The Occurrence of Intersex in Different Populations of the Marine Amphipod Echinogammarus marinus in North-West Brittany - A Longterm-Study.

In the past two decades, an increasing body of studies has been published on the intersex phenomenon in separate-sexed crustaceans from marine and freshwater ecosystems. Various causes are being considered that could have an influence on the occurrence of intersex. Besides genetic factors, environmental conditions such as photoperiodicity, temperature, salinity and parasitism, but also environmental pollution with endocrine disrupting chemicals (EDCs) are discussed. As part of a long-term monitoring (2012 - 2020) in north-west Brittany, we recorded the occurrence of intersex in the marine amphipod Echinogammarus marinus. We quantified the intersex incidence at marine and estuarine sites and analyzed the incidence in relation to the endocrine potential of the sediments. Intersex occurred with mean frequencies between 0.87% and 12%. It was striking that the incidence of intersex increased with increasing distance from the sea. Since the highest incidence was observed at the range boundary of this stenohaline species, we assume that intersex is triggered by endocrine potential and increasing stress due to increasing freshwater content - and thus an interplay of different environmental factors.

The CPCAT as a novel tool to overcome the shortcomings of NOEC/LOEC statistics in ecotoxicology: a simulation study to evaluate the statistical power.

Species reproduction is an important determinant of population dynamics. As such, this is an important parameter in environmental risk assessment. The closure principle computational approach test (CPCAT) was recently proposed as a method to derive a NOEC/LOEC for reproduction count data such as the number of juvenile Daphnia. The Poisson distribution used by CPCAT can be too restrictive as a model of the data-generating process. In practice, the generalized Poisson distribution could be more appropriate, as it allows for inequality of the population mean µ and the population variance σ 2 . It is of fundamental interest to explore the statistical power of CPCAT and the probability of determining a regulatory relevant effect correctly. Using a simulation, we varied between Poisson distribution ( µ = σ 2 ) and generalized Poisson distribution allowing for over-dispersion ( µ < σ 2 ) and under-dispersion ( µ > σ 2 ). The results indicated that the probability of detecting the LOEC/NOEC correctly was ≥ 0.8 provided the effect was at least 20% above or below the mean level of the control group and mean reproduction of the control was at least 50 individuals while over-dispersion was missing. Specifically, under-dispersion increased, whereas over-dispersion reduced the statistical power of the CPCAT. Using the well-known Hampel identifier, we propose a simple and straight forward method to assess whether the data-generating process of real data could be over- or under-dispersed.

Effects of ivermectin application on the diversity and function of dung and soil fauna: Regulatory and scientific background information.

The application of veterinary medical products to livestock can impact soil organisms in manure-amended fields or adversely affect organisms that colonize dung pats of treated animals and potentially retard the degradation of dung on pastures. For this reason, the authorization process for veterinary medicinal products in the European Union includes a requirement for higher-tier tests when adverse effects on dung organisms are observed in single-species toxicity tests. However, no guidance documents for the performance of higher-tier tests are available. Hence, an international research project was undertaken to develop and validate a proposed test method under varying field conditions of climate, soil, and endemic coprophilous fauna at Lethbridge (Canada), Montpellier (France), Zurich (Switzerland), and Wageningen (The Netherlands). The specific objectives were to determine if fecal residues of an anthelmintic with known insecticidal activity (ivermectin) showed similar effects across sites on 1) insects breeding in dung of treated animals, 2) coprophilous organisms in the soil beneath the dung, and 3) rates of dung degradation. By evaluating the effects of parasiticides on communities of dung-breeding insects and soil fauna under field conditions, the test method meets the requirements of a higher-tier test as mandated by the European Union. The present study provides contextual information on authorization requirements for veterinary medicinal products and on the structure and function of dung and soil organism communities. It also provides a summary of the main findings. Subsequent studies on this issue provide detailed information on different aspects of this overall project. Environ Toxicol Chem 2016;35:1914-1923. © 2015 SETAC.

Identification, assessment and management of "endocrine disruptors" in wildlife in the EU substance legislation--discussion paper from the German Federal Environment Agency (UBA).

A discussion paper was developed by a panel of experts of the German Federal Environment Agency (UBA) contributing to the on-going debate on the identification, assessment and management of endocrine disruptors with a view to protect wildlife according to the EU substance legislation (plant protection products, biocides, industrial chemicals). Based on a critical synthesis of the state-of-the-art regarding regulatory requirements, testing methods, assessment schemes, decision-making criteria and risk management options, we advise an appropriate and consistent implementation of this important subject into existing chemicals legislation in Europe. Our proposal for a balanced risk management of endocrine disruptors essentially advocates transparent regulatory decision making based on a scientifically robust weight of evidence approach and an adequate risk management consistent across different legislations. With respect to the latter, a more explicit consideration of the principle of proportionality of regulatory decision making and socio-economic benefits in the on-going debate is further encouraged.

New test strategy for dung beetles during the authorization process of parasiticides.

According to European legislation, an environmental risk assessment (ERA) of veterinary medicinal products (VMPs) for dung fauna is required in the authorization process, if the substance acts as a parasiticide for the treatment of pasture animals. In the past, however, the demonstration of the environmental safety of those VMPs for dung fauna was strongly hampered by the fact that no standardized tests were available. Therefore, starting with recommendations from the Society of Environmental Toxicology and Chemistry (SETAC) advisory group, dung organism toxicity test standardization (DOTTS) test systems for phase II Tier A standardized tests on the mortality of dung fly and dung beetle larvae were developed and published by the Organisation for Economic Co-operation and Development (OECD) in 2008 and 2010. If a risk is identified for dung organisms in phase II Tier A of the ERA process, further tests are required for Tier B. So far, however, no advice is given for such studies in the existing guidelines. Therefore, 4 workshops took place between 2007 and 2009 with international dung fauna experts (Aveiro-Group) to find an appropriate test strategy for dung fauna organisms beyond Tier A mortality testing. For the first time, 2 different Tier B extended laboratory test approaches for dung beetles and test strategies for scenarios beyond Tier B are described in more detail. In case the risk assessment is still not clear, further options for Tier C (i.e., field studies) or Tier D (modeling) are briefly presented. Finally, the role of uncertainty and variability of test results is discussed, including recommendations for assessment factors for the different tiers to be used when assessing the risk of VMPs on dung organisms. The approach, especially the test strategy, will help industry, consultants, and assessors appropriately assess the environmental risk assessments during the authorization procedure of parasiticides.

Toxicity of the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin to photoautotrophic aquatic organisms.

The present study investigated the growth inhibition effect of the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin on four photoautotrophic aquatic species: the freshwater microalga Desmodesmus subspicatus, the cyanobacterium Anabaena flos-aquae, the monocotyledonous macrophyte Lemna minor, and the dicotyledonous macrophyte Myriophyllum spicatum. Both antibiotics, which act by inhibiting the bacterial DNA gyrase, demonstrated high toxicity to A. flos-aquae and L. minor and moderate to slight toxicity to D. subspicatus and M. spicatum. The cyanobacterium was the most sensitive species with median effective concentration (EC50) values of 173 and 10.2 µg/L for enrofloxacin and ciprofloxacin, respectively. Lemna minor proved to be similarly sensitive, with EC50 values of 107 and 62.5 µg/L for enrofloxacin and ciprofloxacin, respectively. While enrofloxacin was more toxic to green algae, ciprofloxacin was more toxic to cyanobacteria. Calculated EC50s for D. subspicatus were 5,568 µg/L and >8,042 µg/L for enrofloxacin and ciprofloxacin, respectively. These data, as well as effect data from the literature, were compared with predicted and reported environmental concentrations. For two of the four species, a risk was identified at ciprofloxacin concentrations found in surface waters, sewage treatment plant influents and effluents, as well as in hospital effluents. For ciprofloxacin the results of the present study indicate a risk even at the predicted environmental concentration. In contrast, for enrofloxacin no risk was identified at predicted and measured concentrations.

Triphenyltin alters lipid homeostasis in females of the ramshorn snail Marisa cornuarietis.

Molluscs are sensitive species to the toxic effects of organotin compounds, particularly to masculinisation. Both tributyltin (TBT) and triphenyltin (TPT) have been recently shown to bind to mollusc retinoid X receptor (RXR). If RXR is involved in lipid homeostasis, exposure to TPT would have an immediate effect on endogenous lipids. To test this hypothesis, the ramshorn snail Marisa cornuarietis was exposed to environmentally relevant concentrations of TPT (30, 125, 500 ng/L as Sn) in a semi-static water regime for 7 days. Percentage of lipids and total fatty acid content decreased significantly in TPT-exposed females while the activity of peroxisomal acyl-CoA oxidase, involved in fatty acid catabolism, increased. In addition, fatty acid profiles (carbon chain length and unsaturation degree) were significantly altered in exposed females but not in males. This work highlights the ability of TPT to disrupt lipid metabolism in M. cornuarietis at environmentally realistic concentrations and the higher susceptibility of females in comparison to males.

Enzyme-triggered and self-cleaving fragrant alcohol precursors.

The high volatility and water solubility of many natural perfumery alcohols leads to their rapid loss in fabric-care and personal-care applications. A dramatically enhanced substantivity is achieved by the use of fragrance precursors as controlled-release systems. In the first part of this article, we present multi-odorant precursors, in which the enzymatic cleavage of esters or carbonates of fragrant alcohols triggers subsequent steps leading to the release of fragrant ketones, lactones, and additional fragrant alcohols. In the second part, a study on oligocarbonates of fragrant alcohols is presented. Therein, the outstanding enzyme-independent performance of gluconolactone oligocarbonate 27 for the long-lasting release of (Z)-hex-3-en-1-ol is highlighted. We show that these polyfunctional compounds undergo complex rearrangements and intramolecular substitution reactions which lead to the observed release kinetics.

Prosobranch snails as test organisms for the assessment of endocrine active chemicals--an overview and a guideline proposal for a reproduction test with the freshwater mudsnail Potamopyrgus antipodarum.

Recently, prosobranch snails have been recommended as promising candidates for test organisms for the assessment of endocrine active chemicals. Three prosobranch snail species, the freshwater mudsnail Potamopyrgus antipodarum, the freshwater ramshorn snail Marisa cornuarietis, and the marine netted whelk Nassarius reticulatus are portrayed and their respective biotests are presented together with results of laboratory experiments and biological effect monitoring surveys in the field. All characterized species are highly sensitive toward xeno-androgens [triphenyltin (TPT), tributyltin (TBT), methyltestosterone (MT) and fenarimol (FEN)], and xeno-estrogens [bisphenol A (BPA), octylphenol (OP), ethinylestradiol], and show effects at environmentally relevant, rather low concentrations in laboratory experiments. For exposure to the xeno-androgen TPT, EC(10) values range between 15.9 and 29.0 ng as Sn/L (sediment 0.03 mug as Sn/kg), for TBT, EC(10) values are found between 3.42 and 37.8 ng as Sn/L (sediment 2.98 microg as Sn/kg) and effect concentrations for FEN are calculated as 18.6 ng/L (EC(10)) and 0.19 microg/kg (EC(50) sediment; EC(10) not calculable). Exposure to xeno-estrogens yielded EC(10 )values of 13.9 ng/L (0.19 microg/kg) for BPA, a NOEC of <1 microg/L (EC(10) of 0.004 microg/kg) for OP and a NOEC of 1 ng/l (EC(10) sediment of 2.2 microg/kg) for ethinylestradiol. Responses to androgens comprised the development of imposex and the reduction of fertility or embryo production, effects of estrogens included the stimulation of egg production and embryo production, and the increased weight of glands. Also, biological effect monitoring studies with P. antipodarum and N. reticulatus in several rivers or estuarine areas revealed the capacity of the biotests to detect an androgenic or estrogenic potential of sediment samples. A comparison of the three test species with regard to sensitivity and practical aspects in routine application favors the freshwater mudsnail P. antipodarum for a standardized procedure, and this reproduction test will be introduced into the OECD guideline program for standardization in the near future.

COMPRENDO: Focus and approach.

Tens of thousands of man-made chemicals are in regular use and discharged into the environment. Many of them are known to interfere with the hormonal systems in humans and wildlife. Given the complexity of endocrine systems, there are many ways in which endocrine-disrupting chemicals (EDCs) can affect the body's signaling system, and this makes unraveling the mechanisms of action of these chemicals difficult. A major concern is that some of these EDCs appear to be biologically active at extremely low concentrations. There is growing evidence to indicate that the guiding principle of traditional toxicology that "the dose makes the poison" may not always be the case because some EDCs do not induce the classical dose-response relationships. The European Union project COMPRENDO (Comparative Research on Endocrine Disrupters--Phylogenetic Approach and Common Principles focussing on Androgenic/Antiandrogenic Compounds) therefore aims to develop an understanding of potential health problems posed by androgenic and antiandrogenic compounds (AACs) to wildlife and humans by focusing on the commonalities and differences in responses to AACs across the animal kingdom (from invertebrates to vertebrates) .

Bisphenol A induces superfeminization in the ramshorn snail Marisa cornuarietis(Gastropoda: Prosobranchia) at environmentally relevant concentrations.

Previous investigations have shown that bisphenol A (BPA) induces a superfeminization syndrome in the freshwater snail Marisa cornuarietis at concentrations as low as 1 microg/L. Superfemales are characterized by the formation of additional female organs, enlarged accessory sex glands, gross malformations of the pallial oviduct, and a stimulation of egg and clutch production, resulting in increased female mortality. However, these studies were challenged on the basis of incomplete experimentation. Therefore, the objective of the current approach was to bridge several gaps in knowledge by conducting additional experiments. In an initial series of experiments, study results from the reproductive phase of the snails were evaluated in the sub-micrograms per liter range. Before and after the spawning season, superfemale responses were observed [NOEC (no observed effect concentration) 7.9 ng/L, EC10 (effective concentration at 10%) 13.9 ng/L], which were absent during the spawning season. A further experiment investigated the temperature dependence of BPA responses by exposing snails at two temperatures in parallel. The adverse effect of BPA was at least partially masked at 27 degrees C (EC10 998 ng/L) when compared with 20 degrees C (EC10 14.8 ng/L). In M. cornuarietis, BPA acts as an estrogen receptor (ER) agonist, because effects were completely antagonized by a co-exposure to tamoxifen and Faslodex. Antiandrogenic effects of BPA, such as a significant decrease in penis length at 20 degrees C, were also observed. Competitive receptor displacement experiments indicate the presence of androgen- and estrogen-specific binding sites. The affinity for BPA of the estrogen binding sites in M. cornuarietis is higher than that of the ER in aquatic vertebrates. The results emphasize that prosobranchs are affected by BPA at lower concentrations than are other wildlife groups, and the findings also highlight the importance of exposure conditions.

Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: the effect of xenoandrogenic compounds.

Molluscs can conjugate a variety of steroids to form fatty acid esters. In this work, the freshwater ramshorn snail Marisa cornuarietis was used to investigate sex differences in endogenous levels of esterified steroids. Testosterone and estradiol were mainly found in the esterified form in the digestive gland/gonad complex of M. cornuarietis, and males had higher levels of esterified steroids than females (4-10-fold). Additionally, the ability of several xenobiotics, namely tributyltin (TBT), methyltestosterone (MT) and fenarimol (FEN) to interfere with the esterification of testosterone and estradiol was investigated. All three compounds induced imposex - appearance of male sexual characteristics in females. Exposure to TBT led to a decrease in both esterified testosterone (60-85%) and estradiol (16-53%) in females after 100 days exposure, but had no effect on males. Exposure to FEN and MT did not alter levels of esterified steroids in males or in females, although exposed females developed imposex after 150 days exposure. The decrease in esterified steroids by TBT could not be directly linked with a decrease in microsomal acyl-CoA:testosterone acyltransferase (ATAT) activity, which catalyzes the esterification of steroids. In fact, ATAT activity was marginally induced in organisms exposed to TBT for 50 days (1.3-fold), and significantly induced in males and females exposed to MT for 50 days (1.8- and 1.5-fold, respectively), whereas no effect on ATAT activity was observed after 150 days exposure.

Evidence for endocrine disruption in invertebrates.

The issue of endocrine disruption (ED) in invertebrates has generated remarkably little interest in the past compared to research with aquatic vertebrates in this area. However, with more than 95% of all known species in the animal kingdom, invertebrates constitute a very important part of the global biodiversity with key species for the structure and function of aquatic and terrestrial ecosystems. Despite the fact that ED in invertebrates has been investigated on a smaller scale than in vertebrates, invertebrates provide some of the best documented examples for deleterious effects in wildlife populations following an exposure to endocrine-active substances. The article provides an overview of the diversity in endocrine systems of invertebrates. The principal susceptibility of invertebrates to endocrine-active compounds is demonstrated with the case studies of tributyltin effects in mollusks and of insect growth regulators, the latter as purposely synthesized endocrine disrupters. The additional evidence for ED in invertebrates from laboratory and field studies is summarized as an update and amendment of the EDIETA report from 1998. Finally, conclusions about the scale and implications of the observed effects are drawn and research needs are defined.