Acute toxicity testing of 6PPD-quinone on the estuarine-dependent sport fish, Sciaenops ocellatus.

Recently, large-scale fish kills in the Pacific Northwest were linked to tire wear particles (TWPs) left on roadways, with the lethality attributed to 6PPD-quinone. which has a median lethal concentration of <1 µg/L for selected salmonids. However, there remains a paucity of 6PPD-quinone toxicity values developed for estuarine fish species, which is particularly significant because estuaries receiving inflows from highly urbanized watersheds are especially vulnerable to TWP contamination. Therefore, the present study aimed to determine the toxicity of 6PPD-quinone to an economically and ecologically important estuarine-dependent fish-red drum (Sciaenops ocellatus). Here, we examined the relative sensitivities of three early life stages within red drum: embryonic, larval, and post-settlement for 24-72 hours, depending on the life stage. Exposure concentrations ranged from 10 µg/L to 500 µg/L. We also assessed the sub-lethal impacts of 6PPD-quinone exposure on development during embryonic and larval stages, including body and organ sizes. Our results indicate that red drum are not acutely sensitive to 6PPD-quinone at each early life stage tested. We also found that yolk-sac larvae did not exhibit sub-lethal morphological impacts in a dose-dependent manner, regardless of exposure during embryonic and larval stages. These data are the first to assess the impacts of 6PPD-quinone on estuarine-dependent non-model fishes.

Warming-induced "plastic floors" improve hypoxia vulnerability, not aerobic scope, in red drum (Sciaenops ocellatus).

Ocean warming is a prevailing threat to marine ectotherms. Recently the "plastic floors, concrete ceilings" hypothesis was proposed, which suggests that a warmed fish will acclimate to higher temperatures by reducing standard metabolic rate (SMR) while keeping maximum metabolic rate (MMR) stable, therefore improving aerobic scope (AS). Here we evaluated this hypothesis on red drum (Sciaenops ocellatus) while incorporating measures of hypoxia vulnerability (critical oxygen threshold; Pcrit) and mitochondrial performance. Fish were subjected to a 12-week acclimation to 20 °C or 28 °C. Respirometry was performed every 4 weeks to obtain metabolic rate and Pcrit; mitochondrial respirometry was performed on liver and heart samples at the end of the acclimation. 28 °C fish had a significantly higher SMR, MMR, and Pcrit than 20 °C controls at time 0, but SMR declined by 36.2 % over the 12-week acclimation. No change in SMR was observed in the control treatment. Contrary to expectations, SMR suppression did not improve AS relative to time 0 owing to a progressive decline in MMR over acclimation time. Pcrit decreased by 27.2 % in the warm-acclimated fishes, which resulted in temperature treatments having statistically similar values by 12-weeks. No differences in mitochondrial traits were observed in the heart - despite a Δ8 °C assay temperature - while liver respiratory and coupling control ratios were significantly improved, suggesting that mitochondrial plasticity may contribute to the reduced SMR with warming. Overall, this work suggests that warming induced metabolic suppression offsets the deleterious consequences of high oxygen demand on hypoxia vulnerability, and in so doing greatly expands the theoretical range of metabolically available habitats for red drum.

Recalibrating the significance of the decline effect in fish ocean acidification research.

The recently described decline effect in ocean acidification impacts on fish behaviour should not be equated with negligible effects. Here, existing mechanistic data are used to argue for continued research and cautions against "throwing the baby out with the bathwater."

Hypoxia acclimation improves mitochondrial efficiency in the aerobic swimming muscle of red drum (Sciaenops ocellatus).

Environmental hypoxia (low dissolved oxygen) is a significant threat facing fishes. As fishes require oxygen to efficiently produce ATP, hypoxia can significantly limit aerobic capacity. However, some fishes show respiratory flexibility that rescues aerobic performance, including plasticity in mitochondrial performance. This plasticity may result in increased mitochondrial efficiency (e.g., less proton leak), increased oxygen storage capacity (increased myoglobin), and oxidative capacity (e.g., higher citrate synthase activity) under hypoxia. We acclimated a hypoxia-tolerant fish, red drum (Sciaenops ocellatus), to 8-days of constant hypoxia to induce a hypoxic phenotype. Fish were terminally sampled for cardiac and red muscle tissue to quantify oxidative phosphorylation, proton leak, and maximum respiration in tissue from both hypoxia-acclimated and control fish. Tissue was also collected to assess the plasticity of citrate synthase enzyme activity and mRNA expression for select oxygen storage and antioxidant pathway transcripts. We found that mitochondrial respiration rates were not affected by hypoxia exposure in cardiac tissue, though citrate synthase activity and myoglobin expression were higher following hypoxia acclimation. Interestingly, measures of mitochondrial efficiency in red muscle significantly improved in hypoxia-acclimated individuals. Hypoxia-acclimated fish had significantly higher OXPHOS Control Efficiency, OXPHOS Capacity and Coupling Control Ratios (i.e., LEAK/OXPHOS). There was no significant change to citrate synthase activity or myoglobin expression in red muscle. Overall, these results suggest that red muscle mitochondria of hypoxia-acclimated fish more efficiently utilize oxygen, which may explain previous reports in red drum of improved aerobic swimming performance in the absence of improved maximum metabolic rate following hypoxia acclimation.

The effects of size on exhaustive exercise and recovery in a marine sportfish, the red drum (Sciaenops ocellatus).

Recreational angling is an economically important activity in many communities around the world. One conservation strategy adopted to offset the population-level consequences of recreational angling is "catch-and-release" (CAR), which is the act of returning fish to the environment following an angling event. While an expansive literature has helped to generalize CAR best practices, species-specific validation of recovery profiles remains a crucial component of species-specific angling guidance. This study sought to define the injury and recovery profiles in the plasma and white muscle following exhaustive exercise in two size classes of a common Gulf of Mexico sportfish, the red drum (Sciaenops ocellatus). The two sizes included a "small" (20-30 cm) and "slot" size (51-74 cm), the latter of which is a common angling target. Both size classes showed a characteristic injury profile that consisted of significantly elevated muscle and plasma lactate, plasma osmolality and haematocrit, as well as decreased muscle ATP and phosphocreatine, and lowered plasma and muscle pH. In small fish, muscle metabolites returned to control values by 1 h post-exercise and plasma metabolites returned to control between 3 and 6 h post-exercise. In contrast, slot sized fish had recovery periods of ≥3 h for all metabolites. The maximum injury effect size was also greater in the slot size class. These data suggest that while red drum conform to typical patterns of post-exercise recovery, larger trophy-sized fish may be more at risk to the ancillary effects of exhaustive exercise owing to greater exercise injury and slower recovery rates.

Functional divergence of teleost carbonic anhydrase 4.

The functional role of membrane-bound carbonic anhydrases (CAs) has been of keen interest in the past decade, and in particular, studies have linked CA in red muscle, heart, and eye to enhanced tissue oxygen extraction in bony fishes (teleosts). However, the number of purported membrane-bound CA isoforms in teleosts, combined with the imperfect system of CA isoform nomenclature, present roadblocks for ascribing physiological functions to particular CA isoforms across different teleost lineages. Here we developed an organizational framework for membrane-bound CAs in teleosts, providing the latest phylogenetic analysis of extant CA4 and CA4-like isoforms. Our data confirm that there are three distinct isoforms of CA4 (a, b, and c) that are conserved across major teleost lineages, with the exception of CA4c gene being lost in salmonids. Tissue distribution analyses suggest CA4a functions in oxygen delivery across teleost lineages, while CA4b may be specialized for renal acid-base balance and ion regulation. This work provides an important foundation for researchers to elucidate the functional significance of CA4 isoforms in fishes.

The role of carbonic anhydrase-mediated tissue oxygen extraction in a marine teleost acclimated to hypoxia.

With the growing prevalence of hypoxia (O2 levels ≤2 mg l-1) in aquatic and marine ecosystems, there is increasing interest in the adaptive mechanisms fish may employ to better their performance in stressful environments. Here, we investigated the contribution of a proposed strategy for enhancing tissue O2 extraction - plasma-accessible carbonic anhydrase (CA-IV) - under hypoxia in a species of estuarine fish (red drum, Sciaenops ocellatus) that thrives in fluctuating habitats. We predicted that hypoxia-acclimated fish would increase the prevalence of CA-IV in aerobically demanding tissues to confer more efficient tissue O2 extraction. Furthermore, we predicted the phenotypic changes to tissue O2 extraction that occur with hypoxia acclimation may improve respiratory and swim performance under 100% O2 conditions (i.e. normoxia) when compared with performance in fish that have not been acclimated to hypoxia. Interestingly, there were no significant differences in relative CA-IV mRNA expression, protein abundance or enzyme activity between the two treatments, suggesting CA-IV function is maintained under hypoxia. Likewise, respiratory performance of hypoxia-acclimated fish was similar to that of control fish when tested in normoxia. Critical swim speed (Ucrit) was significantly higher in hypoxia-acclimated fish but translated to marginal ecological benefits with an increase of ∼0.3 body lengths per second. Instead, hypoxia-acclimated fish may have relied more heavily on anaerobic metabolism during their swim trials, utilizing burst swimming 1.5 times longer than control fish. While the maintenance of CA-IV may still be an important contributor for hypoxia tolerance, our evidence suggests hypoxia-acclimated red drum are using other mechanisms to cope in an O2-depleted environment.

Respiratory plasticity improves aerobic performance in hypoxia in a marine teleost.

Ocean deoxygenation is a pressing concern in the face of climate change. In response to prolonged hypoxia, fishes have demonstrated the ability to dynamically regulate hemoglobin (Hb) expression to enhance oxygen (O2) uptake. Here, we examined hypoxia-inducible Hb expression in red drum (Sciaenops ocellatus) and the subsequent implications on Hb-O2 binding affinity and aerobic scope. Fish were acclimated to 30 % air saturation for 1 d, 4 d, 8 d, 2 w, or 6 w, and red blood cells were collected for gene expression and biochemical profiling. Hypoxia acclimation induced significant up-regulation of one Hb subunit isoform (hbα 2) relative to control by 4 d with consistent upregulation thereafter. Hematocrit increased in hypoxia, with no changes in the allosteric modulator [NTP] at any time point. Changes in Hb expression co-occurred with a reduced Root effect (~26 % in normoxia, ~14 % in hypoxia) at a physiologically relevant pH while increasing O2 binding affinity (i.e., lower P50). These changes correlated with increased maximum metabolic rate and aerobic scope relative to controls when fish were tested in hypoxia. These results demonstrate an important role for Hb multiplicity in improving O2 affinity and maximizing respiratory performance in hypoxia.

Early life-stage Deepwater Horizon crude oil exposure induces latent osmoregulatory defects in larval red drum (Sciaenops ocellatus).

Crude oil is known to induce developmental defects in teleost fish exposed during early-life stages (ELSs). A recent study has demonstrated that zebrafish (Danio rerio) larvae acutely exposed to Deepwater Horizon (DHW) crude oil showed transcriptional changes in key genes involved in early kidney (pronephros) development and function, which were coupled with pronephric morphological defects. Given the osmoregulatory importance of the kidney, it is unknown whether ELS effects arising from short-term crude exposures result in long-term osmoregulatory defects, particularly within estuarine fishes likely exposed to DWH oil following the spill. To address this knowledge gap, an acute 72 h exposure to red drum (Sciaenops ocellatus) larvae was performed using high-energy water-accommodated fractions (HEWAFs) of DWH weathered oil to analyze transcriptional changes in genes involved in pronephros development and function by quantitative PCR. To test the latent effects of oil exposure on osmoregulation ability, red drum larvae were first exposed to HEWAF for 24 h. Larvae were then reared in clean seawater for two weeks and a 96 h acute osmotic challenge test was performed by exposing the fish to waters with varying salinities. Latent effects of ELS crude oil exposure on osmoregulation were assessed by quantifying survival during the acute osmotic challenge test and analyzing transcriptional changes at 14 dpf. Results demonstrated that ELS crude oil exposure reduced survival of red drum larvae when challenged in hypoosmotic waters and that latent transcriptional changes in some target pronephric genes were evident, indicating that an affected kidney likely contributed to the increased mortality.

Pyrene drives reduced brain size during early life exposure in an estuarine fish, the red drum (Sciaenops ocellatus).

Crude oil and the constituent polycyclic aromatic hydrocarbons (PAHs) induce a consistent suite of sub-lethal effects in early life stage fishes. It has been suggested that 3-ring PAHs drive cardiotoxicity and that all other impacts are downstream consequences of these cardiac effects. However, recent studies have documented behavioral alterations that may not be linked to cardiotoxicity. This raises the question of whether the 3-ring PAHs that drive cardiotoxicity are also responsible for the observed neurological impairments. To explore this question, we exposed embryonic red drum (Sciaenops ocellatus) - a species that exhibits greater sensitivity to craniofacial malformations than cardiotoxicity - to individual 2-ring, 3-ring, and 4-ring PAHs for 48 h after which they were assessed for sub-lethal developmental malformations. No effects were observed following exposure to naphthalene, anthracene, dibenzothiophene, phenanthrene and fluorene at doses equivalent to the ΣPAH50 effective concentration 50 for craniofacial malformation in red drum. Conversely, pyrene caused complete lethality at the original dose, and a 5× diluted dose resulted in significantly reduced brain size and spine length. Similar sub-lethal effects were also observed in chrysene at the 1× dose. These results indicate that 4-ring PAHs are driving malformations in developing red drum and suggest oil induced impairments in this species are not a downstream consequence of 3-ring PAH induced cardiac malformations.

Exposure to Deepwater Horizon crude oil increases free cholesterol in larval red drum (Sciaenops ocellatus).

The 2010 Deepwater Horizon oil spill impacted over 2100 km of shoreline along the northern Gulf of Mexico, which coincided with the spawning season of many coastal species, including red drum (Sciaenops ocellatus). Red drum develop rapidly and are sensitive to crude oil exposure during the embryonic and larval periods. This study investigates the predictions from recent transcriptomic studies that cholesterol biosynthetic processes are impacted by oil exposure in fish early life stages. We found that red drum larvae exposed for 72 h to ΣPAH50 3.55-15.45 µg L-1 exhibited significantly increased pericardial area, a cardiotoxicity metric, but the expression of several genes targeted in the cholesterol synthesis pathway was not affected. However, whole-mount staining revealed significant increases in free cholesterol throughout the larval body (ΣPAH50 4.71-16.15 µg L-1), and total cholesterol followed an increasing trend (ΣPAH50 3.55-15.45 µg L-1). Cholesterol plays a critical role in fish embryo development and ion channel function. Therefore, the disruption of cholesterol homeostasis, as observed here, could play a role in the oil toxicity phenotype observed across many fish species.

A review of the toxicology of oil in vertebrates: what we have learned following the Deepwater Horizon oil spill.

In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.

The effects of warming on red blood cell carbonic anhydrase activity and respiratory performance in a marine fish.

Measures of fitness are valuable tools to predict species' responses to environmental changes, like increased water temperature. Aerobic scope (AS) is a measure of an individual's capacity for aerobic processes, and frequently used as a proxy for fitness. However, AS is complicated by individual variation found not only within a species, but within similar body sizes as well. Maximum metabolic rate (MMR), one of the factors determining AS, is constrained by an individual's ability to deliver and extract oxygen (O2) at the tissues. Recently, data has shown that red blood cell carbonic anhydrase (RBC CA) is rate-limiting for O2 delivery in red drum (Sciaenops ocellatus). We hypothesized increased temperature impacts MMR and RBC CA activity in a similar manner, and that an individual's RBC CA activity drives individual variation in AS. Red drum were acutely exposed to increased temperature (+6 °C; 22 °C to 28 °C) for 24 h prior to exhaustive exercise and intermittent-flow respirometry at 28 °C. RBC CA activity was measured before temperature exposure and after aerobic performance. Due to enzymatic thermal sensitivity, acute warming increased individual RBC CA activity by 36%, while there was no significant change in the control (22 °C) treatment. Interestingly, average MMR of the acute warming treatment was 36% greater than that of control drum. However, we found no relationships between individual RBC CA activity and their respective MMR and AS at either temperature. While warming similarly affects RBC CA activity and MMR, RBC CA activity is not a predictor of individual MMR.

Is hypoxia vulnerability in fishes a by-product of maximum metabolic rate?

The metabolic index concept combines metabolic data and known thermal sensitivities to estimate the factorial aerobic scope of animals in different habitats, which is valuable for understanding the metabolic demands that constrain species' geographical distributions. An important assumption of this concept is that the O2 supply capacity (which is equivalent to the rate of oxygen consumption divided by the environmental partial pressure of oxygen: ) is constant at O2 tensions above the critical O2 threshold (i.e. the where O2 uptake can no longer meet metabolic demand). This has led to the notion that hypoxia vulnerability is not a selected trait, but a by-product of selection on maximum metabolic rate. In this Commentary, we explore whether this fundamental assumption is supported among fishes. We provide evidence that O2 supply capacity is not constant in all fishes, with some species exhibiting an elevated O2 supply capacity in hypoxic environments. We further discuss the divergent selective pressures on hypoxia- and exercise-based cardiorespiratory adaptations in fishes, while also considering the implications of a hypoxia-optimized O2 supply capacity for the metabolic index concept.

Behavioral Changes in a Coastal Marine Fish Lead to Increased Predation Risk Following Oil Exposure.

Fishes exposed to crude oil have shown reduced sociability and poor habitat selection, which corresponded with increased predation risk. However, the contribution of oil-induced cardiorespiratory impairments to these findings is uncertain. This study explores the effect of oil exposure on predation risk in a model fish species, Sciaenops ocellatus, across a suite of physiological and behavioral end points to elucidate the mechanisms through which any observed effects are manifested. Using mesocosms to assess group predator avoidance, oil exposure to 36.3 µg l-1 ΣPAH reduced the time to 50% mortality from a mean time of 80.0 (74.1-86.0 95% confidence interval [CI]) min to 39.2 (35.6-42.8 95% CI) min. The influence of oil impaired cardiorespiratory and behavioral pathways on predation risk was assessed based on respiratory performance, swim performance, sociability, and routine activity. Swim trials demonstrated that cardiorespiratory and swim performance were unaffected by exposures to 26.6 or 100.8 µg l-1 ΣPAH. Interestingly, behavioral tests revealed that exposure to 26.6 µg l-1 ΣPAH increased distance moved, speed, acceleration, and burst activity. These data indicate that behavioral impairment is more sensitive than cardiorespiratory injury and may be a more important driver of downstream ecological risk following oil exposure in marine species.

Osmoregulatory plasticity during hypersaline acclimation in red drum, Sciaenops ocellatus.

Prolonged drought and freshwater diversion are making periods of hypersalinity more common in coastal ecosystems. This is especially true in the Laguna Madre system along the Texas coast where salinities can exceed 60 g/kg. As such, the ability to tolerate hypersalinity is critical to the success of endemic species, such as the commercially important red drum (Sciaenops ocellatus). This study evaluated acclimation of red drum to hypersalinity (60 g/kg) using a direct transfer protocol. Hypersalinity exposure resulted in significant impacts on plasma osmolality and muscle water in the first 24 h, but returned to control values coincident with a significant increase in intestinal water volume. Hypersalinity acclimation resulted in significant branchial and intestinal plasticity. The gill showed significant elevated nka α1a, nkcc1 and vha (B subunit) mRNA abundance, as well as NKA enzyme activity. The posterior intestine showed a stronger plasticity signal than the anterior intestine, which included a 12-fold increase in nkcc2 mRNA abundance and significant increases in NKA and VHA enzyme activity. These changes were corroborated by a significant threefold increase in bumetanide-sensitive absorptive short circuit current. These data suggest that the dynamic regulation of NKCC2-mediated intestinal water absorption is an important compliment to HCO3--mediated water absorption during hypersalinity exposure and acclimation.

The effects of temperature on oil-induced respiratory impairment in red drum (Sciaenops ocellatus).

The 2010 Deepwater Horizon (DWH) crude oil spill, among the largest environmental disasters in U.S. history, affected numerous economically important fishes. Exposure to crude oil can lead to reduced cardiac function, limiting oxygen transport, ATP production, and aerobic performance. However, crude oil exposure is not the only stressor that affects aerobic performance, and increasing environmental temperatures are known to significantly increase metabolic demands in fishes. As the DWH spill was active during warm summer months in the Gulf of Mexico, it is important to understand the combined effects of oil and temperature on a suite of metabolic parameters. Therefore, we investigated the effects of 24h crude oil exposure on the aerobic metabolism and hypoxia tolerance of red drum (Sciaenops ocellatus) following 3 week chronic exposure to four ecologically relevant temperatures (18 °C, 22 °C, 25 °C, 28 °C). Our results show that individuals acclimated to higher temperatures had significantly higher standard metabolic rate than individuals at lower temperatures, which resulted in significantly decreased critical oxygen threshold and reduced recovery from exercise. As predicted, crude oil exposure resulted in lower maximum metabolic rates (MMR) across the temperature range, and a significantly reduced ability to recover from exercise. The lowest temperature acclimation showed the smallest effect of oil on MMR, while the highest temperature showed the smallest effect on exercise recovery. Reduced respiratory performance and hypoxia tolerance are likely to have meaningful impacts on the fitness of red drum, especially with climate-induced temperature increases and continued oil exploration in the Gulf of Mexico.

Assessment of hypoxia avoidance behaviours in a eurythermal fish at two temperatures using a modified shuttlebox system.

Behavioural avoidance responses of red drum (Sciaenops ocellatus) to aquatic hypoxia were investigated at 22 and 30°C using a modified shuttlebox system. Fish movement between a control side maintained at normoxia and a hypoxic side with stepwise decreasing water oxygen tension was analysed for entries into the hypoxic side, residence time per entry into the hypoxic side and total time in the hypoxic side. Acclimation to 30°C increased the oxygen threshold for the onset of hypoxia avoidance behaviours for entries and total time, while residence time per entry was unchanged.

The effects of acute crude oil exposure on growth and competition in red drum, Sciaenops ocellatus.

Crude oil is a well-known toxicant that reduces cardiorespiratory performance in acutely exposed fishes. While toxic effects can manifest in death in severe cases, the ecological consequences of sub-lethal exposure remain uncertain. This study investigated the impact of crude oil exposure on long-term social competition, growth, and metabolic performance in a coastal species, the red drum (Sciaenops ocellatus). Fish were acutely exposed to either control or one of two environmentally relevant oil concentrations and reared together in groups of 15 (5 from each exposure concentration) for eight weeks under resource-rich or resource-limited scenarios. Relative to controls, a 41.3% and 45.9% reduction in the specific growth rate was-observed following exposure to 25.3 and 53.4 µg l-1 ΣPAH respectively under resource-limited conditions. These fish were subsequently sampled for metabolic performance and common indicators of social subordination including reduced glucocorticoid receptors in the gill and caudal fin damage. The reduction in specific growth rate coincided with a 15.1% and 17.3% reduction in standard metabolic rate; however, maximum metabolic rate and aerobic scope were unaffected. Additionally, measures of social subordination showed no differences between oil-exposed and control fish. These results reinforce the hypothesis that acute oil exposure can have prolonged sub-lethal effects that compromise the ability of exposed individuals to perform effectively in their environment, including gathering and/or metabolizing food. Furthermore, this work highlights the premise that oil spills can be more detrimental in already at-risk ecosystems.

Red blood cell carbonic anhydrase mediates oxygen delivery via the Root effect in red drum.

Oxygen (O2) and carbon dioxide (CO2) transport are tightly coupled in many fishes as a result of the presence of Root effect hemoglobins (Hb), whereby reduced pH reduces O2 binding even at high O2 tensions. Red blood cell carbonic anhydrase (RBC CA) activity limits the rate of intracellular acidification, yet its role in O2 delivery has been downplayed. We developed an in vitro assay to manipulate RBC CA activity while measuring Hb-O2 offloading following a physiologically relevant CO2-induced acidification. RBC CA activity in red drum (Sciaenops ocellatus) was inhibited with ethoxzolamide by 53.7±0.5%, which prompted a significant reduction in O2 offloading rate by 54.3±5.4% (P=0.0206, two-tailed paired t-test; n=7). Conversely, a 2.03-fold increase in RBC CA activity prompted a 2.14-fold increase in O2 offloading rate (P<0.001, two-tailed paired t-test; n=8). This approximately 1:1 relationship between RBC CA activity and Hb-O2 offloading rate coincided with a similar allometric scaling exponent for RBC CA activity and maximum metabolic rate. Together, our data suggest that RBC CA is rate limiting for O2 delivery in red drum.