Behavioural analysis has been attracting significant attention as a broad indicator of sub-lethal toxicity and has secured a place as an important subdiscipline in ecotoxicology. Among the most notable characteristics of behavioural research, compared to other established approaches in sub-lethal ecotoxicology (e.g. reproductive and developmental bioassays), are the wide range of study designs being used and the diversity of endpoints considered. At the same time, environmental hazard and risk assessment, which underpins regulatory decisions to protect the environment from potentially harmful chemicals, often recommends that ecotoxicological data be produced following accepted and validated test guidelines. These guidelines typically do not address behavioural changes, meaning that these, often sensitive, effects are not represented in hazard and risk assessments. Here, we propose a new tool, the EthoCRED evaluation method, for assessing the relevance and reliability of behavioural ecotoxicity data, which considers the unique requirements and challenges encountered in this field. This method and accompanying reporting recommendations are designed to serve as an extension of the "Criteria for Reporting and Evaluating Ecotoxicity Data (CRED)" project. As such, EthoCRED can both accommodate the wide array of experimental design approaches seen in behavioural ecotoxicology, and could be readily implemented into regulatory frameworks as deemed appropriate by policy makers of different jurisdictions to allow better integration of knowledge gained from behavioural testing into environmental protection. Furthermore, through our reporting recommendations, we aim to improve the reporting of behavioural studies in the peer-reviewed literature, and thereby increase their usefulness to inform chemical regulation.

Vocalizations are an important means for communication within and between all kinds of species. The previous chapters have shown that across species, vocalizations and forms of communications are unique to a given species, from insects to humans. However, there are commonalities in the neuroendocrine mechanisms governing such behavioral responses. Our ability to detect and understand such vocalizations continues to advance. Machine-learning approaches that detect, analyze, and eventually interpret various patterns will assuredly even further our ability and our comprehension as to what vocalizations across ranging taxa mean, identify potential commonalities based on pattern recognition, and even increase our understanding as to the mechanisms regulating varying forms of communication. This approach may even make us rethink how we join and split species when grouping them in evolutionary trees. Sadly, the fact that many of these forms of communication are regulated by steroid and nonsteroid hormones also makes them vulnerable to chemicals that can mimic such hormones, endocrine-disrupting chemicals. Those anthropogenic chemicals were shown to be able to alter critical features of vocalizations in several species or even cease the acoustic signals entirely, which potentially can result in population effects that may affect the whole ecosystem dynamics. It is the hope that by better understanding the normal regulatory mechanisms and disruptions caused by endocrine-disrupting chemical, it might serve as an impetus for countries to ban such chemicals to avoid a “Silent Spring.”

For decades, we have known that chemicals affect human and wildlife behavior. Moreover, due to recent technological and computational advances, scientists are now increasingly aware that a wide variety of contaminants and other environmental stressors adversely affect organismal behavior and subsequent ecological outcomes in terrestrial and aquatic ecosystems. There is also a groundswell of concern that regulatory ecotoxicology does not adequately consider behavior, primarily due to a lack of standardized toxicity methods. This has, in turn, led to the exclusion of many behavioral ecotoxicology studies from chemical risk assessments. To improve understanding of the challenges and opportunities for behavioral ecotoxicology within regulatory toxicology/risk assessment, a unique workshop with international representatives from the fields of behavioral ecology, ecotoxicology, regulatory (eco)toxicology, neurotoxicology, test standardization, and risk assessment resulted in the formation of consensus perspectives and recommendations, which promise to serve as a roadmap to advance interfaces among the basic and translational sciences, and regulatory practices.

To provide guidance on how systematic review authors, guideline developers, and health technology assessment practitioners should approach the use of the risk of bias in nonrandomized studies of interventions (ROBINS-I) tool as a part of GRADE's certainty rating process.The study design and setting comprised iterative discussions, testing in systematic reviews, and presentation at GRADE working group meetings with feedback from the GRADE working group.We describe where to start the initial assessment of a body of evidence with the use of ROBINS-I and where one would anticipate the final rating would end up. The GRADE accounted for issues that mitigate concerns about confounding and selection bias by introducing the upgrading domains: large effects, dose-effect relations, and when plausible residual confounders or other biases increase certainty. They will need to be considered in an assessment of a body of evidence when using ROBINS-I.The use of ROBINS-I in GRADE assessments may allow for a better comparison of evidence from randomized controlled trials (RCTs) and nonrandomized studies (NRSs) because they are placed on a common metric for risk of bias. Challenges remain, including appropriate presentation of evidence from RCTs and NRSs for decision-making and how to optimally integrate RCTs and NRSs in an evidence assessment.

Objective: To provide guidance on how systematic review authors, guideline developers, and health technology assessment practitioners should approach the use of the risk of bias in nonrandomized studies of interventions (ROBINS-I) tool as a part of GRADE's certainty rating process. Study Design and Setting: The study design and setting comprised iterative discussions, testing in systematic reviews, and presentation at GRADE working group meetings with feedback from the GRADE working group. Results: We describe where to start the initial assessment of a body of evidence with the use of ROBINS-I and where one would anticipate the final rating would end up. The GRADE accounted for issues that mitigate concerns about confounding and selection bias by introducing the upgrading domains: large effects, dose-effect relations, and when plausible residual confounders or other biases increase certainty. They will need to be considered in an assessment of a body of evidence when using ROBINS-I. Conclusion: The use of ROBINS-I in GRADE assessments may allow for a better comparison of evidence from randomized controlled trials (RCTs) and nonrandomized studies (NRSs) because they are placed on a common metric for risk of bias. Challenges remain, including appropriate presentation of evidence from RCTs and NRSs for decision-making and how to optimally integrate RCTs and NRSs in an evidence assessment.

BACKGROUND:Guideline developers can: (1) adopt existing recommendations from others; (2) adapt existing recommendations to their own context; or (3) create recommendations de novo. Monetary and nonmonetary resources, credibility, maximization of uptake, as well as logical arguments should guide the choice of the approach and processes.OBJECTIVES:To describe a potentially efficient model for guideline production based on adoption, adaptation, and/or de novo development of recommendations utilizing the Grading of Recommendations Assessment, Development and Evaluation (GRADE) Evidence to Decision (EtD) frameworks.STUDY DESIGN AND SETTING:We applied the model in a new national guideline program producing 22 practice guidelines. We searched for relevant evidence that informs the direction and strength of a recommendation. We then produced GRADE EtDs for guideline panels to develop recommendations.RESULTS:In two waves, a total of 80 EtD frameworks was produced approximately 4 months and 146 EtDs in about 6 months. Use of the EtD frameworks allowed panel members to understand judgments of other guideline groups about the criteria that bear on guideline recommendations and then make their own judgments about those criteria in a systematic approach.CONCLUSION:The "GRADE-ADOLOPMENT" approach to guideline production combines adoption, adaptation, and, as needed, de novo development of recommendations. If guideline developers apply EtD criteria more widely and make their work publically available, this approach should prove even more useful.

BackgroundGuideline developers can: (1) adopt existing recommendations from others; (2) adapt existing recommendations to their own context; or (3) create recommendations de novo. Monetary and nonmonetary resources, credibility, maximization of uptake, as well as logical arguments should guide the choice of the approach and processes.ObjectivesTo describe a potentially efficient model for guideline production based on adoption, adaptation, and/or de novo development of recommendations utilizing the Grading of Recommendations Assessment, Development and Evaluation (GRADE) Evidence to Decision (EtD) frameworks.Study Design and SettingWe applied the model in a new national guideline program producing 22 practice guidelines. We searched for relevant evidence that informs the direction and strength of a recommendation. We then produced GRADE EtDs for guideline panels to develop recommendations.ResultsIn two waves, a total of 80 EtD frameworks was produced approximately 4 months and 146 EtDs in about 6 months. Use of the EtD frameworks allowed panel members to understand judgments of other guideline groups about the criteria that bear on guideline recommendations and then make their own judgments about those criteria in a systematic approach.ConclusionThe "GRADE-ADOLOPMENT" approach to guideline production combines adoption, adaptation, and, as needed, de novo development of recommendations. If guideline developers apply EtD criteria more widely and make their work publically available, this approach should prove even more useful.

Rodent pups use vocalizations to communicate with one or both parents in biparental species, such as California mice (Peromyscus californicus). Previous studies have shown California mice developmentally exposed to endocrine disrupting chemicals, bisphenol A (BPA) or ethinyl estradiol (EE), demonstrate later compromised parental behaviors. Reductions in F-1 parental behaviors might also be due to decreased emissions of F-2 pup vocalizations. Thus, vocalizations of F-2 male and female California mice pups born to F-1 parents developmentally exposed to BPA, EE, or controls were examined. Postnatal days (PND) 2-4 were considered early postnatal period, PND 7 and 14 were defined as mid-postnatal period, and PND 21 and 28 were classified as late postnatal period. EE pups showed increased latency to emit the first syllable compared to controls. BPA female pups had decreased syllable duration compared to control and EE female pups during the early postnatal period but enhanced responses compared to controls at late postnatal period; whereas, male BPA and EE pups showed greater syllable duration compared to controls during early postnatal period. In mid-postnatal period, F-2 BPA and EE pups emitted greater number of phrases than F-2 control pups. Results indicate aspects of vocalizations were disrupted in F-2 pups born to F-1 parents developmentally exposed to BPA or EE, but their responses were not always identical, suggesting BPA might not activate estrogen receptors to the same extent as EE. Changes in vocalization patterns by F-2 pups may be due to multigenerational exposure to BPA or EE and/or reduced parental care received.

Endocrine disruption caused by various anthropogenic compounds is of persisting concern, especially for aquatic wildlife, because surface waters are the main sink of these so-called endocrine disruptors (ED). In the past, research focused on (anti)estrogenic, (anti)androgenic, and (anti)thyroidal substances, affecting primarily reproduction and development in vertebrates; however, other endocrine systems might be also targeted by ED. Environmental gestagens, including natural progestogens (e.g. progesterone (P4)) and synthetic progestins used for contraception, are supposed to affect vertebrate reproduction via progesterone receptors. In the present paper, we review the current knowledge about gestagenic effects in amphibians, focussing on reproduction and the thyroid system. In addition, we support the literature data with results of recent in vitro experiments, demonstrating direct impacts of the gestagens levonorgestrel (LNG) and P4 on sexually differentiated gonads of larval Xenopus laevis. The results showed a higher susceptibility of female over male gonads to gestagenic ED. Only in female gonads LNG, but not P4, had direct inhibitory effects on gene expression of steroidogenic acute regulatory protein and P450 side chain cleavage enzyme, whereas aromatase expression decreased in reaction to both gestagens. Surprisingly, beyond the expected ED effects of gestagens on reproductive physiology in amphibians, LNG drastically disrupted the thyroid system, which resembles direct effects on thyroid glands and pituitary along the pituitary-thyroid axis disturbing metamorphic development. In amphibians, environmental gestagens not only affect the reproductive system but at least LNG can impact also development by disruption of the thyroid system.

Diclofenac (DCF) is a non-steroidal analgesic and antiphlogistic. Due to its tremendous use, DCF can be found in the environment, especially in sewage, but also surface waters, ground and drinking water. Previous studies indicated that DCF can modulate the reproductive physiology of fish by altering the expression of important key enzymes of the hypothalamus-pituitary-gonad-axis (HPG-axis) and might act as an estrogenic endocrine disrupting chemical (EDC). Other studies, however, demonstrated that DCF does not exhibit any estrogenicity. Thus, in the present study we investigated whether an exposure to DCF can affect reproductive behavior and physiology of adult male X. laevis by analyzing DCF effects on the mate calling behavior of the frogs and on gene expression patterns of key biomarkers of the HPG-axis. In addition, plasma sex steroid levels were determined to gain detailed insights into the mechanisms of DCF action. We could demonstrate that DCF can act as EDC by exhibiting slight estrogenic modes of action. In addition, pharmacological impacts on gonadal steroidogenesis could be revealed leading to imbalances in sex steroid levels and ratios. DCF furthermore altered the calling behavior of exposed males, potentially reducing the mating and reproductive success of the frogs, possibly leading to severe population effects.

Rodent species, such as monogamous and biparental California mice, produce vocalizations as a means of communication. A temporal examination of vocalizations produced by California mice pups in isolation was performed. Pup recordings were performed for 3 min at ∼10.00 and 14.00 hrs on early postnatal days (PND) 2-4, 7, 21, and 28. Once initial recordings were finished, pups were returned to the home cage with parents and any siblings for 5 minutes to determine if active biparental responses resulted in an enhanced vocalization response when pups were isolated and retested. We also sought to determine whether potential reduction in vocalizations by older pups might be due to procedure-habituation procedure associated with less anxiety and/or possibly decreased need for parental care. Vocalizations were measured in weanling (30 days of age) "naïve" pups not previously isolated. Results show older pups took significantly longer to vocalize, indicated by increased latency before producing their initial syllable compared to earlier ages. With increasing age, pups demonstrated decreased syllable duration, reduced number and duration of phrases, and decreased number of syllables per phrase. No differences in pup vocalizations were observed before and after being placed back with parents, suggestive biparental potentiation may not exist in California mice pups. Comparison of the naïve to habituated weanling pups indicated the former group had more total calls but no other differences in vocalization parameters were detected between these 2 groups. Collectively, the findings suggest that as California mice pups mature and approach weaning they generally vocalize less in isolation. (PsycINFO Database Record

Multiple anthropogenic stressors cause worldwide amphibian declines. Among several poorly investigated causes is global pollution of aquatic ecosystems with endocrine disrupting compounds (EDCs). These substances interfere with the endocrine system and can affect the sexual development of vertebrates including amphibians. We test the susceptibility to an environmentally relevant contraceptive, the artificial estrogen 17α-ethinylestradiol (EE2), simultaneously in three deeply divergent systematic anuran families, a model-species, Xenopus laevis (Pipidae), and two non-models, Hyla arborea (Hylidae) and Bufo viridis (Bufonidae). Our new approach combines synchronized tadpole exposure to three EE2-concentrations (50, 500, 5,000 ng/L) in a flow-through-system and pioneers genetic and histological sexing of metamorphs in non-model anurans for EDC-studies. This novel methodology reveals striking quantitative differences in genetic-male-to-phenotypic-female sex reversal in non-model vs. model species. Our findings qualify molecular sexing in EDC-analyses as requirement to identify sex reversals and state-of-the-art approaches as mandatory to detect species-specific vulnerabilities to EDCs in amphibians.

Amphibians are undergoing a global decline. One poorly investigated reason could be the pollution of aquatic habitats by endocrine disrupting compounds (EDCs). We tested the susceptibility to the synthetically stabilized estrogen 17α-ethinylestradiol (EE2) in three deeply diverged anuran species, differing in sex determination systems, types of gonadogenesis and larval ecologies. To understand whether data from the amphibian model Xenopus laevis (Pipidae) are analogous and applicable to only distantly related non-model amphibians, tadpoles of X. laevis, Hyla arborea (Hylidae) and Bufo viridis (Bufonidae) were simultaneously exposed to 50, 500 and 5000ng/L EE2 from hatching until completion of metamorphosis, using a flow-through-system under identical experimental conditions. Comparing molecularly established genetic with histologically assessed phenotypic sex in all species, we have recently shown that EE2 provoked numerous genetic-male-to-phenotypic-female sex reversals and mixed sex individuals, confirming overall its expected feminizing effect. In the present study, we focus on the influence of EE2 on gonadal and somatic development. Anatomy and histology revealed several species-specific effects. In both non-model species, H. arborea and B. viridis, high numbers of anatomically impaired gonads were observed. In H. arborea, exposed to 5000ng/L EE2, numerous underdeveloped gonads were detected. Whereas EE2 did not alter snout-to-vent length and body weight of X. laevis metamorphs, H. arborea showed a treatment-dependent decrease, while B. viridis exhibited an increase in body weight and snout-to-vent length. Apart from a concentration-dependent occurrence of yellowish skin color in several H. arborea, no organ-specific effects were detected. Since EE2 ubiquitously occurs in many aquatic ecosystems and affects sexual and somatic development, among EDCs, it may indeed contribute to amphibian decline. The inter-species variation in developmental EE2-effects corroborates species-specific vulnerability differences towards EDCs between deeply diverged amphibian groups.

p,p'-Dichlordiphenyldichloroethylene (DDE) is a metabolite of the insecticide dichlorodiphenyltrichloroethane (DDT), an organochloride which was massively used from its discovery in 1939 until the early 1970's. Due to the tremendous half-life of DDT and DDE, both substances are to date environmentally relevant. Furthermore, DDT is still employed in many African countries in the context of the WHO's antimalaria campaign. In amphibians, DDE was found to act as antiandrogenic endocrine disrupting chemical (EDC), whereas in other species DDE was found to act as an estrogen. To determine the mode of action (MOA) of DDE in adult male Xenopus laevis, we exposed adult male frogs to different concentrations of DDE, as well as to the estrogenic EDC ethinylestradiol (EE2) and the antiandrogenic fungicide vinclozolin (VIN) for four consecutive nights. We then analyzed the mate calling behavior, which was previously shown to be affected by (anti)androgenic and (anti)estrogenic EDC in a MOA-specific manner, in order to assess whether DDE exposure results in estrogen-specific or antiandrogen-specific alterations of the mate calling behavior. Our results demonstrate that DDE alters the reproductive behavior of male X. laevis. Lowered sexual arousal of exposed males was indicated by a decreased production of advertisement calls and higher amounts of calls that suggest a sexually unaroused state of the males. Our results further indicate that DDE can display both, estrogenic and antiandrogenic MOA, either of which can have adverse effects on reproductive physiology and behavior in X. laevis. The disruption of the affected mating behavior, which is crucial for a successful reproduction, might result in a reduced reproductive success of DDE exposed animals.

Different chemical substances, which enter the environment due to anthropogenic influences, can affect the endocrine system and influence development and physiology of aquatic animals. One of these endocrine disrupting chemicals is the synthetic estrogen, 17α-ethinylestradiol (EE2), which is a main component of various oral contraceptives and demonstrably affects many different aquatic vertebrates at extremely low concentrations by feminization phenomena. The aim of the present study was to investigate whether a four week exposure to three different concentrations of EE2 (0.3 ng/L, 29.6 ng/L and 2960 ng/L) affects the catabolism of hemoglobin of the amphibian Xenopus laevis. The results of this study demonstrate for the first time that beside an increase of the hepatic vitellogenin gene expression, exposure to EE2 also decreases the gene expression of the hepatic heme oxygenase 1 and 2 (HO1, HO2), degrading heme of different heme proteins to biliverdin, as well as of the biliverdin reductase A (BLVRA), which converts biliverdin to bilirubin. The results further suggest that EE2 already at the environmentally relevant concentration of (29.6 ng/L) can disrupt hemoglobin catabolism, indicated by decreased gene expression of HO2, which becomes evident at the highest EE2 concentration that led to a severe increase of biliverdin in plasma.

Various synthetic chemicals released to the environment can interfere with the endocrine system of vertebrates. Many of these endocrine disrupting compounds (EDCs) exhibit estrogenic activity and can interfere with sexual development and reproductive physiology. More recently, also chemicals with different modes of action (MOAs), such as antiestrogenic, androgenic and antiandrogenic EDCs, have been shown to be present in the environment. However, to date EDC-research primarily focuses on exposure to EDCs with just one MOA, while studies examining the effects of simultaneous exposure to EDCs with different MOAs are rare, although they would reflect more real, natural exposure situations. In the present study the combined effects of estrogenic and antiestrogenic EDCs were assessed by analyzing the calling behavior of short-term exposed male Xenopus laevis. The estrogenic 17α-ethinylestradiol (EE2), and the antiestrogenic EDCs tamoxifen (TAM) and fulvestrant (ICI) were used as model substances. As previously demonstrated, sole EE2 exposure (10-10 M) resulted in significant alterations of the male calling behavior, including altered temporal and spectral parameters of the advertisement calls. Sole TAM (10-7 M, 10-8 M, 10-10 M) or ICI (10-7 M) exposure, on the other hand, did not affect any of the measured parameters. If frogs were co-exposed to EE2 (10-10 M) and TAM (10-7 M) the effects of EE2 on some parameters were abolished, but co-exposure to EE2 and ICI (10-7 M) neutralized all estrogenic effects. Thus, although EDCs with antiestrogenic MOA might not exhibit any effects per se, they can alter the estrogenic effects of EE2. Our observations demonstrate that there is need to further investigate the combined effects of EDCs with various, not only opposing, MOAs as this would reflect realistic wildlife situations.

The main component of classical contraceptives, 17α-ethinylestradiol (EE2), has high estrogenic activity even at environmentally relevant concentrations. Although estrogenic endocrine disrupting compounds are assumed to contribute to the worldwide decline of amphibian populations by adverse effects on sexual differentiation, evidence for EE2 affecting amphibian mating behaviour is lacking. In this study, we demonstrate that EE2 exposure at five different concentrations (0.296 ng/L, 2.96 ng/L, 29.64 ng/L, 2.96 µg/L and 296.4 µg/L) can disrupt the mating behavior of adult male Xenopus laevis. EE2 exposure at all concentrations lowered male sexual arousal, indicated by decreased proportions of advertisement calls and increased proportions of the call type rasping, which characterizes a sexually unaroused state of a male. Additionally, EE2 at all tested concentrations affected temporal and spectral parameters of the advertisement calls, respectively. The classical and highly sensitive biomarker vitellogenin, on the other hand, was only induced at concentrations equal or higher than 2.96 µg/L. If kept under control conditions after a 96 h EE2 exposure (2.96 µg/L), alterations of male advertisement calls vanish gradually within 6 weeks and result in a lower sexual attractiveness of EE2 exposed males toward females as demonstrated by female choice experiments. These findings indicate that exposure to environmentally relevant EE2 concentrations can directly disrupt male mate calling behavior of X. laevis and can indirectly affect the mating behavior of females. The results suggest the possibility that EE2 exposure could reduce the reproductive success of EE2 exposed animals and these effects might contribute to the global problem of amphibian decline.