Embryonic Exposure to the Benzotriazole Ultraviolet Stabilizer 2-(2H-benzotriazol-2-yl)-4-methylphenol Decreases Fertility of Adult Zebrafish (Danio rerio).

Benzotriazole ultraviolet stabilizers (BUVSs) are emerging contaminants of concern. They are added to a variety of products, including building materials, personal care products, paints, and plastics, to prevent degradation caused by ultraviolet (UV) light. Despite widespread occurrence in aquatic environments, little is known regarding the effects of BUVSs on aquatic organisms. The aim of the present study was to characterize the effects of exposure to 2-(2H-benzotriazol-2-yl)-4-methylphenol (UV-P) on the reproductive success of zebrafish (Danio rerio) following embryonic exposure. Embryos were exposed, by use of microinjection, to UV-P at <1.5 (control), 2.77, and 24.25 ng/g egg, and reared until sexual maturity, when reproductive performance was assessed, following which molecular and biochemical endpoints were analyzed. Exposure to UV-P did not have a significant effect on fecundity. However, there was a significant effect on fertilization success. Using UV-P-exposed males and females, fertility was decreased by 8.75% in the low treatment group and by 15.02% in the high treatment group relative to control. In a reproduction assay with UV-P-exposed males and control females, fertility was decreased by 11.47% in the high treatment group relative to the control. Embryonic exposure to UV-P might have perturbed male sex steroid synthesis as indicated by small changes in blood plasma concentrations of 17ß-estradiol and 11-ketotestosterone, and small statistically nonsignificant decreases in mRNA abundances of cyp19a1a, cyp11c1, and hsd17b3. In addition, decreased transcript abundances of genes involved in spermatogenesis, such as nanos2 and dazl, were observed. Decreases in later stages of sperm development were observed, suggesting that embryonic exposure to UV-P impaired spematogenesis, resulting in decreased sperm quantity. The present study is the first to demonstrate latent effects of BUVSs, specifically on fish reproduction. Environ Toxicol Chem 2024;43:385-397. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Impairment of oocyte maturation as a mechanism of decreased fecundity in Japanese medaka (Oryzias latipes) exposed to the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO).

Inhibition of oocyte maturation is an understudied mechanism by which chemical stressors can impair fecundity of female fishes. The primary objective of the present study was to develop an assay to assess oocyte maturation disruption by chemical stressors in Japanese medaka (Oryzias latipes). First, an in vitro assay to assess maturation inducing hormone (MIH)-stimulated oocyte maturation in zebrafish was validated for use with Japanese medaka. Next, using the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), which previously was shown to decrease fecundity of Japanese medaka and inhibit oocyte maturation in zebrafish, effects on oocyte maturation were quantified using in vitro and in vivo exposure. Adaptation of the protocol for in vitro MIH-stimulated maturation of stage IV oocytes from zebrafish was successful in inducing greater than 80% of stage IX oocytes from female Japanese medaka to mature. To assess effects of in vitro exposure, stage IX oocytes were exposed to 0, 2, 20, and 200 µg/L of TBCO, followed by exposure to MIH. The in vitro exposure caused a significant decrease in maturation of oocytes exposed to 20 and 200 µg/L of TBCO. To assess effects of TBCO on fecundity and oocyte maturation following in vivo exposure, sexually mature fish were fed a control, 100 µg/g, or 1000 µg/g concentration of TBCO-spiked fish food for 21 days, where fecundity was measured daily, and following the exposure, stage IX oocytes were excised to assess MIH-stimulated maturation. Fecundity and oocyte maturation were significantly decreased at either concentration of TBCO. Plasma concentrations of 17ß-estradiol (E2) and hepatic abundances of transcripts of vitellogenin (vtgI and vtgII) were quantified, but there were no significant differences between treatments. Results suggest that inhibition of oocyte maturation is a mechanism by which TBCO decreases fecundity, and that in vitro assays of oocyte maturation might be predictive of fecundity in this species.

Japanese medaka (Oryzias latipes) exposed via maternal transfer to the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), experience decreased fecundity and impaired oocyte maturation.

Early life-stage exposure of fishes to endocrine disrupting chemicals can induce reproductive impairment at sexual maturity. Previously, we demonstrated decreased fecundity of Japanese medaka (Oryzias latipes) exposed via maternal transfer to the novel brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO). However, that study failed to identify the causative mechanism. In other studies we have shown that decreased fecundity of adult fish exposed to dietary TBCO is likely due to impaired oocyte maturation. The goal of the present study was to determine if impaired oocyte maturation is responsible for decreased fecundity of Japanese medaka exposed as embryos to TBCO, via maternal transfer. Sexually mature fish (F0) were fed either a control diet or a low (74.7 µg/g) or high (663 µg/g) diet containing TBCO for 21 days. Eggs (F1) were collected during the final week of exposure and reared to sexual maturity at which point fecundity was assessed using a 21-day reproduction assay. Upon termination of the assay, an ex vivo oocyte maturation assay was used to determine whether maturation inducing hormone (MIH) stimulated oocyte maturation was impaired. Additionally, concentrations of 17ß -estradiol (E2) in blood plasma and expression of genes involved in vitellogenesis and oocyte maturation were quantified. The F1 generation females reared from the low or high F0 treatments experienced a 26.0 % and 56.8 % decrease in cumulative fecundity, respectively. Ex vivo MIH stimulated oocyte maturation from the low and high TBCO treatments were decreased by 23.4 % and 20.0 % respectively. There was no significant effect on concentrations of E2. Transcript abundance of vtgI was significantly decreased in a concentration dependent manner. Transcript abundance of mPRα, pgrmc1, pgrmc2, and igf3 were decreased but effects were not statistically significant. Overall, results suggest that impaired oocyte maturation causes decreased fecundity of Japanese medaka exposed to maternally deposited TBCO.

The brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), causes multigenerational effects on reproductive capacity of Japanese medaka (Oryzias latipes).

Exposure of fishes to endocrine disrupting chemicals (EDCs) during early development can induce multigenerational and transgenerational effects on reproduction. Both in vivo and in vitro studies have demonstrated that the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), is an EDC. The present study investigated whether TBCO has mutigenerational and/or transgenerational effects on the reproductive performance of Japanese medaka (Oryzias latipes). Sexually mature fish (F0 generation) were fed either a control diet or a low (40.6 µg/g) or high (1034.4 µg/g) diet containing TBCO, and three generations of embryos were reared to determine reproductive performance using a standard 21-day reproduction assay. Concentrations of TBCO in eggs (F1 generation) from F0 fish given the low and high diets were 711.3 and 2535.5 ng/g wet weight, respectively. Cumulative fecundity of the F1 generation in the low and high treatment were reduced by 33.9% and 33.3%, respectively, compared to the control. In the F2 generation, cumulative fecundity of the low treatment returned to the level of the controls, but the high treatment was decreased by 29.8%. There was no decrease in cumulative fecundity in the F3 generation compared to the controls. Mechanistically, mRNA abundances of cholesterol side chain cleavage enzyme (cyp11a), aromatase (cyp19a), and luteinizing hormone receptor (lhr) were differentially expressed in gonads from F1 females, suggesting that TBCO might cause developmental reprogramming that disrupts steroidogenesis leading to decreased fecundity. However, concentrations of E2 in plasma and mRNA abundance of vitellogenin in liver were not significantly different compared to controls suggesting a mechanism other than disruption of steroidogenesis or vitellogenesis. Mechanistically, no effects were observed in the F2 or F3 generation. Overall, results suggest that TBCO has multigenerational effects on the reproductive performance of Japanese medaka. However, no transgenerational effects were observed as the F3 generation fully recovered. The mechanism by which multigenerational effects were induced is not known.

Alkylation of Benz[a]anthracene Affects Toxicity to Early-Life Stage Zebrafish and In Vitro Aryl Hydrocarbon Receptor 2 Transactivation in a Position-Dependent Manner.

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.

Inhibition of Oocyte Maturation by Malathion and Structurally Related Chemicals in Zebrafish (Danio rerio) After In Vitro and In Vivo Exposure.

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,20ß-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. Environ Toxicol Chem 2022;41:1381-1389. © 2022 SETAC.