Seasonal variation in aragonite saturation in surface waters of Puget Sound – a pilot study

Introduction In recent years, a significant body of research has shown that increases in atmospheric CO2 concentrations are causing ocean acidification (OA) via ocean uptake of the excess CO2. Among other changes, OA leads to a decrease in pH, and the decrease is expected to continue into the future under various CO2 emissions scenarios (Gattuso et al., 2015). OA also decreases the saturation state of calcium carbonate. A decrease in the saturation state of calcium carbonate has been suggested as a potential negative impact on the health of some organisms (e.g., Bednaršek et al., 2012; Waldbusser et al., 2015a). Aragonite is a metastable form of calcium carbonate that naturally occurs in the shells of many mollusk species. Aragonite has relatively rapid dissolution kinetics, and may become undersaturated in the open ocean by the year 2050 if present trends in OA continue (Orr et al., 2005). The aragonite saturation state in the surface waters of the ocean is changing rapidly with OA as a result of human activities that increase global atmospheric CO2 (Feely et al., 2008). Aragonite under-saturation is likely to affect aragonite-shelled organisms (e.g., Hunt et al., 2008; Bednaršek et al., 2012). The aragonite saturation state in seawater (Ωarag) is the product of calcium ([Ca2+]) and carbonate ([CO3 2–]) ion concentrations at ambient temperature, salinity, and pressure, divided by the apparent solubility product (Ksp) at the ambient conditions:


Introduction
In recent years, a significant body of research has shown that increases in atmospheric CO 2 concentrations are causing ocean acidification (OA) via ocean uptake of the excess CO 2 . Among other changes, OA leads to a decrease in pH, and the decrease is expected to continue into the future under various CO 2 emissions scenarios . OA also decreases the saturation state of calcium carbonate. A decrease in the saturation state of calcium carbonate has been suggested as a potential negative impact on the health of some organisms (e.g., Bednaršek et al., 2012;Waldbusser et al., 2015a). Aragonite is a metastable form of calcium carbonate that naturally occurs in the shells of many mollusk species. Aragonite has relatively rapid dissolution kinetics, and may become undersaturated in the open ocean by the year 2050 if present trends in OA continue (Orr et al., 2005). The aragonite saturation state in the surface waters of the ocean is changing rapidly with OA as a result of human activities that increase global atmospheric CO 2 (Feely et al., 2008). Aragonite under-saturation is likely to affect aragonite-shelled organisms (e.g., Hunt et al., 2008;Bednaršek et al., 2012).
The aragonite saturation state in seawater (Ω arag ) is the product of calcium ([Ca 2+ ]) and carbonate ([CO 3 2-]) ion concentrations at ambient temperature, salinity, and pressure, divided by the apparent solubility product (K sp ) at the ambient conditions: When the aragonite saturation states drop below saturation (Ω arag < 1), carbonate minerals in shells and skeletons may begin to dissolve ). This condition is often described as "corrosive" (e.g., Feely et al., 2010). Recent laboratory studies indicate that impacts on sensitive species can occur at higher aragonite saturation states as organisms expend additional energy during early life stages (e.g., Thomsen et al., 2015;Waldbusser et al., 2015b). In general, surface waters of the open ocean are typically super-saturated with respect to aragonite (Ω arag > 1; e.g., Feely et al., 2009). The coastal region of the Pacific Northwest is strongly influenced by seasonal upwelling of corrosive CO 2 -rich offshore sub-surface water, which typically occurs during April-November. Feely et al. (2010), who sampled in February and August of 2008, found the entire water column of Puget Sound to be under-saturated with respect to aragonite in February, and super-saturated only near the surface in August.
In 2012, the governor of Washington State appointed a Blue Ribbon Panel to summarize the scientific knowledge and to recommend key early actions to address threats to the State's marine species and ecosystems (Washington State Blue Ribbon Panel on Ocean Acidification, 2012). Much of the monitoring focuses on coastal areas in the Pacific Northwest, strongly influenced by the Pacific Ocean, where acidification was first recognized. A key question is the degree to which local contributions exacerbate acidification resulting from global atmospheric CO 2 levels.
Relatively little is known about the magnitudes and variability of carbonate system variables within the inland waters, including Puget Sound, of Washington State. Reum et al. (2014) presented data from five cruises between 2008 and 2011. Feely et al. (2010) presented the results of two cruises in 2008. McLaskey et al. (2016) collected samples from two stations in Hood Canal in April and June 2012. Fassbender et al. (2017) present a summary of various other published data sources of carbonate system chemistry throughout Puget Sound. The present study aims to explore the monthly variation in the carbonate system and aragonite saturation state in the surface waters of Puget Sound, by sampling a spatially distributed set of stations over a full year-long period.

Climate and physical setting
The temperate maritime climate produces strong seasonal variations in sunlight, air temperature, and precipitation. Precipitation reflects strong orographic patterns, ranging from 100 cm year -1 in the Puget Lowland to 380 cm year -1 in the Cascade Mountains and 610 cm year -1 in the Olympic Mountains.
Puget Sound represents one portion of the Salish Sea (Figure 1), which also includes the Strait of Juan de Fuca and the Strait of Georgia, both of which are shared waters with Canada. The exchange flow near the entrance to Puget Sound at the Admiralty Inlet sill is approximately 10,000 to 20,000 m 3 sec -1 (Sutherland et al., 2011), or about 10 to 20 times the average river flow into Puget Sound (Cokelet et al., 1990;Babson et al., 2006). Shallow sills at Admiralty Inlet, Hood Canal, and South Puget Sound restrict circulation and produce differential residence times typically ranging from about 20 to 80 days in the four major basins of Puget Sound (Sutherland et al., 2011). Rivers discharge about 1500 m 3 sec -1 of freshwater into Puget Sound on average. Raindominated river systems peak in the winter while snowdominated systems can peak as late as June. Forested and alpine areas occupy 83% of the watershed and contribute over 90% of the freshwater input. Approximately eight million people live in the Salish Sea watershed, with over four million residents on the US side of the border. The majority is served by centralized wastewater facilities that discharge on average about 14 m 3 sec -1 of treated wastewater with minimal seasonal variation (Mohamedali et al., 2011).

Sampling stations
The Washington State Department of Ecology (Ecology) collected 136 samples from six stations representing a spectrum of ocean and inland influences (Figure 1) monthly from June 2014 through May 2015. No samples were collected during December 2014. For a more detailed description of Ecology's marine flight study area and complete station list from which the six stations were chosen, see Keyzers and Bos (2015). The Admiralty Inlet station (ADM001) represents water entering Puget Sound with the greatest relative influence of the Pacific Ocean. Two stations are in Whidbey Basin, which receives the largest freshwater inflows to Puget Sound: Saratoga Passage (SAR003) and Possession Sound (PSS019). Two other stations represent contrasting variability between the rain-fed Skagit River (SAR003) and the snowmelt-fed Snohomish River (PSS019). The northern-most Hood Canal station (HCB010) strongly reflects Pacific Ocean conditions and was chosen because of its close proximity to commercial oyster beds. Oakland Bay (OAK004) and Totten Inlet (TOT002) in the South Puget Sound were chosen because of their highly productive commercial shellfish beds; they also reflect inland conditions furthest from the Pacific Ocean.

Sampling methods
Total alkalinity (TA), dissolved inorganic carbon (DIC), and nutrient water samples were collected in addition to the routine marine flight sampling described in Keyzers and Bos (2015). Field staff collected triplicate water samples, which were analyzed for TA and DIC from a depth of 5 meters. The average amount of time to conduct triplicate sampling at each station was 30 minutes, including the time for the pump to pull up the water, for each sample container to be filled, and for filters to be changed between samples. Relative standard deviations of replicate samples are presented in Table 1. Sampling procedures specific to TA and DIC are detailed in Keyzers (2014) and were adapted from Bockman and Dickson (2014).
The marine flight CTD (SeaBird SBE25) is equipped with a temperature (SBE3F) and conductivity sensor (SBE4C). The CTD is deployed through a hatch in the belly of the plane, descends to the bottom of the water column, and then returns to the surface, collecting data at eight scans per second. Sampling procedures for all flight-based sampling are detailed in Keyzers (2014), Keyzers and Bos (2015), Bockman and Dickson (2014), and Dickson et al. (2007). CTD measurements of conductivity and temperature were used in the data analysis; we did not use results from the SeaBird SBE 18 pH sensor. The analytical methods, including instrumentation, standardization, and precision, are provided in Table 2. Sampling and analysis of chlorophyll a were performed according to Bos (2008Bos ( , 2010. To assess the quality of our nutrient data, we conducted laboratory performance and analyst proficiency tests of the analytical laboratory, using low nutrient seawater laboratory control samples of known concentrations from Ocean Scientific International Ltd. (GPO) included with every sample batch. Recovery percentage was calculated from these results and used as a measure of analytical accuracy and bias. If results fell outside of established limits, data associated with the batch were flagged as estimates. Any measurement problem that could not be resolved was given a data quality flag.
To analyze DIC and TA, a minimum of 2.5 L of water was collected to produce one 500-mL water sample. The method used a peristaltic pump and filter system to remove particulates (0.45 µm Durapore membrane filters, as in Bockman and Dickson, 2014). We used a Geofilter 350 RPMs peristaltic pump with silicone tubing. The decision to use silicone tubing was based on NOAA PMEL Standard Operating Procedures (SOP) for sampling: "3.1 Drawing tube Tygon® tubing is normally used to transfer the sample from the Niskin to the sample container; however, if dissolved organic carbon samples are being collected from the same Niskins, then it may be necessary to use silicone tubing to prevent contamination from the Tygon®" (Dickson et al. (2007). Once the TA and DIC samples were collected, the glass lids were greased and the containers capped with a rubber band and clamp. The samples were stored in the dark until they were transported back to the laboratory for addition of mercuric chloride. As the samples were collected aboard a small and unstable seaplane, post-sampling fixation was required for safety reasons. Whenever processing could not be completed within 36 hours, samples were preserved by adding 0.2 ml of saturated mercuric chloride solution (50 M) to each 500-ml sample to prevent biological alteration. Leinweber and Gruber (2013) found that a delayed addition of mercuric chloride up to 36 hours after collection was acceptable. The maximum time between the sample collection in the field and mercuric chloride addition in the laboratory was 7 hours, considerably less than the 36 hours in Leinweber and Gruber (2013). Samples were also filtered following Bockmon and Dickson (2014) in an effort to minimize any biological activity during the time between collection and mercuric chloride addition. After preservation, the TA and DIC samples were stored in a dark walk-in cooler at 4-6°C until delivery to the NOAA Pacific Marine Environmental Laboratory (PMEL) Carbon Group for analysis. Nutrient samples were frozen and delivered to the University of Washington (UW) Marine Chemistry Laboratory (MCL) at the end of each month. All samples were analyzed within 3 months of collection.

Calculation of carbonate system variables
Carbonate system calculations were performed using CO2SYS (CO2SYS-Excel-Pelletier, described in Orr et al., 2015, andPelletier et al., 2007). The "best practices" recom-mendations of Orr et al. (2015) were applied with CO2SYS for selection of equilibrium constants and use of total scale for pH. Observed TA and DIC, along with CTD salinity, temperature, and depth, and silicate and phosphate from the discrete nutrient samples, were input values used in CO2SYS. CO2SYS was used to calculate the pH, pCO 2 , and aragonite saturation state for each sample. All of the carbonate system variables, which are inter-related, were calculated from the observed TA and DIC using CO2SYS.

Temperature-normalization of pCO 2
Temperature-normalization of pCO 2 was evaluated to consider whether the seasonal variations in pCO 2 could be explained by seasonal variations in temperature. The following equation was used to temperature-normalize the observed pCO 2 (Takahashi et al., 2002): where pCO 2,Tmean is the pCO 2 normalized to the mean temperature of the observations, pCO 2 is the observed  (1985,1987,1993,1999 pCO 2 at ambient temperature (estimated from CO2SYS), T mean is the mean of the observed temperature data, and T obs is the temperature corresponding to the observed pCO 2 .

Excess DIC due to the net effect of biological activity and mixing
Excess DIC (EDIC) is the difference between observed DIC and DIC computed from total alkalinity and atmospheric CO 2 ; it provides an estimate of CO 2 outgassing if the water mass is upwelled (Borges et al., 2011). EDIC is also an indicator of the gain or loss of DIC due to the net effect of primary production and respiration (Clargo et al., 2015), within Puget Sound and in source waters entering Puget Sound, and mixing of waters from various locations. EDIC was estimated as follows (Borges et al., 2011;Burt et al., 2016;Clargo et al., 2015): DIC atm was calculated with CO2SYS using the same inputs describe above, with the exception that, instead of using observed TA with observed DIC, we used the observed TA with pCO 2 of 400 µatm and calculated the corresponding DIC atm as the output from CO2SYS.

Data availability
The data collected during this study are available to download from the Washington State Department of Ecology Web site at the following link: https://fortress.wa.gov/ ecy/publications/SummaryPages/1603032.html.

Results
Summary statistics for observed temperature, salinity, and carbonate system variables were compared with published averages by Feely et al. (2010) (Table 3). In general the ranges of observed carbonate system variables overlap the ranges of averages reported by Feely et al. (2010). The ranges observed in our study tend to be larger than reported by Feely et al. (2010), which is likely due to several reasons: Feely et al. (2010) tabulated averages, and we are comparing their range of averages to our range of observations; our data represent more months of observation, and during 2014-2015, while their study addressed February and August of 2008; and our data include more inland station locations that may be more influenced by freshwater inflows.
The Ω arag values we obtained indicated corrosive conditions (under-saturated) during the winter season (October-March; Figure 2). Statistically significant differences (p < 0.05) in Ω arag between stations and months were found by a two-factor analysis of variance which was performed on a balanced subset of the data (stations ADM001, PSS019, SAR003, and HCB010 for the months of June, July, August, September, February, and April) using a commercial software package (MATLAB R2016b, The MathWorks Inc., Natick, MA). During April-September, conditions were generally super-saturated. However, stations exhibited much greater variability than in the winter months. For example, August Ω arag ranged from 0.7 to 2.7, Indicates not available. c Excess DIC relative to equilibrium with atmospheric CO 2 , an indicator of the net effect of biological activity (primary production and respiration). d pCO 2 normalized to the mean temperature of the observations. although the salinity range (27 to 30) was relatively less variable among the stations.
The surface waters of Puget Sound typically had greater pCO 2 than the atmosphere during October-March (Figure 3). The pCO 2 values were typically greatest during the winter months of October-March (up to 1417 µatm), and lowest during April-September (as low as 99.9 µatm). The seasonal depletion of pCO 2 during April-September is most likely due to uptake of CO 2 by primary production of phytoplankton during the growing season. Admiralty Inlet tended to have the highest levels of surface water pCO 2 among the stations, probably due to strong vertical mixing and higher pCO 2 at depth. PSEMP Marine Waters Workgroup (2016) reported similar variations in pCO 2 in Puget Sound.
Temperature-normalization of pCO 2 was evaluated to consider whether the seasonal variations in pCO 2 could be explained by seasonal variations in temperature. The pCO 2,Tmean values show about the same seasonal variations (Figure 4) and range (Table 3) as the observed nonnormalized pCO 2 (Figure 3; Table 3), indicating that the observed seasonal variations in pCO 2 cannot be explained by variations in temperature. Seasonal variations in pCO 2 are most likely influenced mainly by biological processes.
The lowest pH occurred during October-March (as low as 7.5), and highest pH during April-September (as high as 8.5; Figure 5). Seasonal variations in pH were also likely strongly influenced by variations in CO 2 uptake by phytoplankton productivity.
EDIC ranged from -206 to 156 µmol kg -1 (Figure 6; Table 3). Positive values of EDIC during October-February indicate outgassing of CO 2 and an increase in ambient DIC due to release of CO 2 by respiration. Negative values of EDIC during March-September indicate reduction of ambient DIC due to primary production and uptake of CO 2 .

Discussion
Data published to date in the Pacific Northwest represent limited locations or coastal conditions that may not provide the full context for acidification levels in Puget Sound and the Salish Sea (e.g., Feely et al., 2010;Ianson et al., 2016). Though this program was designed as a limited pilot study focused on the logistics of monitoring aboard a space-constrained sea plane, the results provide a unique glimpse into both seasonal patterns and spatial variability of acidification in the surface waters of Puget Sound. Ongoing efforts will evaluate the relative influence of local human contributions through water and air pathways using circulation and biogeochemical models (Long et al., 2014;Roberts et al., 2015). Meanwhile, the results of this study provide insight into the major processes involved.
The Pacific Ocean strongly influences conditions in Puget Sound. The deep waters entering Puget Sound originate in the Pacific Ocean and represent a mix of subtropical and subarctic water masses. Upwelling varies in strength and duration, with short-term intrusions over the sill at Admiralty Inlet (Deppe et al., 2013;Khangaonkar et al, 2017). The surface layer is influenced by vertical mixing with deep waters enhanced by circulation around the sills, and also by inputs of freshwater and primary productivity within the euphotic zone. Winter conditions produce the most biologically challenging environments in terms of low Ω arag and low pH.
Primary production of phytoplankton in Puget Sound is greatest in the spring and summer with optimum sunlight and intermediate water column stability and residence time (Strickland, 1983). Uptake of CO 2 by phytoplankton for photosynthesis decreases the surface water pCO 2 , which increases the Ω arag above corrosive conditions. Admiralty Inlet surface layer Ω arag remains among the lowest of the stations because of vertical mixing with more corrosive deep waters, while Hood Canal surface waters are among the highest. However, previous studies have identified Hood Canal bottom waters as highly corrosive throughout the year (Feely et al., 2010). Thus, primary production may either improve or worsen conditions depending on the location within the water column. Uptake of CO 2 to support primary production increases Ω arag in the euphotic zone, but sinking and decomposition of organic matter releases CO 2 back into the deeper water and decreases Ω arag at depth below the euphotic zone. Species that exploit different depth habitats are likely to experience far different conditions. The fundamental question is the relative importance of each of the following on the total anthropogenic influence on the CO 2 system in the Salish Sea: (1) increases in local atmospheric CO 2 ; (2) increases in eutrophication due to local sources of nutrient loading; and (3) increases in inorganic C inputs from the ocean due to global anthropogenic processes. Although the data from this pilot project are not sufficient to answer this over-riding question, they provide some insight on the magnitude of variability of carbonate chemistry in space and time in Puget Sound surface waters.
Washington State leaders have recognized the dominant influence of global atmospheric pCO 2 levels in the acidification of the Pacific Ocean (Washington State Blue Ribbon Panel on Ocean Acidification 2012). The surface layer of Puget Sound appears to be outgassing to the local atmosphere between October and March, as surface water pCO 2 levels were above 400 µatm throughout Puget Sound during this period (with the exception of Hood Canal in March 2015). Regional atmospheric pCO 2 values were higher in the winter than at a global marine reference location (the NOAA Earth System Research Laboratory, Mauna Loa, Hawaii) but can decline below the Hawaiian record during the growing season (PSEMP Marine Waters Workgroup, 2016). Even so, during the summer months, phytoplankton productivity likely decreases pCO 2 to the point where local atmospheric CO 2 represents a source of carbon to most inland surface waters, except Admiralty Inlet where surface layer pCO 2 remains above global and regional atmospheric pCO 2 .
The occurrence of the highest Ω arag and negative EDIC during the phytoplankton growing season suggests that uptake of dissolved CO 2 due to primary production is a very important process to explain seasonal variations in Ω arag . Uptake of CO 2 by primary production could provide a benefit by increasing Ω arag (e.g., Jutterström et al., 2014). The release of dissolved CO 2 due to decay of the organic carbon produced within the Salish Sea, in addition to ocean inputs of waters rich in dissolved CO 2 , contributes to the observed decreases in Ω arag and positive EDIC during the winter months, and could also decrease Ω arag in the deeper waters below the euphotic zone during the growing season.
We cannot determine whether the patterns observed in 2014-2015 were typical for Puget Sound or represent anomalously low or high Ω arag , pCO 2 , or pH. The monitoring period coincided with anomalous Pacific Ocean and climate conditions throughout the Pacific Northwest, but a historical baseline for the carbonate system is lacking. Compared with historical temperature data, 2015 represented extremely warm water temperatures within Puget Sound that resulted from a combination of anomalously warm waters in the Pacific Ocean and anomalously low river base flows resulting from lack of snowpack the previous winter (PSEMP Marine Waters Workgroup, 2016). If the recent decline in chlorophyll a since 1998 continues (PSEMP Marine Waters Workgroup, 2016), decreased phytoplankton productivity could decrease the Ω arag in the surface waters. Anthropogenic nutrient sources have either declined or remained constant throughout the sampling period and thus do not explain the change in chlorophyll a (PSEMP Marine Waters Workgroup, 2016).
Though limited to a one-year sampling period, the resulting data illustrate important seasonal and spatial patterns in the surface waters of Puget Sound. Such distributed monthly monitoring provides a complement to continuous data collection efforts now underway but in limited areas. More information is needed to constrain the carbonate system in Puget Sound. A complementary approach that combines both continuous data of high temporal resolution and a distributed network able to capture broad spatial patterns, as demonstrated in this study, may be an ideal way to obtain the needed information.

Data Accessibility Statement
The data collected during this study are available to download from the Department of Ecology's Web site at the following link: https://fortress.wa.gov/ecy/publications/ SummaryPages/1603032.html.