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The toxicity of single pesticides is likely underestimated when considering complex pesticide mixtures found in agricultural runoff and this is especially true for newer pesticides with little toxicity data on non-target species. The goal of our study was to compare the toxicity of two newer pesticides, imidacloprid (IMI) and chlorantraniliprole (CHL), when an invertebrate and fish were exposed to single compounds, binary mixtures or surface water collected near agricultural fields. A secondary goal was to determine whether changes in select subcellular molecular pathways correspond to the insecticides' mechanisms of activity in aquatic organisms. We conducted acute (96 h) exposures using a dilution series of field water and environmentally relevant concentrations of single and binary mixtures of IMI and CHL. We then evaluated survival, gene expression and the activity of IMI toward the n-acetylcholine receptor (nAChR) and CHL activity toward the ryanodine receptor (RyR). Both IMI and CHL were detected at all sampling locations for May 2019 and September 2019 sampling dates and exposure to field water led to high invertebrate but not fish mortality. Fish exposed to field collected water had significant changes in the relative expression of genes involved with detoxification and neuromuscular function. Exposure of fish to single compounds or binary mixtures of IMI and CHL led to increased relative gene expression of RyR in fish. Furthermore, we found that IMI targets the nAChR in aquatic invertebrates and that CHL can cause overactivation of the RyR in invertebrates and fish. Overall, our finding suggests that IMI and CHL may impact neuromuscular health in fish. Expanding monitoring efforts to include sublethal and molecular assays would allow the detection of subcellular level effects due to complex mixtures present in surface water near agricultural areas.

Although their production was banned in the United States in 1977, polychlorinated biphenyls (PCBs) continue to pose significant risks to the developing nervous system. Perinatal exposure to PCBs is associated with increased risk of neuropsychiatric disorders, perhaps due to altered patterns of dendritic arborization of central neurons. Non-dioxin-like (NDL) PCB congeners enhance dendritic arborization of developing mammalian neurons via sensitization of ryanodine receptors (RYR). Structure-activity relationships (SAR) of RYR sensitization by PCBs have been demonstrated using mammalian and rainbow trout (Oncorhynchus mykiss) tissue homogenates. The purpose of this study is to determine whether this SAR translates to developmental neurotoxicity (DNT) of PCBs in vivo, a question that has yet to be tested. To address this gap, we leveraged a zebrafish model to evaluate the developmental neurotoxicity potential of PCBs 28, 66, 84, 95, 138, and 153, congeners previously shown to have broadly different potencies towards sensitizing RYR. We first confirmed that these PCB congeners exhibited differing potency in sensitizing RYR in zebrafish muscle ranging from negligible (PCB 66) to moderate (PCB 153) to high (PCB 95) RYR activity. Next, enzymatically dechorionated embryos were statically exposed to varying concentrations (0.1-10 μM) of each PCB congener from 6 h post-fertilization to 5 days post-fertilization (dpf). Embryos were observed daily using stereomicroscopy to assess mortality and gross malformations and photomotor behavior was assessed in larval zebrafish at 3, 4, and 5 dpf. The body burden of each PCB was measured by gas chromatography. The key findings are: 1) None of these PCBs caused death or overt teratology at the concentrations tested; 2) A subset of these PCB congeners altered photomotor behavior in larval zebrafish and the SAR for PCB behavioral effects mirrored the SAR for RYR sensitization; and 3) Quantification of PCB levels in larval zebrafish ruled out the possibility that congener-specific effects on behavior were due to differential uptake of PCB congeners. Collectively, the findings from this study provide in vivo evidence in support of the hypothesis that RYR sensitization contributes to the DNT of PCBs.

Legacy and current-use contaminants enter into and accumulate throughout the San Francisco Bay−Delta (Bay−Delta), and are present at concentrations with known effects on species important to this diverse watershed. There remains major uncertainty and a lack of focused research able to address and provide understanding of effects across multiple biological scales, despite previous and ongoing emphasis on the need for it. These needs are challenging specifically because of the established regulatory programs that often monitor on a chemical-by-chemical basis, or in which decisions are grounded in lethality-based endpoints. To best address issues of contaminants in the Bay−Delta, monitoring efforts should consider effects of environmentally relevant mixtures and sub-lethal impacts that can affect ecosystem health. These efforts need to consider the complex environment in the Bay−Delta including variable abiotic (e.g., temperature, salinity) and biotic (e.g., pathogens) factors. This calls for controlled and focused research, and the development of a multi-disciplinary contaminant monitoring and assessment program that provides information across biological scales. Information gained in this manner will contribute toward evaluating parameters that could alleviate ecologically detrimental outcomes. This review is a result of a Special Symposium convened at the University of California−Davis (UCD) on January 31, 2017 to address critical information needed on how contaminants affect the Bay−Delta. The UCD Symposium focused on new tools and approaches for assessing multiple stressor effects to freshwater and estuarine systems. Our approach is similar to the recently proposed framework laid out by the U.S. Environmental Protection Agency (USEPA) that uses weight of evidence to scale toxicological responses to chemical contaminants in a laboratory, and to guide the conservation of priority species and habitats. As such, we also aimed to recommend multiple endpoints that could be used to promote a multi-disciplinary understanding of contaminant risks in Bay−Delta while supporting management needs.

Toxicological studies use "specialty chemicals" and, thus, should assess and report both identity and degree of purity (homogeneity) of the chemicals (or toxicants) under investigation to ensure that other scientists can replicate experimental results. Although detailed reporting criteria for the synthesis and characterization of organic compounds have been established by organic chemistry journals, such criteria are inconsistently applied to the chemicals used in toxicological studies. Biologically active trace impurities may lead to incorrect conclusions about the chemical entity responsible for a biological response, which in turn may confound risk assessment. Based on our experience with the synthesis of PCBs and their metabolites, we herein propose guidelines for the "authentication" of synthetic PCBs and, by extension, other organic toxicants, and provide a checklist for documenting the authentication of toxicants reported in the peer-reviewed literature. The objective is to expand guidelines proposed for different types of biomedical and preclinical studies to include a thorough authentication of specialty chemicals, such as PCBs and their derivatives, with the goal of ensuring transparent and open reporting of scientific results in toxicology and the environmental health sciences.

Non-dioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine receptors (RyR), microsomal Ca2+ channels of broad significance. Teleost fish may be important models for NDL PCB neurotoxicity, and we used sequencing databases to characterize teleost RyR and FK506 binding protein 12 or 12.6kDa (genes FKBP1A; FKBP1B), which promote NDL PCB-triggered Ca2+ dysregulation. Particular focus was placed on describing genes in the Atlantic killifish (Fundulus heteroclitus) genome and searching available RNA-sequencing datasets for single nucleotide variants (SNV) between PCB tolerant killifish from New Bedford Harbor (NBH) versus sensitive killifish from Scorton Creek (SC), MA. Consistent with the teleost whole genome duplication (tWGD), killifish have six RyR genes, corresponding to a and b paralogs of mammalian RyR1, 2 and 3. The presence of six RyR genes was consistent in all teleosts investigated including zebrafish. Killifish have four FKBP1; one FKBP1b and three FKBP1a named FKBP1aa, FKBP1ab, likely from the tWGD and a single gene duplicate FKBP1a3 suggested to have arisen in Atherinomorphae. The RyR and FKBP1 genes displayed tissue and developmental stage-specific mRNA expression, and the previously uncharacterized RyR3, herein named RyR3b, and all FKBP1 genes were prominent in brain. We identified a SNV in RyR3b encoding missense mutation E1458D. In NBH killifish, 57% were heterozygous and 28% were homozygous for this SNV, whereas almost all SC killifish (94%) lacked the variant (n≥39 per population). The outlined sequence differences between mammalian and teleost RyR and FKBP1 together with outlined population differences in SNV frequency may contribute to our understanding of NDL PCB neurotoxicity.

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca2+ channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure-activity relationship and a quantitative structure-activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [3H]Ryanodine ([3H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs.

A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) was developed. Novel immunizing haptens were synthesized by derivatizing at the 4-Cl position of the TCS molecule. Compounds derived from substitutions at 4'-Cl and that replaced the 2'-OH with a Cl atom were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum no. 1155 and a heterologous competitive hapten, where the 2'-OH group was substituted with a Cl atom. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21-6.71 μg/L, respectively. The assay was selective for TCS, providing low cross-reactivity (

Non-dioxin-like polychlorinated biphenyls (NDL PCBs) alter the activity of the ryanodine receptor (RyR), and this activity is linked to developmental neurotoxicity. Most work to date has focused on the activity of single congeners rather than relevant mixtures. The current study assessed the RyR activity of single congeners or binary, tertiary, and complex PCB mixtures. Observed mixture activity was then compared to the expected activity calculated using the concentration addition (CA) model or a RyR-specific neurotoxic equivalency scheme (rNEQ). The predictions of the CA model were consistent with the observed activity of binary mixtures at the lower portion of the concentration-response curve, supporting the additivity of RyR1 active PCBs. Findings also show that minimally active congeners can compete for the RyR1 binding site, and congeners that do not activate the RyR1 do not interfere with the activity of a full agonist. Complex PCB mixtures that mimic PCB profiles detected in indoor air, fish tissue, and the serum of mothers and children activated the RyR1 and displayed similar efficacy and potency regardless of varying congener profiles. Neither the CA model nor the rNEQ perfectly predicted the observed activity of complex mixtures, but predictions were often within one magnitude of change from the observed response. Importantly, PCB mixtures approximating profiles found in environmental samples or human serum displayed RyR1 activity at concentrations reported in published research. The work presented will aid in the development of risk assessment platforms for NDL PCBs, and similar compounds, towards RyR1 activation and related neurotoxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper.