School of Fisheries and Ocean Sciences, Ocean Acidification Research Center

Ocean acidification (OA) is altering the chemistry of the world's oceans at rates unparalleled in the past roughly 1 million years. Understanding the impacts of this rapid change in base-line carbonate chemistry on marine organisms needs a precise, mechanistic understanding of physiological responses to carbonate chemistry. Recent experimental work has shown shell development and growth in some bivalve larvae, have direct sensitivities to calcium carbonate saturation state that is not modulated through organismal acid-base chemistry. To understand different modes of action of OA on bivalve larvae, we experimentally tested how pH, P CO2 , and saturation state independently affect shell growth and development, respiration rate, and initiation of feeding in Mytilus californianus embryos and larvae. We found, as documented in other bivalve larvae, that shell development and growth were affected by aragonite saturation state, and not by pH or P CO2. Respiration rate was elevated under very low pH (~7.4) with no change between pH of ~ 8.3 to ~7.8. Initiation of feeding appeared to be most sensitive to P CO2 , and possibly minor response to pH under elevated P CO2. Although different components of physiology responded to different carbonate system variables , the inability to normally develop a shell due to lower saturation state precludes pH or P CO2 effects later in the life history. However, saturation state effects during early shell development will carry-over to later stages, where pH or P CO2 effects can compound OA effects on bivalve larvae. Our findings suggest OA may be a multi-stressor unto itself. Shell development and growth of the native mussel, M. californianus, was indistinguishable from the Mediterranean mussel, Mytilus galloprovincialis, collected from the southern U.S. Pacific coast, an area not subjected to seasonal upwelling. The concordance in responses suggests a fundamental OA bottleneck during development of the first shell material affected only by saturation state.

Ocean acidification results in co-varying inorganic carbon system variables. Of these, an explicit focus on pH and organismal acid–base regulation has failed to distinguish the mechanism of failure in highly sensitive bivalve larvae. With unique chemical manipulations of seawater we show definitively that larval shell development and growth are dependent on seawater saturation state, and not on carbon dioxide partial pressure or pH. Although other physiological processes are aaected by pH, mineral saturation state thresholds will be crossed decades to centuries ahead of pH thresholds owing to nonlinear changes in the carbonate system variables as carbon dioxide is added. Our findings were repeatable for two species of bivalve larvae could resolve discrepancies in experimental results, are consistent with a previous model of ocean acidification impacts due to rapid calcification in bivalve larvae, and suggest a fundamental ocean acidification bottleneck at early life-history for some marine keystone species. O cean acidification (OA) is described as an imbalance between the acidic influence of rapidly accelerating anthropogenic CO 2 emissions and the slow buffering response due to weathering of continental rock and carbonate marine sediment, causing increased acidity of marine waters 1,2. The release of CO 2 from fossil fuel emissions and cement production, and decreasing uptake efficiency of CO 2 by land and sea has resulted in the fastest increase in p CO 2 (partial pressure of carbon dioxide) in the past 800,000 years 3. Conversely the natural mechanisms that buffer acidic perturbations from increasing p CO 2 occur over timescales of hundreds of thousands to millions of years 1,2. Modern anthropogenic changes in the open ocean have tightly coupled aqueous p CO 2 , pH and mineral solubility responses, but it was not always thus. Previous instances of elevated p CO 2 in the geologic record, such as the Cretaceous, seem to coincide with significantly elevated alkalinity 4 , and were fairly benign with respect to OA, with elevated p CO 2 not indicative of low pH or mineral corrosivity. Throughout the geologic record and in many coastal habitats the marine carbonate system decouples, resulting in changes in pH, p CO 2 and saturation state that do not follow the co-variance assumed for modern open-ocean average surface waters 5. Effects of ocean acidification on a suite of marine organisms have been the subject of significant recent work. Although many experimental results have shown equivocal impacts when taken in composite, the process of calcification has mostly exhibited negative sensitivity to OA (ref. 6). Physiological processes that may experience OA sensitivity occur across all taxa in nearly all natural waters; however, persistent calcified structures can elevate species that precipitate calcium carbonate to keystone status in marine waters. Bivalves, which provide a number of critical ecosystem services, have been noted as particularly sensitive to OA (refs 7–10). Some experiments have even found OA impacts at present-day, compared with pre-industrial, p CO 2 levels 11. Marine bivalves seem to be sensitive to OA owing to the limited degree to which they regulate the ionic balance and pH of their haemolymph (blood) 12–15 , and acute sensitivities at specific, short-lived, life-history stages that may result in carryover effects later in life 16–20. Bivalve larvae are particularly sensitive to OA during the hours-to days-long bottleneck when initial shell (called prodissoconch I or PDI) is formed during embryogenesis 17. Before PDI shell formation, larvae lack robust feeding and swimming appendages and must rely almost exclusively on maternal energy from eggs; and during calcification of PDI the calcification surfaces are in greater contact with ambient seawater than during following shell stages 17. Failure of larvae to complete shell formation before exhausting maternal energy reserves leads to eventual mortality, as seen in well-documented oyster hatchery failures 18. So far, the prevailing physiological mechanism identified for OA effects on organisms has been in their ability to regulate internal acid–base status; however, short-term exposure impacts and carryover effects documented in bivalve larvae 18–21 and greater exposure of PDI calcification to ambient seawater 17 points to another mechanism for the early larval sensitivity not captured by regulation of internal acid–base chemistry 22. In most natural waters the dissolved inorganic carbon (DIC) system controls both pH and the thermodynamic mineral solubility (saturation state), but in different ways. pH is determined by the ratio of dissolved concentrations of CO 2 to carbonate ions, whereas saturation state is predominantly controlled by absolute carbonate ion concentration. The potential that organisms will respond differently to pH (ratio) or mineral saturation state (abundance), highlights how the decoupling of carbonate system variables in coastal zones 5 or geologic time 1,2 provides a formidable challenge in interpreting and predicting organismal responses to OA. The seemingly simple experimental perturbation of CO 2 bubbling results in the

Complex biogeochemical processes in the coastal oceans lead to highly variable carbonate chemistry that is further modified by the shifting baseline of pCO2 caused by ocean acidification. Unfavorable carbonate chemistry, which is pervasive in near-shore, shallow-water deposits, due to high rates of organic matter remineralization and the oxidation of reduced metabolites, has been shown to negatively affect benthic calcifying organisms. For settling infaunal bivalve larvae, such as the hard clam Mercenaria mercenaria, pore waters high in CO2 exert a physiological stress on early post-larval development and homeostasis. The effects of acidification have been shown to increase mortality and reduce calcification for ‘just settled’ juvenile M. mercenaria, a life stage that heavily affects adult clam abundance. To better understand the effects of acidification and temperature on this sensitive life stage, we constructed a post-larval stage-based development model that investigated how varying degrees of aragonite saturation state affect post-settlement survival of juvenile M. mercenaria. Initial model simulations predict a similar consistency and trend to field experiments that examined the survival of juvenile Mya arenaria residing in buffered and unbuffered sediments. According to our model, the magnitude of acidification had a large effect on post-larval stage duration, which translated to a 60% decrease in total survivors under high variability compared to low variability saturation state scenarios. By modifying temperature-dependent growth rates, we were able to progress juveniles out of the more sensitive life stages faster and, therefore, determined scenarios in which faster growth can reduce exposure to acidification during more sensitive stages.

The commercially available Sea-Bird SeaFET ™ provides an accessible way for a broad community of researchers to study ocean acidification and obtain robust measurements of seawater pH via the use of an in situ autonomous sensor. There are pitfalls, however, that have been detailed in previous best practices for sensor care, deployment , and data handling. Here, we took advantage of two distinctly different coastal settings to evaluate the Sea-Bird SeaFET ™ and examine the multitude of scenarios in which problems may arise confounding the accuracy of measured pH. High-resolution temporal measurements of pH were obtained during 3-to 5-month field deployments in three separate locations (two in south-central Alaska, USA, and one in British Columbia, Canada) spanning a broad range of nearshore temperature and salinity conditions. Both the internal and external electrodes onboard the SeaFET ™ were evaluated against robust benchtop measurements for accuracy using the factory calibration, an in situ single-point calibration , or an in situ multi-point calibration. In addition, two sensors deployed in parallel in Kasitsna Bay, Alaska, USA, were compared for inter-sensor variability in order to quantify other factors contributing to the sensor's intrinsic inaccuracies. Based on our results, the multi-point calibration method provided the highest accuracy (< 0.025 difference in pH) of pH when compared against benchtop measurements. Spectral analysis of time series data showed that during spring in Alaskan waters, a range of tidal frequencies dominated pH variability, while seasonal oceanographic conditions were the dominant driver in Canadian waters. Further, it is suggested that spectral analysis performed on initial deployments may be able to act as an a posteriori method to better identify appropriate calibration regimes. Based on this evaluation, we provide a comprehensive assessment of the potential sources of uncertainty associated with accuracy and precision of the SeaFET ™ electrodes.