Bivalves are an economically and ecologically important group of organisms. They are ecosystem engineers that create habitat structure, modify phytoplankton communities, and can increase species richness (Borthagaray and Carranza, 2007; Gutiérrez et al., 2003; Peterson and Black, 1987), while their shellfisheries are worth $2B to the US economy (NOAA, 2019). Reductions in bivalve populations can have wide ranging effects through in ecosystem functionality (Gutiérrez et al., 2003; Wijsman et al., 2019). Bivalves can be negatively impacted by multiple anthropogenic stressors such as hypoxia, acidification, or harmful algal blooms (Breitburg et al., 2018; Diaz and Rosenberg, 2008; Tomasetti and Gobler, 2020; Shumway, 1990).

Eutrophication is the progressive overload of organic matter to a coastal ecosystem, often driven by excessive nutrient loading that increases organic matter production by algae (Nixon, 1995). Microbial respiration of excessive organic matter can lead to the formation of low oxygen, hypoxic zones (Diaz and Rosenberg, 2008; Breitburg et al., 2018). Additionally, many of the primary producers that respond to increased nutrient and organic matter loading may be mixotrophic and, therefore, may display strong respiration and exacerbate nocturnal hypoxia (Burkholder, 1998; Burkholder et al., 2008; Heisler et al., 2008). Hypoxia affects coastal systems around the world and has intensified with time (Diaz and Rosenberg, 2008; Steckbauer et al., 2011). The severity and length of hypoxic conditions can vary tidally, on day-night cycles, and seasonally and is driven by multiple biological, physical, and chemical conditions (Baumann et al., 2015; Tyler et al., 2009; Wallace and Gobler, 2021).

Dissolved oxygen (DO) can display extreme diel cycles in eutrophic coastal waters with hyperoxic conditions found during the day and hypoxia occurring at night (Baumann and Smith, 2018; Gobler and Baumann, 2016; Tyler et al., 2009; Wallace et al., 2021). Diel cycling of DO is generally most severe during mid/late summer when temperatures and photosynthetically active radiation are maximal (Breitburg, 1990; Caffrey, 2004; Wallace et al., 2014, 2021). Physical processes such as tidally flushing may moderate or intensify conditions in estuaries; more intensive tidal flushing can raise the DO via increased mixing of non-hypoxic, ocean end-member water, while reduced tidal flow may maximize the influence of localized respiration rates on DO levels (Baumann et al., 2015; Tyler et al., 2009).

As concentrations of DO decline, organisms may transition to anaerobic respiration and experience impaired physiological performance (Miller et al., 2016; Pörtner, 2012; Stevens and Gobler, 2018). Hypoxic conditions generally occur as a vertical gradient with more severe conditions in bottom waters, although in shallow systems, hypoxic conditions can appear throughout the water column (Beck and Bruland, 2000; Tyler et al., 2009; Wallace et al., 2021). As sessile or semi-sessile benthic organisms, bivalves are often more vulnerable to hypoxia than mobile animals (Diaz and Rosenberg, 2008; Vaquer-Sunyer and Duarte, 2008; Wang and Widdows, 1993).

While previous research has provided important information on the impacts of static hypoxic on shellfish (Steckbauer et al., 2015; Tomasetti et al., 2018; Vaquer-Sunyer and Duarte, 2008; Wang and Widdows, 1993), such experiments are not necessarily representative of dynamic estuarine ecosystems. Recent studies have explored diel cycling of hypoxia and have shown that brief periods of low DO can reduce the growth and/or survival of several species of early life stage shellfish (Clark and Gobler, 2016; Keppel et al., 2016; Miller et al., 2016; Tomasetti et al., 2021). Few studies, however, have considered how naturally occurring diel cycles of hypoxia in estuaries may impact juvenile bivalves.

The objective of this study was to determine how juvenile bivalves are affected by natural, diel cycling of hypoxia in an estuarine setting. Nine experiments were performed over three years with three species of North Atlantic bivalves: Argopecten irradians (bay scallops), Crassostrea virginica (Eastern oysters), and Mercenaria mercenaria (hard clams). Juvenile bivalves were grown in estuarine waters experiencing nocturnal hypoxia or in the same source water with hypoxia experimentally ameliorated. Differences in growth and survival rates were assessed and trends were considered in light of the intensity and duration of nocturnal hypoxia. We hypothesized nocturnal hypoxia would reduce the growth and survival of each bivalve species.

Hypoxia and acidification are well-known co-stressors that can have additive and synergistic negative effects on marine life (Alter et al., 2015; Gobler and Baumann, 2016; Waldbusser and Salisbury, 2014) and studies have shown that diel cycles of low pH and low DO negatively impact survival and growth of early life stage bivalves (Clark and Gobler, 2016; Gobler et al., 2017; Wallace et al., 2021). Here, experiments ameliorated nocturnal hypoxia allowing for the effects of this stressor, but not acidification, to be resolved. Oysters and scallops were found to be more responsive to the amelioration of nocturnal hypoxia compared to hard clams that, in contrast, appeared more resilient to this stressor. Importantly, the intensity of hypoxia during this study was relatively mild, with DO levels below 2 mg L^-1 rarely occurring, suggesting that extended exposure to even mild hypoxia can have significantly negative effects on bivalves. Collectively, these findings bring new insight toward understanding how nocturnal hypoxia may affect bivalve populations in coastal ecosystems.

In these experiments, DO cycling was ameliorated by vigorous aeration with microbubbles coupled with an extended residence time of seawater via a maze. In contrast, pH was unaffected. pH is influenced by multiple chemical species (CO₂, H₂CO₃, HCO₃ ^-, CO₃ ^2-, HSO₄, etc.) (Sarmiento and Gruber, 2006) that react to changes in carbonate chemistry and ultimately change pH (Sarmiento and Gruber, 2006; Tomasetti et al., 2023a; Wallace et al., 2021). In contrast, oxygen, which in an aqueous environment is in the form of a dissolved gas (O_2(aq)) was directly and rapidly influenced by forced aeration. Regardless, the amelioration of nocturnal hypoxia alone allowed for experiments to isolate the effects of dissolved oxygen on bivalves.

Hard clams (Mercenaria mercenaria) are known to be less sensitive to low oxygen conditions than other temperate bivalves such as oysters and scallops (Levin et al., 2009; Morrison, 1971; Stevens and Gobler, 2018). As infaunal organisms, hard clams live within sediments that can be anoxic (Nitsche et al., 2007; Ripley, 1998). These bivalves may sometimes even benefit from hypoxia on a population level due to reduced predation pressure (Altieri, 2008; Galligan et al., 2022). A lower sensitivity to hypoxia would account for the small and non-significant changes in growth rates seen in these experiments. On the other hand, hard clam juvenile growth and development has been shown to be significantly impacted by high CO₂ conditions (Talmage and Gobler, 2010, 2011; Waldbusser et al., 2010). Had pH cycling been ameliorated, there may have been a larger difference in growth rates compared to the clams exposed to lower nocturnal pH values.

In contrast to hard clams, oysters and scallops significantly benefited from the amelioration of nocturnal hypoxia. Their growth rates significantly increased in the majority of experiments, with aeration-induced increases in growth significantly correlated with the duration of nocturnal hypoxia, and average nocturnal DO concentrations. This is congruent with other studies that have found juvenile oysters and scallops experience decreased growth rates when exposed to static or cycling hypoxia (Gobler et al., 2017, 2014; Johnson et al., 2009; Levin et al., 2009; Stevens and Gobler, 2018; Tomasetti et al., 2023b). Reduced growth rates of bivalves occurred even at hypoxia thresholds of > 3 mg L^-1, which is higher than hypoxia thresholds set by most US states (2 mg L^-1) (Tomasetti and Gobler, 2020). Extrapolation of results to other locations across NY demonstrated hypoxia can be significantly more sever than this ( Supplementary Table 1 ) and thus the benefits of aeration can be larger (up to 240% increase in bivalve growth rates). To provide managers with a better understanding of the impacts of diel cycles of hypoxia and acidification, more studies need to be conducted on other ecologically and economically important species and across more locations.

Prior research has demonstrated that stressors such as ocean acidification, warming, and hypoxia can be overcome in some bivalves if there is adequate food, providing the excess energy needed upregulate physiological pathways to mitigate stress (Thomsen et al., 2013; Mackenzie et al., 2014; Schwaner et al., 2023). During this study, however, the levels of chlorophyll a were, on average, ~20 µg per L or higher for 8 of the 9 experiments, a level known to be sufficient for the maximal performance of the bivalves studied here (Coutteau et al., 1994; Weiss et al., 2007; Wall et al., 2013). There was a single experiment (scallop experiment 3) when chlorophyll a levels averaged < 5 µg per L, a level known to potentially restrict the growth of bivalves (Coutteau et al., 1994; Weiss et al., 2007; Wall et al., 2013). However, this was also the only scallop experiment where the control outgrew the treatment and there was no nocturnal hypoxia. This experiment also had the highest control DO levels of any of the four scallop experiments, 5.92 ± 1.19 mg L^-1, a level not known to impair bay scallop performance (Gurr et al., 2018; Tomasetti et al., 2023b). Moreover, a regression analyses of the mean chlorophyll a level present during each experiment and the increase in growth rate as a consequence of aeration depicted no relationship between these parameters ( Supplementary Figure 3 ). While chlorophyll a may not be a perfect proxy for food availability, it was been a useful predictor in NY estuaries in the past (Weiss et al., 2007; Wall et al., 2013). Regardless, while food supply can be important for ameliorating stress in coastal bivalves, it appears food levels as estimated by chlorophyll a were adequate for nearly all experiments performed here and were unrelated to improved growth rates bivalves experienced as a consequence of higher DO levels.

As temperatures continue to increase across marine habitats, so will oxygen demand, which could lead to oxygen and capacity-limited thermal tolerance (OCLT) (Pörtner and Knust, 2007). Every species has a specific thermal tolerance range; just outside of this optimal range is the pejus range, where conditions will become disadvantageous to the organism. Higher average temperatures and heat waves could have an impact on bivalve populations by limiting their aerobic scope due to higher metabolism rates (Pörtner and Peck, 2010). Most of the experiments performed here were during maximal water annual temperatures in NY, typically around 25°C. When coupled with nocturnal hypoxia, increased temperatures may force organisms into of their respective pejus range on a daily basis (Pörtner and Peck, 2010). At lower DO concentrations, the pejus temperatures are increased, decreasing optimal temperature ranges. The faster growth rates observed within the aerated treatment suggest that more energy was available for processes other than maintaining homeostasis translating to increased growth (Pörtner and Peck, 2010). Due to low DO and lack of excess energy, some proteins may not be produced in the necessary amounts to combat environmental stresses such as heat-shock (Pörtner and Knust, 2007). Over time reductions in an organism’s fitness due to low oxygen supply and high demand coupled with an increased likelihood of experiencing conditions inside of the pejus range will eventually cause reduced population abundances (Pörtner and Peck, 2010).

The results of this study suggest that diel cycling of hypoxia may have significant impacts on fisheries and ecological services in eutrophic coastal regions (Diaz and Rosenberg, 2008; Vaquer-Sunyer and Duarte, 2008). Reductions in juvenile bivalve growth rates due to nocturnal hypoxia would increase the increase risk of predation for individuals prior to reaching predation refuge sizes (Juanes, 1992; Summerson and Peterson, 1984; Wong et al., 2010) increasing population level stressors (Johnson and Smee, 2012). Detrimental nocturnal hypoxia will become more severe as climate change and eutrophication increase (Nixon, 2009; Sunda and Cai, 2012; Tomasetti and Gobler, 2020; Wootton et al., 2008). Rising temperatures could extend the season of severe diel cycling earlier and create more extreme daily maxima and nightly minima due to higher metabolic rates and lower oxygen saturation concentrations (Baumann and Smith, 2018; Sunda and Cai, 2012; Tyler et al., 2009). Increased nutrient loading can promote algal blooms that can also intensify nocturnal hypoxia and the magnitude of diel cycles (Nixon, 2009; Oelsner and Stets, 2019; Paerl et al., 2006; Steel, 1991). Reductions in populations of coastal bivalves due to extreme diel DO cycles would have impacts on important fisheries by reducing harvest and ecosystems services (Newell, 2004).

Current dissolved oxygen regulations are not representative of lethal/sub-lethal oxygen minimum thresholds for many taxa (Vaquer-Sunyer and Duarte, 2008). It has been shown that 61% and 43% of taxa tested experienced mortality and sub-lethal effects, respectively, above a 2 mg L^-1 threshold commonly cited in literature (Tomasetti and Gobler, 2020; Vaquer-Sunyer and Duarte, 2008). It has been suggested that a new threshold of 4.6 mg L^-1 should be adopted to be representative of the 90^th percentile of mean lethal concentrations (Steckbauer et al., 2011; Vaquer-Sunyer and Duarte, 2008). Based on the regression analyses used in this study ( Figure 4 ), however, even at a threshold of 4.6 mg L^-1, Eastern oysters and bay scallops could experience a decrease in growth rates of 39%. Further research is needed on other coastal taxa to determine their susceptibility to repeated, short bouts of hypoxia to establish similar representative thresholds. Nocturnal extremes are often not considered within managerial frameworks and, therefore, may allow detrimental conditions to persist without regulatory action (Tomasetti and Gobler, 2020). For example, historically, most dissolved oxygen monitoring has occurred during the day, and during this study hypoxia was generally absent after 0800, even when DO minimums of < 2 mg L^-1 occurred only an hour earlier. Advancements in continuous remote monitoring have refined our understanding of nocturnal hypoxia and may, therefore, facilitate future management reform.

Restoration of bivalve populations has received significant attention recently (Duarte et al., 2020; Gobler et al., 2022), with a focus on ecosystem services such as improved water clarity, increased habitat, and reduced algal biomass (Duarte et al., 2020; Kreeger et al., 2018). Two of the main roadblocks to restoring bivalve populations are water quality issues and low natural abundance (Duarte et al., 2020). Diel cycling of hypoxia and acidification affect many of these habitats and may make restoration efforts more difficult due to lower larval survival and slower juvenile growth rates as seen in this study (Wallace et al., 2021). Aquaculture of bivalves may serve as an intermediate step to population and habitat recovery by allowing for more control over conditions and improving water quality (Kreeger et al., 2018). Gobler et al. (2022) described the recovery of a hard clam population in a coastal lagoon in NY using spawner sanctuaries. Given other bivalves species (oysters and scallops) are less tolerant of nocturnal hypoxia compared to M. mercenaria, improved water quality might be needed for such species to experience similar restoration recoveries.

Aquaculture is one of the fastest growing food production sectors in the world with lower trophic levels, including shellfish, constituting about half of all production (Subasinghe et al., 2009; van der Schatte Olivier et al., 2020), and many studies focused on the means to increase aquaculture output (Creswell and McNevin, 2008). Given that oxygenation in flow through systems significantly increased growth rates of Eastern oysters and bay scallops in this study similar results might be achieved in larger but localized aquaculture settings. For example, the inclusion of aeration using floating upweller systems (FLUPSY; (RaLonde, 1999) or similar environmental aeration in semi-enclosed bodies of water could be incorporated to ameliorate nocturnal hypoxia. In the experiments presented here, bivalve densities were low at 20 individuals per replicate (~50 individuals m^-2) and high survival was found in both treatments. Given that substantially higher densities (>250 individuals m^-2) are common in hatchery and farm settings (Tobi and Ward, 2019; Widman and Rhodes, 1991), it is possible that increased survival could also be achieved via such aeration.

Recent studies have explored co-culturing macroalgae to improve water quality on commercial bivalve farms (Fernández et al., 2019; Young and Gobler, 2018; Young et al., 2022). Young et al. (2022) found that co-culture with sugar kelp (Saccharina latissimi) increased seawater pH and the growth rates of Eastern oysters whereas Young and Gobler (2018) demonstrated that sea lettuce (Ulva spp.) could increase calcium carbonate saturation states and bivalve growth rates. Such studies, however, have not fully investigated the changes in diel cycles of DO nor the manner in which higher DO may benefit bivalves. The introduction of additional photosynthetic organisms could improve oxygen conditions during the day, but respiration could also cause greater declines in nocturnal concentrations. A better understanding of diel cycles during macroalgae co-culture could be achieved via experiments that investigate all water chemistry parameters including DO on a continuous basis. Artificially perturbing diel cycles in the presence of macroalgal co-cultures, by aerating for example, may create an excellent opportunity to investigate this interaction.

Climate change and eutrophication fueled diel cycles of hypoxia and acidification result in organisms being repeatedly subjected to environmental extremes. The experiments presented here demonstrated that amelioration of these conditions improves juvenile bivalve growth compared to individuals experiencing diel DO cycles. Given that experiments were performed with water that displayed only mild nocturnal hypoxia (mean nocturnal DO across all experiments was 4.7 mg L^-1), the growth and potentially survival improvement via aeration in more severe hypoxic zones would likely be greater than observed here. Scallop and oyster growth rates were improved on average by 23% and 30%, respectively, and by up to 70% relative to the controls, demonstrating the impact of nocturnal hypoxia on these species. Extrapolation of results to 19 other locations across NY suggested substantially larger benefits at some location with increases in bivalve growth exceeding 100% for half of the locations. While mitigating nocturnal hypoxia via aeration was effective on a small scale, reducing coastal eutrophication to improve DO levels will be needed for sustainable, large-scale improvement.