From Bioavailability Science to Regulation of Organic Chemicals.

The bioavailability of organic chemicals in soil and sediment is an important area of scientific investigation for environmental scientists, although this area of study remains only partially recognized by regulators and industries working in the environmental sector. Regulators have recently started to consider bioavailability within retrospective risk assessment frameworks for organic chemicals; by doing so, realistic decision-making with regard to polluted environments can be achieved, rather than relying on the traditional approach of using total-extractable concentrations. However, implementation remains difficult because scientific developments on bioavailability are not always translated into ready-to-use approaches for regulators. Similarly, bioavailability remains largely unexplored within prospective regulatory frameworks that address the approval and regulation of organic chemicals. This article discusses bioavailability concepts and methods, as well as possible pathways for the implementation of bioavailability into risk assessment and regulation; in addition, this article offers a simple, pragmatic and justifiable approach for use within retrospective and prospective risk assessment.

Vitellogenin content in European eel (Anguilla anguilla) in Flanders, Belgium.

As part of a large-scale monitoring program of bioaccumulating contaminants in the European eel (Anguilla anguilla) in Flanders (Belgium), we investigated potential effects of xenoestrogens in these fish. The present paper describes the results of the plasma vitellogenin (VTG) content, measured in 142 eels sampled at 20 different locations, in relation to the internal pollution levels. To validate the blood VTG assays, a small number of eels (n=8) was exposed to 10 microg ethinylestradiol/l (EE2) for 9 days. In this experiment, VTG was detected as a protein with a molecular weight of 214 kDa and confirmed by Western blotting. Compared with the solvent controls, significantly higher concentrations of VTG were measured in EE2 exposed eel. However, the VTG content was relatively low compared with other fish species exposed to high concentrations of estrogens. The plasma VTG content of eels from the field study was very low, despite a very high internal load of endocrine disrupters. These results, together with previously published studies, suggest that immature yellow European eel might not be the best sentinel species to study the effects of estrogenic compounds on VTG levels of wild fish populations.

Estrogenic and toxic effects of methoxychlor on zebrafish (Danio rerio).

Although zebrafish (Danio rerio) have been suggested as a good candidate for screening potential endocrine disruptors, little information is available on the effects of weak estrogens on this species. We investigated the sensitivity of different life stages of zebrafish toward toxic and estrogenic properties of methoxychlor (MXC). Short-term tests with adults resulted in a sex-specific 96-h lethal concentration for 50% (LC50) of the test animals of 36 microg/L for males and 129 microg/L for females. To determine the estrogenic capacity of MXC, adult zebrafish were exposed to 0, 0.5, 5, and 50 microg MXC/L for 14 d. Induction of vitellogenin ([VTG] measured with protein electrophoresis and Western blot) in males was detected at 5 and 50 microg MXC/L. Females, however, did not exhibit higher blood VTG concentrations at the tested MXC concentrations. In a second series of experiments, juvenile zebrafish were exposed to 0, 0.05, 0.5, and 5 microg MXC/L for 33 d. Survival, length, weight, and condition of larvae were examined as indicators of toxic stress and the VTG content in whole body homogenates of juveniles was measured to determine xenoestrogenic effects. No effects of the tested concentrations of MXC were observed. Finally, the effect of MXC on zebrafish eggs, exposed to 0, 1, 10, and 32 microg MXC/L, was examined. Hatching and survival of hatched zebrafish were affected at 10 and 32 microg MXC/L. This study demonstrated that adult male zebrafish are sensitive toward the estrogenic effects of MXC. However, the use of VTG induction to detect effects of (xeno)estrogens in early life stages has to be further investigated, as low concentrations of VTG were detectable in exposed as well as unexposed juvenile fish.

In vitro and in vivo estrogenicity and toxicity of o-, m-, and p-dichlorobenzene.

The estrogenicity of o-, m-, and p-dichlorobenzene (DCB) was evaluated with a yeast estrogen screen (YES) and zebrafish (Danio rerio) vitellogenin (VTG) assays. With the YES, p-DCB and m-DCB were found to be estrogenic in a concentration-responsive manner. The relative potency measured with the YES (relative to 17beta-estradiol) was 2.2 x 10(-7) for p-DCB and 1.04 x 10(-8) for m-DCB. Following acute toxicity tests with the zebrafish, plasma VTG production was measured to examine the in vivo estrogenic activity of the three compounds after a 14-d exposure. Adult zebrafish were exposed to different concentrations of o-, m- and p-DCB, ranging from 0.1 to 32 mg/L; ethynylestradiol ([EE2]; 5 ng/L, 10 ng/L, 50 ng/L, and 100 ng/L) was used as a positive control. After exposure, blood samples were taken and protein electrophoresis was performed to determine the relative VTG content. Gonadosomatic indices (GSI) and condition factors (CF) were also calculated. Elevated VTG levels and decreased female GSIs were found in fish exposed to > or = 5 ng EE2/L and in fish exposed to > or = 10 mg p-DCB/L. Low GSIs coincided with high levels of VTG in the blood of female zebrafish. This relation was not only found in fish exposed to EE2 but also in controls and fish exposed to DCB. Therefore, a direct or indirect effect of VTG on the GSI is suggested rather than a direct toxic effect of the tested compounds on the gonads.

Analysis of the ecotoxicity data submitted within the framework of the REACH Regulation. Part 1. General overview and data availability for the first registration deadline.

REACH(1) entered into force in June 2007 and has hence been operational for six years. With the first registration deadline in November 2010, the European Chemicals Agency (ECHA(2)) has received a large amount of scientific and administrative information related to chemical substances. In order to understand what type of data on ecotoxicity endpoints was submitted under the REACH framework a detailed analysis of the availability and content of relevant information was performed. To avoid unnecessary testing, the REACH Regulation provides registrants with the possibility to build testing strategies and to adopt the standard information requirements based on the specific conditions listed in the regulation. The types of information submitted by registrants to fulfil data requirements for aquatic, sediment and terrestrial toxicity endpoints were analysed. The REACH database analysis confirms large differences in the availability of experimental aquatic versus sediment and soil ecotoxicity data. Information requirements on aquatic organisms are mainly covered by experimental data, while those for sediment and soil are mostly waived.

Analysis of the ecotoxicity data submitted within the framework of the REACH Regulation. Part 2. Experimental aquatic toxicity assays.

This paper summarises the aquatic ecotoxicity data submitted in the REACH(1) registration dossiers and disseminated by the European Chemicals Agency (ECHA(2)). The analysis describes both the guidelines and the species mostly used by registrants. Non-OECD guidelines have been extensively used, in particular in covering of fish and aquatic invertebrate studies, but the main concern is that in 22-36% of the cases, depending on the endpoint, no information on the methodological approach and potential equivalences to test guidelines has been provided. As expected, most studies were conducted with those species typically used in laboratory ecotoxicity testing; nevertheless, the database provides a broad range of available species, covering the most relevant taxonomic groups for both freshwater and marine systems, although most are just occasionally used. This species diversity is essential for higher tier testing strategies, including the use of Species Sensitivity Distribution approaches. The assessment suggests that collecting available information has been the main approach used by registrants to fulfil their REACH information requirements for this first REACH registration deadline. Many studies are disclosed for the first time, and all are available through searchable web tools.

Analysis of the ecotoxicity data submitted within the framework of the REACH Regulation: part 4. Experimental terrestrial toxicity assays.

This paper summarises the terrestrial ecotoxicity data submitted in the REACH registration dossiers and disseminated by ECHA. The analysis describes both the guidelines and the test species mostly used by registrants. REACH information requirements in relation to the effects on terrestrial organisms encompass three trophic levels; invertebrates, plants and micro-organisms, and the study of both long and short-term exposure. The results observed for soil invertebrates showed that on one hand there was a clear prevalence for testing on the species recommended by the standard test guidelines. On the other, the reporting included a large variety of species from very different families, demonstrating the feasibility for conducting toxicity tests on a number of relevant groups e.g. for species sensitivity distribution approaches. Standard toxicity testing with terrestrial plants under REACH follows a different approach and requires simultaneous testing on several species, using the same test conditions, adapted to each species, if needed. The test methods used to conduct the studies were only reported for 30% of cases. The most extensively reported test guidelines for terrestrial plants were OECD 208, ISO 11269-1 and ISO 11269-1. Information requirements for soil micro-organisms under REACH are related to the analysis of functional endpoints instead of on species or taxa. As recommended in REACH, OECD 216 and OECD 217 were the most often used test methods for soil micro-organisms. But overall, the test method was reported for only about 40% of the experimental studies. Moreover, it is noted that information on potential effects on soil micro-organisms is available for a limited number of REACH registered substances. The assessment suggests that providing waiving justifications and collecting available information, which in many cases might be well used for covering standard REACH data requirements, have been the main approaches used by registrants for the first REACH registration deadline.

Analysis of the ecotoxicity data submitted within the framework of the REACH Regulation. Part 3. Experimental sediment toxicity assays.

For the first REACH registration deadline, companies have submitted registrations with relevant hazard and exposure information for substances at the highest tonnage level (above 1000 tonnes per year). At this tonnage level, information on the long-term toxicity of a substance to sediment organisms is required. There are a number of available test guidelines developed and accepted by various national/international organisations, which can be used to investigate long-term toxicity to sediment organisms. However instead of testing, registrants may also use other options to address toxicity to sediment organisms, e.g. weight of evidence approach, grouping of substances and read-across approaches, as well as substance-tailored exposure-driven testing. The current analysis of the data provided in ECHA database focuses on the test methods applied and the test organisms used in the experimental studies to assess long-term toxicity to sediment organisms. The main guidelines used for the testing of substances registered under REACH are the OECD guidelines and OSPAR Protocols on Methods for the Testing of Chemicals used in the Offshore Oil Industry: "Part A: A Sediment Bioassay using an Amphipod Corophium sp." explaining why one of the mostly used test organisms is the marine amphipod Corophium sp. In total, testing results with at least 40 species from seven phyla are provided in the database. However, it can be concluded that the ECHA database does not contain a high enough number of available experimental data on toxicity to sediment organisms for it to be used extensively by the scientific community (e.g. for development of non-testing methods to predict hazards to sediment organisms).

Identification of chemical hazards for terrestrial plants in the regulatory context: comparison of OECD and ISO guidelines.

Standardized test protocols are used in the regulatory context for identifying the hazardous properties of chemicals, wastes, and contaminated materials. This paper compares the relevance of two guidelines measuring effects on terrestrial plants, the OECD TG 208 and the ISO TG 22030 and presents the scientific basis for a recent decision of the European Chemicals Agency (ECHA) under the European chemicals regulation REACH. If there are no specific phytotoxicity alerts, both guidelines are considered suitable for assessing long-term hazards, providing that a sufficient number of species is included in the OECD protocol, the recommended minimum number is six, which offer a reasonably broad selection of species to account for interspecies sensitivity. The proposed methodology, based on a combination of probabilistic assessments using Monte Carlo analysis, can be adapted for supporting similar decisions under specific regulatory processes; for example, for assessing contaminated soils or pesticides' applications.

Experimental parameters affecting sensitivity and specificity of a yeast assay for estrogenic compounds: results of an interlaboratory validation exercise.

In vitro assays are considered as the first step in a tiered approach to compound screening for hormonal activity. Although many new assays have been developed in recent years, little attention has been paid towards assay validation. Our objective was to identify critical experimental parameters in a yeast estrogen screen (YES) that affect its sensitivity and specificity. We investigated the role of incubation time, solvent type, yeast inoculum growth stage and concentration on the outcome of the YES. Compounds tested included new and established agonists, antagonists and negative controls, and results were evaluated according to prefixed statistical criteria. In addition, we assessed the assay's performance in a blind interlaboratory validation exercise (IVE). An incubation time of five days was necessary to positively identify the estrogenic properties of all agonists tested, when dissolved in DMSO. Longer incubation times were required when using an ethanol protocol. Similar estrogenic activity was reported for benzyl butyl phthalate, bisphenol-A, methoxychlor, permethrin and genistein in the IVE. One out of the three laboratories did not classify alpha,beta-endosulfan, dissolved in DMSO, as an estrogen. The same was true for 4,4'-DDE and lindane, dissolved in ethanol, a result that might be attributable to an inappropriate yeast start concentration and/or growth stage. These validation experiments show that under appropriate experimental conditions the YES yields sensitive, specific and reliable results. Therefore it fulfills the requirements as a first step screening assay to evaluate the capacity of chemicals to interact with the estrogen receptor.

Xenoestrogenic effects of ethinylestradiol in zebrafish (Danio rerio).

To assess the estrogenic effects of ethinylestradiol on zebrafish, zebrafish at different developmental stages (embryos, juveniles, and adults) were exposed to the synthetic hormone ethinylestradiol (EE2) in concentrations of 1, 10, and 100 ng/L for up to 33 days. Survival, hatching, length, weight, growth, condition, hepatosomatic index, gonadosomatic index, and vitellogenin (VTG) production were examined. Exposure of zebrafish juveniles and embryos to 100 ng EE2/L for up to 33 days had significant effects on survival, growth, and hatching. Two VTG fragments with molecular weights of approximately 140 and 170 kDa were detected with protein electrophoresis and Western blotting in the blood of exposed males and exposed and unexposed females, as well as in whole-body homogenates of exposed and unexposed juveniles. Significantly higher VTG concentrations (compared to controls) were measured in adults exposed to 10 and 100 ng EE2/L for 14 days, but not in fish exposed to 1 ng EE2/L. This study demonstrated that (1) zebrafish juveniles, larvae, and embryos are sensitive to the toxic effects of the endocrine disrupter EE2; (2) the effects on VTG production in adults are detected after exposure to environmentally relevant concentrations of EE2; (3) unexposed juvenile zebrafish produce measurable concentrations of VTG.

Induction of vitellogenesis in 17alpha-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.