Exploring BPA alternatives - Environmental levels and toxicity review.

Bisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety. The EU PARC project highlights BPA alternatives as priority chemicals and consolidates information on BPA alternatives, with a focus on environmental relevance and on the identification of the research gaps. The review highlighted aspects and future perspectives. In brief, an extension of environmental monitoring is crucial, extending it to cover BPA alternatives to track their levels and facilitate the timely implementation of mitigation measures. The biological activity has been studied for BPA alternatives, but in a non-systematic way and prioritized a limited number of chemicals. For several BPA alternatives, the data has already provided substantial evidence regarding their potential harm to the environment. We stress the importance of conducting more comprehensive assessments that go beyond the traditional reproductive studies and focus on overlooked relevant endpoints. Future research should also consider mixture effects, realistic environmental concentrations, and the long-term consequences on biota and ecosystems.

The generation gap in endocrine disruption: Can the integrated fish endocrine disruptor test (iFEDT) bridge the gap by assessing intergenerational effects of thyroid hormone system disruption?

Thyroid hormones (THs) act early in ontogenesis, even prior to the differentiation of thyrocytes. Maternal transfer of THs is therefore known to play an essential role in early development. Current OECD test guidelines for the assessment of TH system disruption (THSD) do not address inter- or transgenerational effects. The integrated fish endocrine disruptor test (iFEDT), a test combining parental and developmental exposure of filial fish, may fill this gap. We tested the ability of the iFEDT to detect intergenerational effects in zebrafish (Danio rerio): Parental fish were exposed to propylthiouracil (PTU), an inhibitor of TH synthesis, or not exposed. The offspring was submitted to a crossed experimental design to obtain four exposure scenarios: (1) no exposure at all, (2) parental exposure only, (3) embryonic exposure only, and (4) combined parental and embryonic exposure. Swim bladder inflation, visual motor response (VMR) and gene expression of the progeny were analysed. Parental, but not embryonic PTU exposure reduced the size of the swim bladder of 5 d old embryos, indicating the existence of intergenerational effects. The VMR test produced opposite responses in 4.5 d old embryos exposed to PTU vs. embryos derived from exposed parents. Embryonic exposure, but not parental exposure increased gene expression of thyroperoxidase, the target of PTU, most likely due to a compensatory mechanism. The gene expression of pde-6h (phosphodiesterase) was reduced by embryonic, but not parental exposure, suggesting downregulation of phototransduction pathways. Hence, adverse effects on swim bladder inflation appear more sensitive to parental than embryonic exposure and the iFEDT represents an improvement in the testing strategy for THSD.

Comprehensive mapping of the AOP-Wiki database: identifying biological and disease gaps.

Introduction: The Adverse Outcome Pathway (AOP) concept facilitates rapid hazard assessment for human health risks. AOPs are constantly evolving, their number is growing, and they are referenced in the AOP-Wiki database, which is supported by the OECD. Here, we present a study that aims at identifying well-defined biological areas, as well as gaps within the AOP-Wiki for future research needs. It does not intend to provide a systematic and comprehensive summary of the available literature on AOPs but summarizes and maps biological knowledge and diseases represented by the already developed AOPs (with OECD endorsed status or under validation). Methods: Knowledge from the AOP-Wiki database were extracted and prepared for analysis using a multi-step procedure. An automatic mapping of the existing information on AOPs (i.e., genes/proteins and diseases) was performed using bioinformatics tools (i.e., overrepresentation analysis using Gene Ontology and DisGeNET), allowing both the classification of AOPs and the development of AOP networks (AOPN). Results: AOPs related to diseases of the genitourinary system, neoplasms and developmental anomalies are the most frequently investigated on the AOP-Wiki. An evaluation of the three priority cases (i.e., immunotoxicity and non-genotoxic carcinogenesis, endocrine and metabolic disruption, and developmental and adult neurotoxicity) of the EU-funded PARC project (Partnership for the Risk Assessment of Chemicals) are presented. These were used to highlight under- and over-represented adverse outcomes and to identify and prioritize gaps for further research. Discussion: These results contribute to a more comprehensive understanding of the adverse effects associated with the molecular events in AOPs, and aid in refining risk assessment for stressors and mitigation strategies. Moreover, the FAIRness (i.e., data which meets principles of findability, accessibility, interoperability, and reusability (FAIR)) of the AOPs appears to be an important consideration for further development.

Development of the integrated fish endocrine disruptor test (iFEDT)-Part A: Merging of existing fish test guidelines.

There has been increasing interest in endocrine-disrupting chemicals (EDCs) among scientists and public authorities over the last 30 years, notably because of their wide use and the increasing evidence of detrimental effects on humans and the environment. However, test systems for the detection of potential EDCs as well as testing strategies still require optimization. Thus, the aim of the present project was the development of an integrated test protocol that merges the existing OECD test guidelines (TGs) 229 (fish short-term reproduction assay) and 234 (fish sexual development test) and implements thyroid-related endpoints for fish. The integrated fish endocrine disruptor test (iFEDT) represents a comprehensive approach for fish testing, which covers reproduction, early development, and sexual differentiation, and will thus allow the identification of multiple endocrine-disruptive effects in fish. Using zebrafish (Danio rerio) as a model organism, two exposure tests were performed with well-studied EDCs: 6-propyl-2-thiouracil (PTU), an inhibitor of thyroid hormone synthesis, and 17α-ethinylestradiol (EE2), an estrogen receptor agonist. In part A of this article, the effects of PTU and EE2 on established endpoints of the two existing TGs are reported, whereas part B focuses on the novel thyroid-related endpoints. Results of part A document that, as expected, both PTU and EE2 had strong effects on various endocrine-related endpoints in zebrafish and their offspring. Merging of TGs 229 and 234 proved feasible, and all established biomarkers and endpoints were responsive as expected, including reproductive and morphometric changes (PTU and EE2), vitellogenin levels, sex ratio, gonad maturation, and histopathology (only for EE2) of different life stages. A validation of the iFEDT with other well-known EDCs will allow verification of the sensitivity and usability and confirm its capacity to improve the existing testing strategy for EDCs in fish. Integr Environ Assess Manag 2024;20:817-829. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Development of the integrated fish endocrine disruptor test-Part B: Implementation of thyroid-related endpoints.

Given the vital role of thyroid hormones (THs) in vertebrate development, it is essential to identify chemicals that interfere with the TH system. Whereas, among nonmammalian laboratory animals, fish are the most frequently utilized test species in endocrine disruptor research, for example, in guidelines for the detection of effects on the sex hormone system, there is no test guideline (TG) using fish as models for thyroid-related effects; rather, amphibians are used. Therefore, the objective of the present project was to integrate thyroid-related endpoints for fish into a test protocol combining OECD TGs 229 (Fish Short-Term Reproduction Assay) and 234 (Fish Sexual Development Test). The resulting integrated Fish Endocrine Disruption Test (iFEDT) was designed as a comprehensive approach to covering sexual differentiation, early development, and reproduction and to identifying disruption not only of the sexual and/or reproductive system but also the TH system. Two 85-day exposure tests were performed using different well-studied endocrine disruptors: 6-propyl-2-thiouracil (PTU) and 17α-ethinylestradiol (EE2). Whereas the companion Part A of this study presents the findings on effects by PTU and EE2 on endpoints established in existing TGs, the present Part B discusses effects on novel thyroid-related endpoints such as TH levels, thyroid follicle histopathology, and eye development. 6-Propyl-2-thiouracil induced a massive proliferation of thyroid follicles in any life stage, and histopathological changes in the eyes proved to be highly sensitive for TH system disruption especially in younger life stages. For measurement of THs, further methodological development is required. 17-α-Ethinylestradiol demonstrated not only the well-known disruption of the hypothalamic-pituitary-gonadal axis, but also induced effects on thyroid follicles in adult zebrafish (Danio rerio) exposed to higher EE2 concentrations, suggesting crosstalk between endocrine axes. The novel iFEDT has thus proven capable of simultaneously capturing endocrine disruption of both the steroid and thyroid endocrine systems. Integr Environ Assess Manag 2024;20:830-845. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Thyroid-like hormone signaling in invertebrates and its potential role in initial screening of thyroid hormone system disrupting chemicals.

This review examines the presence and evolution of thyroid-like systems in selected aquatic invertebrates to determine the potential use of these organisms in screens for vertebrate thyroid hormone axis disrupting chemicals (THADCs). Such a screen might support the phasing out of some vertebrate testing. Although arthropods including crustaceans do not contain a functional thyroid signaling system, elements of such a system exist in the aquatic phyla mollusks, echinoderms, tunicates, and cephalochordates. These phyla can synthesize thyroid hormone, which has been demonstrated in some groups to induce the nuclear thyroid hormone receptor (THR). Thyroid hormone may act in these phyla through interaction with a membrane integrin receptor. Thyroid hormone regulates inter alia metamorphosis but, unlike in vertebrates, this does not occur via receptor activation by the ligands triiodothyronine (T3) and thyroxine (T4). Instead, the unliganded nuclear receptor itself controls metamorphosis in mollusks, echinoderms, and tunicates, whereas the T3 derivative tri-iodothyroacetic acid (TRIAC) acts as a THR ligand in cephalochordates. In view of this, it may be possible to develop an invertebrate-based screen that is sensitive to vertebrate THADCs that interfere with thyroid hormone synthesis or metabolism along with interaction with membrane receptors. The review makes some recommendations for the need to develop an appropriate test method. Integr Environ Assess Manag 2023;19:63-82. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Microplastic contamination in seawater across global marine protected areas boundaries.

Despite the relatively rich literature on the omnipresence of microplastics in marine environments, the current status and ecological impacts of microplastics on global Marine Protected Areas (MPAs) are still unknown. Their ubiquitous occurrence, increasing volume, and ecotoxicological effects have made microplastic an emerging marine pollutant. Given the critical conservation roles of MPAs that aim to protect vulnerable marine species, biodiversity, and resources, it is essential to have a comprehensive overview of the occurrence, abundance, distribution, and characteristics of microplastics in MPAs including their buffer zones. Here, extensive data were collected and screened based on 1565 peer-reviewed literature from 2017 to 2020, and a GIS-based approach was applied to improve the outcomes by considering boundary limits. Microplastics in seawater samples were verified within the boundaries of 52 MPAs; after including the buffer zones, 1/3 more (68 MPAs) were identified as contaminated by microplastics. A large range of microplastic levels in MPAs was summarized based on water volume (0-809,000 items/m3) or surface water area (21.3-1,650,000,000 items/km2), which was likely due to discrepancy in sampling and analytical methods. Fragment was the most frequently observed shape and fiber was the most abundant shape. PE and PP were the most common and also most abundant polymer types. Overall, 2/3 of available data reported that seawater microplastic levels in MPAs were higher than 12,429 items/km2, indicating that global MPAs alone cannot protect against microplastic pollution. The current limitations and future directions were also discussed toward the post-2020 Global Biodiversity Framework goals.

Investigation of the in vivo estrogenicity of the UV-filters 4-methylbenzylidene camphor and octyl methoxy cinnamate in rainbow trout (Oncorhynchus mykiss).

The estrogenic activity of the chemical UV-filters, 4-methylbenzylidene camphor (4-MBC) and octyl methoxy cinnamate (OMC) was investigated in an in vivo rainbow trout (Oncorhynchus mykiss) assay. Plasma vitellogenin concentrations were quantified by means of an Enzyme Linked Immuno Sorbent Assay (ELISA) in juvenile rainbow trout before and after intraperitoneal injection of the test compounds. Injection of 4-MBC on day 0, 3, 6 and 10 in the exposure period caused dose and time dependent increases in the concentration of plasma vitellogenin. Significant elevation of vitellogenin concentrations in plasma was demonstrated from 151 mg 4-MBC kg-1 injection-1. Logistic regression analysis relating the percentage of responding fish to the injected dose of 4-MBC resulted in ED10, ED50 and ED90 values of 37, 115 and 194 mg kg-1 injection-1, respectively, after 14 days of exposure (4 injections). Injections with OMC (up to 202 mg kg-1 injection-1) did not result in a statistically significant response in groups of exposed fish, although some individual fish showed elevated concentrations of vitellogenin in plasma. The results confirm that 4-MBC is estrogenic in fish in vivo.

Thymus development in the zebrafish (Danio rerio) from an ecoimmunology perspective.

The thymus is present in all gnathostome vertebrates and is an essential organ for the adaptive immune system via the generation of functional mature T-cells. Over the life span of mammals, the thymus undergoes morphological and functional alterations, including an age-related involution, which in humans starts in early life. Life history tradeoffs have been suggested as possible reasons for thymus involution. While in teleost fish, only a few studies have investigated alterations of thymus structure and function over different life stages, resulting in a fragmented database. Here, we investigated the thymus growth of zebrafish (Danio rerio) from early life, throughout puberty and reproductive stage, up to 1-year-old. We assessed thymus growth by histological and morphometric analyses and thymocyte numbers. Thymus function was assessed by measuring the transcripts of the thymocyte marker genes, ikaros, tcrα, and tcrδ. Additionally, we analyzed gonad maturity and tail homogenate vitellogenin concentrations to align thymus status with the status of the reproductive system. Our results showed that the zebrafish thymus, in contrast to the human thymus, grew strongly during early life and puberty but started to undergo involution when the fish reached the reproductive age. The involution was characterized by reduced thymus area and thymocyte number, altered histoarchitecture, and decreasing thymocyte marker gene transcript levels. Our findings suggest that age-related changes of the zebrafish thymus do exist and could be partly explained in terms of resource tradeoffs, but also in terms of the ontogenetically late development of a functional adaptive immune system in teleosts.

Toward an AOP Network-Based Tiered Testing Strategy for the Assessment of Thyroid Hormone Disruption.

A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone disrupting chemicals. We provide an example of how the adverse outcome pathway (AOP) framework and associated data generation can address current testing challenges in the context of fish early life stage tests, and fish tests in general. We demonstrate how a suite of assays covering biological processes involved in the underlying toxicological pathways can be implemented in a tiered screening and testing approach for thyroid hormone disruption, using the levels of assessment of the OECD's Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals as a guide.

ERGO: Breaking Down the Wall between Human Health and Environmental Testing of Endocrine Disrupters.

ERGO (EndocRine Guideline Optimization) is the acronym of a European Union-funded research and innovation action, that aims to break down the wall between mammalian and non-mammalian vertebrate regulatory testing of endocrine disruptors (EDs), by identifying, developing and aligning thyroid-related biomarkers and endpoints (B/E) for the linkage of effects between vertebrate classes. To achieve this, an adverse outcome pathway (AOP) network covering various modes of thyroid hormone disruption (THD) in multiple vertebrate classes will be developed. The AOP development will be based on existing and new data from in vitro and in vivo experiments with fish, amphibians and mammals, using a battery of different THDs. This will provide the scientifically plausible and evidence-based foundation for the selection of B/E and assays in lower vertebrates, predictive of human health outcomes. These assays will be prioritized for validation at OECD (Organization for Economic Cooperation and Development) level. ERGO will re-think ED testing strategies from in silico methods to in vivo testing and develop, optimize and validate existing in vivo and early life-stage OECD guidelines, as well as new in vitro protocols for THD. This strategy will reduce requirements for animal testing by preventing duplication of testing in mammals and non-mammalian vertebrates and increase the screening capacity to enable more chemicals to be tested for ED properties.

Does hepatotoxicity interfere with endocrine activity in zebrafish (Danio rerio)?

Vitellogenin (VTG), a well-established biomarker for the diagnosis of endocrine activity in fish, is used in multiple OECD test guidelines (TG) to identify activities of chemicals on hormonal pathways. However, the synthesis of VTG may not only be modified by typical endocrine-related pathways, but also through non-endocrine-mediated processes. In particular, hepatotoxicity, i.e. toxicant-induced impairment of liver structure and function, might influence VTG as a biomarker, since VTG is synthesized in hepatocytes. An intimate understanding of the interplay between endocrine-related and non-endocrine-related pathways influencing VTG production is crucial for the avoidance of erroneous diagnoses in hazard assessment for regulatory purposes of chemical compounds. In order to investigate whether hepatotoxicity may interfere with hepatic VTG synthesis, adult zebrafish (Danio rerio) were exposed to three well-known hepatotoxicants, acetaminophen, isoniazid and acetylsalicylic acid, according to OECD TG 230. Various hepatotoxicity- and endocrine system-related endpoints were recorded: mRNA expression of selected endocrine- and hepatotoxicity-related marker genes in the liver; VTG levels in head/tail homogenates; and liver histopathology. All three test compounds induced significant, but mild single cell necrosis of hepatocytes and transcriptional changes of hepatotoxicity-related marker genes, thus confirming hepatotoxic effects. A positive correlation between hepatotoxicity and reduced hepatic VTG synthesis was not observed, with the single exception of a weak increase in female zebrafish exposed to APAP. This suggests that - in studies conducted according to OECD TG 229 or 230 - it is unlikely that hepatotoxic chemicals will interfere with the hepatic capacity for VTG synthesis.

Use of standard test organisms for sound validation of UV-based ballast water treatment systems.

To challenge ballast water treatment system (BWTS) efficacy for organisms in the size-class 10-50 µm, intake concentration during tests must reach certain minimum requirements. Often, natural concentrations are too low to meet intake requirements and standard test organisms (STOs) are added. We tested the robustness of Tetraselmis suecica and Odontella sp. to a range of UV-treatments to explore fluences needed to meet the IMO discharge standard (<10 org. ml-1) evaluated using two viability assessment methods. To meet discharge standards, fluences of >1000 mJ cm-2 were required using vital stain whereas 135-500 mJ cm-2 were needed using regrowth assays. Besides, results suggest that T. suecica and Odontella sp. were at least as robust as natural algae towards UV-treatments. We suggest the advantageous use of these species as STOs in test water to support intake water requirements and to obtain more conservative validation of UV-based BWTS to ensure more environmental protective procedures.

UV fluences required for compliance with ballast water discharge standards using two approved methods for algal viability assessment.

This study investigates the extra UV fluence needed to meet the International Maritime Organisation's ballast water discharge standards for the 10-50 µm size-class using the approved vital stain (VS) method compared to the Most Probable Number (MPN) method for organism viability assessment. Low- and medium pressure UV collimated beam treatments were applied to natural algae collected in temperate and tropical water environments and analysed using both methods. About 10 times higher UV fluence was required to meet discharge standards when using VS compared to MPN. Implementing a dark-hold period after UV treatments decreased algal viability. Length of dark-hold period to meet discharge standards decreased with increasing UV fluence. No significant differences between temperate and tropical samples were observed. The results showed that UV treated algae assessed using the VS method could meet discharge standards by increasing fluence and/or introducing a dark-hold period.

Investigation of the potential endocrine effect of nitrate in zebrafish Danio rerio and brown trout Salmo trutta.

Nitrate has the potential to affect steroid production. Nitrate concentrations in streams in agricultural areas may exceed concentrations showing effects in laboratory studies. The effects of nitrate and/or nitrite on endocrine relevant endpoints were tested in zebrafish and brown trout. Zebrafish were exposed in two experiments to nitrate (8.8 to 89 mg NO3-/L) and nitrite (3.6 to 19 mg NO2-/L) during the period of sexual differentiation and sex ratios were determined. Vitellogenin concentrations were determined in the second experiment. The sex ratio was unaffected by the exposure to nitrate and nitrite. Vitellogenin concentrations were slightly elevated in males (but not females) in all of the groups exposed to nitrate. Juvenile brown trout were exposed to 5.7, 14, and 31 mg NO3-/L for 8 days and vitellogenin levels in liver were determined. Vitellogenin concentrations in the females were not affected by exposure, but in the males, there was an overall statistically significant effect of exposure to nitrate with the group exposed to 5.7 mg NO3-/L showing a trend of higher vitellogenin concentrations than the control group; levels in the males of the groups exposed to 14 and 31 mg NO3-/L were not statistically different from those of the control group. In conclusion, some marginal effect of nitrate in male fish on endocrine activity was observed but the present results for zebrafish, using environmentally relevant concentrations, do not define nitrate and nitrite as endocrine disrupting chemicals according to the generally accepted WHO/IPCS definition because no adverse effects (altered sex ratios) were demonstrated.

A critical review of the environmental occurrence and potential effects in aquatic vertebrates of the potent androgen receptor agonist 17ß-trenbolone.

Trenbolone acetate is widely used in some parts of the world for its desirable anabolic effects on livestock. Several metabolites of the acetate, including 17ß-trenbolone, have been detected at low nanograms per liter concentrations in surface waters associated with animal feedlots. The 17ß-trenbolone isomer can affect androgen receptor signaling pathways in various vertebrate species at comparatively low concentrations/doses. The present article provides a comprehensive review and synthesis of the existing literature concerning exposure to and biological effects of 17ß-trenbolone, with an emphasis on potential risks to aquatic animals. In vitro studies indicate that, although 17ß-trenbolone can activate several nuclear hormone receptors, its highest affinity is for the androgen receptor in all vertebrate taxa examined, including fish. Exposure of fish to nanograms per liter water concentrations of 17ß-trenbolone can cause changes in endocrine function in the short term, and adverse apical effects in longer exposures during development and reproduction. Impacts on endocrine function typically are indicative of inappropriate androgen receptor signaling, such as changes in sex steroid metabolism, impacts on gonadal stage, and masculinization of females. Exposure of fish to 17ß-trenbolone during sexual differentiation in early development can greatly skew sex ratios, whereas adult exposures can adversely impact fertility and fecundity. To fully assess ecosystem-level risks, additional research is warranted to address uncertainties as to the degree/breadth of environmental exposures and potential population-level effects of 17ß-trenbolone in sensitive species. Environ Toxicol Chem 2018;37:2064-2078. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Vitellogenin concentrations in feral Danish brown trout have decreased: An effect of improved sewage treatment in rural areas?

Feminization of male and juvenile fish because of exposure to estrogens or estrogenic chemicals in effluents from central wastewater treatment plants (WWTPs) is a worldwide issue of concern. Intersex and induction of the female yolk protein, vitellogenin, in male and juvenile fish are robust biomarkers for estrogenic exposure, and feminized fish have been observed downstream of WWTP outlets in many countries. Danish central WWTPs reduce effluent estrogenicity effectively by advanced sewage treatment, and feminizations have not been observed downstream of central WWTP outlets. However, between 2000 and 2004, investigations of Danish streams not receiving sewage from central WWTPs revealed a high variation in vitellogenin concentrations of male juvenile brown trout (Salmo trutta); some individuals had high concentrations, probably as a result of estrogenic point sources, and the plasma concentration was >50 ng mL-1 in 79% of the juvenile males. The streams were reinvestigated in 2010 to 2016, and the average male level had decreased to a hitherto unseen baseline level; in 2010 only 0.7% (one individual) of the males had a vitellogenin concentration >50 ng mL-1 , which could indicate that the estrogenicity of the streams decreased after 2004. We examined possible estrogenic sources in streams unaffected by central WWTP effluents, and found that the reduced vitellogenin levels are most likely explained by a national effort to improve on-site wastewater treatment in scattered houses not connected to central WWTPs. Environ Toxicol Chem 2018;37:839-845. © 2017 SETAC.

Two common mild analgesics have no effect on general endocrine mediated endpoints in zebrafish (Danio rerio).

Mild analgesics such as acetylsalicylic acid (ASA) and acetaminophen (APAP) exert their pain-relieving effect in humans by inhibition of prostaglandin synthesis. Prostaglandins play key roles in developmental and reproductive processes in vertebrates, and in recent years, it has been suggested that weak analgesics might also act as endocrine disrupters. In a set of experiments we investigated if ASA and APAP affect well-established endocrine endpoints in zebrafish (Danio rerio), which is a commonly used model organism in the investigation of endocrine disrupting chemicals. Zebrafish were exposed to APAP (0.22, 2.3, and 30mgL-1) or ASA (0.2, 0.5, 1.7, and 8.2mgL-1) from hatch to sexual maturity in a test design resembling the OECD Fish Sexual Development Test. No effects on sex ratio and vitellogenin levels were observed. Adult zebrafish were exposed to high concentrations (mgL-1) of ASA or APAP for eight or 14days. ASA reduced the levels of prostaglandin E2, but had no effect on the concentration of 11-ketotestosterone and vitellogenin. Overall, ASA decrease prostaglandin E2 concentrations, but well-established endpoints for endocrine disruption in zebrafish are generally not affected by aquatic exposure neither during development nor adulthood. According to the WHO/IPCS definition of an endocrine disrupter, the present results do not define APAP and ASA as endocrine disrupters.