International round-robin study on the Ames fluctuation test.

An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%.

Electrophoretic determination of estrogen-induced protein in fish exposed to synthetic and naturally occurring chemicals.

In a first step we investigated whether Leuciscus idus, a fish routinely used to assess the quality of surface water in Germany, is suitable to indicate the presence of endocrine-disrupting chemicals. An electrophoretic method was used to determine estrogen-induced protein in fish serum (presumed vitellogenin). This method was tested by exposing juvenile L. idus to 17 alpha-ethinylestradiol, 17 beta-estradiol and estrone. Nominal concentrations of the steroid were compared with concentrations determined analytically by using GC/MS techniques. Under flow-through conditions, 7 days of exposure to 6 ng/l EE2 significantly induced serum vitellogenin in juvenile L. idus. In juvenile rainbow trout (Oncorhynchus mykiss) 4 ng/l EE2 induced significantly elevated levels of serum vitellogenin. In Danio rerio oral application of 40 micrograms 4-nonylphenol/fish per day during a 3-week exposure period caused a major increase of serum vitellogenin. Physiological protein induction could also be measured in Abramis brama, Cyprinus carpio, Oncorhynchus mykiss, Perca fluviatilis, Rutilus rutilus obtained from natural habitats and in Rhodeus sericeus. The minimum amount of blood required for electrophoretic analysis was 1 microliter which should allow monitoring of small sized fish species as well as juvenile fish.

Endocrine disrupters in the aquatic environment.

Possible mechanisms to explain endocrine effects on reproduction and sex differentiation are presented for selected pharmaceuticals, agrochemicals, industrial chemicals and plant sterols which are known to be present in the aquatic environment. Disruptions of the hormonal coordination can be induced by xenobiotics on various levels of the hierachically organised endocrine system of vertebrates. Phthalate plasticisers, for example, may disrupt the pituitary control of gonadal functions; prenatal/larval exposure to synthetic estrogen impairs sex differentiation and neuroendocrine sexual determination of the central nervous system; phenylurea herbicides block the androgen receptor; the biotransformation of weakly estrogenic plant sterol components of paper mill wastewater (e.g. beta-sitosterol) may lead to androgenic compounds. The effect of hypolipidemic drugs on lipid homeostasis (peroxysom proliferation) is transmitted via a receptor protein that seems to be closely related to the endocrine system both functionally as well as phylogenetically; possible interferences with the neuroendocrine control of sex differentiation are also discussed. In invertebrates, tributyltin is known to effect the biosynthesis of steroidal sexual hormones. PCBs are suspected to be competitive inhibitors of the steroid catabolism. In order to identify potential risks caused by chemicals to the reproductive capacities of aquatic animals and to the quality of drinking water, methods should be established to detect endocrine disrupters at the various levels of the endocrine system.