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The emerging presence of environmental obesogens, chemicals that disrupt energy balance and contribute to adipogenesis and obesity, has become a major public health challenge. Molecular initiating events (MIEs) describe biological outcomes resulting from chemical interactions with biomolecules. Machine learning models based on MIEs can predict complex toxic end points due to chemical exposure and improve the interpretability of models. In this study, a system was constructed that integrated six MIEs associated with adipogenesis and obesity. This system showed high accuracy in external validation, with an area under the receiver operating characteristic curve of 0.78. Molecular hydrophobicity (SlogP_VSA) and direct electrostatic interactions (PEOE_VSA) were identified as the two most critical molecular descriptors representing the obesogenic potential of chemicals. This system was further used to predict the obesogenic effects of chemicals on the candidate list of substances of very high concern (SVHCs). Results from 3T3-L1 adipogenesis assays verified that the system correctly predicted obesogenic or nonobesogenic effects of 10 of the 12 SVHCs tested, and identified four novel potential obesogens, including 2-benzotriazol-2-yl-4,6-ditert-butylphenol (UV-320), 4-(1,1,5-trimethylhexyl)phenol (p262-NP), 2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethanol (OP1EO) and endosulfan. These validation data suggest that the screening system has good performance in adipogenic prediction.

Perfluorohexanesulfonic acid (PFHxS), an emerging short-chain per- and polyfluoroalkyl substance, has been frequently detected in aquatic environments. Adverse outcome pathway studies have shown that perfluorinated compounds impair lipid homeostasis through peroxisome proliferator activated receptors (PPARs). However, many of these studies were performed at high concentrations and may thus be a result of overt toxicity. To better characterize the molecular and key events of PFHxS to biota, early life-stage zebrafish (Danio rerio) were exposed to concentrations detected in the environment (0.01, 0.1, 1, and 10 μg/L). Lipidomic and transcriptomic evaluations were integrated to predict potential molecular targets. PFHxS significantly impaired lipid homeostasis by the dysregulation of glycerophospholipids, fatty acyls, glycerolipids, sphingolipids, prenol lipids, and sterol lipids. Informatic analyses of the lipidome and transcriptome indicated alterations of the PPAR signaling pathway, with downstream changes to retinol, linoleic acid, and glycerophospholipid metabolism. To assess the role of PPARs, potential binding of PFHxS to PPARs was predicted and animals were coexposed to a PPAR antagonist (GW6471). Molecular simulation indicated PFHxS had a 27.1% better binding affinity than oleic acid, an endogenous agonist of PPARα. Antagonist coexposures rescued impaired glycerophosphocholine concentrations altered by PFHxS. These data indicate PPARα activation may be an important molecular initiating event for PFHxS.

Deltamethrin (DM) is a widely used insecticide that has demonstrated developmental toxicity in the early life stages of fish. To better characterize the underlying mechanisms, embryos from Tg(cmlc2:RFP), Tg(apo14:GFP), and Tg(mpx:GFP) transgenic strains of zebrafish were exposed to nominal DM concentrations of 0.1, 1, 10, 25, and 50 μg/L until 120 h post-fertilization (hpf). Heart size increased 56.7%, and liver size was reduced by 17.1% in zebrafish exposed to 22.7 and 24.2 μg/L DM, respectively. RNA sequencing and bioinformatic analyses predicted that key biological processes affected by DM exposure were related to inflammatory responses. Expression of IL-1 protein was increased by 69.0% in the 24.4 μg/L DM treatment, and aggregation of neutrophils in cardiac and hepatic histologic sections was also observed. Coexposure to resatorvid, an anti-inflammatory agent, mitigated inflammatory responses and cardiac toxicity induced by DM and also restored liver biomass. Our data indicated a complex proinflammatory mechanism underlying DM-induced cardiotoxicity and hepatotoxicity which may be important for key events of adverse outcomes and associated risks of DM to early life stages of fish.

Many contaminants of emerging concern (CECs) have reactive functional groups and may readily undergo biotransformations, such as methylation and demethylation. These transformations have been reported to occur during human metabolism and wastewater treatment, leading to the propagation of CECs. When treated wastewater and biosolids are used in agriculture, CECs and their transformation products (TPs) are introduced into soil-plant systems. However, little is known about whether transformation cycles, such as methylation and demethylation, take place in higher plants and hence affect the fate of CECs in terrestrial ecosystems. In this study, we explored the interconversion between four common CECs (acetaminophen, diazepam, methylparaben, and naproxen) and their methylated or demethylated TPs in Arabidopsis thalianacells and whole wheat seedlings. The methylation–demethylation cycle occurred in both plant models with demethylation generally taking place at a greater degree than methylation. The transformation rate of demethylation or methylation was dependent on the bond strength of R-CH3, with demethylation of methylparaben or methylation of acetaminophen being more pronounced. Although not explored in this study, these interconversions may exert influences on the behavior and biological activity of CECs, particularly in terrestrial ecosystems. The study findings demonstrated the prevalence of transformation cycles between CECs and their methylated or demethylated TPs in higher plants, contributing to a more complete understanding of risks of CECs in the human-wastewater-soil-plant continuum.

With increasing water scarcity, many utilities are considering the potable reuse of wastewater as a source of drinking water. However, not all chemicals are removed in conventional wastewater treatment, and disinfection byproducts (DBPs) can form from these contaminants when disinfectants are applied during or after reuse treatment, especially if applied upstream of advanced treatment processes to control biofouling. We investigated the chlorination of seven priority emerging contaminants (17β-estradiol, estrone, 17α-ethinylestradiol, bisphenol A (BPA), diclofenac, p-nonylphenol, and triclosan) in ultrapure water, and we also investigated the impact of chlorination on real samples from different treatment stages of an advanced reuse plant to evaluate the role of chlorination on the associated cytotoxicity and estrogenicity. Many DBPs were tentatively identified via liquid chromatography (LC)- and gas chromatography (GC)-high resolution mass spectrometry, including 28 not previously reported. These encompassed chlorinated, brominated, and oxidized analogs of the parent compounds as well as smaller halogenated molecules. Chlorinated BPA was the least cytotoxic of the DBPs formed but was highly estrogenic, whereas chlorinated hormones were highly cytotoxic. Estrogenicity decreased by ∼4–6 orders of magnitude for 17β-estradiol and estrone following chlorination but increased 2 orders of magnitude for diclofenac. Estrogenicity of chlorinated BPA and p-nonylphenol were ∼50% of the natural/synthetic hormones. Potential seasonal differences in estrogen activity of unreacted vs reacted advanced wastewater treatment field samples were observed.

Contaminants of emerging concern (CECs) in the environment undergo various transformations, leading to the formation of transformation products (TPs) with a modified ecological risk potential. Although the environmental significance of TPs is increasingly recognized, there has been relatively little research to understand the influences of such transformations on subsequent ecotoxicological safety. In this study, we used four pairs of CECs and their methylated or demethylated derivatives as examples to characterize changes in bioaccumulation and acute toxicity in Daphnia magna, as a result of methylation or demethylation. The experimental results were further compared to quantitative structure–activity relationship (QSAR) predictions. The methylated counterpart in each pair generally showed greater acute toxicity in D. magna, which was attributed to their increased hydrophobicity. For example, the LC50values of methylparaben (34.4 ± 4.3 mg L–1) and its demethylated product (225.6 ± 17.3 mg L–1) differed about eightfold in D. magna. The methylated derivative generally exhibited greater bioaccumulation than the demethylated counterpart. For instance, the bioaccumulation of methylated acetaminophen was about 33-fold greater than that of acetaminophen. In silicopredictions via QSARs aligned well with the experimental results and suggested an increased persistence of the methylated forms. The study findings underline the consequences of simple changes in chemical structures induced by transformations such as methylation and demethylation and highlight the need to consider TPs to achieve a more holistic understanding of the environmental fate and risks of CECs.

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are ubiquitous contaminants that can be formed through oxidation of parent PAHs. Our previous studies found 2-hydroxychrysene (2-OHCHR) to be significantly more toxic to Japanese medaka embryos than 6-hydroxychrysene (6-OHCHR), an example of regioselective toxicity. We have also previously identified a sensitive developmental window to 2-OHCHR toxicity that closely coincided with liver development, leading us to hypothesize that differences in metabolism may play a role in the regioselective toxicity. To test this hypothesis, Japanese medaka embryos were treated with each isomer for 24 h during liver development (52–76 hpf). Although 6-OHCHR was absorbed 97.2 ± 0.18% faster than 2-OHCHR, it was eliminated 57.7 ± 0.36% faster as a glucuronide conjugate. Pretreatment with cytochrome P450 inhibitor, ketoconazole, reduced anemia by 96.8 ± 3.19% and mortality by 95.2 ± 4.76% in 2-OHCHR treatments. Formation of chrysene-1,2-diol (1,2-CAT) was also reduced by 64.4 ± 2.14% by ketoconazole pretreatment. While pretreatment with UDP-glucuronosyltransferase inhibitor, nilotinib, reduced glucuronidation of 2-OHCHR by 52.4 ± 2.55% and of 6-OHCHR by 63.7 ± 3.19%, it did not alter toxicity for either compound. These results indicate that CYP-mediated activation, potentially to 1,2-CAT, may explain the isomeric differences in developmental toxicity of 2-OHCHR.

Due to the detection frequencies and measured concentrations in surface water, the type I pyrethroid insecticide, bifenthrin, has been of particular concern within the Sacramento-San Joaquin Delta in California. Concentrations have been detected above levels previously reported to impair neuroendocrine function and induce neurotoxicity to several species of salmonids. Metabolomic and transcriptomic studies indicated impairment of cellular signaling within the brain of exposed animals and potential alteration of lipid metabolism. To better understand the potential impacts of bifenthrin on brain lipids, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to mean bifenthrin concentrations of 28 or 48 ng/L for 14 days, and non-targeted lipidomic profiling in the brain was conducted. Brain tissue sections were also assessed for histopathological insult following bifenthrin treatment. Bifenthrin-exposed trout had a concentration-dependent decrease in the relative abundance of triglycerides (TGs) with levels of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) significantly altered following 48 ng/L bifenthrin exposure. An increased incidence of histopathological lesions, such as focal hemorrhages and congestion of blood vessels, was noted in the brains of bifenthrin-treated animals, suggesting an association between altered lipid metabolism and neuronal cell structure and integrity.

Concerns related to environmental risks associated with pharmaceuticals and personal care products (PPCPs) have led researchers to seek methods for assessing and monitoring these contaminants in the aquatic environment. Identifying and validating risk assessment tools that can evaluate ecological concerns and risks associated with PPCPs is critical. Herein, the suitability of a dose-related risk and effect assessment model, which estimates predicted environmental concentrations and allowed comparisons with predicted no effect concentrations determined, in combination with in vitro analyses of the whole effluent toxicity, was verified for the characterization of a PPCP hazard. Concentrations of the most utilized PPCPs in Norway were measured in influent and effluent samples and used to parameterize the fate model.

The immunosuppressive effects of antibiotics and the potential associations with the intestinal microbiota of the host have been increasingly recognized in recent years. However, the detailed underlying mechanisms of immune interference of antibiotics in environmental organisms remain unclear, particularly at the early life stage of high sensitivity. To better understand the gut microbiome and immune function interactions, the vertebrate model, zebrafish, was treated with environmentally relevant concentrations of a frequently detected antibiotic, enrofloxacin (ENR), ranging from 0.01 to 100 μg/L. 16S ribosomal RNA sequencing indicated diminished diversity, richness, and evenness of intestinal flora following ENR treatment. Twenty-two taxa of gut bacteria including Rickettsiales, Pseudomonadales, and Flavobacterialeswere significantly correlated with immunosuppressive biomarkers, including a significant decrease in the abundance of macrophages and neutrophils. To validate the immunomodulatory effects due to altered intestinal microbial populations, zebrafish reared under sterile and non-sterile husbandry conditions were compared after ENR treatment. A significant inhibitory effect was induced by ENR treatment under non-sterile conditions, while the number of macrophages and neutrophils, as well as biomarkers of immunosuppressive effects, were significantly salved in zebrafish under sterile conditions, confirming for the first time that immunosuppression by ENR was closely mediated through alterations of the intestinal microbiome in fish.

Two commonly used insecticides, bifenthrin and fipronil, can accumulate in the prey of juvenile Chinook salmon, yet the effects of dietary exposure are not understood. Therefore, to better characterize the effect of a dietary exposure route, juvenile Chinook salmon were fed chironomids dosed with a concentration of 9 or 900 ng/g of bifenthrin, fipronil, or their mixture for 25 days at concentrations previously measured in field-collected samples. Chinook were assessed for maximum swimming performance (Umax) using a short-duration constant acceleration test and biochemical responses related to energetic processes (glucose levels) and liver health (aspartate aminotransferase (AST) activity). Chinook exposed to bifenthrin and bifenthrin and fipronil mixtures had a significantly reduced swimming performance, although not when exposed to fipronil alone. The AST activity was significantly increased in bifenthrin and mixture treatments and glucose levels were increased in Chinook following a mixture treatment, although not when exposed to fipronil alone. These findings suggest that there are different metabolic processes between bifenthrin and fipronil following dietary uptake that may influence toxicity. The significant reductions in swimming performance and increased levels of biochemical processes involved in energetics and fish heath could have implications for foraging activity and predator avoidance in wild fish at sensitive life stages.

The extensive and increasing global use of antibiotics results in the ubiquitous presence of antibiotics in the environment, which has made them “pseudo persistent organic contaminants.” Despite numerous studies showing wide adverse effects of antibiotics on organisms, the chronic environmental risk of their exposure is unknown, and the molecular and cellular mechanisms of antibiotic toxicity remain unclear. Here, we systematically quantified transgenerational immune disturbances after chronic parental exposure to environmental levels of a common antibiotic, chlortetracycline (CTC), using zebrafish as a model. CTC strongly reduced the antibacterial activities of fish offspring by transgenerational immunosuppression. Both innate and adaptive immunities of the offspring were suppressed, showing significant perturbation of macrophages and neutrophils, expression of immune-related genes, and other immune functions. Moreover, these CTC-induced immune effects were either prevented or alleviated by the supplementation with PDTC, an antagonist of nuclear factor-κB (NF-κB), uncovering a seminal role of NF-κB in CTC immunotoxicity. Our results provide the evidence in fish that CTC at environmentally relevant concentrations can be transmitted over multiple generations and weaken the immune defense of offspring, raising concerns on the population hazards and ecological risk of antibiotics in the natural environment.

Excess dietary seleno-l-methionine (Se-Met) induces various adverse effects in fish inhabiting the Se-contaminated environments. However, there is an extreme paucity of data on the effects of excess dietary Se-Met on the microbiota in the gastrointestinal (GI) tract in fish. In this study, Japanese medaka Oryzias latipes(three months old) were fed the Se-Met enriched diets at environmentally relevant concentrations: 2.90 (Control: (C), 6.69 (L), 11.89 (M), and 27.05 (H) μg Se/g dw) for 60 d. Histopathological, high throughput sequencing, and biochemical approaches were used to investigate the alterations in histology and microbial communities of the GI tract, enzymatic activity, and transcripts of closely related genes. The results showed that the fish weight was reduced at ∼13% from the L and H treatments. Decreased height and thickness of villus in the GI tract were observed in the H treatment. Meanwhile, the level of D-lactate and activity of diamine oxidase (DAO), protease, and lipase were inhibited in the H treatment. The transcripts of the genes related to the inflammation (i.e., IL-1βand IL-8) were elevated, while those of the genes related to the intestinal barrier (i.e., cdh1, ZO-1, ocln, and cldn7) were inhibited in the H treatment. In addition, alpha diversity at the genus level was higher in the L treatment than the control, and the composition of the microbial community was altered by dietary Se-Met. Furthermore, 5 genera (Rhodobacter, Cloacibacterium, Bdellovibrio, Shinella, and Aeromonas) exhibited the largest variation in abundance among treatments. This study has demonstrated that excess dietary Se-Met inhibits growth, causes hispathological damage to the GI tract, and alters the composition of the microbial community in Oryzias latipes.

Previous studies have shown that altered expression of a family of small noncoding RNAs (microRNAs, or miRs) regulates the expression of downstream mRNAs and is associated with diseases and developmental disorders. miR133b is highly expressed in mammalian cardiac and skeletal muscle, and aberrant expression is associated with cardiac disorders and electrophysiological changes in cardiomyocytes. Similarly, cardiac dysfunction has been observed in early life-stage mahi-mahi (Coryphaena hippurus) exposed to crude oil, a phenotype that has been associated with an upregulation of miR133b as well as subsequent downregulation of a delayed rectifier potassium channel (IKr) and calcium signaling genes that are important for proper heart development during embryogenesis. To examine the potential role of miR133b in oil-induced early life-stage cardiotoxicity in fish, cleavage-stage zebrafish (Danio rerio) embryos were either (1) microinjected with ∼3 nL of negative control miR (75 μM) or miR133b (75 μM) or (2) exposed to a treatment solution containing 5 μM benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, as a positive control. At 72 h post fertilization (hpf), miR133b-injected fish exhibited BaP-like cardiovascular malformations, including a significantly increased pericardial area relative to negative control miR-injected embryos, as well as a significantly reduced eye area. qPCR revealed that miR133b microinjection decreased the abundance of cardiac-specific IKrkcnh6at 5 hpf, which may contribute to action potential elongation in oil-exposed cardiomyocytes. Additionally, ryanodine receptor 2, a crucial calcium receptor in the sarcoplasmic reticulum, was also downregulated by miR133b. These results indicate that an oil-induced increase in miR133b may contribute to cardiac abnormalities in oil-exposed fish by targeting cardiac-specific genes essential for proper heart development.

Bifenthrin (BF) is a widely used pyrethroid that has been frequently detected in surface waters. Previous studies indicated that BF had antiestrogenic activity in zebrafish embryos but estrogenic activity in posthatch fish. To determine whether age-related differences in metabolism contribute to the endocrine effects in developing fish, embryos from zebrafish and Japanese medaka were exposed to BF before and after liver development. Since the commercial mixture of BF is an isomer-enriched product containing two enantiomers (1R-cis-BF and 1S-cis-BF), enantioselective metabolism was also evaluated. The estrogenic metabolite, 4-hydroxybifenthrin (4-OH-BF) was identified in zebrafish embryos, and formation was higher in animals after liver development (>48 hpf). Treatments with β-glucuronidase indicated that 4-OH-BF underwent conjugation in embryos. Formation was reduced by cotreatment of the cytochrome P450 (CYP450) inhibitor, ketoconazole. Formation of 4-OH-BF was greater when treated with 1R-cis-BF compared to the S-enantiomer. However, metabolites were not observed in medaka embryos. These data indicate enantioselective oxidation of BF to an estrogenic metabolite occurs in zebrafish embryos and, since it is increased after liver development, may partially explain estrogenic activity observed in older animals. The lack of activity in medaka suggests species-specific effects with BF metabolism and may influence risk assessment strategies in wildlife.

Boscalid is a succinate dehydrogenase inhibitor fungicide and is frequently detected in surface water. Due to the frequent detection of boscalid, we evaluated its impact on the reproduction of adult zebrafish following a 21 d exposure to 0, 0.01, 0.1, and 1.0 mg/L. Following exposure to boscalid, the fertility of female zebrafish and fertilization rate of spawning eggs were reduced in a concentration-dependent manner up to a respective 87% and 20% in the highest concentration. A significant 16% reduction in the percentage of late vitellogenic oocytes was noted in ovaries, and a significant 74% reduction in the percentage of spermatids in testis was also observed after treatment with 1.0 mg/L. 17β-Estradiol (E2) concentrations decreased significantly in females (34% decrease) but significantly increased in males (15% increase) following 1.0 mg/L boscalid treatment. The expression of genes (such as era, er2b, cyp19a, and cyp19b) related to the hypothalamus-pituitary-gonad-liver (HPGL) axis was significantly altered and positively correlated with E2 concentrations in female and male zebrafish (p< 0.05). Molecular docking results revealed that the binding modes between boscalid and target proteins (ER and CYP19) of zebrafish were similar to that of the reference compounds and the target proteins. The binding energies indicate that boscalid may have a weak estrogen-like binding effect or CYP19 inhibition, potentially altering the HPGL axis, thereby reducing E2 concentrations and fecundity in females. In contrast, boscalid caused significant induction of E2 steroidogenesis and subsequent feminization of gonads in males, indicating gender-specific adverse outcome pathways.

The pyrethroid insecticide, bifenthrin, is frequently measured at concentrations exceeding those that induce acute and chronic toxicity to several invertebrate and fish species residing in the Sacramento-San Joaquin Delta of California. Since the brain is considered to be a significant target for bifenthrin toxicity, juvenile steelhead trout (Oncorhynchus mykiss) were treated with concentrations of bifenthrin found prior to (60 ng/L) and following (120 ng/L) major stormwater runoff events with nontargeted metabolomics used to target transcriptomic alterations in steelhead brains following exposure. Predicted responses were involved in cellular apoptosis and necrosis in steelhead treated with 60 ng/L bifenthrin using the software Ingenuity Pathway Analysis. These responses were predominately driven by decreased levels of acetyl-l-carnitine (ALC), docosahexaenoic acid (DHA), and adenine. Steelhead treated with 120 ng/L bifenthrin had reductions of lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), and increased levels of betaine, which were predicted to induce an inflammatory response. Several genes predicted to be involved in apoptotic (caspase3 and nrf2) and inflammatory (miox) pathways had altered expression following exposure to bifenthrin. There was a significantly increased expression of caspase3 and mioxin fish treated with 120 ng/L bifenthrin with a significant reduction of nrf2 in fish treated with 60 ng/L bifenthrin. These data indicate that bifenthrin may have multiple targets within the brain that affect general neuron viability, function, and signaling potentially through alterations in signaling fatty acids.

Formation of halogenated disinfection byproducts (DBPs) from pharmaceutically active compounds has been observed in water supply systems following wastewater chlorination. Although research has been limited thus far, several studies have shown that halogenated DBPs may elicit increased toxicity compared to their parent compounds. For example, the lipid regulator gemfibrozil has been shown to form chlorogemfibrozil (Cl-gemfibrozil) and bromogemfibrozil (Br-gemfibrozil) following chlorination, which are more potent antiandrogens in male Japanese medaka (Oryzias latipes) compared to their parent compounds. In the present study, we aimed to characterize the bioaccumulative ability of halogenated gemfibrozil DBPs in marine polychaetes viachronic sediment exposures and, consequently, to assess the chronic and acute toxicity of halogenated gemfibrozil DBPs through sediment (in vivo) and aqueous (in vivoand in silico) toxicity evaluations. Following 28 day sediment exposures, Cl-gemfibrozil and Br-gemfibrozil bioaccumulated within Neanthes arenaceodentata, with both compounds reducing survival and growth. The biota–sediment accumulation factors determined for Cl-gemfibrozil and Br-gemfibrozil were 2.59 and 6.86, respectively. Furthermore, aqueous 96 h toxicity tests with N. arenaceodentataindicated that gemfibrozil DBPs elicited increased toxicity compared to the parent compound. While gemfibrozil had an acute LC50 value of 469.85 ± 0.096 mg/L, Cl-gemfibrozil and Br-gemfibrozil had LC50 values of 12.34 ± 0.085 and 9.54 ± 0.086 mg/L, respectively. Although acute toxicity is relatively low, our results indicate that halogenated gemfibrozil DBPs are bioaccumulative and may elicit effects in apex food web organisms prone to accumulation following lifelong exposures.

Soil is a primary sink for plastics, but the influence of microplastics as carriers on terrestrial cycling of persistent contaminants is poorly understood as compared to aquatic systems. Studies to date have disregarded the potential fact that microplastics are generally contaminated before their entry into soil. In this study, earthworm Eisenia fetidawas incubated for 28 d in a soil amended with five common types of microplastics precontaminated with polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) to elucidate contaminant transfer. Accumulation of HOCs in E. fetidavaried greatly among different plastic types and HOCs. The freely dissolved concentration (Cfree) of HOCs showed that desorption of HOCs from microplastics into soil was closely related to plastic types and HOC hydrophobicity and was much slower for polystyrene or polypropylene than polyethylene. Biodynamic model analysis suggested that ingestion of microplastics could act as a significant pathway for some microplastics, likely due to HOCs on the plastics being in an “over-equilibrium” state. This was in contrast with mixing clean microplastics into HOC-contaminated soil, where the microplastics decreased bioaccumulation. Therefore, whether microplastics serve as facilitators or inhibitors of HOC bioaccumulation depends on the fugacity gradient of HOCs between microplastics and soil, which highlights the importance of considering the sequence of contamination between the plastics and soil. These findings also question the validity of short-term experiments because of the generally very slow partition kinetics of HOCs on plastics.

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil are known to impair visual development in fish. However, the underlying mechanism of PAH-induced toxicity to the visual system of fish is not understood. Embryonic zebrafish (Danio rerio) at 4 h post fertilization were exposed to weathered crude oil and assessed for visual function using an optokinetic response, with subsequent samples taken for immunohistochemistry and gene expression analysis. Cardiotoxicity was also assessed by measuring the heart rate, stroke volume, and cardiac output, as cardiac performance has been proposed to be a contributing factor to eye-associated malformations following oil exposure. Larvae exposed to the highest concentrations of crude oil (89.8 μg/L) exhibited an increased occurrence of bradycardia, though no changes in stroke volume or cardiac output were observed. However, genes important in eye development and phototransduction were downregulated in oil-exposed larvae, with an increased occurrence of cellular apoptosis, reduced neuronal connection, and reduced optokinetic behavioral response in zebrafish larvae.

Bisphenol S (BPS), an alternative for bisphenol A (BPA) that is present in thermal paper and numerous consumer products, has been linked to estrogenic, cytotoxic, genotoxic, neurotoxic, and immunotoxic responses. However, the mechanisms of BPS toxicity remain poorly understood. Here, following exposure to environmentally relevant concentrations ranging from 0.1 to 100 μg/L BPS, transcriptional changes evaluated by enriched gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity Pathway Analysis (IPA) predicted cardiac disease and impairment of immune function in zebrafish at the embryo-to-larvae stage. Consistent with impacts predicted by transcriptional changes, significant sublethal impacts were observed ranging from reduced heart rate [8.7 ± 2.4% reductions at 100 μg/L BPS treatment; P< 0.05] to abnormal cardiac morphology [atrial/ventricle area significantly increased; 36.2 ± 9.6% at 100 μg/L BPS treatment; P< 0.05]. RNA-sequencing analysis results also indicated changes in nitric oxide synthetase (NOS2) and interleukin 12 (IL12) after BPS treatment, which was confirmed at the protein level. Increased expression of other cytokine genes was observed in larvae, suggesting inflammatory responses may be contributing to cardiac impairment by BPS. BPS caused cardiotoxicity, which temporally corresponded with inflammatory responses as predicted from RNA sequencing and confirmed at the protein and cellular levels of biological organization. Additional study is needed to find causal linkages between these responses.

Ecotoxicological risks of neonicotinoid insecticides are raising significant concerns, including their potential role in bee population declines. Neonicotinoids are water-soluble, systemic insecticides, and exposure of nontarget organisms such as pollinators occurs mainly through residues in nectar and pollens of flowering plants. To better elucidate the underlying mechanisms for such nontarget exposure, it is highly valuable to develop analytical capabilities for in vivo monitoring of neonicotinoids in live plants. In this study, we developed a novel biomimetic water-swelling solid-phase microextraction (SPME) probe, with limits of detection for neonicotinoids as low as 0.03 ng mL–1, and applied it for in vivo detection of seven neonicotinoids in plant sap. The preparation of this fiber was simple and free of stringent or complex physical–chemical reactions. Equilibrium in neonicotinoid accumulation on the fiber was reached in <10 min, allowing for near instantaneous sampling. The water-swelling fiber displayed much greater sampling capacity than the commercially available polydimethylsiloxane and polyacrylate fibers, good reproducibility (RSD of inter- and intrafiber <8.9% and 7.8%, respectively), and antibiofouling property (no loss in performance after 20 use cycles). After treating lettuce (Lactuca sativaL.) by foliar spray and soybean (Glycine maxM.) by seed soaking, the in vivo assays provided a wealth of information, including changes in levels and distribution of neonicotinoids over time in the same plants. Kinetics and distribution patterns suggested that after treatment at the same level, neonicotinoids differed significantly in their levels in the sap. The in vivo sampling and monitoring of neonicotinoids in live plants may provide unique and much needed information in achieving breakthrough understanding of the connection between neonicotinoid use and pollinator exposure.

Smoked cigarettes are the most prevalent form of litter worldwide, often finding their way into oceans and inland waterways. Cigarette smoke contains more than 4000 individual chemicals, some of them carcinogenic or otherwise toxic. We examined the cytotoxicity, genotoxicity, aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and p53 response pathways of smoked cigarette leachate in vitro. Both seawater and freshwater leachates of smoked cigarettes were tested. Cytotoxicity and genotoxicity were negligible at 100 smoked cigarettes/L, while statistically significant AhR, ER, and p53 responses were observed in the extracts of both leachates, suggesting a potential risk to human health through exposure to cigarette litter in the environment. To identify responsible chemicals for the AhR response, an effect directed analysis approach was coupled with nontargeted chemical analysis based on comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC/TOF-MS). Eleven compounds potentially responsible for the AhR response were identified. Among them, 2-methylindole was partially responsible for the AhR response.

Insecticides such as pyrethroids and fipronil are used in large amounts in both agricultural and urban settings and have the potential to elicit toxicity to nontarget aquatic organisms. In California, like in many other regions of the world, urban centers are located along the coast, and it is documented that urban-use insecticides enter the marine environment, where little is known about their occurrence and consequences. In this study, we measured the spatial distribution of pyrethroids and fipronil (and its metabolites) on the Palos Verdes Shelf off the coast of Los Angeles. Total pyrethroid levels ranged from nd to 170 μg/kg (dry weight), and fipronil sulfide levels ranged from 1.8 to 5.6 μg/kg. Two pyrethroids were traced to wastewater effluent discharge, while two others and fipronil sulfide were traced to to surface runoff. Toxicity units (TUs) were estimated for benthic invertebrates, which ranged from no toxicity (nt) to 146 for total pyrethroids, and 0.09 to 1.6 and 4.2 to 75 for fipronil sulfide, depending on the indicator species. Therefore, near-shore deposition of urban-use insecticides due to wastewater discharge and surface runoff poses a significant risk to marine benthic invertebrates and highlights the importance of monitoring near-shore ocean environments and developing mitigation strategies to reduce seaward movement.

Organophosphate esters (OPEs) have been in use as flame retardants for many decades, with their actual usage varying over time. Knowledge of the emission history of OPEs is valuable for improving our prediction of their environmental loadings and associated risks. In this study, concentrations and compositions of 10 OPEs were measured in three dated sediment cores from the Palos Verdes Shelf (PVS) off the coast of Los Angeles, which has been impacted by wastewater treatment plant (WWTP) effluents for over a century. The total OPE concentrations varied from 0.68 to 1064 ng/g along the sediment profile, with two apparent peaks. The first peak occurred in the 1970s, coinciding with peak emissions from WWTPs. The second peak appeared in the 2000s and was possibly attributed to increased consumption of OPEs as replacement flame retardants. Since downward movement of OPEs in the PVS sediment bed was retarded by their slow desorption, the reconstructed history likely provided an accurate picture of OPE emissions in Southern California and North America. These findings suggest that the near-shore marine sediments affected by WWTP effluents could serve as an environmental proxy documenting history in OPE use and emissions.

The dopaminergic effect of PAH and PFAS mixtures, prepared according to environmentally relevant concentrations, has been studied in juvenile female Atlantic cod (Gadus morhua). Benzo[a]pyrene, dibenzothiophene, fluorene, naphthalene, phenanthrene, and pyrene were used to prepare a PAH mixture, while PFNA, PFOA, PFOS, and PFTrA were used to prepare a PFAS mixture. Cod were injected intraperitoneally twice, with either a low (1×) or high (20×) dose of each compound mixture or their combinations. After 2 weeks of exposure, levels of plasma 17β-estradiol (E2) were significantly elevated in high PAH/high PFAS treated group. Brain dopamine/metabolite ratios (DOPAC/dopamine and HVA+DOPAC/dopamine) changed with E2 plasma levels, except for high PAH/low PFAS and low PAH/high PFAS treated groups. On the transcript levels, thmRNA inversely correlated with dopamine/metabolite ratios and gnrh2mRNA levels. Respective decreases and increases of drd1and drd2aafter exposure to the high PAH dose were observed. Specifically, high PFAS exposure decreased both drds, leading to high plasma E2 concentrations. Other studied end points suggest that these compounds, at different doses and combinations, have different toxicity threshold and modes of action. These effects indicate potential alterations in the feedback signaling processes within the dopaminergic pathway by these contaminant mixtures.

Plastic is ingested by over 100 bird species and 40 fish species. Once ingested, plastic may release endocrine-disrupting plastic additives in the animal; however, amounts transferred are poorly characterized. We exposed 16 commonly ingested plastic items to fish and seabird laboratory gut mimic models using the digestive enzyme pepsin at pH 2 and shook them for 16 h at either 28 °C (in saltwater) for fish or 40 °C (in freshwater) for seabirds. Gut liquid was then evaluated for estrogen receptor activity using an in vitro cell line, and plastic-additive concentrations were quantified using ultrahigh-performance liquid chromatography/tandem mass spectrometry. Both seabird (p< 0.0001) and fish gut conditions (p< 0.0001) significantly enhanced the biological estrogenicity of expanded polystyrene, polyethylene shopping bag, and polypropylene string relative to controls, resulting in up to a 10.6-fold increase in estrogenicity. Out of 12 plastic additives analyzed, bisphenol A (BPA) (204 ± 129%) and diethylhexyl phthalate (DEHP) (175 ± 97%) concentrations were significantly increased in seabird gut conditions relative to control and butylbenzyl phthalate (BBP) (132 ± 68%) was significantly increased in fish gut conditions relative to control. BPA, DEHP, and BBP did not adequately account for the increase in biological estrogenicity, suggesting that uncharacterized plastic additives may have been enhanced by gut conditions.

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil can cause global gene dysregulation and developmental impairment in fish. However, the mechanisms that alter gene regulation are not well understood. In this study, larval red drum (Sciaenops ocellatus) were exposed to water accommodated fractions of source oil (6.8, 13.7, and 35.9 μg/L total PAHs) and weathered slick oil (4.7, 8.1, and 18.0 μg/L total PAHs) from the Deepwater Horizon(DWH) oil spill. The global mRNA-microRNA functional networks associated with the toxicity of DWH oil were explored by next-generation sequencing and in-depth bioinformatics analyses. Both source and slick oil significantly altered the expression of miR-18a, miR-27b, and miR-203a across all exposure concentrations. Consistent with the observed concentration-dependent morphological changes, the target mRNAs of these microRNAs were predominantly involved in neuro-cardio system development processes and associated key signaling pathways such as axonal guidance signaling, cAMP-response-element-binding protein signaling in neurons, calcium signaling, and nuclear-factor-of-activated T cells signaling in cardiac hypertrophy. The results indicated that the developmental toxicity of crude oil may result from the abnormal expression of microRNAs and associated target genes, especially for the nervous system. Moreover, we provide a case study for systematic toxicity evaluation leveraging mRNA-microRNA-seq data using nonmodel species.

Perfluoroethylcyclohexane sulfonate (PFECHS) is an emerging per- and polyfluoroalkyl substance used to replace perfluorooctane sulfonate (PFOS), mainly in aircraft hydraulic fluids. However, previous research indicates the potential neurotoxicity of this replacement chemical. In this study, marine medaka (Oryzias melastigma) was exposed to environmentally relevant concentrations of PFECHS (concentrations: 0, 0.08, 0.26, and 0.91 μg/L) from the embryonic stage for 90 days. After exposure, the brain and eyes of the medaka were collected to investigate the bioconcentration potential of PFECHS stereoisomers and their effects on the nervous systems. The determined bioconcentration factors (BCFs) of PFECHS ranged from 324 ± 97 to 435 ± 89 L/kg and from 454 ± 60 to 576 ± 86 L/kg in the brain and eyes of medaka, respectively. The BCFs of trans-PFECHS were higher than those of cis-PFECHS. PFECHS exposure significantly altered γ-aminobutyric acid (GABA) levels in the medaka brain and disrupted the GABAergic system, as revealed by proteomics, implying that PFECHS can disturb neural signal transduction like PFOS. PFECHS exposure resulted in significant alterations in multiple proteins associated with eye function in medaka. Abnormal locomotion was observed in PFECHS-exposed medaka larvae, which was rescued by adding exogenous GABA, suggesting the involvement of disrupted GABA signaling pathways in PFECHS neurotoxicity.

In this study, we performed a systematic evaluation of global microRNA–mRNA interactions associated with the developmental toxicity of Deepwater Horizonoil using a combination of integrated mRNA and microRNA deep sequencing, expression profiling, gene ontology enrichment, and functional predictions by a series of advanced bioinformatic tools. After exposure to water accommodated fraction (WAF) of both weathered slick oil (0.5%, 1%, and 2%) and source oil (0.125%, 0.25%, and 0.5%) from the Deep Water Horizonoil spill, four dose-dependent miRNAs were identified, including three up-regulated (miR-23b, miR-34b, and miR-181b) and one down-regulated miRNAs (miR-203a) in mahi-mahi hatchings exposed from 6 h postfertilization (hpf) to 48 hpf. Consistent with morphological, physiological, and behavioral changes, the target genes of these miRNAs were largely involved in the development of the cardiovascular, visual, nervous system and associated toxicity pathways, suggesting that miRNAs play an essential role in regulating the responses to oil exposure. The results obtained from this study improve our understanding of the role of miRNAs and their target genes in relation to dose-dependent oil toxicity and provide the potential of using miRNAs as novel biomarkers in future oil studies.

This study investigated the significant influence of HCO3–on the degradation of contaminants of emerging concern (CECs) during nitrate photolysis at 254 nm for water reuse applications. The second-order rate constants for the reactions between selected contaminants with carbonate radical (CO3•–) were determined at pH 8.8 and T= 20 °C: estrone ((5.3 ± 1.1) × 108M–1s–1), bisphenol A ((2.8 ± 0.2) × 108M–1s–1), 17α-ethynylestradiol ((1.6 ± 0.3) × 108M–1s–1), triclosan ((4.2 ± 1.4) × 107M–1s–1), diclofenac ((2.7 ± 0.7) × 107M–1s–1), atrazine ((5.7 ± 0.1) × 106M–1s–1), carbamazepine ((4.2 ± 0.01) × 106M–1s–1), and ibuprofen ((1.2 ± 1.1) × 106M–1s–1). Contributions from UV, reactive nitrogen species (RNS), hydroxyl radical (•OH), and CO3•–to the CEC decomposition in UV/NO3–in the presence and absence of HCO3–were investigated. In addition, possible transformation products and degradation pathways of triclosan, diclofenac, bisphenol A, and estrone in UV/NO3–/HCO3–were proposed based on the mass (MS) and MS2spectra. Significant reduction in the cytotoxicity of bisphenol A was observed after the treatment with UV/NO3–/HCO3–.

Biotransformation is a notable modulator of the fate, bioaccumulation, and toxicity of contaminants in the environment. However, it is often formidable to identify unknown biotransformation products in the absence of reference standards, and this analytical challenge is particularly true for contaminants of emerging concern (CECs) that are mostly polar molecules without characteristic structures (e.g., Cl and Br) and in complex matrices such as plants. In this study, using the fibrate drug gemfibrozil as a model CEC and Arabidopsis thalianaas a model plant, we developed and demonstrated a novel analytical framework coupling deuterium stable isotope labeling with high-resolution mass spectrometry (SILAMS) in identifying plant biotransformation products. When exposed in A. thalianacells, gemfibrozil was quickly taken up into the cells and extensively metabolized. The use of nonlabeled and deuterated gemfibrozil at a 3:1 ratio created unique diagnostic patterns in mass spectra, enabling the identification of 11 novel phase II amino acid/peptide conjugates. Similarity in mass fragmentation patterns and chromatographic behaviors was then employed to establish the probable structures. Two major metabolites were further confirmed as glutamate and glutamine conjugates using authentic standards. Most of the identified conjugates were also detected in the whole A. thalianaplant. Therefore, SILAMS offers unique advantages by excluding false matrix positives and helping discern unknown metabolites, including polar conjugates with endogenous biomolecules, with a high degree of confidence. This novel framework may be readily applied to other CECs for high-throughput metabolite screening in plants to improve our understanding of their food safety and human health risks and potential deleterious effects on other species living on plants.

The degradation kinetics and cytotoxicity of two commonly detected contaminants of emerging concern (CECs), diclofenac and triclosan, in UV/H2O2system were investigated in this study. The second-order rate constants of hydroxyl radical (OH) with diclofenac (kOH,diclofenac) and triclosan (kOH,triclosan) varied at different reaction pH (5.3–8.5) in the range of 7.6–9.1 × 109M−1s−1and 7.0–4.4 × 109M−1s−1, respectively. The pH plays a crucial role in the UV/H2O2treatment for the destruction of diclofenac, triclosan, and four additional CECs (estrone, 17β-estradiol, 17α-ethynylestradiol, and bisphenol A), affecting the ionic state of the CECs (based on pKa) and scavenging the OH by increasing the concentration of hydroxide ion. The impacts of H2O2concentration, common inorganic ions (i.e., HCO3−, NO3−, Cl−, and SO42−), and natural organic matter (NOM) were studied as well. Field water samples from the local water works and Lake Harsha were utilized as reaction matrices to assess the possibility of applying UV/H2O2to decompose diclofenac and triclosan in surface water. Cytotoxicity of diclofenac and triclosan was not reduced during treatment even though concentrations of the compounds were diminished, indicating the formation of toxic transformation products. Overall, UV/H2O2is useful to degrade CECs, such as diclofenac and triclosan, in both Milli-Q water and field water samples, but higher UV fluence might be needed to reduce the cytotoxicity of CECs after UV/H2O2treatment.

Ultraviolet-driven advanced oxidation processes (UV/AOPs) are integral steps in water reuse treatment trains. The toxicity of trace organic transformation products during a UV/AOP is critical to its implementation. This study examined the cyto- and geno-toxicity of transformation products of 1,4-dioxane (1,4-D), a trace organic contaminant commonly found in secondary wastewater, in extracts using the CellSensor p53RE-bla HCt-116 cell assay, following UV photolysis at 254 nm with three oxidants, hydrogen peroxide (H2O2), persulfate (S2O82−) and monochloramine (NH2Cl). 1,4-D was transformed into six major oxidation by-products, including ethylene glycol diformate, formaldehyde, glycolaldehyde, glycolic acid, formic acid, and methoxyacetic acid. Formaldehyde and glycolaldehyde were the most geno- and cyto-toxic, while 1,4-D had weak genotoxicity and no cytotoxicity. The order for cytotoxicity on the basis of EC50values is as follows: glycolaldehyde > formaldehyde > formic acid > glycolic acid > 1,4-D > ethylene glycol diformate methoxyacetic acid, with glycolaldehyde and formaldehyde showing high genotoxicity. With the three UV/AOPs, genotoxicity expressed as mitomycin equivalency quotient (MEQ) increased significantly by 10 to 100 fold with a UV dosage of 720 mJ cm−2, mainly due to the formation of glycolaldehyde. UV/S2O82−reduced the MEQ with an increased UV dosage of 1440 mJ cm−2, due to the transformation of toxic aldehydes to less toxic organic acids. In contrast, UV/H2O2increased the MEQ with UV dosage, resulting from the accumulation of aldehyde products. UV/NH2Cl showed the lowest MEQ due to its slow removal of 1,4-D. This study suggests that oxidants and UV dosage can affect the toxicological responses of treatments for recycled water.

Glucocorticoid (GC) release into the environment has led to widespread detection of glucocorticoid receptor (GR) activity in water resources that has been shown to persist throughout conventional and some advanced wastewater treatment processes. Here, we used high performance liquid chromatography, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy to explore the reaction of natural (cortisone, cortisol) and synthetic (prednisone, prednisolone, dexamethasone) GCs with free chlorine (HOCl) to simulate their fate during chemical disinfection of water and wastewater. Generally, GCs react slowly (t1/2∼ 7–200 h) with HOCl when compared to other steroid classes, but they yield complex mixtures of transformation products, with at times the majority of product mass comprising structurally identifiable and likely bioactive steroids. For example, we frequently observed chlorination at the C-9 position (e.g., 9-chloro-prednisone), a reaction known to increase GC activity 4-fold. We also identified reaction products in the adrenosterone family of androgens produced viacleavage of the C-17 side-chain on many GCs. Another common transformation pathway was the conversion of endogenous GCs to their more potent synthetic analogs viaoxidation at the C-1/C-2 positions, with unsaturation reported to increase GR activity 4-fold (e.g., cortisol to prednisolone). Despite identification of such products, in vitroassays generally suggest GR activity decreases with extent of parent decay during chlorination. Cortisol was the exception, with GR activity only decreasing 2-fold in product mixtures (based on measured EC50values) despite a 95% reduction in parent concentration, a result attributable to formation of the more potent prednisolone during chlorination. Furthermore, our assay likely underestimates product bioactivity as it did not account for the activity of several identified GC byproducts that first require in vivoactivation viaC-11 reduction, nor did it consider androgen receptor (AR) activity associated with byproducts from the adrenosterone family. To avoid formation of product mixtures with conserved bioactivity, advanced chemical oxidation processes may represent a more promising approach; we show that GCs react much more rapidly with ozone (t1/2∼ 0.4–1.3 min) and produce no observable UV-active products. This suggests disruption of the GC conjugated π-electron and ring systems, thereby likely mitigating biological activity.

Carbonaceous materials are often proposed for use in restoring soils or sediments contaminated with hydrophobic organic contaminants (HOCs). However, the contamination of most sites is a result of historical events, where HOCs have resided in the solid compartment for many years or decades. The prolonged contact time, or aging, leads to reduced contaminant availability and likely diminished effectiveness of using sorbents. In this study, three different carbonaceous sorbents, i.e., biochars, powdered activated carbon, and granular activated carbon, were amended to a Superfund site marine sediment contaminated with DDT residues from decades ago. The amended sediments were incubated in seawater for up to 1 year, and the freely dissolved concentration (Cfree) and the biota-sediment accumulation factors (BSAFs) for a native polychaete (Neanthes arenaceodentata) were measured. Even though the bulk sediment concentrations were very high (6.4–154.9 μg/g OC), both Cfreeand BSAFs were very small, ranging from nd to 1.34 ng/L and from nd to 0.024, respectively. The addition of carbonaceous sorbents, even at 2% (w/w), did not consistently lead to reduced DDT bioaccumulation. The limited effectiveness of carbonaceous sorbents was attributed to the low DDT availability due to prolonged aging, highlighting the need for considering contaminant aging when using sorbents for remediation.

Tilapia have relatively rapid osmoregulatory abilities, but the combined stress due to extreme temperature and salinity can negatively affect the survival of the fish. This study compared the tolerance of the California Mozambique Tilapia hybrid (Mozambique Tilapia Oreochromis mossambicus× Wami Tilapia O. urolepis; CA hybrid) from aquaculture and three species of tilapia that currently live in the Salton Sea area: Blue Tilapia O. aureus, Redbelly Tilapia Tilapia zillii, and CA hybrid collected from the sea. The CA hybrid tilapia (both cultured and feral) was the most resistant species across all treatments and could survive salinities up to 60 g/L (‰). The CA hybrid from aquaculture was the most tolerant to higher temperatures (33–38°C), while the one from the Salton Sea was the most tolerant to colder temperatures (11–16°C). In terms of salinity regimes, the aquaculture CA hybrid and Blue Tilapia survived better at 20‰, while the CA hybrid from the sea did best at 45‰. The different stocks of tilapia showed very different tolerances, and these results provide valuable information for the future management of the Salton Sea.

AbstractTilapia have relatively rapid osmoregulatory abilities, but the combined stress due to extreme temperature and salinity can negatively affect the survival of the fish. This study compared the tolerance of the California Mozambique Tilapia hybrid (Mozambique Tilapia Oreochromis mossambicus× Wami Tilapia O. urolepis; CA hybrid) from aquaculture and three species of tilapia that currently live in the Salton Sea area: Blue Tilapia O. aureus, Redbelly Tilapia Tilapia zillii, and CA hybrid collected from the sea. The CA hybrid tilapia (both cultured and feral) was the most resistant species across all treatments and could survive salinities up to 60 g/L (‰). The CA hybrid from aquaculture was the most tolerant to higher temperatures (33–38°C), while the one from the Salton Sea was the most tolerant to colder temperatures (11–16°C). In terms of salinity regimes, the aquaculture CA hybrid and Blue Tilapia survived better at 20‰, while the CA hybrid from the sea did best at 45‰. The different stocks of tilapia showed very different tolerances, and these results provide valuable information for the future management of the Salton Sea.Received October 28, 2013; accepted February 1, 2014

Catalytically active human flavin-containing monooxygenase isoform 2 (FMO2.1) is encoded by an allele detected only in individuals of African or Hispanic origin. Genotyping and haplotyping studies indicate that S195L and N413K occasionally occur secondary to the functional FMO2*1 allele encoding reference protein Gln472. Sulfoxygenation under a range of conditions reveals the role these alterations may play in individuals expressing active FMO2 and provides insight into FMO structure. Expressed S195L lost rather than gained activity as pH was increased or when cholate was present. The activity of S195L was mostly eliminated after heating at 45°C for 5 min in the absence of NADPH, but activity was preserved if NADPH was present. By contrast, Gln472 was less sensitive to heat, a response not affected by NADPH. A major consequence of the S195L mutation was a mean 12-fold increase in Kmfor NADPH compared with Gln472. Modeling an S213L substitution, the equivalent site, in the structural model of FMO from the Methylophaga bacterium leads to disruption of interactions with NADP+. N413K had the same pattern of activity as Gln472 in response to pH, cholate, and magnesium, but product formation was always elevated by comparison. N413K also lost more activity when heated than Gln472; however, NADPH attenuated this loss. The major effects of N413K were increases in velocity and kcatcompared with Gln472. Although these allelic variants are expected to occur infrequently as mutations to the FMO2*1 allele, they contribute to our overall understanding of mammalian FMO structure and function.

A cDNA encoding a hepatic isoform of flavin-containing monooxygenase (hFMO) (EF063736) containing an open reading frame of 1792 base pairs (bp) and encoding 554 amino acids was cloned and sequenced from liver mRNA of rainbow trout (Oncorhynchus mykiss). The genomic sequence of hFMOwas also characterized and was 4.379 kilobases, possessing 10 exons and 9 introns (EU519462). Structural analysis of the promoter region showed several cis-acting elements including putative glucocorticoid and osmoregulatory response elements, which have been reported to be functionally related to induction of flavin-containing monooxygenase (FMO) proteins in vertebrates. The amino acid sequence showed 74% identity to a putative FMO gene from fugu (Takifugu rubripes; Q6ZZY9), 52 to 55% to zebrafish (Brachydanio rerio; Q5RGM6, Q5RGM3, Q6TLD2, Q7T1D7) FMO5, and 54 and 50% to human FMO1 (Q01740), FMO3 (P49326), and FMO5 (P49326). Southern blot analysis using a 180-bp fragment of the hFMO cDNA indicated at least seven potential genes. Treatment of primary trout hepatocytes with cortisol and sodium chloride for 24 h enhanced hFMOexpression. Expression of hFMOwas not detected in untreated or solute-treated primary cultures of gill epithelial cells, suggesting tissue-specific expression of hFMO. Induction of hFMOis consistent with the occurrence of cis-osmoregulatory and glucocorticoid response elements identified in the 5′-upstream sequence, indicating regulation of hFMOin response to hypersaline conditions and the osmoregulatory hormone cortisol.

A cDNA encoding a hepatic isoform of flavin-containing monooxygenase (hFMO) (EF063736) containing an open reading frame of 1792 base pairs (bp) and encoding 554 amino acids was cloned and sequenced from liver mRNA of rainbow trout (Oncorhynchus mykiss). The genomic sequence of hFMO was also characterized and was 4.379 kilobases, possessing 10 exons and 9 introns (EU519462). Structural analysis of the promoter region showed several cis-acting elements including putative glucocorticoid and osmoregulatory response elements, which have been reported to be functionally related to induction of flavin-containing monooxygenase (FMO) proteins in vertebrates. The amino acid sequence showed 74% identity to a putative FMO gene from fugu (Takifugu rubripes; Q6ZZY9), 52 to 55% to zebrafish (Brachydanio rerio; Q5RGM6, Q5RGM3, Q6TLD2, Q7T1D7) FMO5, and 54 and 50% to human FMO1 (Q01740), FMO3 (P49326), and FMO5 (P49326). Southern blot analysis using a 180-bp fragment of the hFMO cDNA indicated at least seven potential genes. Treatment of primary trout hepatocytes with cortisol and sodium chloride for 24 h enhanced hFMO expression. Expression of hFMO was not detected in untreated or solute-treated primary cultures of gill epithelial cells, suggesting tissue-specific expression of hFMO. Induction of hFMO is consistent with the occurrence of cis-osmoregulatory and glucocorticoid response elements identified in the 5'-upstream sequence, indicating regulation of hFMO in response to hypersaline conditions and the osmoregulatory hormone cortisol.

We analyzed flavin-containing monooxygenase 3 (FMO3) polymorphisms, haplotype structure, and linkage disequilibrium (LD) in 256 Han Chinese and 50 African-American individuals to compare their haplotype frequencies and LD with other world populations. For the Han Chinese, genotyping of three haplotype tag single nucleotide polymorphisms (E158K, V257M, and E308G) was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. For the African-Americans, genotyping of all coding exons was performed by modified PCR-single strand conformational polymorphism. Haplotype frequencies, LD, and evolutionary rates were inferred and estimated computationally. There were significant differences in haplotype frequency distribution and LD pattern among Han Chinese, African-Americans, and other world populations. Four major haplotypes of Han Chinese were EVE, KVE, EME, and EVG. Two major haplotypes of African-Americans were EVE and KVE. We found that sites 158 and 257 are in significant LD in both populations. This is the first report comparing FMO haplotypes and LD of Han Chinese with African-Americans. The data presented here justify further pharmacogenetic studies for potentially optimizing recommended drug dosages and evaluating relationships with disease processes.

The cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes are the major oxidative enzymes in phase I metabolism. Many organophosphate and carbamate thioether compounds are excellent substrates for these enzymes. Stereoselective sulfoxidation of fenthion and methiocarb by human liver, kidney, and microsomes was investigated. A high level of stereoselectivity in the formation of fenthion +-sulfoxide was observed in kidney and intestinal microsomes. This activity was not inhibited by the P450 inhibitor 1-aminobenzotriazole but was dramatically reduced following mild heat treatment. In liver, fenthion was metabolized to its sulfoxide in a nonstereoselective manner, and the activity was sensitive to both 1-aminobenzotriazole and heat treatment. The carbamate pesticide methiocarb also was sulfoxidated with a high degree of stereoselectivity in human kidney microsomes. Human liver microsomes formed both stereoisomers in equal amounts. Sulfoxide formation in kidney was not inhibited by 1-aminobenzotriazole but was abolished in liver microsomes. Formation of methiocarb sulfoxides was not observed in intestinal microsomes. The relative contribution of FMO1 and FMO3 to the sulfoxidation of carbophenothion, demeton-O, ethiofencarb, fonofos, and methiocarb also was investigated by using baculovirus-expressed recombinant proteins. FMO1 showed the highest catalytic activity for all pesticides. This study indicates that FMO1 may have a bigger role in extrahepatic metabolism than previously thought.

The use of biocides to control blooms of cyanobacteria (blue-green algae) is important for the aquaculture industry because cyanobacteria produce chemicals that cause off-flavors in fish. However, the consequences of exposure to biocides for fish health will determine the effective use of a biocide treatment. Two novel cyanobactericides, anthraquinone-19 and anthraquinone-59, were evaluated for acute toxicity to fingerlings of channel catfish Ictalurus punctatus. Acute toxicity was determined through measurement of 50% mortality following 96 h of aqueous exposure (96- h LC50). In addition, the no-observed-effect concentration (NOEC) was determined for each compound. Following a 1-week acclimation, groups of catfish fingerlings were exposed at each of six different concentrations ranging from 0.01 mg/L to 20 mg/L for each compound. Two groups of unexposed fingerlings served as the controls. The 96-h LC50 value for anthraquinone-19 was 2.51 ± 0.51 mg/L (mean ± SD) and that for anthraquinone-59 was 2.06 ± 0.31 mg/L. Among catfish exposed to the sublethal doses, no differences were detected in body size (weight and length) and gill and liver weight. The NOEC was determined to be 1 mg/L for both test compounds. Compared with anthraquinone-19, anthraquinone-59 has a greater margin between concentrations that are toxic to channel catfish and concentrations that reduce undesirable cyanobacteria in catfish ponds.

Trimethylaminuria is a rare metabolic disorder that is associated with abnormal amounts of the dietary-derived trimethylamine. Excess unmetabolized trimethylamine in the urine, sweat and other body secretions confers a strong, foul body odor that can affect the individual's ability to work or engage in social activities. This review summarizes the biochemical aspects of the condition and the classification of the disorder into: 1) primary genetic form, 2) acquired form, 3) childhood forms, 4) transient form associated with menstruation, 5) precursor overload and 6) disease states. The genetic variability of the flavin-containing monooxygenase (form 3) that is responsible for detoxication and deodoration of trimethylamine is discussed and put in context with other variant forms of the flavin-containing monooxygenase (forms 1-5). The temporal-selective expression of flavin-containing monooxygenase forms 1 and 3 is discussed in terms of an explanation for childhood trimethylaminuria. Information as to whether variants of the flavin-containing monooxygenase form 3 contributes to hypertension and / or other diseases are presented. Discussion is provided outlining recent bioanalytical approaches to quantify urinary trimethylamine and trimethylamine N-oxide and plasma choline as well as data on self-reporting individuals tested for trimethylaminuria. Finally, trimethylaminuria treatment strategies and nutritional support are described including dietary sources of trimethylamine, vitamin supplementation and drug treatment and issues related to trimethylaminuria in pregnancy and lactation are discussed. The remarkable progress in the biochemical, genetic, clinical basis for understanding the trimethylaminuria condition is summarized and points to needs in the treatment of individuals suffering from trimethylaminuria.

Copper sulfate (CuSO4) is applied periodically to commercial channel catfish (Ictalurus punctatus) ponds as an algicide or parasiticide. Current understanding of the chemistry of copper in soil–water systems suggests that copper may accumulate in pond sediments, although the forms and potential bioavailability of copper in catfish pond sediments are not known. This study investigated the accumulation and distribution of copper in the sediment of catfish ponds receiving periodic additions of CuSO4· 5H2O. All ponds were constructed in Sharkey (very‐fine, smectitic, thermic Chromic Epiaquert) soil. Nine 0.40‐ha ponds received 59 applications of 2.27 kg CuSO4· 5H2O per application per pond over 3 yr; no CuSO4· 5H2O applications were made to nine additional ponds. Total Cu concentration in the sediments of CuSO4· 5H2O‐amended catfish ponds (172.5 mg kg−1) was four to five times higher than that in the sediments of nonamended ponds (36.1 mg kg−1). Copper accumulated in catfish pond sediments at a rate of 41 μg kg−1dry sediment for each 1 kg ha−1of CuSO4· 5H2O applied to ponds. Copper in the sediments of amended ponds was mainly in the organic matter–bound (30.7%), carbonate‐bound (31.8%), and amorphous iron oxide–bound (22.1%) fractions with a considerable fraction (3.4%; 3 to 8 mg kg−1) in soluble and exchangeable fractions. This indicates that Cu accumulates differentially in various fractions, with proportionally greater initial accumulation in potentially bioavailable forms. However, toxicity bioassays with amphipods (Hyallela azteca) and common cattail (Typha latifoliaL.) indicated that the effect of exposure to amended or nonamended pond sediments was not different.

2-Methylisoborneol (MIB) and structurally related terpenoid compounds are responsible for millions of dollars of lost revenue to catfish farmers. In an attempt to determine enzymatic pathways of biotransformation and elimination of MIB, the in vitrometabolism of MIB was examined in the Ulvade strain of channel catfish (Ictalurus punctatus). Although cytochrome P450 (CYP) activities were observed and correlated with expression of specific isoforms (i.e. steroid hydroxylation and CYP3A expression), no metabolites of MIB were observed. To determine whether extrahepatic biotransformation may be occurring the in vivo metabolism and disposition of 14C-MIB was examined in Uvalde, USDA-103 channel catfish, and a channel catfish X blue catfish (Ictalurus furcatus) hybrid species. Confirming in vitrohepatic studies, no metabolites were observed in plasma from animals treated with an intra-arterial dose of 14C-MIB. 14C-MIB elimination was predicted using a two compartment model in each strain of fish. There was no significant difference in terminal half-lives between strains but possible differences in total body clearance and apparent volumes of distribution which may be related to higher lipid content in the hybrids. Results of these studies indicate biotransformation has no involvement in MIB elimination and that other physiological processes may play a more significant role in MIB disposition within Ictalurid fish species.