Co-Exposure of Phenanthrene and the cyp-Inducer 3-Methylchrysene Leads to Altered Biotransformation and Increased Toxicity in Fish Egg and Larvae.

Polycyclic aromatic hydrocarbons (PAHs) have frequently been suspected of governing crude oil toxicity because of similar morphological defects in fish. However, PAH concentrations are often not high enough to explain the observed crude oil toxicity. We hypothesize that one PAH can enhance the metabolism and toxicity of another PAH when administered as a mixture. Early life stage Atlantic haddock (Melanogrammus aeglefinus) were in this study exposed to phenanthrene in the presence and absence of 3-methylchrysene that is known to induce the metabolic enzyme cytochrome P450 1A via cyp1a gene expression. Uptake, metabolism, and multiple toxicity endpoints were then measured in a time-course study up to 3 days post-hatching. Passive dosing provided aqueous concentrations ≈180 µg/L for phenanthrene and ≈0.6 µg/L for 3-methylchrysene, which resulted in tissue concentrations ≈60 µg/g ww for phenanthrene and ≈0.15 µg/g ww for 3-methylchrysene. The low concentration of 3-methylchrysene led to the elevated expression of cyp1a but no toxicity. Levels of phenanthrene metabolites were 5-fold higher, and morphological defects and cardiotoxicity were consistently greater when co-exposed to both compounds relative to phenanthrene alone. This work highlights the metabolic activation of PAH toxicity by a co-occurring PAH, which can lead to excess toxicity, synergistic effects, and the overproportional contribution of PAHs to crude oil toxicity.

Seasonal variation of pituitary gonadotropin subunit, brain-type aromatase and sex steroid receptor mRNAs, and plasma steroids during gametogenesis in wild sablefish.

Pituitary-hormone signaling plays critical roles in the onset and progression of gametogenesis in vertebrates. This study characterized expression patterns of pituitary gonadotropin beta-subunits (fshb and lhb), brain-type aromatase (cyp19a1b), androgen (ar1, ar2) and estrogen receptors (esr1, esr2a, esr2b), and changes in plasma steroid levels by liquid chromatography/tandem mass spectrometry in wild sablefish (Anoplopoma fimbria, order Scorpaeniformes) during a complete reproductive cycle. Transcripts for fshb increased during early gametogenesis and peaked in late vitellogenic females and late recrudescent males, while expression of lhb reached maximum levels in periovulatory and spermiating fish. Pituitary levels of cyp19a1b and ar1 were strongly correlated with those of lhb in females and males, increasing during gametogenesis and reaching maximum levels prior to spawning. By contrast, expression of ar2, and the three estrogen receptors differed between female and male sablefish. 17ß-estradiol (E2) was the dominant steroid in females during vitellogenesis, while a range of at least 6 steroids (11ß-hydroxyandrostenedione, testosterone [T], E2, 11-ketotestosterone [11KT], 11-deoxycortisol, and 17α,20ß,21-trihydroxyprogesterone) were detected at similar levels in males during testicular development. Prior to spawning, a marked increase in 4-androstenedione, T, 11KT and E2 was found in both periovulatory females and spermiating males. In conclusion, the concomitant changes in plasma androgen levels and pituitary ar1 expression during gametogenesis suggest a specific role for androgens in pituitary hormone regulation of reproduction in sablefish. Further, our data highlight the importance of E2 during final stages of maturation in this species, which may regulate the transcription of pituitary lhb in a paracrine fashion.

Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria).

Efforts to establish sustainable and efficient aquaculture production of sablefish (Anoplopoma fimbria) have been constrained by delayed puberty in cultured females. This study integrates a series of experiments aimed at gaining an understanding of the reproductive physiology of puberty in female sablefish. We detected transcripts for the dopamine D2 receptor (drd2) in brain, pituitary and ovary of sablefish, and prepubertal females exhibited significantly elevated brain and pituitary drd2 expression relative to wild maturing females. Treatments with sustained-release cholesterol pellets containing testosterone (T) and the dopamine D2 receptor antagonist, metoclopramide (Met), stimulated expression of pituitary luteinizing hormone beta subunit (lhb) and follicle-stimulating hormone beta subunit (fshb), respectively, in prepubertal females, whereas a combination of T and gonadotropin-releasing hormone agonist (GnRHa) had a strong synergistic effect on lhb expression (2000-fold higher than control). Although T induced a significant increase in the maximum ovarian follicle volume, none of the treatments tested stimulated onset of vitellogenesis. Using liquid chromatography/tandem mass spectrometry, we demonstrated that Met stimulated production of T by previtellogenic ovarian follicles in vitro, whereas gonadotropin preparations enhanced 17α-hydroxyprogesterone, androstenedione (A4), T and 17ß-estradiol (E2) production. Treatment with T increased production of A4, 11ß-hydroxyandrostenedione, 11ß-hydroxytestosterone, E2, 11-ketotestosterone, and 5α-dihydrotestosterone (DHT). Interestingly, in the presence of high doses of T the previtellogenic ovary preferentially produced A4 and DHT over any other metabolite. Our data suggest the existence of dopamine inhibition of the reproductive axis in female sablefish. Treatments with Met and T elevated gonadotropin mRNAs in prepubertal females but failed to stimulate the transition into vitellogenic growth, suggesting a possible failure in pituitary gonadotropin protein synthesis/release. Previtellogenic ovarian follicles of sablefish are equipped to synthesize steroids, including those required for vitellogenic growth, and DHT, a steroid hormone whose role in reproduction of fishes remains unknown.

Diarrhetic shellfish toxins and other lipophilic toxins of human health concern in Washington State.

The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs) in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB) partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 µg okadaic acid (OA) + dinophysistoxins (DTXs)/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1) with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2) and yessotoxin (YTX) with azaspiracid-2 (AZA-2) also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1) and azaspiracid-3 (AZA-3) were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.

Assessing hydroxylated polycyclic aromatic hydrocarbon (OHPAH) metabolites in bile of English sole (Parophrys vetulus) from Puget Sound, WA, USA by liquid chromatography/tandem mass spectrometry (LC-MS/MS).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that are widely monitored in marine biota from urbanized areas, due to their toxicity to aquatic organisms. Teleost fish can quickly metabolize PAHs into hydroxylated forms (OHPAHs) that, in some cases, are more toxic than the parent (unmetabolized) PAHs. But due to this fast metabolism, monitoring traditional parent PAHs in fish can cause underestimation on assessing PAH exposure. In addition, environmental levels of individual OHPAH metabolites are lacking in the literature worldwide. Therefore, we developed a rapid and accurate analytical method in which a number of individual OHPAHs metabolites are measured simultaneously in fish bile, via liquid chromatography coupled with tandem mass spectrometry, including low and high molecular weight mono- and diol-OHPAHs. We analyzed bile samples of 119 English sole (Parophrys vetulus) collected from 14 Puget Sound, WA, USA, sites, which has multiple sources of PAHs, including urban stormwater runoff, wastewater effluents, as well as an inactive creosote facility. The mean (± SD) biliary summed OHPAH (∑OHPAH) concentrations determined in English sole from urban, near-urban, and non-urban sites were 790 ± 1400 (n = 46), 310 ± 330 (n = 44) and 130 ± 200 (n = 29) ng/mL, respectively, with a maximum reaching 9400 ng/mL in a sample from an urban site. We compared these novel biliary OHPAH metabolite data with parent PAHs measured in stomach content of the same individual sole. Biliary ∑OHPAH concentrations were significantly correlated with the levels of ∑PAH in stomach content, however, with major differences in their distribution. We also demonstrated that biliary OHPAH metabolite data in English sole can potentially be used to distinguish different sampling sites due to a specific variety and intensity of PAH sources in the aquatic environment, which makes this a very important analytical approach for assessing PAH exposure in the environment.

Temporal trends in aluminum smelter-derived polycyclic aromatic hydrocarbons in outmigrant juvenile Chinook salmon from Kitimat, British Columbia, Canada.

Aluminum smelter-derived polycyclic aromatic hydrocarbons (PAHs) in outmigrant juvenile Chinook salmon were evaluated in Kitimat Arm, British Columbia, Canada from 2000 to 2004, and in 2015. Decades of continual smelter operations by Rio Tinto resulted in PAH contamination of marine sediments at levels associated with adverse effects in juvenile salmon. Recently, smelter operations have undergone process changes to reduce PAH input to the environment. The PAH concentrations in juvenile Chinook salmon observed in 2000 to 2004, at sites nearest the smelter were comparable to salmon in other urban areas where reduced disease resistance was observed; the levels were lower in 2015 than 2000-2004 suggesting that the recent process changes were effective. Further, these data establish a benchmark for assessing any future changes affecting PAH input and the potential risks to the receiving environment.

Metabolites in bile of fish from São Sebastião Channel, São Paulo, Brazil as biomarkers of exposure to petrogenic polycyclic aromatic compounds.

This study reports the analysis of polycyclic aromatic compound (PAC) metabolites, as biomarkers of exposure to PACs in marine environment. PAC metabolites were measured in bile samples from 14 species of demersal fish caught in the São Sebastião Channel (SSC), SE Brazilian coastline. Naphthalene (NPH) equivalents, phenanthrene (PHN) equivalents, and benzo[a]pyrene (BaP) equivalents were quantified using a reverse-phase high-performance liquid chromatography coupled with fluorescence detection method. For all samples, the means (+/-standard deviation, n=37) of concentrations obtained for NPH, PHN, and BaP equivalents were, respectively, 290,000+/-200,000 ng/g, 18,000+/-14,000 ng/g, and 970+/-1900 ng/g. These results indicate recent exposure of these fish to PACs in their environment. In addition, two species (Cyclichthys spinosus and Prionotus nudigula) of fish were analyzed in order to investigate local sources of PAC contamination in the SSC and the influence of the petroleum terminal in fish caught in remote areas. The results showed that these fish species potentially migrate along the channel, especially P. nudigula. Correlations among groups of PAC metabolites indicate the same petrogenic source for NPH and PHN equivalents and a combustion source (e.g., automobile, ships) for BaP equivalents. The ratio BaP/PHN equivalents (0.05+/-0.07, n=37) confirms the predominance of petrogenic PACs for contamination by these chemicals in this region.

Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

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 17ß-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.

Endocrine disrupting chemicals in fish bile: a rapid method of analysis using English sole (Parophrys vetulus) from Puget Sound, WA, USA.

This study describes a recently developed and rapid method to measure bisphenol A (BPA), 17ß-estradiol (E2) and 17α-ethynylestradiol (EE2) in bile of fish using enzymatic hydrolysis of samples followed by solid-phase extraction and ultra-performance liquid chromatography with tandem mass spectrometry. The limits of quantitation (LOQ) for BPA, EE2 and E2 were 6.3ngmL(-1), 12.5ngmL(-1) and 6.3ngmL(-1), respectively. These compounds were analyzed in bile of male English sole (Parophrys vetulus) collected from urban and non-urban sites in Puget Sound, WA, USA. The BPA and E2 concentrations (and occurrence) ranged from

Polycyclic aromatic hydrocarbons and petroleum biomarkers in São Sebastião Channel, Brazil: assessment of petroleum contamination.

Polycyclic aromatic hydrocarbons (PAHs) and non-aromatic hydrocarbons (NAHs), including n-alkanes, isoprenoids and petroleum biomarkers (terpanes, hopanes, steranes and diasteranes), were quantified by gas chromatography with flame ionization and mass spectrometer detectors in sediment samples collected from the São Sebastião Channel (SSC), Brazil, where the largest Brazilian maritime petroleum terminal is located. The concentrations of total PAHs, total n-alkanes and petroleum biomarkers ranged from below the detection limits to 370ngg(-1), 28microgg(-1), 2200ngg(-1) (dry weight), respectively. The analysis of PAH distribution suggested combustion sources of PAHs as the main input for these compounds with smaller amount from petroleum contamination. The distribution of petroleum biomarkers undoubtedly demonstrated petroleum as a source of anthropogenic contamination throughout the region. The assessment of petrogenic sources of contamination in marine sediment is more challenging if only PAH analysis were carried out, which demonstrates that more stable hydrocarbons such as petroleum biomarkers are useful for investigating potential presence of petroleum.