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Following the Deepwater Horizon (DWH) oil spill, the Natural Resource Damage Assessment conducted comprehensive health assessments on common bottlenose dolphins Tursiops truncatus in Barataria Bay (BB), Louisiana, in 2011, 2013 and 2014, as well as in Mississippi Sound (MS) in 2013, to assess potential health effects from exposure to oil compared to Sarasota Bay (SB), Florida dolphins not exposed to DWH oil. Immune functions demonstrated a consistent increase in T (BB 2011) and B (BB 2011 and 2013) lymphocyte proliferation compared to SB. Cytokine concentrations varied considerably in wild dolphin populations, and no significant differences were found; however, interesting trends were observed. The Th1 cytokines IL-2, IL-12, and IFNγ, and the Th2 cytokines IL-5, IL-10, and IL-13, were 2- to 50-fold lower, and IL-4 was 3-fold higher, in BB 2011 compared to SB. Overall, the changes observed were compatible with those documented in other species following exposure to oil or PAHs and were most pronounced in BB 2011, at the place and time most affected by oil. The changes in T cell functions, and the trend towards a cytokine balance tilted towards a Th2, rather than a Th1 response, are compatible with intra-cellular bacterial infections such as Brucella, which has been identified as one of the potential contributory factors to perinatal dolphin mortalities, and changes in B cell functions are compatible with an increase in extra-cellular bacterial infections and primary bacterial pneumonia.

Timely detection and understanding of causes for population decline are essential for effective wildlife management and conservation. Assessing trends in population size has been the standard approach, but we propose that monitoring population health could prove more effective. We collated data from 7 bottlenose dolphin (Tursiops truncatus) populations in the southeastern United States to develop a method for estimating survival probability based on a suite of health measures identified by experts as indices for inflammatory, metabolic, pulmonary, and neuroendocrine systems. We used logistic regression to implement the veterinary expert system for outcome prediction (VESOP) within a Bayesian analysis framework. We fitted parameters with records from 5 of the sites that had a robust network of responders to marine mammal strandings and frequent photographic identification surveys that documented definitive survival outcomes. We also conducted capture-mark-recapture (CMR) analyses of photographic identification data to obtain separate estimates of population survival rates for comparison with VESOP survival estimates. The VESOP analyses showed that multiple measures of health, particularly markers of inflammation, were predictive of 1- and 2-year individual survival. The highest mortality risk 1 year following health assessment related to low alkaline phosphatase (odds ratio [OR] = 10.2 [95% CI: 3.41-26.8]), whereas 2-year mortality was most influenced by elevated globulin (OR = 9.60 [95% CI: 3.88-22.4]); both are markers of inflammation. The VESOP model predicted population-level survival rates that correlated with estimated survival rates from CMR analyses for the same populations (1-year Pearson's r = 0.99, p = 1.52 × 10 -5 ; 2-year r = 0.94, p = 0.001). Although our proposed approach will not detect acute mortality threats that are largely independent of animal health, such as harmful algal blooms, it can be used to detect chronic health conditions that increase mortality risk. Random sampling of the population is important and advancement in remote sampling methods could facilitate more random selection of subjects, obtainment of larger sample sizes, and extension of the approach to other wildlife species., (© 2023 The Authors. Conservation Biology published by Wiley Periodicals LLC on behalf of Society for Conservation Biology.)

Following the 2010 Deepwater Horizon disaster and subsequent unusual mortality event, adverse health impacts have been reported in bottlenose dolphins in Barataria Bay, LA including impaired stress response and reproductive, pulmonary, cardiac, and immune function. These conditions were primarily diagnosed through hands-on veterinary examinations and analysis of standard diagnostic panels. In human and veterinary medicine, gene expression profiling has been used to identify molecular mechanisms underlying toxic responses and disease states. Identification of molecular markers of exposure or disease may enable earlier detection of health effects or allow for health evaluation when the use of specialized methodologies is not feasible. To date this powerful tool has not been applied to augment the veterinary data collected concurrently during dolphin health assessments. This study examined transcriptomic profiles of blood from 76 dolphins sampled in health assessments during 2013-2018 in the waters near Barataria Bay, LA and Sarasota Bay, FL. Gene expression was analyzed in conjunction with the substantial suite of health data collected using principal component analysis, differential expression testing, over-representation analysis, and weighted gene co-expression network analysis. Broadly, transcript profiles of Barataria Bay dolphins indicated a shift in immune response, cytoskeletal alterations, and mitochondrial dysfunction, most pronounced in dolphins likely exposed to Deepwater Horizon oiling. While gene expression profiles in Barataria Bay dolphins were altered compared to Sarasota Bay for all years, profiles from 2013 exhibited the greatest alteration in gene expression. Differentially expressed transcripts included genes involved in immunity, inflammation, reproductive failure, and lung or cardiac dysfunction, all of which have been documented in dolphins from Barataria Bay following the Deepwater Horizon oil spill. The genes and pathways identified in this study may, with additional research and validation, prove useful as molecular markers of exposure or disease to assist wildlife veterinarians in evaluating the health of dolphins and other cetaceans., Competing Interests: The authors have declared that no competing interests exist.

The 2010 Deepwater Horizon (DWH) oil spill exposed common bottlenose dolphins (Tursiops truncatus) in Barataria Bay, Louisiana to heavy oiling that caused increased mortality and chronic disease and impaired reproduction in surviving dolphins. We conducted photographic surveys and veterinary assessments in the decade following the spill. We assigned a prognostic score (good, fair, guarded, poor, or grave) for each dolphin to provide a single integrated indicator of overall health, and we examined temporal trends in prognostic scores. We used expert elicitation to quantify the implications of trends for the proportion of the dolphins that would recover within their lifetime. We integrated expert elicitation, along with other new information, in a population dynamics model to predict the effects of observed health trends on demography. We compared the resulting population trajectory with that predicted under baseline (no spill) conditions. Disease conditions persisted and have recently worsened in dolphins that were presumably exposed to DWH oil: 78% of those assessed in 2018 had a guarded, poor, or grave prognosis. Dolphins born after the spill were in better health. We estimated that the population declined by 45% (95% CI 14-74) relative to baseline and will take 35 years (95% CI 18-67) to recover to 95% of baseline numbers. The sum of annual differences between baseline and injured population sizes (i.e., the lost cetacean years) was 30,993 (95% CI 6607-94,148). The population is currently at a minimum point in its recovery trajectory and is vulnerable to emerging threats, including planned ecosystem restoration efforts that are likely to be detrimental to the dolphins' survival. Our modeling framework demonstrates an approach for integrating different sources and types of data, highlights the utility of expert elicitation for indeterminable input parameters, and emphasizes the importance of considering and monitoring long-term health of long-lived species subject to environmental disasters. Article impact statement: Oil spills can have long-term consequences for the health of long-lived species; thus, effective restoration and monitoring are needed., (© 2021 The Authors. Conservation Biology published by Wiley Periodicals LLC on behalf of Society for Conservation Biology.)

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.

Perfluorooctanoic acid (PFOA), a member of the Per- and polyfluoroalkyl substances, is a highly persistent "forever" chemicals with increasing concern for its potential health effects. However, the mechanisms of PFOA immunotoxic effects are poorly understood. We assessed the antibody response to a physiological antigen stimulation and associated cytokine response upon PFOA exposure. The significant decrease in the IgM antibody response to the T cell dependent antigen keyhole limpet hemocyanin (KLH) at a dose lower than the previously documented LOAEL was accompanied by a significant reduction of the Th2 serum cytokines IL-5 and IL-13, a non-significant dose-response reduction of IL-4, a significant reduction of the Th1 cytokine IL-12, and a non-significant dose-response increase in IL-2 and IFNγ. PFOA significantly decreased the pro-inflammatory cytokines IL-17α and IL-1α, decreased (non-significantly but dose-response) IL-6, and a significantly increased TNFα. Overall, the modulation of serum Th1/Th2 cytokines could explain the reduction in antibody response, pointing to a potential role for T helper cells in the immunotoxicity of PFOA. Further, the higher than anticipated weight loss and increased liver weight, compared to previous studies using similar doses, highlight the potential importance of the route and duration of exposure, contributing to the total accumulated dose, in assessing the toxicity of PFOA., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Increased nutrient loading has led to eutrophication of coastal shelf waters which has resulted in increased prevalence of persistent hypoxic zones - areas in which the dissolved oxygen content of the water drops below 2 mg/L. The northern Gulf of Mexico, fed primarily by the Mississippi River watershed, undergoes annual establishment of one of the largest hypoxic zones in the world. Exposure to hypoxia can induce physiological impacts in fish cardiac systems that include bradycardia, changes in stroke volume, and altered cardiovascular vessel development. While these impacts have been addressed at the functional level, there is little information regarding the molecular basis for these changes. This study used transcriptomic analysis techniques to interrogate the effects of hypoxia exposure on the developing cardiovascular system in newly hatched larvae of two estuarine species that occupy the same ecological niche - the sheepshead minnow (Cyprinodon variegatus) and the Gulf killifish (Fundulus grandis). Results suggest that while differential gene expression is largely distinct between the two species, downstream impacts on pathways and functional responses such as reduced cardiac hypertrophy, modulation of blood pressure, and increased incidence of apoptosis appear to be conserved. Further, differences in the magnitude of these conserved responses may suggest that the length of embryonic development could impart a level of resiliency to hypoxic perturbation in early life stage fish., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Per- and polyfluoroalkyl substances (PFAS) make up a large group of persistent anthropogenic chemicals which are difficult to degrade and/or destroy. PFAS are an emerging class of contaminants, but little is known about the long-term health effects related to exposure. In addition, technologies to identify levels of contamination in the environment and to remediate contaminated sites are currently inadequate. In this opinion-type discussion paper, a team of researchers from the University of Connecticut and the University at Albany discuss the scientific challenges in their specific but intertwined PFAS research areas, including rapid and low-cost detection, energy-saving remediation, the role of T helper cells in immunotoxicity, and the biochemical and molecular effects of PFAS among community residents with measurable PFAS concentrations. Potential research directions that may be employed to address those challenges and improve the understanding of sensing, remediation, exposure to, and health effects of PFAS are then presented. We hope our account of emerging problems related to PFAS contamination will encourage a broad range of scientific experts to bring these research initiatives addressing PFAS into play on a national scale., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Health assessments were conducted on bottlenose dolphins in Barataria Bay, Louisiana, USA, during 2011 to 2018, to assess potential health effects following the Deepwater Horizon oil spill, compared to the unoiled Sarasota Bay, Florida, USA, reference dolphin population. We previously reported significant increases in T-lymphocyte proliferation, as well as lower T helper 1 (Th1) cytokines, higher Th2 cytokine IL-4, and lower T regulatory (Treg) cytokine IL-10 in Barataria Bay in 2011 compared to Sarasota Bay, consistent with Deepwater Horizon oil exposure. Although values between 2013 and 2016 were more similar to those observed in Sarasota Bay, T-cell proliferation was again elevated and cytokine balance tilted toward Th2 in Barataria Bay during 2017-2018. In 2018, Barataria Bay dolphins had significantly more circulating Treg cells than Sarasota Bay dolphins. Mice experimentally exposed to oil also had significantly increased T-lymphocyte proliferation and circulating Treg cell number, including effects in their unexposed progeny. In vitro stimulation resulted in greater Th2 responsiveness in Barataria Bay compared to Sarasota Bay dolphins, and in vitro oil exposure of Sarasota Bay dolphin cells also resulted in enhanced Th2 responsiveness. Evidence points to Treg cells as a potential target for the immunomodulatory effects of oil exposure. The immunological trends observed in Barataria Bay appeared exaggerated in dolphins born after the spill, suggesting the possibility of continued oil exposure or multigenerational health consequences of exposure to oil, as observed in mice. Environ Toxicol Chem 2021;40:1308-1321. © 2021 SETAC., (© 2021 SETAC.)

As an emerging contaminant, per- and polyfluoroalkyl substances (PFASs) make up a large group of persistent anthropogenic chemicals, which are difficult to degrade in the environment. Notwithstanding their wide range of applications in consumer products and industrial processes, PFASs have been detected in the environment as well as in human body. Due to their potential adverse human health effects, the U.S. Environmental Protection Agency (EPA) set the combined concentration of PFOA and PFOS in drinking water at 70 ng/L or 70 ppt (parts per trillion) as a lifetime health advisory level. Current standard detection methods for PFASs heavily rely on chromatographic techniques coupled with mass spectrometry. Although these methods provide accurate, specific, and sensitive measurements, their applications are greatly limited in advanced analytical laboratories because it necessitates expensive instrumentations, professional operators, complicated sample pretreatment, and considerable analysis time. Therefore, other detection methods beyond chromatographic based techniques, such as optical and electrochemical techniques, have also been extensively explored for simple, accessible, inexpensive, rapid, and sensitive detection of PFASs, particularly PFOA and PFOS. The purpose of this review is to provide recent progress in alternative detection platforms relying on non-MS based techniques for PFASs analysis. Starting with a brief introduction about the importance of monitoring PFASs, recent advances in various PFASs detection methods are grouped and discussed based on the difference of signals, with an emphasis on the working principles of different techniques, the sensing mechanism, and the sensing performance. The review is closed with the conclusion and discussion of future trends., (Copyright © 2020. Published by Elsevier B.V.)

Following the 2010 Deepwater Horizon oil spill, extensive research has been conducted on the toxicity of oil and polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. Many studies have identified the toxicological effects of PAHs in estuarine and marine fishes, however, only recently has work begun to identify the combinatorial effect of PAHs and abiotic environmental factors such as hypoxia, salinity, and temperature. This study aims to characterize the combined effects of abiotic stressors and PAH exposure on the cardiac transcriptomes of developing Fundulus grandis larvae. In this study, F. grandis larvae were exposed to varying environmental conditions (dissolved oxygen (DO) 2, 6 ppm; temperature 20, 30 °C; and salinity 3, 30 ppt) as well as to a single concentration of high energy water accommodated fraction (HEWAF) (∑PAHs 15 ppb). Whole larvae were sampled for RNA and transcriptional changes were quantified using RNA-Seq followed by qPCR for a set of target genes. Analysis revealed that exposure to oil and abiotic stressors impacts signaling pathways associated with cardiovascular function. Specifically, combined exposures appear to reduce development of the systemic vasculature as well as strongly impact the cardiac musculature through cardiomyocyte proliferation resulting in inhibited cardiac function and modulated blood pressure maintenance. Results of this study provide a holistic view of impacts of PAHs and common environmental stressors on the cardiac system in early life stage estuarine species. To our knowledge, this study is one of the first to simultaneously manipulate oil exposure with abiotic factors (DO, salinity, temperature) and the first to analyze cardiac transcriptional responses under these co-exposures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Age is an important parameter to better understand wildlife populations, and is especially relevant for interpreting data for fecundity, health, and survival assessments. Estimating ages for marine mammals presents a particular challenge due to the environment they inhabit: accessibility is limited and, when temporarily restrained for assessment, the window of opportunity for data collection is relatively short. For wild dolphins, researchers have described a variety of age-determination techniques, but the gold-standard relies upon photo-identification to establish individual observational life histories from birth. However, there are few populations with such long-term data sets, therefore alternative techniques for age estimation are required for individual animals without a known birth period. While there are a variety of methods to estimate ages, each involves some combination of drawbacks, including a lack of precision across all ages, weeks-to-months of analysis time, logistical concerns for field applications, and/or novel techniques still in early development and validation. Here, we describe a non-invasive field technique to determine the age of small cetaceans using periapical dental radiography and subsequent measurement of pulp:tooth area ratios. The technique has been successfully applied for bottlenose dolphins briefly restrained during capture-release heath assessments in various locations in the Gulf of Mexico. Based on our comparisons of dental radiography data to life history ages, the pulp:tooth area ratio method can reliably provide same-day estimates for ages of dolphins up to about 10 years old., Competing Interests: J.M.H. owns and operates Companion Animal Dental Services, which has no commercial interests in the work described in this manuscript, and has no involvement in product development or marketing. This does not alter our adherence to PLOS ONE policies on sharing data and materials

The area and timing of the Deepwater Horizon oil spill highlight the need to study oil and hypoxia exposure in early life stage fishes. Though critical to health, little research has targeted the effect of oil and hypoxia exposure on developing immune systems. To this end, we exposed sheepshead minnows (Cyprinodon variegatus) at three early life stages: embryonic; post-hatch; and post-larval, to a high energy water accommodated fraction (HEWAF) of oil, hypoxia, or both for 48 hours. We performed RNAseq to understand how exposures alter expression of immune transcripts and pathways. Under control conditions, the embryonic to post-hatch comparison (first transition) had a greater number of significantly regulated immune pathways than the second transition (post-hatch to post-larval). The addition of oil had little effect in the first transition, however, hypoxia elicited changes in cellular and humoral immune responses. In the second transition, oil exposure significantly altered many immune pathways (43), and while hypoxia altered few pathways, it did induce a unique signature of generally suppressing immune pathways. These data suggest that timing of exposure to oil and/or hypoxia matters, and underscores the need to further investigate the impacts of multiple stressors on immune system development in early life stage fishes.

Oil spills have polluted the marine environment for decades and continue to be a major source of polycyclic aromatic hydrocarbons (PAHs) to marine ecosystems around the globe, for example during the 2010 Deepwater Horizon spill. Although the toxicity of PAHs to fish has been well studied, their effects combined with abiotic stressors are poorly understood. The goal of this study was to describe the combined impacts of crude oil and environmental stressors on fish larvae, a sensitive life stage. Gulf killifish (Fundulus grandis) larvae (<24 h post-hatch) were exposed for 48 h to high energy water accommodated fractions (HEWAF; total PAHs 0-125 ppb) of Macondo oil from the Deepwater Horizon spill under different combinations of environmental conditions (dissolved oxygen 2, 6 ppm; temperature 20, 25, 30 °C; salinity 3, 10, 30 ppt). Even under optimal environmental conditions (25 °C, 10 ppt, 6 ppm) larval survival and development were negatively affected by PAHs, starting with the lowest concentration tested (∼15 ppb). Hypoxia and high temperature each increased the adverse effects of HEWAF on development and mortality. In contrast, salinity had little effect on any of the endpoints measured. Importantly, expression of the detoxifying gene cyp1a was highly induced in PAH-exposed larvae under normoxic conditions, but not under hypoxic conditions, potentially explaining the enhanced toxicity observed under hypoxia. This work highlights the importance of considering how suboptimal environmental conditions can exacerbate the effects of pollution on fish early life stages., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The 2010 Deepwater Horizon oil spill released approximately 780 million liters of crude oil contaminating coastal habitats from Texas to Florida which are important habitats for many fish species during early life stages. These diverse habitats are also prone to rapid fluctuations in water quality, such as dissolved oxygen concentration and salinity. The consequence of combined exposure to crude oil and suboptimal environmental conditions during early life stage development of fish is still largely unknown. The objective of this project was to investigate the impacts of exposure to crude oil in combination with varying environmental stressors on developing Cyprinodon variegatus survival, growth, and gene expression. Three life stages (embryonic, post-hatch, and post-larval) were exposed to four nominal concentrations (6.25%, 12.5%, 50% and 100% with actual polycyclic aromatic hydrocarbon (PAH) concentrations ranging from 0 to 512 μg/L) of high energy water accommodated fractions (HEWAF) under different oxic (2.0 or >5.0 mg/L) and salinity (10 or 30 ppt) regimes at 30 °C for 48 h. We found that the post-larval developmental stage was the most sensitive to oil toxicity. Median lethal concentrations during the post-larval exposures followed a treatment-dependent pattern with the highest mortality observed under hypoxic-high salinity conditions (64.55 μg/L). Real-time PCR analysis identified down regulation of target genes, encoding cytochrome P450-1α (cyp1a1), erythropoietin (epo), and the aryl hydrocarbon receptor nuclear translocator (arnt1) only when oil exposure occurred under hypoxic-high salinity conditions in treatments with PAH concentrations greater than 226 μg/L. The target genes measured in this experiment are involved in the aryl hydrocarbon receptor signaling pathway which modulates metabolism of PAHs (a major component of crude oil), and the hypoxia inducible 1-α signaling pathway which is responsible for resilience to hypoxic stress, and it is known that disruption of these pathways can lead to an array of acute and chronic effects. Our results indicated that sheepshead minnow are most sensitive to oil exposure during the post-larval developmental stage. Survival data from this age-stage also indicate that oil toxicity response is exacerbated in hypoxic and high salinity environments. The increased mortality observed during the post-larval developmental stage might be attributed to the suppression of the aryl hydrocarbon receptor signaling and the hypoxia inducible 1-α signaling pathways which is evident in by the down-regulated expression of cyp1a1, epo, and arnt1. These findings provide more information about interactions between oil and abiotic factors which enable us to make better assumptions of the ecological impacts of DWH on coastal estuaries., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The explosion of the Deepwater Horizon (DWH) oil exploration platform on April 20, 2010 began a catastrophic leak of approximately 640 million liters crude oil into the northern Gulf of Mexico (GOM), affecting more than 2100 km of coastline, including wetlands and estuaries that provide habitat and nursery for many aquatic species. Estuaries of the GOM are dynamic environments, with constant fluctuations in salinity and dissolved oxygen, including large hypoxic zones during summer months. Spawning fish in northern GOM estuaries following the DWH incident were at significant risk of oil exposure, and adverse environmental conditions at the time of exposure, such as hypoxia and low salinity, could have exacerbated developmental effects in the offspring. The present study investigated the effects of F 0 parental oil exposure in different environmental scenarios on development of F 1 sheepshead minnow (SHM) offspring. Adult SHM were exposed to the high-energy water accommodated fraction (HEWAF) of crude oil in three environmental scenarios: normoxic (NORM), hypoxic (HYP), and hypoxic with low salinity (HYP-LS). Parental HEWAF exposure in the NORM scenario resulted in developmental effects in F 1 offspring, including altered heart rate, decreased length at hatch, and impaired prey capture. Co-exposure of F 0 SHM to HEWAF and adverse environmental conditions altered HEWAF effects on F 1 heart rate, hatch rate, prey capture, and survival. Time to hatch was not significantly impacted by parental HEWAF in any environmental scenario. The present study demonstrates that parental exposure to HEWAF results in developmental changes in F 1 embryos, and co-exposure to adverse environmental conditions altered the effects for several developmental endpoints. These data suggest that SHM exposed to oil in estuaries experiencing hypoxia or low salinity may produce offspring with worsened outcomes. These developmental effects, in addition to previously reported reproductive effects in adult fish, could lead to long-term population level impacts for SHM., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Considerable efforts have been made to better understand the immune system of bottlenose dolphins in view of the common environmental challenges they encounter, such as exposure to polychlorinated biphenyls, oil spills, or harmful algal bloom biotoxins. However, little is known about the identity and functionality of the Th1, Th2, and Treg T helper cell subsets in bottlenose dolphins. The present study aimed at validating assays and reagents to identify T helper cell subsets and their functions in a subset of dolphins from Sarasota Bay, Florida, USA, which have been long studied and often used as a reference population. A population of CD4+ FOXP3+ lymphocytes was identified representing an average <1% of blood lymphocyte population, which is in the range observed in for Treg cells in other species. The use of porcine reagents to measure TGFß, one of the key Treg cytokines, was further validated using the relatively high-throughput and highly standardized Luminex technology. The proportion of circulating Treg cells was not correlated with the serum concentrations of the Treg effector cytokines TGFß and IL-10, nor could it significantly contribute to predicting the variability of T lymphocyte proliferation, suggesting that not all dolphin circulating Treg cells are functional and active. However, stimulation of dolphin lymphocytes with TGFß and IL-2 increased the expression of the gene for TGFß and IL-10, and stimulation with IL-12 and IFNγ induced a robust increase in the expression of the gene for IFNγ, suggesting the potential for polarization and differentiation of dolphin T helper cells toward a Treg and Th1 response, respectively. The lack of an increase in the expression of the genes for the Th2 cytokines IL-4 and IL-13 upon stimulation with IL-4 may be due to the requirement for IL-2 for a Th2 polarization as described in mice. However, regression analysis and PCA suggested the potential ability of both the Th1 and Th2 response to be triggered upon acute inflammatory signals. These results may be useful in better understanding the mechanisms by which the dolphin immune system is affected upon exposure to environmental challenges and how it responds to pathogen challenges.

Estuaries of the northern Gulf of Mexico are dynamic environments, with fluctuations in salinity and dissolved oxygen, including areas of seasonal hypoxia. Fish that reside and reproduce in these estuaries, including sheepshead minnow (Cyprinodon variegatus; SHM), were at significant risk of oil exposure following the Deepwater Horizon oil spill. It is poorly understood how differences in environmental conditions during oil exposure impact its toxicity. The present study investigated the effects of crude oil high-energy water accommodated fraction (HEWAF) on SHM reproduction in three environmental scenarios (normoxic, hypoxic, and hypoxic with low salinity) to determine if differences in salinity (brackish vs low salinity) and dissolved oxygen (normoxia vs hypoxia) could exacerbate the effects of HEWAF-derived polycyclic aromatic hydrocarbons (PAHs). We observed that HEWAF exposure significantly increased liver somatic index of SHM compared to control, but this effect was not exacerbated by hypoxia or low salinity. HEWAF exposure also significantly decreased egg production and egg fertilization rate, but only in the hypoxic and hypoxic with low salinity scenarios. A significant correlation existed between body burdens of PAHs and reproductive endpoints, providing substantial evidence that oil exposure reduced reproductive capacity in SHM, across a range of environmental conditions. These data suggest that oil spill risk assessments that fail to consider other environmental stressors (i.e. hypoxia and salinity) may be underestimating risk., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The Deepwater Horizon oil spill resulted in the release of over 640 million L of crude oil into the Gulf of Mexico, affecting over 2000 km of shoreline, including estuaries that serve as important habitats and nurseries for aquatic species. Cyprinodon variegatus (sheepshead minnow) are small-bodied fish that inhabit northern Gulf of Mexico estuaries, are easily adaptable to laboratory conditions, and are commonly used in toxicological assessment studies. The purpose of the present study was to determine the somatic, reproductive, and developmental effects of an environmentally relevant polycyclic aromatic hydrocarbon (PAH) mixture, the oil high-energy water accommodated fraction (HEWAF), on experimentally exposed sheepshead minnow (F 0 ) as well as 2 generations of offspring (F 1 and F 2 ) without additional exposure. The F 0 generation exposed to HEWAF had increased liver somatic indices, altered egg production, and decreased fertilization. Several developmental endpoints in the F 1 were altered by F 0 HEWAF exposure. As adults, low HEWAF-exposed F 1 females demonstrated decreased weight and length. Both the F 1 and F 2 generations derived from high HEWAF-exposed F 0 had deficits in prey capture compared to control F 1 and F 2 , respectively. Correlations between endpoints and tissue PAHs provide evidence that the physiological effects observed were associated with hydrocarbon exposure. These data demonstrate that PAHs were capable of causing physiological changes in exposed adult sheepshead minnow and transgenerational effects in unexposed offspring, both of which could have population-level consequences. Environ Toxicol Chem 2019;38:638-649. © 2018 SETAC., (© 2018 SETAC.)

Given their particle feeding behavior, sessile nature, and abundance in coastal zones, bivalves are at significant risk for exposure to oil and oil dispersant following environmental disasters like the Deepwater Horizon oil spill. However, the effects of oil combined with oil dispersants on the health of oysters are not well studied. Therefore, eastern oysters (Crassostrea virginica) were exposed in vivo to Corexit ® 9500, crude oil (high-energy water accommodated fraction; HEWAF), and a Corexit ® /oil mixture (chemically-enhanced water accommodated fraction; CEWAF) to evaluate potential toxic effects on immunological (phagocytosis and respiratory burst), physiological (feeding rate), and histological endpoints. Phagocytosis was significantly increased following CEWAF exposure only. Respiratory burst was significantly decreased following Corexit ® exposure, but significantly increased following exposure to the highest concentration of CEWAF. Oyster feeding rates were significantly decreased following exposure to Corexit ® , HEWAF, and CEWAF, and were most sensitive to CEWAF exposure. These modulations of important immunological and physiological functions could result in serious health outcomes for oysters, such as increased parasitism and decreased growth. Our experiments showed that subtle, sub-lethal effects occurred following acute in vivo exposure to Corexit ® , HEWAF, and CEWAF, though oysters were not equally sensitive to the three components. Data from this study can be used for more accurate risk assessment concerning the impact of oil and Corexit ® on the health of oysters., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Killer whales ( Orcinus orca ) are among the most highly polychlorinated biphenyl (PCB)-contaminated mammals in the world, raising concern about the health consequences of current PCB exposures. Using an individual-based model framework and globally available data on PCB concentrations in killer whale tissues, we show that PCB-mediated effects on reproduction and immune function threaten the long-term viability of >50% of the world's killer whale populations. PCB-mediated effects over the coming 100 years predicted that killer whale populations near industrialized regions, and those feeding at high trophic levels regardless of location, are at high risk of population collapse. Despite a near-global ban of PCBs more than 30 years ago, the world's killer whales illustrate the troubling persistence of this chemical class., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In the present study, we examined how sensitivity to oil changes in combination with environmental stressors in Fundulus grandis embryos. We exposed embryos (<24 h post fertilization) to a range of high-energy water accommodated fraction (HEWAF) concentrations (0-50 parts per billion [ppb] total polycyclic aromatic hydrocarbons [PAHs]) made from Macondo crude oil in conjunction with various environmental conditions (temperature: 20 and 30 °C; salinity: 3, 7, and 30 practical salinity units [PSU]; and dissolved oxygen: 2 and 6 mg/L). Endpoints included mortality, hatching rates, and expression of cytochrome p450 1a and 1c (cyp1a, cyp1c) in hatched larvae. There was 100% mortality for all fish under the 2 parts per million (ppm) dissolved oxygen regimes. For the 6 mg/L dissolved oxygen treatments, mortality and median lethal time (LT50) were generally higher in the 30 °C treatments versus the 20 °C treatments. Oil increased mortality in fish exposed to the highest concentration in the 20-3-6 (°C-PSU-mg/L), 25-7-6, and 30-30-6 conditions. Hatching was driven by environmental conditions, with oil exposure having a significant impact on hatching in only the 25-7-6 and 30-30-6 groups at the greatest HEWAF exposure. Expression of cyp1a was up-regulated in most treatment groups versus the controls, with cyp1c expression exhibiting a similar pattern. These data suggest interactive effects among temperature, salinity, and PAHs, highlighting a need to further assess the effects of oil exposure under various environmental conditions. Environ Toxicol Chem 2018;37:1916-1925. © 2018 SETAC., (© 2018 SETAC.)

The ubiquity of pharmaceuticals in the aquatic environment and the accumulation in organisms of lower trophic levels have been documented. The immunotoxicity of these xenobiotics has however been little investigated. This study assessed the effects of pharmaceuticals on the immune responses of harbor seal lymphocytes. Peripheral blood mononuclear cells isolated from harbor seal pups were exposed to varying concentrations of 17α-ethinyl estradiol (250-50,000μg/L), naproxen (500-100,000μg/L), carbamazepine (500-100,000μg/L), erythromycin (750-150,000μg/L) and binary mixtures thereof in vitro. All individual compounds and mixtures inhibited lymphocyte proliferation. Mixture effects were non-additive and predictive values overestimated the inhibition of proliferation. Male pups were more sensitive to erythromycin exposure. Comparison with the sensitivity of the 11B7501 cell line showed a higher sensitivity of pups to individual compounds and the inverse trend for mixtures. Based on our results, we hypothesize that pharmaceuticals may have the potential to interrupt immune functions in harbor seals., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Most controlled toxicity studies use single chemical exposures that do not represent the real world situation of complex mixtures of known and unknown natural and anthropogenic substances. In the present study, complex contaminant cocktails derived from the blubber of polar bears (PB; Ursus maritimus) and killer whales (KW; Orcinus orca) were used for in vitro concentration-response experiments with PB, cetacean and seal spp. immune cells to evaluate the effect of realistic contaminant mixtures on various immune functions. Cytotoxic effects of the PB cocktail occurred at lower concentrations than the KW cocktail (1 vs 16 μg/mL), likely due to differences in contaminant profiles in the mixtures derived from the adipose of each species. Similarly, significant reduction of lymphocyte proliferation occurred at much lower exposures in the PB cocktail (EC 50 : 0.94 vs 6.06 μg/mL; P < 0.01), whereas the KW cocktail caused a much faster decline in proliferation (slope: 2.9 vs 1.7; P = 0.04). Only the KW cocktail modulated natural killer (NK) cell activity and neutrophil and monocyte phagocytosis in a concentration- and species-dependent manner. No clear sensitivity differences emerged when comparing cetaceans, seals and PB. Our results showing lower effect levels for complex mixtures relative to single compounds suggest that previous risk assessments underestimate the effects of real world contaminant exposure on immunity. Our results using blubber-derived contaminant cocktails add realism to in vitro exposure experiments and confirm the immunotoxic risk marine mammals face from exposure to complex mixtures of environmental contaminants.

The potential risk of pharmaceuticals in the environment to top-predators is still largely unknown. In this study, we assessed the immunotoxic effects of ten pharmaceuticals individually and as mixtures on a harbor seal (Phoca vitulina) B lymphoma cell line. A significant reduction in lymphocyte transformation was observed following an exposure to 12,500μg/L 17α-ethinyl estradiol and 25,000μg/L naproxen. Exposure to 12,500μg/L 17α-ethinyl estradiol decreased the percentage of cell in the G 0 /G 1 phase of the cell cycle while increasing the percentage of cells in the S phase. Carbamazepine exposure increased the amount of cells in the G 2 /M phase. Binary mixtures showed synergistic effects in lymphocyte transformation, cell cycle and apoptosis assays. Concentrations inducing toxic effects in the cell line were similar to those affecting fish in previous studies. A reduction of functional activities of the immune system may lead to altered host resistance to pathogens in free-ranging pinnipeds., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To better elucidate the potential immune-related health effects of exposure to environmentally persistent organic pollutants (POP), such as polychlorinated biphenyls (PCBs) and perfluoroalkyl substances (PFASs), in ringed seals (Pusa hispida), a sentinel Arctic species, we assessed 1) associations between mitogen-induced lymphocyte proliferation and in vivo tissue contaminant burdens, and 2) the concentration-response effects of in vitro exposure to PFASs and PCB congeners on mitogen-induced lymphocyte proliferation. Upon in vitro contaminant exposure, the non-coplanar PCB congeners CB 138, 153, and 180, but not the coplanar CB 169, significantly reduced lymphocyte proliferation between 10 and 20µgg -1 ww. The respective in vitro EC 50 values for these congeners were 13.3, 20.7, 20.8, and 54.6µgg -1 ww. No modulation of lymphocyte proliferation was observed upon in vitro exposure to two individual PFASs, perfluorooctane sulphonic acid (PFOS) and perfluorooctanoic acid (PFOA), at concentrations up to 1000ngg-1. In addition, no significant correlations were found between lymphocyte proliferation and any blood or blubber contaminant measured. Taken together, these data suggest this population of ringed seals is not currently at high risk of altered lymphocyte proliferation from exposure to the POPs or PFASs in this study., (Copyright © 2016. Published by Elsevier Inc.)

Several marine mammal epizootics have been closely linked to infectious diseases, as well as to the biotoxins produced by harmful algal blooms (HABs). In two of three saxitoxin (STX) associated mortality events, dolphin morbillivirus (DMV) or phocine distemper virus (PDV) was isolated in affected individuals. While STX is notorious for its neurotoxicity, immunotoxic effects have also been described. This study investigated the role of STX in altering immune function, specifically T lymphocyte proliferation, in harbor seals (Phoca vitulina concolor) upon in-vitro exposure. In addition, the study also examined whether exposure to STX could alter the susceptibility of harbor seal immune cells to PDV infection upon in-vitro exposure. STX caused an increase in harbor seal lymphocyte proliferation at 10ppb and exposure to STX significantly increased the amount of virus present in lymphocytes. These results suggest that low levels of STX within the range of those reported in northeast U.S. seals may affect the likelihood of systemic PDV infection upon in-vivo exposure in susceptible seals. Given the concurrent increase in morbillivirus epizootics and HAB events in the last 25 years, the relationship between low level toxin exposure and host susceptibility to morbillivirus needs to be further explored., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In the last 30 years, several large-scale marine mammal mortality events have occurred, often in close association with highly polluted regions, leading to suspicions that contaminant-induced immunosuppression contributed to these epizootics. Some of these recent events also identified morbillivirus as a cause of or contributor to death. The role of contaminant exposures regarding morbillivirus mortality is still unclear. The results of this study aimed to address the potential for a mixture of polychlorinated biphenyls (PCBs), specifically Aroclor 1260, to alter harbor seal T-lymphocyte proliferation and to assess if exposure resulted in increased likelihood of phocine distemper virus (PDV USA 2006) to infect susceptible seals in an in vitro system. Exposure of peripheral blood mononuclear cells to Aroclor 1260 did not significantly alter lymphocyte proliferation (1, 5, 10, and 20 ppm). However, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), lymphocytes exposed to 20 ppm Aroclor 1260 exhibited a significant decrease in PDV replication at day 7 and a significant increase at day 11 compared with unexposed control cells. Similar and significant differences were apparent on exposure to Aroclor 1260 in monocytes and supernatant. The results here indicate that in harbor seals, Aroclor 1260 exposure results in a decrease in virus early during infection and an increase during late infection. The consequences of this contaminant-induced infection pattern in a highly susceptible host could result in a greater potential for systemic infection with greater viral load, which could explain the correlative findings seen in wild populations exposed to a range of persistent contaminants that suffer from morbillivirus epizootics.

Due to their marine ecology and life-history, marine mammals accumulate some of the highest levels of environmental contaminants of all wildlife. Given the increasing prevalence and severity of diseases in marine wildlife, it is imperative to understand how pollutants affect the immune system and consequently disease susceptibility. Advancements and adaptations of analytical techniques have facilitated marine mammal immunotoxicology research. Field studies, captive-feeding experiments and in vitro laboratory studies with marine mammals have associated exposure to environmental pollutants, most notable polychlorinated biphenyls (PCBs), organochlorine pesticides and heavy metals, to alterations of both the innate and adaptive arms of immune systems, which include aspects of cellular and humoral immunity. For marine mammals, reported immunotoxicology endpoints fell into several major categories: immune tissue histopathology, haematology/circulating immune cell populations, functional immune assays (lymphocyte proliferation, phagocytosis, respiratory burst, and natural killer cell activity), immunoglobulin production, and cytokine gene expression. Lymphocyte proliferation is by far the most commonly used immune assay, with studies using different organic pollutants and metals predominantly reporting immunosuppressive effects despite the many differences in study design and animal life history. Using combined field and laboratory data, we determined effect threshold levels for suppression of lymphocyte proliferation to be between b0.001-10 ppm for PCBs, 0.002-1.3 ppm for Hg, 0.009-0.06 for MeHg, and 0.1-2.4 for cadmium in polar bears and several pinniped and cetacean species. Similarly, thresholds for suppression of phagocytosis were 0.6-1.4 and 0.08-1.9 ppm for PCBs and mercury, respectively. Although data are lacking for many important immune endpoints and mechanisms of specific immune alterations are not well understood, this review revealed a systemic suppression of immune function in marine mammals exposed to environmental contaminants. Exposure to immunotoxic contaminants may have significant population level consequences as a contributing factor to increasing anthropogenic stress in wildlife and infectious disease outbreaks.