Establishing Chronic Toxicity Effect Levels for Zebrafish (Danio rerio) Exposed to Perfluorooctane Sulfonate.

Zebrafish (Danio rerio) are among the aquatic species most sensitive to perfluorooctane sulfonate (PFOS). Environmental regulatory agencies and researchers use effect benchmarks from laboratory zebrafish PFOS toxicity studies in PFOS-spiked water to calculate PFOS aquatic life criteria. Threshold values as low as 0.7 µg/L (identified in an early, limited scope study) have been used in criteria derivation and site-specific aquatic ecological risk assessments. The present study reviews PFOS effects benchmarks for lethality, growth, and reproduction endpoints from more than 20 zebrafish toxicity studies, including a recent multigenerational study conducted by the United States Army Corps of Engineers Engineer Research & Development Center. Our review of 12 key studies examining long-term, chronic exposures (including multigenerational exposures of 300 days or more) indicated that 0.7 µg/L should not be used as a conservative screening threshold given that effects could not be repeated at this concentration by the recent enhanced multigenerational study. Based on this finding and multiple chronic sublethal studies on PFOS in zebrafish, chronic effects on lethality, growth, and reproduction occur at concentrations two orders of magnitude higher than 0.7 µg/L. Overall, the present review indicates a no-effect screening level of 31 µg/L and a low-effect screening level of 96 µg/L should be used to develop PFOS aquatic life criteria and to inform site-specific ecological risk assessments that are charged with evaluating risks to freshwater fish. Environ Toxicol Chem 2024;43:7-18. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Survival, Growth, and Reproduction Responses in a Three-Generation Exposure of the Zebrafish (Danio rerio) to Perfluorooctane Sulfonate.

A prior multigenerational perfluorooctane sulfonic acid (PFOS) exposure investigation in zebrafish reported adverse effects at 0.734 µg/L, among the lowest aquatic effect levels for PFOS reported to date. The present three-generation PFOS exposure quantified survival, growth, reproduction, and vitellogenin (VTG; egg yolk protein) responses in zebrafish, incorporating experimental design and procedural improvements relative to the earlier study. Exposures targeting 0.1, 0.6, 3.2, 20, and 100 µg/L in parental (P) and first filial (F1) generations lasted for 180 days post fertilization (dpf) and the second filial generation (F2) through 16 dpf. Survival decreased significantly in P and F2 generation exposures, but not in F1, at the highest PFOS treatment (100 µg/L nominal, 94-205 µg/L, measured). Significant adverse effects on body weight and length were infrequent, of low magnitude, and occurred predominantly at the highest exposure treatment. Finally, PFOS had no significant effects on P or F1 egg production and survival or whole-body VTG levels in P or F1 male fish. Overall, the predominance and magnitude of adverse PFOS effects at <1 µg/L reported in prior research were largely nonrepeatable in the present study. In contrast, the present study indicated a threshold for ecologically relevant adverse effects in zebrafish at 117 µg/L (SE 8 µg/L, n = 10) for survival and 47 µg/L (SE 11 µg/L, n = 19) for all statistically significant negative effects observed. Environ Toxicol Chem 2024;43:115-131. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

A Critical Review of Amphibian Per- and Polyfluoroalkyl Substance Ecotoxicity Research Studies: Identification of Screening Levels in Water and Other Useful Resources for Site-Specific Ecological Risk Assessments.

With the goal of aiding risk assessors conducting site-specific risk assessments at per- and polyfluoroalkyl substance (PFAS)-contaminated sites, this critical review synthesizes information on the ecotoxicity of PFAS to amphibians in 10 amphibian species and 16 peer-reviewed publications. The studies in this review consisted of spiked-PFAS chronic toxicity experiments with perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and 6:2 fluorotelomer sulfonate (6:2 FTS) that evaluated apical endpoints typical of ecological risk-based decision making (survival, growth, and development). Body mass was the most sensitive endpoint, showing clear and biologically meaningful population level adverse effect sizes (≥20% adverse effects). From these results, we recommend chronic no observed effect concentration (NOEC) screening levels of 590 µg/L for PFOS and 130 µg/L for PFOA. At or above recommended chronic lowest observed effect concentration screening levels of 1100 µg/L PFOS and 1400 µg/L PFOA, there is an increased chance of adverse biologically relevant chronic effects. Biologically relevant adverse effects were not observed for PFHxS and 6:2 FTS, so unbounded NOECs of 1300 µg/L PFHxS and 1800 µg/L 6:2 FTS are recommended. Screening levels are also provided for the concentration of PFAS in an amphibian diet, amphibian tissue, and moss substrate. In addition, we recommend bioconcentration factors that can be useful to predict concentrations of PFAS in amphibians using concentrations in water; these values are useful for food web modeling to understand risks to vertebrate wildlife that prey on amphibians. Overall, the present study provides a guide to the wealth of ecotoxicological research on PFAS conducted by our research group and highlights the need for additional work that would improve the understanding of chemical risks to amphibians. Environ Toxicol Chem 2023;42:2078-2090. © 2023 SETAC.

Transcriptomic Changes and the Roles of Cannabinoid Receptors and PPARγ in Developmental Toxicities Following Exposure to Δ9-Tetrahydrocannabinol and Cannabidiol.

Human consumption of cannabinoid-containing products during early life or pregnancy is rising. However, information about the molecular mechanisms involved in early life stage Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) toxicities is critically lacking. Here, larval zebrafish (Danio rerio) were used to measure THC- and CBD-mediated changes on transcriptome and the roles of cannabinoid receptors (Cnr) 1 and 2 and peroxisome proliferator activator receptor γ (PPARγ) in developmental toxicities. Transcriptomic profiling of 96-h postfertilization (hpf) cnr+/+ embryos exposed (6 - 96 hpf) to 4 µM THC or 0.5 µM CBD showed differential expression of 904 and 1095 genes for THC and CBD, respectively, with 360 in common. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in the THC and CBD datasets included those related to drug, retinol, and steroid metabolism and PPAR signaling. The THC exposure caused increased mortality and deformities (pericardial and yolk sac edemas, reduction in length) in cnr1-/- and cnr2-/- fish compared with cnr+/+ suggesting Cnr receptors are involved in protective pathways. Conversely, the cnr1-/- larvae were more resistant to CBD-induced malformations, mortality, and behavioral alteration implicating Cnr1 in CBD-mediated toxicity. Behavior (decreased distance travelled) was the most sensitive endpoint to THC and CBD exposure. Coexposure to the PPARγ inhibitor GW9662 and CBD in cnr+/+ and cnr2-/- strains caused more adverse outcomes compared with CBD alone, but not in the cnr1-/- fish, suggesting that PPARγ plays a role in CBD metabolism downstream of Cnr1. Collectively, PPARγ, Cnr1, and Cnr2 play important roles in the developmental toxicity of cannabinoids with Cnr1 being the most critical.

An 'omics approach to investigate the growth effects of environmentally relevant concentrations of guanylurea exposure on Japanese medaka (Oryzias latipes).

Metformin is a widely prescribed pharmaceutical used in the treatment of numerous human health disorders, including Type 2 Diabetes, and as a results of its widespread use, metformin is thought to be the most prevalent pharmaceutical in the aquatic environment by weight. The removal of metformin during the water treatment process is directly related to the formation of its primary degradation product, guanylurea, generally present at higher concentrations in surface waters relative to metformin. Growth effects observed in 28-day early life stage (ELS) Japanese medaka exposed to guanylurea were found to be similar to growth effects in 28-day ELS medaka exposed to metformin; however, effect concentrations were orders of magnitude below those of metformin. The present study uses a multi-omics approach to investigate potential mechanisms by which low-level, 1 ng · L-1 nominal, guanylurea exposure may lead to altered growth in 28-day post hatch medaka via shotgun metabolomics and proteomics and qPCR. Specifically, analyses show 6 altered metabolites, 66 altered proteins and 2 altered genes. Collectively, metabolomics, proteomics, and gene expression data (using qPCR) indicate that developmental exposure to guanylurea exposure alters a number of important pathways related to the overall health of ELS fish, including biomolecule metabolism, cellular energetics, nervous system function/development, cellular communication and structure, and detoxification of reactive oxygen species, among others. To our knowledge, this is the first study to both report the molecular level effects of guanylurea on non-target aquatic organisms, and to relate molecular-level changes to whole organism effects.

Developmental exposure to Δ9-tetrahydrocannabinol (THC) causes biphasic effects on longevity, inflammation, and reproduction in aged zebrafish (Danio rerio).

Increased availability of cannabis and cannabinoid-containing products necessitates the need for an understanding of how these substances influence aging. In this study, zebrafish (Danio rerio) were exposed to different concentrations of THC (0.08, 0.4, 2 µM) during embryonic-larval development and the effects on aging were measured 30 months later and in the offspring of the exposed fish (F1 generation). Exposure to 0.08 µM THC resulted in increased male survival at 30 months of age. As the concentration of THC increased, this protective effect was lost. Treatment with the lowest concentration of THC also significantly increased egg production, while higher concentrations resulted in impaired fecundity. Treatment with the lowest dose of THC significantly reduced wet weight, the incidence of kyphosis, and the expression of several senescence and inflammatory markers (p16ink4ab, tnfα, il-1ß, il-6, pparα and pparγ) in the liver, but not at higher doses indicating a biphasic or hormetic effect. Exposure to THC did not affect the age-related reductions in locomotor behavior. Within the F1 generation, many of these changes were not observed. However, the reduction in fecundity due to THC exposure was worse in the F1 generation because offspring whose parents received high dose of THC were completely unable to reproduce. Together, our results demonstrate that a developmental exposure to THC can cause significant effects on longevity and healthspan of zebrafish in a biphasic manner.

Developmental exposure to cannabidiol (CBD) alters longevity and health span of zebrafish (Danio rerio).

Consumption of cannabinoid-containing products is on the rise, even during pregnancy. Unfortunately, the long-term, age-related consequences of developmental cannabidiol (CBD) exposure remain largely unknown. This is a critical gap given the established Developmental Origins of Health and Disease (DOHaD) paradigm which emphasizes that stressors, like drug exposure, early in life can instigate molecular and cellular changes that ultimately lead to adverse outcomes later in life. Thus, we exposed zebrafish (Danio rerio) to varying concentrations of CBD (0.02, 0.1, 0.5 µM) during larval development and assessed aging in both the F0 (exposed generation) and their F1 offspring 30 months later. F0 exposure to CBD significantly increased survival (~ 20%) and reduced size (wet weight and length) of female fish. While survival was increased, the age-related loss of locomotor function was unaffected and the effects on fecundity varied by sex and dose. Treatment with 0.5 µM CBD significantly reduced sperm concentration in males, but 0.1 µM increased egg production in females. Similar to other model systems, control aged zebrafish exhibited increased kyphosis as well as increased expression markers of senescence, and inflammation (p16ink4ab, tnfα, il1b, il6, and pparγ) in the liver. Exposure to CBD significantly reduced the expression of several of these genes in a dose-dependent manner relative to the age-matched controls. The effects of CBD on size, gene expression, and reproduction were not reproduced in the F1 generation, suggesting the influence on aging was not cross-generational. Together, our results demonstrate that developmental exposure to CBD causes significant effects on the health and longevity of zebrafish.

Developmental and Full-Life Cycle Exposures to Guanylurea and Guanylurea-Metformin Mixtures Results in Adverse Effects on Japanese Medaka (Oryzias latipes).

Metformin is currently thought to be the highest drug by weight released into the aquatic environment, as a direct result of its widespread use in the treatment of a number of human health disorders. The removal of metformin from wastewaters is directly related to the formation of guanylurea (metformin's only known persistent degradation product), which is generally present at higher concentrations in surface waters than the parent compound. With metformin use rising steadily, it is important to characterize the effects of guanylurea on nontarget aquatic organisms. We recently demonstrated the effects of developmental exposure to environmentally relevant concentrations of metformin on the growth of early life stage (ELS) medaka as well as effects on the body weight of adult male fish following full-life cycle exposures. In the present study, we describe similar effects of guanylurea exposure on these endpoints and life stages. Guanylurea led to effects on growth in a 28-d ELS assessment that were similar to those of metformin; however, these effects occurred at concentrations in the ng/L range compared with the µg/L range for metformin. A possible sex-dependent association with body weight changes was also observed in adults following a 165-d full-life cycle exposure to guanylurea alone or in a mixture with metformin. To our knowledge, the present is the first study to report the toxicity of guanylurea to nontarget aquatic organisms. Environ Toxicol Chem 2019;00:1-6. © 2019 SETAC.

Multigenerational consequences of early-life cannabinoid exposure in zebrafish.

While Δ9-tetrahydrocannabinol (THC) has been widely studied in the realm of developmental and reproductive toxicology, few studies have investigated potential toxicities from a second widely used cannabis constituent, cannabidiol (CBD). CBD is popularized for its therapeutic potential for reducing seizure frequencies in epilepsy. This study investigated developmental origins of health and disease (DOHaD) via multigenerational gene expression patterns, behavior phenotypes, and reproductive fitness of a subsequent F1 following an F0 developmental exposure of zebrafish (Danio rerio) to THC (0.024, 0.12, 0.6 mg/L; 0.08, 0.4, 2 µM) or CBD (0.006, 0.03, 0.15 mg/L; 0.02, 0.1, 0.5 µM). Embryonic exposure at these concentrations did not cause notable morphological abnormalities in either F0 or F1 generations. However, during key developmental stages (14, 24, 48, 72, and 96 h post fertilization) THC and CBD caused differential expression of c-fos, brain-derived neurotrophic factor (bdnf), and deleted-in-azoospermia like (dazl), while in F1 larvae only CBD differentially expressed dazl. Larval photomotor behavior was reduced (F0) or increased (F1) by THC exposure, while CBD had no effect on F0 larvae, but decreased activity in the unexposed F1 larvae. These results support our hypothesis of cannabinoid-related developmental neurotoxicity. As adults, F0 fecundity was reduced, but it was not in F1 adults. Conversely, in the adult open field test there were no significant effects in F0 fish, but a significant reduction in the time in periphery was seen in F1 fish from the highest THC exposure group. The results highlight the need to consider long-term ramifications of early-life exposure to cannabinoids.

Effects of environmentally relevant metformin exposure on Japanese medaka (Oryzias latipes).

Metformin is one of the most prevalent pharmaceuticals in both surface and waste waters, yet little is known about the bioavailability and/or effects of developmental exposure on early life stage (ELS) fish. Here, we demonstrate that embryo-larval stages of medaka are capable of taking up metformin from the aquatic environment, provided exposure occurs prior to chorion hardening (∼6-hpf). Once transferred to clean water, ELS medaka are able to completely depurate metformin in <24-hours. Furthermore, ELS medaka exposed to a range of relevant concentrations of waterborne metformin (from 6 hpf through 28-days post hatch) had significantly reduced growth metrics, altered metabolomes, and changes in the expression of genes associated with cell growth. The range of concentrations investigated were 1.0, 3.2, 10, 32, and 100 µg·L-1. To examine effects of chronic, low level metformin exposure across the full medaka life-cycle, we exposed newly fertilized embryos to 3.2 µg L-1 waterborne metformin for 165-days. The weight and length of adult fish were examined, as were effects on the production of some steroid hormones, specifically a significant increase (control females: 0.161 ± 0.023 pg/mg; metformin treated females: 3.42 ± 0.543) in the production of 11-ketotestosterone was observed in adult female medaka. Collectively, these results suggest that current environmental exposure scenarios may be sufficient to cause effects on developing fish.

Pharmaceuticals and personal care products: A critical review of the impacts on fish reproduction.

Research in environmental toxicology involving pharmaceuticals and personal care products (PPCPs) has increased greatly over the last 10-15 years. Much research has been focused on the endocrine-disrupting potential of PPCPs, as they relate to negative population impacts of aquatic organisms. This review assesses the current data on the reported effects of PPCPs on fish reproduction with an emphasis on fecundity, a predictor of population effects. Studies of both individual PPCPs and PPCP mixtures are presented. As the majority of individual PPCP studies reviewed demonstrate negative effects on fish fecundity, we relate these findings to detected surface water concentrations of these compounds. Very few studies involving PPCP mixtures have been conducted; however, the need for these types of studies is warranted as fish are most likely exposed to mixtures of PPCPs in the wild. In addition, laboratory and field assessments of wastewater treatment plant (WWTP) effluents, a major source of PPCPs, are reviewed. Much of the data provided from these assessments are variable and do not generally demonstrate negative impacts on reproduction, or the studies are unable to directly associate observed effects with WWTP effluents. Finally, future research considerations are outlined to provide an avenue into understanding how wild populations of fish are affected by PPCPs. These considerations are aimed at determining the adaptation potential of fish exposed to mixtures of PPCPs over multiple generations. As global use of PPCPs continually rises, the need to discern the effects of chronic exposure to PPCPs is greatly increased.

Effects of pulp and paper mill effluent extracts on liver anaerobic and aerobic metabolic enzymes in rainbow trout.

This study investigates whether pulse exposure to Chilean pulp and paper mill effluent solid phase extracted (SPE) extracts via intraperitoneal injection (IP), would result in changes in the activities of the respiratory metabolic enzymes citrate synthase (CS) and lactate dehydrogenase (LDH) in rainbow trout livers. It also investigated if an alteration in liver metabolic capacity influenced the liver detoxification processes and estrogenic effects previously reported. Besides, a comparison of those enzymatic activities with fish IP injected with SPE extracts of two model effluents coming from industries that process 100% different type of feedstock (softwood, SW and hardwood, HW) was also evaluated. An initial induction of the anaerobic metabolism (increase in LDH enzymatic activity) was detected in all Chilean pulp mill effluent extracts evaluated, contrary to the initial unaltered aerobic metabolism (CS enzymatic activity) observed. A compensatory relationship in energy metabolism (Pasteur effect) was observed when comparing both enzymatic activities of fish exposed to those effluent extracts. LDH and CS activities observed in fish injected with Chilean extracts seem to be related to the effects observed in fish injected with SW extracts. This study showed that intraperitoneal injection of pulp and paper mill effluent extracts affected the anaerobic and aerobic metabolic capacities in rainbow trout livers, but this metabolic alteration did not affect detoxification capability or estrogenic effect previously reported.