Tim Verslycke

The toxicity of metal mixtures to the estuarine mysid Neomysis integer (Crustacea: Mysidacea) under changing salinity

Water quality criteria are mainly based on data obtained in toxicity tests with single toxicants. Several authors have demonstrated that this approach may be inadequate as the joint action of the chemicals is not taken into account. In this study, the combined effects of six metals on the European estuarine mysid Neomysis integer (Leach, 1814) were examined. Acute 96-h toxicity tests were performed with mercury, copper, cadmium, nickel, zinc and lead, and this as single compounds and as a mixture of all six. The concentrations of the individual metals of the equitoxic mixtures were calculated using the concentration-addition model. The 96-h LC50s for the single metals, at a salinity of 5 per thousand, ranged from 6.9 to 1140 microg/l, with the following toxicity ranking: Hg>Cd>Cu>Zn>Ni>Pb. Increasing the salinity from 5 to 25 per thousand resulted in lower toxicity and lower concentrations of the free ion (as derived from speciation calculations) for all metals. This salinity effect was strongest for cadmium and lead and could be attributed to complexation with chloride ions. The toxicity of nickel, copper and zinc was affected to a smaller extent by salinity. The 96-h LC50 for mercury was the same for both salinities. In order to evaluate the influence of changing salinity conditions on the acute toxicity of metal mixtures, tests were performed at different salinities (5, 10, 15 and 25 per thousand ). The 96-h LC50 value (1.49 T.U.) of the metal mixture, at a salinity of 5 per thousand, was clearly lower than the expected value (6 T.U.) based on the non-additive hypothesis, thus confirming the additive effect of these metals in the marine/estuarine environment. Changing salinity had a profound effect on the toxicity of the mixture. The toxicity clearly decreased with increasing salinity until 15 per thousand. Higher salinities (25 per thousand ) had no further influence on the 96-h LC50 of the mixture which is situated at a value between 4.4 and 4.6. Finally, the relative sensitivity to the selected metals was compared with the relative sensitivity of the commonly used mysid Americamysis (=Mysidopsis) bahia.

Induction of vitellogenesis in 17α-ethinylestradiol-exposed rainbow trout (Oncorhynchus mykiss): a method comparison

Juvenile rainbow trout, Oncorhynchus mykiss, were exposed to the synthetic estrogen 17alpha-ethinylestradiol (EE(2)) through injection (1, 10, 25 and 50 microg EE(2)/g fish/week) and via water exposure (1, 10 and 100 ng EE(2)/l). After seven (injection and water exposure) and 14 days (only for water exposure), blood and plasma vitellogenin concentrations were quantified using indirect endpoints, i.e. plasma alkaline-labile phosphorus (ALP), plasma protein and plasma calcium. In addition, the relative gonad (GSI) and liver weight (HSI) were recorded. Actual plasma vitellogenin concentrations were measured with an enzyme immunoassay. Only fish injected with 50 microg EE(2)/g fish had a significantly higher gonad weight. No concentration-dependent changes in the HSI were detected in fish exposed via the water, but a significant dose-dependent increase of the HSI was observed in fish injected with EE(2). Exposure of rainbow trout to EE(2) had a significant effect on all tested plasma parameters. Plasma protein, phosphoprotein and calcium concentrations were significantly higher after two weeks exposure to 100 ng EE(2)/l. Fish injected with 10, 25 and 50 microg EE(2)/g fish exhibited increased plasma protein concentrations after 1 week. Compared to the controls, plasma ALP and calcium levels were significantly higher in all injected fish. A significant and positive correlation was observed between all three plasma parameters and between these indirect parameters and the actual plasma vitellogenin concentrations. These findings indicate that both the plasma ALP and the plasma calcium assay have a similar sensitivity as that of available antibody-based assays (EIA), at least in EE(2) exposure studies, and thus these assays can provide a rapid, simple and cost-effective alternative to available immunoassays.

Effects of 17α-ethinylestradiol on sexual development of the amphipod Hyalella azteca

The effects of the synthetic estrogen 17a-ethinylestradiol (EE) on sexual development of the freshwater amphipod Hyalella azteca was investigated. Organisms were exposed in a multigeneration experiment to EE concentrations ranging from 0.1 to 10mg/L and the development of both external and internal sexual characteristics were studied. Second-generation male H. azteca exposed from gametogenesis until adulthood to 0.1 and 0.32mg EE/L developed significantly smaller second gnathopods. The sex ratio of the populations exposed to EE for more than two generations tended, although not statistically significantly, to be in favor of females. Histological aberrations of the reproductive tract, i.e., indications of hermaphroditism, disturbed maturation of the germ cells, and disturbed spermatogenesis, of post-F1-generation males were observed in all EE exposures. These findings provide evidence that sexual development of H. azteca is affected by exposure to sublethal concentrations of EE. r 2002 Elsevier Science (USA). All rights reserved.

Effects of a changing abiotic environment on the energy metabolism in the estuarine mysid shrimp Neomysis integer (Crustacea: Mysidacea)

Adaptations to life in an estuary include a wide salinity tolerance, an extremely efficient osmoregulatory and respiratory physiology. These adaptive mechanisms are energy-consuming and relatively little data is available on the combined effects of abiotic stress factors on the energy metabolism of mysid shrimp. A new methodology (cellular energy allocation, CEA) to assess the energy budget was adopted for the estuarine crustacean Neomysis integer (Crustacea: Mysidacea). The biochemical composition of N. integer was determined: protein (7.39F1.81% wet weight), lipid (3.99F1.05% ww) and sugar (0.42F0.18% ww). To assess the effect of natural variability on the energy metabolic processes in N. integer, a fractional factorial test design was set up with different naturally (Westerscheldt estuary, The Netherlands) occurring combinations of temperature, salinity and dissolved oxygen. The different abiotic factors had no significant effect on the energy metabolism of N. integer within the tested range. Temperature explained the decrease in lipid, protein and total energy reserves. Temperature, in general, had the most adverse effect on the CEA. Salinity was the most important factor explaining the effects on sugar reserves, with higher salinities causing an increased sugar demand. By modeling the influence of these abiotic stresses on the energy metabolism (CEA) of N. integer, it will be possible to use the CEA as an ecologically relevant biomarker of exposure to pollutants in estuaries. D 2002 Elsevier Science B.V. All rights reserved.

Cellular energy allocation in the estuarine mysid shrimp Neomysis integer (Crustacea: Mysidacea) following tributyltin exposure

Recently, we described the cellular energy allocation (CEA) methodology to asses the effects of abiotic stress on the energy metabolism of the estuarine crustacean Neomysis integer (Crustacea: Mysidacea) [J. Exp. Mar. Biol. Ecol. 279 (2002) 61]. This short-term assay is based on the biochemical assessment of changes in the energy reserves (total carbohydrate, protein and lipid content) and the energy consumption (electron transport activity), and has been shown to be predictive of effects at the population level in daphnids [J. Aquat. Ecosyst. Stress Recovery 6 (1997) 43]. In the present study, the CEA methodology was evaluated using adult N. integer exposed for 96 h to the antifoulant tributyltinchloride (TBTCl). From a range-finding experiment with juvenile N. integer, a 96-h LC50 of 164 ng TBTCl/l was calculated. The energy metabolism of N. integer, as summarized by the CEA, was significantly altered by TBTCl exposure. Mysids exposed to 10, 100 and 1000 ng TBTCl/l consumed less energy and had lower respiration rates (in 10 and 1000 ng TBTCl/l treatments) than the control, resulting in a lower CEA. These changes at the cellular level occurred at environmentally relevant concentrations of the toxicant TBTCl which were an order of magnitude lower than reported effect concentrations for scope for growth in other marine invertebrates.

Mysid crustaceans as potential test organisms for the evaluation of environmental endocrine disruption: a review

Anthropogenic chemicals that disrupt the hormonal systems (endocrine disruptors) of wildlife species recently have become a widely investigated and politically charged issue. Invertebrates account for roughly 95% of all animals, yet surprisingly little effort has been made to understand their value in signaling potential environmental endocrine disruption. This omission largely can be attributed to the high diversity of invertebrates and the shortage of fundamental knowledge of their endocrine systems. Insects and crustaceans are exceptions and, as such, appear to be excellent candidates for evaluating the environmental consequences of chemically induced endocrine disruption. Mysid shrimp (Crustacea: Mysidacea) may serve as a viable surrogate for many crustaceans and have been put forward as suitable test organisms for the evaluation of endocrine disruption by several researchers and regulatory bodies (e.g., the U.S. Environmental Protection Agency). Despite the long-standing use of mysids in toxicity testing, little information exists on their endocrinology, and few studies have focused on the potential of these animals for evaluating the effects of hormone-disrupting compounds. Therefore, the question remains as to whether the current standardized mysid endpoints can be used or adapted to detect endocrine disruption, or if new procedures must be developed, specifically directed at evaluating hormone-regulated endpoints in these animals. This review summarizes the ecological importance of mysids in estuarine and marine ecosystems, their use in toxicity testing and environmental monitoring, and their endocrinology and important hormone-regulated processes to highlight their potential use in assessing environmental endocrine disruption.

Ecdysteroid receptor from the American lobster Homarus americanus: EcR/RXR isoform cloning and ligand-binding properties.

In arthropods, ecdysteroids regulate molting by activating a heterodimer formed by the ecdysone receptor (EcR) and retinoid X receptor (RXR). While this mechanism is similar in insects and crustaceans, variation in receptor splicing, dimerization and ligand affinity adds specificity to molting processes. This study reports the EcR and RXR sequences from American lobster, a commercially and ecologically important crustacean. We cloned two EcR splice variants, both of which specifically bind ponasterone A, and two RXR variants, both of which enhance binding of ponasterone A to the EcR. Lobster EcR has high affinity for ponasterone A and muristerone and moderately high affinity for the insecticide tebufenozide. Bisphenol A, diethyl phthalate, and two polychlorinated biphenyls (PCB 29 and PCB 30), environmental chemicals shown to interfere with crustacean molting, showed little or no affinity for lobster EcR. These studies establish the molecular basis for investigation of lobster ecdysteroid signaling and signal disruption by environmental chemicals.

Testosterone and energy metabolism in the estuarine mysid Neomysis integer (Crustacea: Mysidacea) following exposure to endocrine disruptors

A diverse set of reference compounds suspected of having an endocrine-disrupting mode of action (i.e., testosterone, flutamide, ethinylestradiol, precocene, nonylphenol, fenoxycarb, and methoprene) were tested for acute toxicity to the estuarine mysid Neomysis integer (Crustacea: Mysidacea). Neomysis integer was very sensitive to all tested compounds, with 96-h median lethal concentrations in a narrow range between 0.32 and 1.95 mg/L. The pesticides methoprene and fenoxycarb, both synthetic insect juvenile hormone analogs, were most toxic to N. integer. In addition, the short-term sublethal effects of methoprene and nonylphenol (an estrogen agonist) on the energy and steroid metabolism of N. integer were evaluated. Both compounds significantly affected energy and testosterone metabolism of N. integer at concentrations below acute toxicity levels. Energy consumption in methoprene- and nonylphenol-exposed mysids was significantly induced at 100 microg/L, resulting in a lower cellular energy allocation in these animals. Testosterone phase I metabolism was affected at 10 microg/L, whereas glycosylation was the most important phase II pathway affected in mysids exposed to 100 microg/L of both compounds. Methoprene exposure resulted in a concentration-dependent increase in the metabolic androgenization ratio. Mysids exposed to nonylphenol at 10 microg/L had a significantly higher metabolic androgenization ratio. The present study indicates that energy and testosterone metabolism of mysids, as endpoints, are able to detect endocrine-disruptive activity of chemicals after short-term exposure to environmentally realistic levels of endocrine disruptors.

Alternative to vertebrate animal experiments in the study of metabolism of illegal growth promotors and veterinary drugs

Abstract The continuous production of new illegal veterinary drugs and related products requires residue laboratories to initiate research into developing fast and accurate extraction and detection methods for the identification (and/or quantification) of the major analyte or metabolites of these compounds. In practice, animal experiments are carried out in which vertebrate animals (bovine, porcine, …) are treated orally or intramuscularly with the illegal compound. Different matrices (urine, faeces, blood) are collected over 2 or 3 weeks until the animal is sacrificed. Edible matrices (meat, liver, kidney,…) are collected. Because of the complexity of the animal experiment and the method development, a lot of valuable time and money is consumed. Recent studies have shown that some of these vertebrate experiments can be replaced by invertebrates metabolism studies. Vertebrate-type steroids such as testosterone have been used as substrates to study enzyme systems (cytP450) for the oxidative metabolism in invertebrates. Results from these studies provide information on the degree of similarity to the enzyme systems in vertebrates. These findings are of great importance to the research of illegally used substances but also to the downscaling of vertebrate animal experiments and their considerable cost factors. The invertebrate Neomysis integer (Crustacea, Mysidacea) has been used as an alternative model for the partial replacement of vertebrate animals in metabolism studies with illegal growth promotors and veterinary drugs. The principle of this assay and some examples are described.

Recommended approaches to the scientific evaluation of ecotoxicological hazards and risks of endocrine-active substances

A SETAC Pellston Workshop® “Environmental Hazard and Risk Assessment Approaches for Endocrine-Active Substances (EHRA)” was held in February 2016 in Pensacola, Florida, USA. The primary objective of the workshop was to provide advice, based on current scientific understanding, to regulators and policy makers; the aim being to make considered, informed decisions on whether to select an ecotoxicological hazard- or a risk-based approach for regulating a given endocrinedisrupting substance (EDS) under review. The workshop additionally considered recent developments in the identification of EDS. Case studies were undertaken on 6 endocrine-active substances (EAS—not necessarily proven EDS, but substances known to interact directly with the endocrine system) that are representative of a range of perturbations of the endocrine system and considered to be data rich in relevant information at multiple biological levels of organization for 1 or more ecologically relevant taxa. The substances selected were 17α-ethinylestradiol, perchlorate, propiconazole, 17β-trenbolone, tributyltin, and vinclozolin. The 6 case studies were not comprehensive safety evaluations but provided foundations for clarifying key issues and procedures that should be considered when assessing the ecotoxicological hazards and risks of EAS and EDS. The workshop also highlighted areas of scientific uncertainty, and made specific recommendations for research and methods-development to resolve some of the identified issues. The present paper provides broad guidance for scientists in regulatory authorities, industry, and academia on issues likely to arise during the ecotoxicological hazard and risk assessment of EAS and EDS. The primary conclusion of this paper, and of the SETAC Pellston Workshop on which it is based, is that if data on environmental exposure, effects on sensitive species and life-stages, delayed effects, and effects at low concentrations are robust, initiating environmental risk assessment of EDS is scientifically sound and sufficiently reliable and protective of the environment. In the absence of such data, assessment on the basis of hazard is scientifically justified until such time as relevant new information is available.