Assessing the toxicity and teratogenicity of pond water in north-central minnesota to amphibians

BACKGROUND: Incidence of amphibian deformities have increased in recent years, especially in the northern region of the United States. While many factors have been proposed as being responsible for generating deformities (e.g., contaminants, ultraviolet radiation [UV], parasites), no single cause has been definitively established. METHODS: To determine whether waterborne chemicals are responsible for amphibian deformities in ponds in north-central Minnesota, we deployed semipermeable membrane devices (SPMDs) in an impacted and a reference site to accumulate lipophilic contaminants. We then exposed native tadpoles (northern leopard frogs; Rana pipiens) to the SPMD extracts combined with two agricultural pesticides (atrazine, carbaryl) at two levels of UV radiation. RESULTS AND DISCUSSION: UV radiation alone caused a slight increase in hatching success and tadpole growth rate. Deformity rate among hatchlings was high following exposure to SPMD extracts from the reference site in the absence of UV, suggesting that chemicals present at this site are broken down by UV to less harmful forms, or become less bioavailable. Conversely, impacted site SPMD extracts caused hatchling deformities only in the presence of UV, suggesting that UV potentiates the teratogenicity of the compounds present there. Impacted site SPMD extracts significantly increased the number of bony triangles among metamorphs, a common deformity observed at this site. The incidence of skin webbings increased significantly with SPMD extracts from both sites as well as with our pesticide control containing atrazine and carbaryl alone. CONCLUSIONS: Higher deformity rates among tadpoles reared in the presence of UV radiation and SPMD extracts from sites where deformities are common indicates a chemical compound (or compounds) in the water at this site may be causing the deformities. RECOMMENDATIONS AND OUTLOOK: It is important to examine the effects of chemical stressors in the presence of other natural stressors (e.g., UV radiation) to gain a better understanding of how multiple stressors work to impact amphibians and amphibian populations.     

Environmental signals: synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis

According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS1500 was applied. The in vivo results showed that HS1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSHbeta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals.     

Inter- and intra-specific variation on sensitivity of larval amphibians to nitrite

Several authors have suggested that nitrogen-based fertilizers may be contributing to the global amphibian decline. We have studied the impact of sodium nitrite on early aquatic stages of Epidalea calamita, Pelophylax perezi and Hyla meridionalis larvae from Do?ana National Park (coastal wetland) and P. perezi from Gredos Mountain (high mountain ponds), exposed during 10 to 16 days. After 8 days of exposure all P. perezi larvae from Do?ana presented 100% mortality at 5 mg l(-1)N-NO2(-) while E. calamita larvae mortality rates were significantly lower at that concentration after 15 days. However, for H. meridionalis at day 15 no deaths were registered at 5 mg l(-1)N-NO2(-) and at 20 mg l(-1)N-NO2(-) presented intermediate mortality rates. In Do?ana the 10 d LC50 of older H. meridionalis larvae was between 20 and 30 mg l(-1)N-NO2(-) whilst for P. perezi it was below 5 mg l(-1)N-NO2(-). These results indicate inter-specific variation of the sensitivity of larval amphibians to nitrite. Gredos Mountain P. perezi larvae exposed since the egg stage were highly sensitive to nitrite, with a 16 d LC50 below 0.5 mg l(-1)N-NO2(-). The same species in Do?ana had a 15 d LC50 between 5 and mg l(-1)N-NO2(-). These results suggest that there is also intra-specific variation in sensitivity of amphibian larvae to nitrite: mountain amphibian populations appear to be more sensitive to polluted environments than coastal populations. Geographic and genetic variation and evolutionary adaptation of tolerance may also be the keys to variation amongst populations of the same species.     

Effects of agricultural pesticides on the health of Rana pipiens frogs sampled from the field

There is evidence that over the last 30 years, there have been mass declines in diverse geographic locations among amphibian populations due to disease outbreaks. Multiple causes have been suggested to explain this increase in disease incidence. Among these, climate changes, environmental pollution and reduced water quality are gaining attention. Indeed, some chemicals of environmental concerns are known to alter the immune system. It is possible that exposure to these pollutants could alter the immune system of anurans and render them more susceptible to different pathogens. In this study, we sampled Rana pipiens in five different sites near St. Lawrence River (Quebec, Canada) during the months of July and September in 2001. Two of these sites were protected areas, in which low levels of pesticides were detected, while the remaining three sites were located in areas with intensive corn and soybeans cultivations. Our results demonstrated that frogs living in agricultural regions are smaller in size and weight than frogs living in areas with lower levels of pesticides at both sampling times. Moreover, we have observed a significant decrease in the number of splenocytes (cellularity) and the phagocytic activity in frogs sampled in impacted sites. Taken together, these results suggest that frogs living in agricultural regions might be more vulnerable to infections and diseases through their smaller size and alteration of their immune system. Our results also contribute to the overall discussion on factors involved in amphibian declines.     

Polychlorinated biphenyls, sex steroid hormones and liver retinoids in adult male European common frogs Rana temporaria

Declines in amphibian populations and species biodiversity during the last decades has called for an assessment of possible threats to these animals. Polychlorinated biphenyls (PCBs) are known endocrine disrupting contaminants and are found in high levels in some populations of wild living amphibians. To evaluate the endocrine disrupting potential of PCBs in adult frogs, Aroclor 1,254 were subcutaneously injected into male European common frogs Rana temporaria. The injected doses ranged from 0.01 to 100 mg/kg body mass, resulting in liver concentrations between 74 and 133,619 microg/kg ww. After 14 days, serum testosterone (T), estradiol (E) and hepatic retinol (R) and retinylpalmitate (RP) were easured. No dose dependent effects were found on levels of hormones or retinoids. However, a significantly higher within-group variation in the E-T ratio in the exposed groups may indicate that the sex-hormone homeostasis of male R. temporaria is affected by PCBs shortly after arousal from hibernation, but that the effects are subtle and that several different mechanisms are involved. The lack of direct effect on T, E, R and RP may be due to the timing of exposure (shortly after arousal from hibernation), or due to a relatively short exposure time to Aroclor 1,254. Based on the results, we propose that future research should focus on effects of PCBs in relation to the different physiological phases frogs experience throughout the year (hibernation, reproduction etc.).     

The effects of aluminum and other metals on amphibians

This review discusses the effects of aluminum and other metals associated with acidification on amphibians. The majority of research has centered around aluminum although some information is available regarding copper, cadmium, iron, zinc and lead. The major habitat types in which amphibians live and breed are listed and the mechanisms by which these habitats may become acidified and contaminated with metals are discussed. Three lines of investigation into acid and metal toxicity are reviewed in detail: (1) field surveys of water chemistry, (2) laboratory toxicity tests, (3) field studies which include in situ toxicity tests, and surveys of amphibian distribution and survival relative to water chemistry. As a result of recent information from each of these three lines of research, it appears that aluminum toxicity is an extremely complex phenomenon which is influenced by the hardness, pH, DOC of the water, and the developmental stage and species of amphibian.     

First report on development of quantitative interspecies structure-carcinogenicity relationship models and exploring discriminatory features for rodent carcinogenicity of diverse organic chemicals using OECD guidelines

Different regulatory agencies in food and drug administration and environmental protection worldwide are employing quantitative structure-activity relationship (QSAR) models to fill the data gaps related with properties of chemicals affecting the environment and human health. Carcinogenicity is a toxicity endpoint of major concern in recent times. Interspecies toxicity correlations may provide a tool for estimating sensitivity towards toxic chemical exposure with known levels of uncertainty for a diversity of wildlife species. In this background, we have developed quantitative interspecies structure-carcinogenicity correlation models for rat and mouse [rodent species according to the Organization for Economic Cooperation and Development (OECD) guidelines] based on the carcinogenic potential of 166 organic chemicals with wide diversity of molecular structures, spanning a large number of chemical classes and biological mechanisms. All the developed models have been assessed according to the OECD principles for the validation of QSAR models. Consensus predictions for carcinogenicity of the individual compounds are presented here for any one species when the data for the other species are available. Informative illustrations of the contributing structural fragments of chemicals which are responsible for specific carcinogenicity endpoints are identified by the developed models. The models have also been used to predict mouse carcinogenicities of 247 organic chemicals (for which rat carcinogenicities are present) and rat carcinogenicities of 150 chemicals (for which mouse carcinogenicities are present). Discriminatory features for rat and mouse carcinogenicity values have also been explored.     

Model selection and evaluation for risk assessment of dioxin-contaminated sites

The general European population has a total intake of dioxins and dioxin-like chemicals near the limit recommended by the European Union, making additional exposure above background levels undesirable. For populations living near dioxin-contaminated sites, additional exposure may occur by intake of locally produced food, inhalation of particles, dermal contact with soils, or other exposure pathways. Risk assessment tools are required to estimate risks associated with contaminated sites and to set priorities for site remediation. Here, we review several multimedia models that can be applied as tools to support risk assessment. We then present a strategy to select, apply, evaluate, and adapt a model to address a specific situation. The case study we consider is a risk assessment of generic background dioxin exposure in Sweden, and we compare the predictions with environmental observations and exposure data from Sweden. Arguments are presented for selecting the CalTOX model for this case study. We demonstrate the application, evaluation, and adaptation of the model and discuss the requirements for extending the analysis to conduct risk assessment for subpopulations living near dioxin-contaminated sites.     

Adaptation to osmotic stress provides protection against ammonium nitrate in Pelophylax perezi embryos

The negative effects of pollution on amphibians are especially high when animals are additionally stressed by other environmental factors such as water salinity. However, the stress provoked by salinity may vary among populations because of adaptation processes. We tested the combined effect of a common fertilizer, ammonium nitrate (0-90.3 mg N-NO₃NH₄/L), and water salinity (0-2‰) on embryos of two Pelophylax perezi populations from ponds with different salinity concentrations. Embryos exposed to the fertilizer were up to 17% smaller than controls. Survival rates of embryos exposed to a single stressor were always below 10%. The exposure to both stressors concurrently increased mortality rate (>95%) of embryos from freshwater. Since the fertilizer was lethal only when individuals were stressed by the salinity, it did not cause lethal effects on embryos naturally adapted to saline environments. Our results underscore the importance of testing multiple stressors when analyzing amphibian sensitivity to environmental pollution.     

The global re-cycling of persistent organic pollutants is strongly retarded by soils

'Persistent organic pollutants' (POPs) are semi-volatile, mobile in the environment and bioaccumulate. Their toxicity and propensity for long-range atmospheric transport (LRAT) has led to international bans/restrictions on their use/release. LRAT of POPs may occur by a 'single hop' or repeated temperature-driven air-surface exchange. It has been hypothesised that this will result in global fractionation and distillation-with condensation and accumulation in polar regions. Polychlorinated biphenyls (PCBs)--industrial chemicals banned/restricted in the 1970s--provide a classic illustration of POP behaviour. A latitudinally-segmented global PCB inventory has been produced, which shows that approximately 86% of the 1.3 x 10(6) tonnes produced was used in the temperate industrial zone of the northern hemisphere. A global survey of background surface soils gives evidence for 'fractionation' of PCBs. More significantly, however, very little of the total inventory has 'made the journey' via primary emission and/or air-surface exchange and LRAT out of the heavily populated source regions, in the 70 years since PCBs were first produced. Soils generally occlude PCBs, especially soils with dynamic turnover of C/bioturbation/burial mechanisms. This limits the fraction of PCBs available for repeated air-soil exchange. The forested soils of the northern hemisphere, and other C-rich soils, appear to be playing an important role in 'protecting' the Arctic from the advective supply of POPs. Whilst investigations on POPs in remote environments are important, it is imperative that researchers also seek to better understand their release from sources, persistence in source regions, and the significant loss mechanisms/global sinks of these compounds, if they wish to predict future trends.     

Abiotic reduction reactions of anthropogenic organic chemicals in anaerobic systems: A critical review

This review is predicated upon the need for a detailed process-level understanding of factors influencing the reduction of anthropogenic organic chemicals in natural aquatic systems. In particular, abiotic reductions of anthropogenic organic chemicals are reviewed. The most important reductive reaction is alkyl dehalogenation (replacement of chloride with hydrogen) which occurs in organisms, sediments, sewage sludge, and reduced iron porphyrin model systems. An abiotic mechanism involving a free radical intermediate has been proposed. The abstraction of vicinal dihalides (also termed dehalogenation) is another reduction that may have an abiotic component in natural systems. Reductive dehalogenation of aryl halides has recently been reported and further study of this reaction is needed. Several other degradation reactions of organohalides that occur in anaerobic environments are mentioned, the most important of which is dehydrohalogenation. The reduction of nitro groups to amines has also been thoroughly studied. The reactions can occur abiotically, and are affected by the redox conditions of the experimental system. However, a relationship between nitro-reduction rate and measured redox potential has not been clearly established. Reductive dealkylation of the N- and O-heteroatom of hydrocarbon pollutants has been observed but not investigated in detail. Azo compounds can be reduced to their hydrazo derivatives and a thorough study of this reaction indicates that it can be caused by extracellular electron transfer agents. Quinone-hydroquinone couples are important reactive groups in humic materials and similar structures in resazurin and indigo carmine make them useful as models for environmental redox conditions. The interconversion of sulfones, sulfoxides, and sulfides is a redox process and is implicated in the degradation of several pesticides though the reactions need more study. Two reductive heterocyclic cleavage reactions are also mentioned. Finally, several difficulties (both semantic and experimental) that recur in the studies reviewed are discussed. The subtle effects of various sterilization techniques on extracellular biochemicals and complex chemical reducing agents in sediment have stifled attempts to separate abiotic from biological degradation reactions. The characterization of redox conditions in a natural system is still problematic since measured redox potential is not adequate. Suggestions for future research toward a process-level understanding of abiotic chemical reductions are made.     

Do herbicide treatments reduce the sensitivity of mosquito larvae to insecticides?

Invasive mosquitoes are economic and sanitary concerns especially in Europe and America. Most work has emphasized the role of resistance [Berrada, S., Fournier, D., Cuany, A., Nguyen, T.X., 1994. Identification of resistance mechanisms in a selected laboratory strain of Cacopsylla pyri (Homoptera: Psyllidae): altered acetylcholinesterases and detoxifying oxidases. Pesticide Biochemistry and Physiology 48, 41-47; Hemingway, J., Hawkes, N.J., McCarroll, L., Ranson, H., 2004. The molecular basis of insecticide resistance in mosquitoes. Insect Biochemistry and Molecular Biology 34, 653-665] to insecticides. Compounds acting on larval sensitivity to insecticides are not well studied and their action remains poorly understood. Among several residual chemicals in ecosystems, particularly in wetlands, we identified a possible interaction of an herbicide on larval resistance to an insecticide. Our work contributes to the global control of mosquito populations by identifying possible pathways of resistance to insecticides of these vectors. Resistance or tolerance to insecticide treatments might contribute to successful invasion by mosquitoes. Here we report an ecotoxicological approach to test the hypothesis of an indirect effect of atrazine on mortality of an invasive vector. A brief contact (48h) between Aedes aegypti mosquito larvae and atrazine led to a modification of larval sensitivity to an insecticide: using atrazine as an inducer led to a decrease in the mortality of larvae treated with Bacillus thuringiensis var. israelensis (Bti).     

An overview of brominated flame retardants in the environment

The presence of brominated flame retardant (BFR) chemicals, and particularly polybrominated diphenyl ethers (PBDEs), tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD), has become of increasing concern to scientists over the past decade. Environmental studies conducted primarily in Europe, Japan and North America indicate that these chemicals are ubiquitous in sediment and biota. The levels of PBDEs seem to be increasing, and several trends, including in humans, indicate that this increase may be rapid. The occurrence of high concentrations of certain PBDE isomers may be sufficient to elicit adverse effects in some wildlife. There is also concern that levels could cause adverse effects in sensitive human populations such as young children, indigenous peoples, and fish consumers. However, our knowledge about these chemicals, their sources, environmental behavior, and toxicity is limited, making risk assessment difficult. In this paper, the current state of knowledge is reviewed and areas for further research recommended to improve future monitoring and risk assessment efforts.     

Bioaccumulation of trace elements in omnivorous amphibian larvae: implications for amphibian health and contaminant transport

Despite the influence that amphibians have on the flow of energy and nutrients in ecological systems, the role that amphibians play in transporting contaminants through food webs has received very little attention. This study was undertaken to investigate bioaccumulation of trace elements in amphibians relative to other small aquatic organisms in a contaminated wetland. We collected bullfrog larvae (Rana catesbeiana) along with three other species of small vertebrates and four species of invertebrates from a site contaminated with a wide array of trace elements and analyzed them for trace element concentrations and stable nitrogen and carbon isotope composition. We found that amphibian larvae accumulated the highest concentrations of most trace elements, possibly due to their feeding ecology. These results suggest that omnivorous amphibian larvae can serve as a critical link for trace element trophic transfer. Their propensity to accumulate trace elements may have important implications for amphibian health in contaminated environments and should be further investigated.     

油田常用化学品对含油废水处理效果的影响

通过对江汉油田采油废水水质分析以及混凝实验研究,分析采油作业返排液中主要化学组分对含油废水混凝处理效果的影响,探讨了混凝剂、常用油田化学品与浊度及油含量去除率的关系,初步分析了它们的作用机理。研究表明,在钻井液中,单宁、磺化酚醛树脂(SMP)等对含油废水处理的影响很大,在压裂液中瓜胶、重铬酸钾等的影响较大,酸化液中乙酸和柠檬酸等的影响较大,射孔液中盐酸等的影响较大。本研究的结果对于消除返排液对油田生产的不良影响,保证系列增储上产措施的实施及其有效性,避免环境污染,具有十分重要的参考价值。     

Problems in testing and risk assessment of endocrine disrupting chemicals with regard to developmental toxicology.

Endocrine disrupting chemicals (EDCs) may affect mammalian development either indirectly (by impairing implantation, placental development, lactation, etc.) or directly, altering the maturation of target tissues. Current regulatory tests for reproductive/developmental toxicity should be carefully evaluated with regard to risk assessment of EDCs, considering hazard identification (are relevant endpoints being assessed?) and dose-response assessment (are sensitive NOEL/dose-response curves being provided?). Many in vitro and in vivo assays for sex steroid disruption are available; provided that the metabolic capacities of the assays are defined, they could be integrated in a sensitive battery for early detection of steroid-disrupting potentials. The screening battery should address further regulatory in vivo tests (e.g. what specific parameters have to be investigated). As regards dose-response, qualitative differences may be observed between lower and higher exposures, showing primary hormone-related effects and frank embryotoxicity, respectively. Other problems concern (a) the identification of critical developmental windows, according to hormone concentrations and/or receptor levels in the developing target tissues; (b) the potential for interactions between chemicals with common mechanism/target (e.g. xenoestrogens); (c) most important, besides sex steroids more attention should be given to other mechanisms of endocrine disruption, e.g., thyroid effects, which can be highly relevant to prenatal and postnatal development.     

Do aquatic effects or human health end points govern the development of sediment-quality criteria for nonionic organic chemicals ?

The equilibrium partitioning theory may be used to describe the partitioning of nonionic organic chemicals between water, sediment, and aquatic biota. This paradigm was employed to compare the relative magnitudes of organic carbon-normalized sediment-quality criteria that are intended to protect either benthic organisms from the direct toxic effects of sediment-associated chemicals or humans from the indirect health effects posed by the ingestion of contaminated aquatic animals. Comparison of calculated sediment-quality criteria for a variety of hydrophobic chemicals suggests that human health-based end points often result in more restrictive criteria than aquatic effects-based values. Review of published field data indicates that the equilibrium partitioning paradigm may, depending on contaminant class, either over- or underestimate the extent to which sediment-associated contaminations are bioaccumulated. Despite the limitations of adopting this simple theory for criteria development, calculations reveal that regulatory decisions involving sediments contaminated with such chemicals may be dictated by human health concerns if current risk assessment methodologies are applied.     

The effect of low molecular weight chelators on iron chelation and free radical generation as studied by ESR measurement

The purpose of this work was to improve our current knowledge of the non-enzymatic mechanisms involved in brown rot fungal decay of wood, but also to improve our understanding of chelator-mediated reactions that may occur in other biological systems where low pH conditions may prevail. Several key steps (iron chelation and reduction) during early non-enzymatic wood decay processes have been studied by using electron spin resonance (ESR). It has been proposed that low molecular weight chelators as well as Fenton reagents are involved in brown rot decay, at least in early non-enzymatic stages. In this work, the binding between a catecholate model chelator and ferric iron was studied by ESR spectroscopy. The effects of the model chelator, Fenton reagents, as well as the reaction conditions on free radical generation were also studied using ESR spin-trapping techniques. The results indicate: (1) The relative quantity of the chelator-iron complex can be determined by measurement of the intensities of the characteristic g = 4.3 ESR signal. (2) The effects of the chelator:iron ratio, the pH, and other reaction parameters on the hydroxyl radical generation in a Fenton type system can be determined using ESR spin-trapping techniques. (3) Data support the hypothesis that superoxide radicals are involved in chelator-mediated Fenton processes.     

Breast cancer and persistent organic pollutants (excluding DDT): a systematic literature review

Persistent organic pollutants (POPs) are a group of heterogeneous compounds of both natural and anthropogenic origin with highly persistent and bioaccumulative properties. They cause a range of adverse effects to human health and the environment around the world. There is growing concern that POPs may increase breast cancer risk due to their xenoestrogenic properties. The aim of this systematic literature review is to summarize and integrate the risks of breast cancer following environmental exposure to POPs (other than DDT) from primary epidemiological studies published between 2006 and 2015. After searching various databases, 14 case-control studies and one cohort study were included. Evidence of an association between increased breast cancer risk and environmental exposure to these chemicals is inconsistent and inadequate to conclude with certainty. However, most of the studies have examined exposure to the pollutants after diagnosis of breast cancer, overlooking exposure during critical windows of vulnerability. They have also largely focused on individual chemicals but ignored the combined effects of different chemicals. Therefore, major data gaps remain in examining exposure during critical windows of vulnerability and assessing combined effects of multiple chemicals. Development of better exposure assessment methods addressing these gaps is required for future research.     

The zebrafish embryo model in environmental risk assessment—applications beyond acute toxicity testing

BACKGROUND, AIM, AND SCOPE: The use of fish embryos is not regulated by current legislations on animal welfare and is therefore considered as a refinement, if not replacement of animal experiments. Fish embryos represent an attractive model for environmental risk assessment of chemicals since they offer the possibility to perform small-scale, high-throughput analyses. MAIN FEATURES: Beyond their application for determining the acute toxicity, fish embryos are also excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible adverse and long-term effects. Therefore, we have reviewed the scientific literature in order to indicate alternative applications of the fish embryo model with focus on embryos of the zebrafish. RESULTS AND DISCUSSIONS: The analysis of the mode of action is important for the risk assessment of environmental chemicals and can assist in indicating adverse and long-term effects. Toxicogenomics present a promising approach to unravel the potential mechanisms. Therefore, we present examples of the use of zebrafish embryos to study the effect of chemicals on gene and protein patterns, and the potential implications of differential expression for toxicity. The possible application of other methods, such as kinase arrays or metabolomic profiling, is also highlighted. Furthermore, we show examples of toxicokinetic studies (bioconcentration, ABC transporters) and discuss limitations that might be caused by the potential barrier function of the chorion. Finally, we demonstrate that biomarkers of endocrine disruption, immune modulation, genotoxicity or chronic toxicity could be used as indicators or predictors of sub-acute and long-term effects. CONCLUSIONS: The zebrafish embryo represents a model with an impressive range of possible applications in environmental sciences. Particularly, the adaptation of molecular, system-wide approaches from biomedical research is likely to extend its use in ecotoxicology. RECOMMENDATIONS AND PERSPECTIVES: Challenges for future research are (1) the identification of further suitable molecular markers as indicators of the mode of action, (2) the establishment of strong links between (molecular) effects in short-term assays in embryos and long-term (toxic) effects on individuals, (3) the definition of limitations of the model and (4) the development of tests that can be used for regulatory purposes.