The recent rise of 'omics and other molecular research technologies alongside improved techniques for tissue preservation have broadened the scope of marine mammal research. Collecting biological samples from wild marine mammals is both logistically challenging and expensive. To enhance the power of marine mammal research, great effort has been made in both the field and the laboratory to ensure the scientific integrity of samples from collection through processing, supporting the long-term use of precious samples across a broad range of studies. However, identifying the best methods of sample preservation can be challenging, especially as this technological toolkit continues to evolve and expand. Standardizing best practices could maximize the scientific value of biological samples, foster multi-institutional collaborative efforts across fields, and improve the quality of individual studies by removing potential sources of error from the collection, handling, and preservation processes. With these aims in mind, we summarize relevant literature, share current expert knowledge, and suggest best practices for sample collection and preservation. This manuscript is intended as a reference resource for scientists interested in exploring collaborative studies and preserving samples in a suitable manner for a broad spectrum of analyses, emphasizing support for 'omics technologies.

Chemical pollution can degrade aquatic ecosystems. Chinook salmon in contaminated habitats are vulnerable to health impacts from toxic exposures. Few studies have been conducted on adverse health outcomes associated with current levels and mixtures of contaminants. Fewer still address effects specific to the juvenile life-stage of salmonids. The present study evaluated contaminant-related effects from dietary exposure to environmentally relevant concentrations and mixture profiles in juvenile Chinook salmon from industrialized waterways in the U.S. Pacific Northwest using two end points: growth assessment and disease susceptibility. The dose and chemical proportions were reconstituted based on environmental sampling and analysis using the stomach contents of juvenile Chinook salmon recently collected from contaminated, industrialized waterways. Groups of fish were fed a mixture with fixed proportions of 10 polychlorinated biphenyls (PCBs), 3 dichlorodiphenyltrichloroethanes (DDTs), and 13 polycyclic aromatic hydrocarbons (PAHs) at five concentrations for 35 days. These contaminant compounds were selected because of elevated concentrations and the widespread presence in sediments throughout industrialized waterways. Fork length and otolith microstructural growth indicators were significantly reduced in fish fed environmentally relevant concentrations of these contaminants. In addition, contaminant-exposed Chinook salmon were more susceptible to disease during controlled challenges with the pathogen Aeromonas salmonicida. Our results indicate that dietary exposure to contaminants impairs growth and immune function in juvenile Chinook salmon, thereby highlighting that current environmental exposure to chemicals of potential management concern threatens the viability of exposed salmon.

Contaminant studies in cetaceans can provide information about pollutant levels and patterns in a given region. Due to the confounding effects of reproductive status and maternal offloading in females, these studies typically focus on males. However, an improved understanding of contaminant burdens in female cetaceans is needed to better assess potential impacts to populations. The objectives of this study were to characterize concentrations of persistent organic pollutants (POPs) in blubber of female humpback whales across age classes and to also better characterize maternal offloading of these pollutants to their offspring. A total of 36 blubber biopsy samples of female humpback whales (Megaptera novaeangliae) from the Gulf of Maine were analyzed to examine contaminant loads across females of different ages. Sampled individuals were individually-identified from longitudinal studies and assigned to age class (i.e., adult, subadult, juvenile, calf). Analysis was performed using gas chromatography/mass spectrometry (GC/MS) of POPs including polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethanes (DDTs), chlordanes (CHLDs), polybrominated diphenyl ethers (PBDEs), hexachlorocyclohexanes (HCHs). The most abundant POPs were PCB congeners, with summed values ranging from 280 to 12,000 ng/g, lipid weight, which is above recent estimates of the threshold for adverse health effects. We found significant differences in mean values between adults and juveniles and between adults and subadults, with the exception of the less persistent HCHs for the latter. We also found significant differences in mean levels of ∑HCHs between the juveniles and subadults. Changes over age are consistent with maternal offloading and potentially important for evaluating population health and viability.

The pituitary gland is a central regulator of reproduction, producing two gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), which regulate gonadal development, sex steroid synthesis, and gamete maturation. The present study sought to optimize an in vitro test system using pituitary cells isolated from previtellogenic female coho salmon and rainbow trout, focusing on fshb and lhb subunit gene expression. Initially, we optimized culture conditions for duration and benefits of culturing with and without addition of endogenous sex steroids (17β-estradiol [E2] or 11-ketotestosterone) or gonadotropin-releasing hormone (GnRH). The results suggest that culturing with and without E2 was valuable because it could mimic the (+) feedback effects on Lh that are observed from in vivo studies. After optimizing assay conditions, a suite of 12 contaminants and other hormones was evaluated for their effects on fshb and lhb gene expression. Each chemical was tested at four to five different concentrations up to solubility limitations in cell culture media. The results indicate that more chemicals alter lhb synthesis than fshb. The more potent chemicals were estrogens (E2 and 17α-ethynylestradiol) and the aromatizable androgen testosterone, which induced lhb. The estrogen antagonists 4-OH-tamoxifen and prochloraz decreased the E2-stimulated expression of lhb. Among several selective serotonin reuptake inhibitors tested, the sertraline metabolite norsertraline was notable for both increasing fshb synthesis and decreasing the E2 stimulation of lhb. These results indicate that diverse types of chemicals can alter gonadotropin production in fish. Furthermore, we have shown that pituitary cell culture is useful for screening chemicals with potential endocrine-disrupting activity and can support the development of quantitative adverse outcome pathways in fish. Environ Toxicol Chem 2023;42:1730-1742. © 2023 SETAC.

Electronic waste (E-waste) recycling is a rapidly growing occupation in the USA with the potential for elevated exposure to flame retardants and metals associated with electronic devices. We previously measured polybrominated diphenyl ethers (PBDEs) in plasma from E-waste workers and found them similar to non-E-waste workers. This study focused on structurally related PBDE derivatives, the hydroxylated (OH-PBDEs) and methoxylated (MeO-PBDEs) forms along with metals known to occur in E-waste. Humans can metabolize PBDEs and some MeO-PBDEs into OH-PBDEs, which is a concern due to greater health risks associated with OH-PBDEs. We measured 32 different OH-PBDEs and MeO-PBDEs in plasma samples provided by 113 volunteers living in the greater Puget Sound region of Washington State, USA. We measured 14 metals in a subset of 10 E-waste and 10 non-E-waste volunteers. Volunteers were selected based on occupational and dietary habits: work outdoors and consume above average amounts of seafood (outdoor), electronic waste recycling (E-waste) or non-specific indoor occupations (indoor). A two-week food consumption diary was obtained from each volunteer prior to blood sampling. OH-PBDEs were detected in all volunteers varying between 0.27 and 102 ng/g/g-lipid. The MeO-PBDEs were detected in most, but not all volunteers varying between n.d. 60.4 ng/g/g-lipid. E-waste recyclers had OH-PBDE and MeO-PBDE plasma levels that were similar to the indoor group. The outdoor group had significantly higher levels of MeO-PBDEs, but not OH-PBDEs. Comparison of plasma concentrations of BDE-47 with its known hydroxylated metabolites suggested OH-PBDE levels were likely determined by biotransformation and at least two subpopulations identified differing in their apparent rates of OH-PBDE formation. The metals analysis indicated no significant differences between E-waste workers and non-E-waste workers. Our results indicate E-waste workers do not have elevated plasma levels of these contaminants relative to non-E-waste workers. (C) 2020 Elsevier B.V. All rights reserved.

Endogenous estrogens play major roles in many aspects of female reproductive development in fish. In order to develop a relatively high-throughput assay to determine the potential impact on reproductive development, vitellogenic rainbow trout ovarian follicles were exposed to a suite of contaminants in vitro and then assessed for the ability to produce estradio1-17 beta (E2) after a 500 ng/ml salmon gonadotropin (sGTH) challenge. There was a positive correlation between ovarian follicle size and E2 production, but an inverse correlation between size and responsiveness to sGTH. Significant impacts on E2 levels were observed following treatment with different endocrine disrupting chemicals, such as 17 alpha-ethinylestradiol (EE2), prochloraz, or trenbolone. EE2 was remarkably potent and significantly reduced ovarian follicle responsiveness to sGTH at concentrations as low as 0.1 nM. Of the other contaminants tested, only tamoxifen impacted E2 levels, and only at concentrations near the limits of solubility. Flutamide, fluoxetine, 4-hydroxy tamoxifen, hydroxyflutamide, and norfluoxetine had little or no impact. Quantitative PCR analyses of steroidogenesis-related genes were carried out on EE2 treated ovarian follicles, but significant transcriptional responses to EE2 were not observed. Overall, this study suggests that xenoestrogens and anti-estrogens are more likely to interfere with ovarian E2 synthesis than other classes of EDCs. This also provides a template for further testing of the effects of EDCs on ovarian function.

Linking organismal-level processes to underlying suborganismal mechanisms at the molecular, cellular and organ level constitutes a major challenge for predictive ecological risk assessments. This challenge can be addressed with the simple bioenergetic models in the family of dynamic energy budget (DEB), which consist of a small number of state equations quantifying universal processes, such as feeding, maintenance, development, reproduction and growth. Motivated by the need for process-based models to evaluate the impact of endocrine disruptors on ecologically relevant endpoints, this paper develops and evaluates two general modelling modules describing demand-driven feedback mechanisms within the DEB modelling framework exerted by gonads on the allocation of resources to production of reproductive matter. These modules describe iteroparous, semelparous and batch-mode reproductive strategies. The modules have a generic form with both positive and negative feedback components; species- and sex-specific attributes of endocrine regulation can be added without changing the core of the modules. We demonstrate that these modules successfully describe time-resolved measurements of wet weight of body, ovaries and liver, egg diameter and plasma content of vitellogenin and oestradiol in rainbow trout (Oncorynchus mykiss) by fitting these models to published and new data, which require the estimation of less than two parameters per data type. We illustrate the general applicability of the concept of demand-driven allocation of resources to reproduction by evaluating one of the modules with data on growth and seed production of an annual plant, the common bean (Phaseolisvulgaris). A plain language summary is available for this article.

Polybrominated diphenyl ethers (PBDEs) were widely used as flame retardants in consumer products including electronic devices. Important routes of human exposure are contaminated food and contact with dust. In this study, we measured twelve PBDEs in household/workplace dust and blood plasma samples provided by 113 volunteers living in the Puget Sound region, WA and working at electronic waste (E-waste) recycling sites (n = 29) or non-specific indoor (n = 57) or outdoor occupations (n = 27). The volunteers in the outdoor group were also selected because of a history of high seafood consumption habits. Results indicated the sum PBDE levels varied between <2.5 and up to 310 ng g-1 lipid. E-waste recyclers were predominantly men, generally consumed low amounts of seafood, and had PBDE blood levels (geometric mean, GM = 26.56 ng g-1 lipid) that were similar to indoor workers (GM = 27.17 ng g-1 lipid). The sum PBDE levels were highest in the outdoor group (GM = 50.63 ng g-1 lipid). Dust samples from E-waste sites were highly enriched with BDE-209 and BDE-153 relative to non-E-waste businesses and homes. The concentrations of these BDE congeners in dust at E-waste sites were ∼32-39 times higher than in dust from other sites. However, the detection rate of BDE-209 in plasma was low across all groups (13%) and no statistical comparisons were made. Our results suggest that E-waste recyclers in this study population did not have elevated PBDE levels in comparison to volunteers working in other types of occupations.

Synthetic polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental contaminants and known to occur in most food items. Consumer fish products have been identified as having some of the highest PBDE levels found in USA food sources. Natural formation of hydroxylated (OH-) and methoxylated (MeO-) PBDEs are also known to occur in simple marine organisms, which may be bioaccumulated by seafood. In this study, we report findings of an initial survey of PBDE, OH-PBDE and MeO-PBDE content in common seafood items available to residents living in the Puget Sound region of Washington State. Seafood samples were either purchased from local grocery stores or caught off the coast of SE Alaska and in Puget Sound. The edible portions of the seafood were analyzed, which for finfish was white muscle (skinless fillets) and for shellfish, either the entire soft tissue (bivalves) or processed meat (calamari, shrimp and scallops). Results indicated that finfish typically had higher levels of PBDEs compared to shellfish with BDE-47 and BDE-99 as the most common congeners detected. Among shellfish, bivalves (clams and mussels) were notable for having much higher levels of OH- and MeO-PBDEs compared to other types of seafood with 6′-OH-BDE-47 and 2′-MeO-BDE-68 being the more common OH- and MeO- congeners, respectively. Based on our results and recent updates to daily fish consumption rates, estimated intake rates for Washington State residents will be between 34 and 644ngPBDEs/day, depending on species consumed. For the OH- and MeO- forms, daily exposure is much more variable but typically would range between 15 and 90ng/day for most seafood types. If shellfish are primarily consumed, OH-PBDE intake could be as high as 350ng/day. These daily intake rates for PBDEs are higher than most dietary intake rates calculated for populations in other world regions.

A working group at the National Institute for Mathematical and Biological Synthesis (NIMBioS) explored the feasibility of integrating 2 complementary approaches relevant to ecological risk assessment. Adverse outcome pathway (AOP) models provide "bottom-up" mechanisms to predict specific toxicological effects that could affect an individual's ability to grow, reproduce, and/or survive from a molecular initiating event. Dynamic energy budget (DEB) models offer a "top-down" approach that reverse engineers stressor effects on growth, reproduction, and/or survival into modular characterizations related to the acquisition and processing of energy resources. Thus, AOP models quantify linkages between measurable molecular, cellular, or organ-level events, but they do not offer an explicit route to integratively characterize stressor effects at higher levels of organization. While DEB models provide the inherent basis to link effects on individuals to those at the population and ecosystem levels, their use of abstract variables obscures mechanistic connections to suborganismal biology. To take advantage of both approaches, we developed a conceptual model to link DEB and AOP models by interpreting AOP key events as measures of damage-inducing processes affecting DEB variables and rates. We report on the type and structure of data that are generated for AOP models that may also be useful for DEB models. We also report on case studies under development that merge information collected for AOPs with DEB models and highlight some of the challenges. Finally, we discuss how the linkage of these 2 approaches can improve ecological risk assessment, with possibilities for progress in predicting population responses to toxicant exposures within realistic environments. Integr Environ Assess Manag 2018;14:615-624. © 2018 SETAC.

The gut microbiome is often termed “the second genome” because it can interact with the host genome to regulate important metabolic pathways such as xenobiotic biotransformation and nutrient homeostasis. The polybrominated diphenyl ethers (PBDEs) that are previously used as flame retardants are among the most abundant and persistent environmental contaminants in the human population. Especially, the 2,2′,4,4′‐tetrabromodiphenyl (BDE‐47) and 2,2′,4,4′,5‐pentabromodiphenyl ether (BDE‐99) congeners are prevalent in seafood and breast milk at worrisome levels. Very little is known about how the gut microbiome affects the hepatic metabolism of PBDEs in vivo . Therefore, the goal of the present study was to utilize germ‐free (GF) mice to investigate the interactions between the gut microbiome and the host hepatic biotransformation pathways of PBDEs. Three‐month‐old male C57BL/6J conventional (CV) or GF mice were treated with vehicle (corn oil, 10ml/kg, oral gavage), BDE‐47 (100μmol/kg) or BDE‐99 (100μmol/kg) once daily for four days. The hepatic transcriptome was determined using RNA‐Seq. The differentially expressed drug‐processing genes (DPGs) were defined as FDR< 0.05 in at least one of the chemical‐treated groups as compared to the vehicle‐treated group. No apparent hepatic injury was observed in any treatment groups (serum alanine aminotransferase and H&E staining). PBDE targeted metabolomics (GC‐MS‐NCI) revealed that the major hydroxylated PBDE metabolites in livers of both CV and GF mice were 5‐OH‐BDE‐47 and 5′‐OH‐BDE‐99 for BDE‐47 and BDE‐99, respectively. Following BDE‐47 treatment, livers of GF mice had higher levels of 5‐OH‐BDE‐47, and lower levels of 4 other minor metabolites (3‐OH‐BDE‐47, 4′‐OH‐BDE‐49, 4′‐OH‐BDE‐17, and 2′‐OH‐BDE‐28) than CV mice. Following BDE‐99 treatment, livers of GF mice had lower levels of 4 minor metabolites (4′‐OH‐BDE‐101, 4′‐OH‐BDE‐49, 6‐OH‐BDE‐85, and 4′‐OH‐BDE‐103), as compared to livers of PBDE‐treated CV mice. RNA‐Seq demonstrated that certain DPGs were differentially regulated in livers of GF mice under both basal and PBDE‐treated conditions. For example, under basal condition, many Cyp2c genes ( Cyp2c40, 2c50, 2c54, 2c67, 2c68, 2c69, and 2c70) were up‐regulated, whereas Cyp3a genes ( Cyp3a11, 3a16, 3a41a, 3a41b, 3a44, and 3a59 ) were down‐regulated in GF mice compared with CV mice. Interestingly, after BDE‐99 treatment, more Cyp2d genes ( Cyp2d9, 2d10, 2d12, 2d26, and 2d40 ) were significantly down‐regulated in GF condition. Moreover, the lack of gut microbiome sensitized the liver to BDE‐99 mediated up‐regulation of many DPGs such as Phase I ( Cyp1a, 2a4, 2a5, 2b10, 2b13, 2c29, 2c37, 2c50, 2c54, 2c55, 2d13, 2j9, 3a11, 3a16, 3a25, 3a41a, 3a41b, 3a44, and 3a59 ), Phase II ( Ugt2b1, Ugt2b5, Ugt2b34, Ugt2b35, Ugt3b36, Ugt3b38, Ugdh and Ugp2; Sult1d1, Sult1e1, Papss2; Gsta1, Gsta2, Gstm1, Gstm2, Gstm3, Gstm4, Gstt3, and Gclc ), and transporter ( Slco1a4, Slco1b2, Abcb1a, Abcc2, Abcc4, and Abcc10 ) genes. In summary, our study demonstrated a novel interaction between the gut microbiome and the hepatic biotransformation of PBDEs in vivo .

The disposition and metabolism of fluoxetine in the European shore crab and the Dungeness crab were assessed. Crabs received intracardiac doses of either 0.13 μg/kg or 0.5 mg/kg fluoxetine, respectively. In addition, fluoxetine was administered to Metacarcinus cancer by oral gavage at 7.8 mg/kg. The distribution of fluoxetine was quantified in haemolymph and digestive gland for both crabs, as well as brain, muscle, and testis of Carcinus maenas, over 12 days. The metabolite norfluoxetine, was also measured in C. maenas. Fluoxetine was mainly found in lipid rich tissues. Distribution coefficients increased for digestive gland until three days after fluoxetine administration and then decreased until the end of the observations. The highest distribution coefficients were obtained for brain. Norfluoxetine displayed continuously high levels in digestive gland and brain. The strong decrease in fluoxetine and the concomitant increase in norfluoxetine demonstrates that decapod crustaceans metabolise fluoxetine into the more biologically active norfluoxetine. Fluoxetine levels in the haemolymph of M. cancer declined within 20 h, but showed a second peak 25 h later, suggesting remobilisation from tissues sequestering the compound. The steady state volume distribution and the total body clearance of fluoxetine were high, consistent with high diffusion of fluoxetine into the peripheral tissues and biotransformation as an important elimination pathway. Oral administration of fluoxetine prolonged its half-life in M. cancer, but bioavailability was low. These results confirm the high distribution into nervous tissue, extensive biotransformation into the highly active norfluoxetine and a half-life similar to that observed in vertebrates.

It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17β-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to estrogenic activity of the WWTP effluents. These results suggest that lhb gene expression may be a sensitive index of acute exposure to estrogenic chemicals in juvenile coho salmon. Further work is needed to determine the kinetics and specificity of lhb induction to evaluate its utility as a potential indicator of estrogen exposure in immature fish.

Reproduction in fishes and other vertebrates represents the timely coordination of many endocrine factors that culminate in the production of mature, viable gametes. In recent years there has been rapid growth in understanding fish reproductive biology, which has been motivated in part by recognition of the potential effects that climate change, habitat destruction and contaminant exposure can have on natural and cultured fish populations. New approaches to understanding the impacts of these stressors are being developed that require a systems biology approach with more biologically accurate and detailed mathematical models. We have developed a multi-scale mathematical model of the female rainbow trout hypothalamus-pituitary-ovary-liver axis to use as a tool to help understand the functioning of the system and for extrapolation of laboratory findings of stressor impacts on specific components of the axis. The model describes the essential endocrine components of the female rainbow trout reproductive axis. The model also describes the stage specific growth of maturing oocytes within the ovary and permits the presence of sub-populations of oocytes at different stages of development. Model formulation and parametrization was largely based on previously published in vivo and in vitro data in rainbow trout and new data on the synthesis of gonadotropins in the pituitary. Model predictions were validated against several previously published data sets for annual changes in gonadotropins and estradiol in rainbow trout. Estimates of select model parameters can be obtained from in vitro assays using either quantitative (direct estimation of rate constants) or qualitative (relative change from control values) approaches. This is an important aspect of mathematical models as in vitro, cell-based assays are expected to provide the bulk of experimental data for future risk assessments and will require quantitative physiological models to extrapolate across biological scales.

The rainbow trout endocrine system is sensitive to changes in annual day length, which is likely the principal environmental cue controlling its reproductive cycle. This study focuses on the endocrine regulation of vitellogenin (Vg) protein synthesis, which is the major egg yolk precursor in this fish species. We present a model of Vg production in female rainbow trout which incorporates a biological pathway beginning with sex steroid estradiol-17β levels in the plasma and concluding with Vg secretion by the liver and sequestration in the oocytes. Numerical simulation results based on this model are compared with experimental data for estrogen receptor mRNA, Vg mRNA, and Vg in the plasma from female rainbow trout over a normal annual reproductive cycle. We also analyze the response of the model to parameter changes. The model is subsequently tested against experimental data from female trout under a compressed photoperiod regime. Comparison of numerical and experimental results suggests the possibility of a time-dependent change in oocyte Vg uptake rate. This model is part of a larger effort that is developing a mathematical description of the endocrine control of reproduction in female rainbow trout. We anticipate that these mathematical and computational models will play an important role in future regulatory toxicity assessments and in the prediction of ecological risk.