Polycyclic aromatic hydrocarbons (PAHs) are structurally diverse organic chemicals that can have adverse effects on the health of fishes through activation of aryl hydrocarbon receptor 2 (AhR2). They are ubiquitous in the environment, but alkyl PAHs are more abundant in some environmental matrices. However, relatively little is known regarding the effects of alkylation on the toxicity of PAHs to fishes in vivo and how this relates to potency for activation of AhR2 in vitro. Therefore, the objectives of the present study were to determine the toxicity of benz[a]anthracene and three alkylated homologs representing various alkylation positions to early life stages of zebrafish (Danio rerio) and to assess the potency of each for activation of the zebrafish AhR2 in a standardized in vitro AhR transactivation assay. Exposure of embryos to each of the PAHs caused a dose‐dependent increase in mortality and malformations characteristic of AhR2 activation. Each alkyl homolog had in vivo toxicities and in vitro AhR2 activation potencies different from those of the parent PAH in a position‐dependent manner. However, there was no statistically significant linear relationship between responses measured in these assays. The results suggest a need for further investigation into the effect of alkylation on the toxicity of PAHs to fishes and greater consideration of the contribution of alkylated homologs in ecological risk assessments. Environ Toxicol Chem 2022;41:1993–2002. © 2022 SETAC

Dioxin-like compounds (DLCs) cause early life stage mortality of vertebrates through activation of the aryl hydrocarbon receptor (AhR). A prior study developed a cross-species quantitative adverse outcome pathway (qAOP) which can predict full dose-response curves of early life stage mortality for any species of bird or fish exposed to DLCs using the species-and chemical-specific 50% effect concentration (EC50) from an in vitro AhR transactivation assay with COS-7 cells. However, calculating a reliable EC50 for input into this qAOP requires the maximal response of the concentration-response curve to be known, which is not always possible for low-potency agonists, such as some polychlorinated biphenyls (PCBs). To enable predictions for these low-potency agonists, the present study revised this qAOP to use the effect concentration threshold (EC Threshold) from the in vitro AhR transactivation assay as input. Significant linear relationships were demonstrated between EC Threshold and the dose to cause 0, 10, 50, or 100% mortality among early life stages of 3 species of birds and 7 species of fish for 4 DLCs: 2,3,7,8-tetrachlorodibenzo-p-dioxin, PCB 126, PCB 77, and PCB 105. These 4 linear relationships were combined to form the revised qAOP. This qAOP using the EC Threshold enables prediction of experimental dose-response curves for lower-potency agonists to within an order of magnitude on average, but the prior qAOP using EC50 predicts experimental dose-response curves for higher-potency agonists with greater accuracy.

Polycyclic aromatic hydrocarbons (PAHs) are naturally occurring or anthropogenic organic chemicals that can activate the aryl hydrocarbon receptor 2 (AhR2) and induce toxicity in fishes. Alkyl PAHs are more abundant than nonalkylated PAHs in certain environmental matrices and there is growing evidence that alkylation can increase potency, dependent on the position of alkylation. However, it is unknown if the effect of alkylation on potency is conserved across species. In addition, relatively little is known regarding the extent of interspecies variation in sensitivity to PAHs and alkyl PAHs. Therefore, objectives of the present study were to characterize potency of benz[a]anthracene (BAA) and three alkylated homologues representing different alkylation positions in nine phylogenetically diverse species of fish using a standardized in vitro AhR2 transactivation assay. BAA and each alkylated homologue activated the AhR2 in a concentrationdependent manner in each species. Position-dependent effects on potency were observed in every species, but these effects were not consistent across species. Interspecies variation in sensitivity to AhR2 activation by each PAH was observed and ranged by up to 561-fold. Alkylation both increased and decreased the range of interspecies variation and sensitivity, but the potency of each alkylated homologue relative to BAA ranged by less than an order of magnitude among species. These results represent an early step toward the consideration of alkylated homologues for more objective ecological risk assessments of PAHs to native fishes.

Oogenesis is the process by which a primary oocyte develops into a fertilizable oocyte, making it critical to successful reproduction in fish. In zebrafish (Danio rerio), there are five stages of oogenesis. During the final step (oocyte maturation), the maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (MIH) activates the membrane progestin receptor, inducing germinal vesicle breakdown. Using in vitro assays, it has been shown that anthropogenic stressors can dysregulate MIH-induced oocyte maturation. However, it is unknown whether the in vitro assay is predictive of reproductive performance after in vivo exposure. We demonstrate that a known inhibitor of oocyte maturation, malathion, and a structurally related chemical, dimethoate, inhibit oocyte maturation. However, malaoxon and omethoate, which are metabolites of malathion and dimethoate, did not inhibit oocyte maturation. Malathion and dimethoate inhibited maturation to a similar magnitude when oocytes were exposed for 4 h in vitro or 10 days in vivo, suggesting that the in vitro zebrafish oocyte maturation assay might be predictive of alterations to reproductive performance. However, when adult zebrafish were exposed to malathion for 21 days, there was no alteration in fecundity or fertility in comparison with control fish. Our study supports the oocyte maturation assay as being predictive of the success of in vitro oocyte maturation after in vivo exposure, but it remains unclear whether inhibition of MIH-induced oocyte maturation in vitro correlates to decreases in reproductive performance.

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