this range was achieved within the ± 0.05 error of the HOBO pH loggers used in these experiments, which reached a maximum pH_NBS of 8.04, 8.05, and 8.09 during the 30 s, 1 min, and 5 min exposure periods, respectively.

The lowest peak pH_NBS measured at 8.04 in this study corresponded to a dilution factor of ~143, which is estimated to occur within the acute mixing zone at ~5.8 m and 3.5 m from the outfall, assuming an ambient current of 0.02 and 0.05 m/s, respectively. By the edge of the acute mixing zone at 6.3 m, pH_NBS is predicted between 8.01 and 8.04. The pH ranges achieved in this study therefore generally represent predicted conditions within the further half of the acute mixing zone moving away from the alkaline outfall. We note that comparison of absolute pH values across modeled field conditions and laboratory measurements is limiting. The peak pH values achieved in the laboratory study were approximately 0.4 units above the ambient pH into which alkalinity was released, and modeled field data projects an increase in pH of ~0.29-0.52 at 3 m from the outfall and 0.23-0.26 at 6.3 m from the outfall, indicating that the lab study represents a point between these two locations.

A full description of Project Macoma operations and field data is out of scope of this study, which used dilution model results to test impacts on coho salmon in advance of alkalinity release into the field, but the mixing zone analysis is further discussed in the Supplementary material. In advance of the submission of field data to a future publication, we note that there has been no identified shift in seawater pH at 3-6.3 m from the outfall, as assessed by sensor and sample data during the release of alkalinity from mid-August 2025 to the submission of this manuscript. The discrepancy between field and model data is likely due to (1) release of effluent at a pH below the modeled maximum permitted value of 9.8, and (2) the conservative nature of the dilution model.

A dye was released within the test chamber to determine the optimum placement of the alkaline discharge port to represent Project Macoma, as well as to assess appropriate timelines for alkaline dosing, exposure, and flushing in this experiment (see Supplementary material). While the pH loggers at the bottom of each test chamber recorded slightly higher pH than the surface loggers during the alkaline exposure experiments, with a maximum difference in pH of ~0.05 during the 30 s exposure experiment, these variations occurred within error of the loggers. The alkaline sample was fairly evenly distributed during the exposure period of each experiment, and was further homogenized during the flow-through flushing phase of each experiment. This ensured that each salmon experienced similar conditions throughout each test, representative of the fish remaining in this portion of the mixing zone for the duration of each alkaline exposure experiment. Beyond the tested exposure periods, the fish remained in seawater at elevated pH for an extended duration while the test chambers were slowly flushed. As such, these experiments conservatively represented juvenile fish swimming within the acute mixing zone for at least 30 s, 1 min, and 5 min, but also remaining within a pH solution elevated above the baseline for periods ranging from 42 to 131 min.

Placing these data in the context of Project Macoma (with average pH_NBS ~7.8 and TA ~2200 μmol/kg) and among TA treatments used in other OAE studies, a discharge of alkalinity-enhanced seawater at pH_NBS 9.8 may represent an increase in estimated TA on order of ~1950 μmol/kg at the point of discharge, which rapidly dilutes into surrounding waters. Assuming DIC remains unchanged in the initial dilution, a CO₂sys calculation (assuming 12C and 30 ppt, using constants from Lueker et al., 2000; Dickson, 1990) using the mixing analysis predicted pH at 3 m from the outfall indicates an increase in TA of ~ 88-194 μmol/kg, depending on assumptions regarding ambient currents. While it is not entirely straightforward to infer the results from the laboratory experiments to predict alkalinity and pH conditions resulting from OAE in the field, given complexity in tides, currents, outfall design, and other factors, it is useful to consider the spatial extent of OAE conditions when extrapolating laboratory results to species and eventually ecosystem level predictions. We note that the ambient seawater used in this study, sourced from the laboratory's flow through system in Port Gamble, WA, had a lower pH_NBS than observed in the field at Project Macoma (e.g. 7.62-7.70 vs. 7.8, respectively). The peak pH observed using this method would be higher if we started from an elevated ambient seawater pH, which may warrant future testing.

These results indicate the pulse of alkalinity into a test chamber followed by slow seawater flushing is an appropriate method to represent the exposure to elevated pH on mobile fish species (such as the regionally important coho salmon) swimming through the Project Macoma mixing zone. This is not a sufficient study design to understand impacts on slow-moving or sessile organisms that remain within the mixing zone for extended periods of time or that might be more sensitive to the exposure. It is also difficult to extrapolate to mobile species that remain in the mixing zone for longer periods of time. While we assume that mobile species will leave an effluent mixing zone if they experience discomfort, they may remain for reasons such as foraging or predator avoidance, or if it takes some time for the fish to feel discomfort. Evaluating the habitats and biodiversity within OAE sites, using visual survey methods, offers valuable context for understanding potential responses of the marine ecosystem. Other surveys have been performed at the proposed site that may provide context into how salmon may use this area including a video and sonar survey followed by a dive survey (September 2024; Memorandum by Baxter et al., 2024) that indicated limited quality of aquatic vegetation in the mixing zone. The lack of native or nonnative eelgrass and sparsity of other vegetation in the region suggest limited cause for salmon to remain within the mixing zone for purposes of foraging or avoiding predators.

While this study focused on pH measurements, other seawater carbonate chemistry changes, including a decrease in pCO₂ and shift toward bicarbonate and carbonate ions due to the addition of alkalinity, may impact marine species in multiple ways– both directly through physiological impacts on any given species studied, and indirectly through impacts on both predator and prey species. This study design could be modified to include measurement of another carbonate parameter to assess additional chemical changes, which are particularly important for calcifying shellfish and plankton and for aquatic vegetation.

We note that this study does not address implications of OAE outside of the mixing zone. At this pilot stage, there is limited expectation that DIC will increase enough to be measurable against the background of natural variability except in highly controlled settings, as has been noted in regional OAE modeling studies (Wang et al., 2023; Khangaonkar et al., 2024; Ho et al., 2023). However, as mCDR efforts increase toward climate relevant scales, marine organisms may experience additional shifts in carbonate chemistry that require further study. This has been considered in recent NaOH-based OAE ecological safety studies (e.g., Bach et al., 2019; Marín-Samper et al., 2024; Guo et al., 2025) through designation of “equilibrated” treatments (resulting in minor shifts in CO₂ and pH) and “unequilibrated” treatments representing the low CO₂ and elevated pH resulting from alkalinity addition before air-sea equilibration of CO₂ drives atmospheric carbon uptake and storage in seawater. For the purposes of understanding the potential environmental impacts of specific OAE projects, we stress that consideration of realistic exposures to altered seawater chemistry including mixing zone analyses that consider dilution, near-field measurements, and evaluation of the species that are likely to be present within the footprint of an OAE deployment over time and space scales relevant to the site are critical to advancing mCDR.

4.2 Impacts of elevated pH on juvenile coho salmon health and behavior

Coho salmon are of major importance to the Pacific Northwest across cultural, economic, and ecological lenses (Noakes, 2014; Pearsall and Schmidt, 2024; Rapaport, 2024). Coho salmon play an important role in nutrient cycling and tie together multiple food webs, from grazing on plankton, fish, and benthic and pelagic invertebrates, to serving as prey species for both terrestrial and marine species (Pearsall et al., 2021; Losee et al., 2014; Couture et al., 2024; Quinn and Losee, 2022).

For these reasons, understanding the potential impact of Project Macoma on juvenile salmon passing through the shallow mixing zone is critical to the operational design and social license of this OAE research pilot.

The results of physical inspection and video observation from this study indicate that temporary exposure to alkaline conditions representing Project Macoma's acute mixing zone does not appear to harm juvenile coho salmon. Fish behavior under alkaline exposure was normal relative to the control before, during, and after alkaline exposure. The salmon did not attempt to avoid the control or alkaline solution as it was discharged into the test chambers, indicating lack of additional stress or physical discomfort, and they exhibited normal predator evasion responses during netting for physical examination at the end of each experiment. The duration of exposure under alkaline conditions may exceed exposure to Project Macoma effluent in the field where fish may swim through the mixing zone freely.

Coho salmon spawn in freshwater coastal streams and river tributaries and may live in either saltwater or brackish estuarine conditions outside of spawning periods. Juveniles may traverse freshwater and marine systems for many months after leaving spawning grounds (Munsch et al., 2025). As such, they experience diverse physical and chemical conditions in the natural environment. Preliminary measurements of surface pH_NBS within Port Angeles Harbor (February 2024-2025, see Supplementary material) indicate an average ambient pH_NBS of 7.86 and range of 7.43-8.48 in the coastal nearshore. pH in the coastal environment varies based on multiple factors including terrestrial runoff, tidal fluctuations, ocean acidification and biogeochemical cycles. While the pH conditions that were tested in this study, based on conservative mixing analyses, fall within the range of natural variability for marine species in the mixing zone of Project Macoma, it is important to note that consistent release of alkalinity resulting in an elevated pH over time, even within the range of natural conditions, could be anomalous in the coastal ocean, warranting continued modeling and testing on both the potential harms and co-benefits of OAE deployments. With the natural variability of the near-shore in mind, it is not surprising that juvenile coho salmon tested in conditions of elevated pH and alkalinity did not exhibit any difference in physical impacts or behavior relative to those tested in a control over the duration of these experiments.

It is known that fish can be highly sensitive to ${\text{HCO}}_3^{-}$, CO₂, and H⁺, which are altered by both ocean acidification and OAE (Perry and Gilmour, 2006; Melzner et al., 2009; Tresguerres et al., 2020). Acidification has been linked to respiratory, behavioral, and reproductive issues in salmon (Crozier and Siegel, 2023). (Williams et al. 2018) investigated the impacts of elevated CO₂ on adult ocean-phase coho salmon. The authors concluded that acidifying conditions in Puget Sound may result in altered neural signaling pathways in olfactory-mediated behavioral responses, which may impact coho salmon's ability to avoid predators, find prey, and migrate to their natal streams. An earlier study by Perry, 1982 noted efficient and rapid regulation of hypercapnic acidosis, or the buildup of CO₂ in the bloodstream, by coho salmon, indicating some tolerance to shifting acid-base regimes.

There are few direct studies on the impact of elevated pH on fish, most of which are focused on aquaculture and the influence of algae on pH impacting fish incubation. During incubations of Pacific herring eggs (Clupea harengus pallasi), Kelley (1946) reported that eggs hatched one day sooner at pH 8.68, but did not find a difference in survival relative to control at pH 7.93. Brownell (1980) investigated the impact of elevated pH on 8 fish larvae species, reporting a decrease in first-feeding incidences of larvae exposed to pH_NBS above 8.4 after 24 h exposure, and higher tolerance to elevated pH during gradual exposures. Parra and Yúfera (2002) incubated Gilthead seabream (Sparus aurata) and Senegal sole (Solea senegalensis) larvae under altered pH, recommending avoidance of pH above 8.6 and 8.9, respectively. Alderson and Howell (1973) noted an increased growth rate in juvenile sole (Solea solea) raised for 13 days above pH 8.4, though this is likely due more to the effectiveness of algae in removing dissolved ammonia than to pH in this case. Jordan and Llyod (1964) acclimated adult rainbow trout (Salmo gardnerii) and roach (Rutilus rutilus) to solutions of pH 6.55-8.40 for 5 days, then exposed them to NaOH-based alkaline solutions at pH ranging from 9.86-10.15. They found that fish which were acclimatized to pH 8.40 before exposure to increased pH were significantly more resistant to alkaline solutions than those acclimatized to lower initial pH solutions, and indicate that both species may survive if exposed to pH >9.0 for several months.

We are only aware of one complete research study testing the impacts of OAE on fish, Goldenberg et al. (2024). Larval and juvenile coastal species including Atlantic herring (Clupea harengus) and cod (Gadus morhua) were exposed to increased calcium or silicate based mineral alkalinity (+600 μmol/kg, pH + 0.7 units) for 49 days. There were no significant negative physiological or behavioral impacts due to the alkaline treatments, and the authors noted an increase in fish biomass under alkalization.

Collectively, these results indicate that there may be significant variations in physiological and behavioral responses between and within species exposed to increased pH conditions under OAE, requiring consideration of adaptations to environmental conditions across life stages. Coho salmon experience various pH conditions within dynamic coastal environments (migration and spawning as adults, and eggs through smolting during early life stages) and more stable open ocean conditions (adult ocean growth). OAE is likely to be more stressful on fish that are not adapted to dynamic environments or that experience additional stressors such as increasing temperatures, acidification, and hypoxia, but even in environmentally resilient species, reproductive cells and embryos may be impacted in different ways by the elevation of pH, requiring further study (Goldenberg et al., 2024; Melzner et al., 2009; Dahlke et al., 2020). OAE may also indirectly impact coho salmon through interactions with both predator and prey species. Continued research across marine species and communities targeted to the realistic impacts on water chemistry is necessary to advance the use of OAE for mCDR, both for current pilot research projects and scaled operations.

4.3 Summary and recommendations for future work

This study demonstrated a laboratory method to test the impact of elevated pH and alkalinity on juvenile coho salmon, using a standard dilution model in advance of OAE in the field. The study represented the routine discharge of electrochemically-generated alkalinity into the acute mixing zone of Project Macoma in Port Angeles, WA. A pulse of alkalinity was released into test chambers at a dilution factor predicted at a given distance from the outfall. Each test chamber containing 10 juvenile coho salmon each was held for an exposure time ranging from 30 s-5 mins, based on conservative assumptions of the amount of time required for a juvenile salmon to swim through the mixing zone. The test chambers were then slowly flushed with ambient seawater, during which time salmon were still exposed to a seawater solution with enhanced alkalinity but continually decreasing pH due to dilution. The alkalinity-enhanced seawater discharge, initially at pH_NBS of 10.0, diluted into ambient test chamber seawater to reach a peak of 8.04-8.09, which is predicted at a distance of ~3-6.3 m from the outfall, depending on assumptions of ambient current and receiving water pH. The exposure to this solution did not impact juvenile coho salmon behavior, survival, or physical impacts on gills, eyes, or external body tissues. Given the conservative nature of the dilution model and Project design, these results indicate that Ebb Carbon's Project Macoma research pilot presents minimal risk to juvenile salmon within the acute mixing zone of the alkaline effluent.

The design of this study targets exposure to altered seawater chemistry at a given location and concentration relative to a point source outfall, bridging a gap between traditional ecotoxicology dilution series studies and the realistic footprint of an OAE deployment. The study is designed for transient exposure, as of a mobile species swimming or floating through a portion of an OAE mixing zone. The methodology may be altered to test multiple concentrations (for mapping out potential impacts across a mixing zone) or for different timelines (targeting various exposure periods that species may experience within the footprint of an OAE project based on their swimming speed, foraging habits, or other considerations). To help contextualize specific OAE deployments, the study could be modified to address alkalinity additions into seawater at the extremes of naturally varying pH. For example, this study tested salmon responses when alkalinity was added to ambient seawater in the pH_NBS ~7.62-7.70 range. One year of weekly discrete surface sampling at Project Macoma (October 2024-2025) indicated an average pH_NBS of 7.83, ranging from ~7.43-8.20 (see Supplementary material), and the dilution model used in permitting the Project included a sensitivity study with ambient seawater pH ranging from 7.5-8.5. Testing salmon responses with alkalinity added to ambient seawater across this range would help flesh out connections between the dilution model, laboratory experiments, and field observations.

We note that as a species-specific study at a particular life stage (in this case, smolted juvenile coho), this methodology is not sufficient to consider impacts of OAE on a given species' reproductive traits or other life stages, food webs, or other ecosystem dimensions. This study is also limited in its total length, such that it does not test chronic or repeated exposures (for example if a fish returned to the outfall every day), nor does it capture potential physical or behavioral impacts that may develop more than 3 h after a given exposure.

As an unblinded study, we note that there are limitations in assessing bias in physical and behavioral observations, particularly in the small test chambers with limited test subjects. Researchers should take care to limit stress during fish handling and observational periods.

One of the advantages of this study is that it was designed to be conducted within a commercial ecotoxicology laboratory, allowing for rapid assessment of species response to support regulatory decision making and OAE project development in a manner that is additional to whole effluent toxicology tests (US Environmental Protection Agency, 1996, 2018). However, we note that measurements conducted during standard ecotoxicology studies rarely cover seawater carbonate chemistry parameters relevant to mCDR, which should include at least two of the following parameters: pH, total alkalinity, dissolved inorganic carbon, and pCO2. During this study, we collected pH data via electrode at 1 min intervals, estimated initial TA from average seawater conditions, and calculated the addition of alkalinity from the known concentration and volume of the effluent. This provides a basic understanding of carbonate chemistry, but would have been strengthened by improved precision in pH measurement and by direct analysis of TA before and after alkalinity addition. Carbonate parameters should be well quantified following best practices (e.g., Dickson et al., 2007), and changes to these parameters should be tracked throughout the experiment as appropriate to the length of the experiment. The chemistry of flow-through ambient seawater may change over time, and for longer studies, uptake of CO₂ into alkalinity-enhanced seawater will result in decreasing pH and TA and increasing pCO₂ and DIC over time (Hartmann et al., 2022; Ringham et al., 2024; Suitner et al., 2025). Alkalinity addition can result in secondary mineral precipitation, including brucite and aragonite. Experimental design should account for appropriate mixing and quantification of mineral precipitation, which can impact pH and alkalinity values either temporarily (e.g., until brucite is redissolved) or permanently (in the case of carbonate precipitation).

Finally, we note that building trust and transparency in independent data is crucial to the development of the mCDR industry. There are inherent limitations to the independence of ecological assessments of mCDR at this pilot stage, given the necessity of representing realistic in-water conditions for which mCDR supplier input is necessary. Commercial laboratories are well primed to rapidly collect actionable data in a context that is familiar and useful to regulators. However, eco-toxicological datasets are not always transferred to the scientific literature. We recommend that OAE suppliers and researchers leverage both standard and novel ecotoxicology methods to accelerate understanding of potential impacts on marine species. Further, we recommend that the OAE community invest in publication of these data in the scientific record, specifically including null results alongside significant findings.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

Ethics statement

Ethical approval was not required for the study involving animals in accordance with the local legislation and institutional requirements because EcoAnalysts is a commercial toxicology lab. It follows an internally-derived animal care SOP that was modeled from the Institutional Animal Care and Use Committees and the American Veterinary Medical Association (AVMA) for holding, care, and euthanasia of vertebrates (fish). The study did not employ client-owned animals, however the animals (juvenile Coho salmon) are also not commercially available. They were provided by a local Tribe hatchery (Lower Elwha Klallam Tribe) that raises them and donated them to this study.

Author contributions

MR: Conceptualization, Methodology, Writing – original draft, Writing – review & editing. MG: Conceptualization, Methodology, Writing – original draft, Writing – review & editing. MK: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. JL: Conceptualization, Investigation, Methodology, Project administration, Writing – review & editing. TM: Conceptualization, Methodology, Project administration, Writing – review & editing. TP: Conceptualization, Funding acquisition, Methodology, Project administration, Writing – review & editing. NS: Conceptualization, Methodology, Writing – original draft, Writing – review & editing. KW: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Writing – review & editing. JW: Conceptualization, Data curation, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This study was funded by Ebb Carbon, Inc. The funder was involved in the study design and discussion of results, but had no role in data collection or laboratory analysis. Interpretation of the results and manuscript preparation were conducted collaboratively by the authors.

Acknowledgments

We are grateful for Matthew Eisaman, whose contributions were invaluable in shaping the direction of this research. We thank Emily Carrington of the University of Washington and Lenaïg Hemery of Pacific Northwest National Laboratory for their review and feedback on the study design. Finally, we thank the Lower Elwha Klallam Tribe for their feedback on experimental design and analysis, and for providing salmon for this study.

Conflict of interest

MR, JL, TM, TD and KW are employees of Ebb Carbon, Inc, which sponsored the research. MG and NS are employees of Anchor QEA, an environmental consultancy that contributed to the study design and interpretation of the results. MK and JW are employees of Spheros Environmental, formerly EcoAnalysts, an independent contract laboratory that conducted the toxicological study. The commercial affiliations of the authors did not affect the objectivity, analysis, or reporting of the research.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fclim.2025.1717924/full#supplementary-material

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