Ecotoxicological characterization of the estrogenic mycotoxin zearalenone and environmental samples : effects on zebrafish development and reproduction

Endocrine disruption occurs when specific chemicals interact with internal endocrine signalling pathways in organisms and appears to be particularly widespread in freshwater fish throughout the world. Effects ranged from subtle changes in the physiology and sexual behaviour of fish to permanently altered sexual differentiation and growth, and reproductive impairment. Most attention has been directed towards identifying the main estrogenic chemicals, because many of the effects reported in wildlife appear to be a consequence of ‘feminization’ of males. Environmental estrogens can be anthropogenic, such as certain pharmaceuticals, pesticides and industrial chemicals or natural, such as phyto- and mycoestrogens.
Resorcyclic acid lactones (RALs) are the only known class of estrogenic mycotoxins and show estrogenic activity in the range of natural steroid estrogens. RALs occur as secondary metabolites of Fusarium spec. fungi growing on a variety of cereals. Zearalenone (ZON) belongs to the chemical group of RALs and is one of the worldwide most common mycotoxins. The occurrence of mycotoxins has been studied extensively in food, feed products, and domestic animals. Agricultural products around the world exhibited ZON contaminations of up to 69 % of the tested samples. However, only little is known about the ecotoxicological effects of ZON at concentrations found in the environment.
In this research project, the ecotoxicological potential of ZON was evaluated. Effects on fish caused by an exposure to ZON were assessed using the zebrafish (Danio rerio) as a model organism. An embryo toxicity test was performed to investigate toxic effects of ZON on fish development. This study was complemented with a 30 day zebrafish early life-stage experiment to assess the influence of ZON on juvenile growth. In a second step the estrogenic potency of ZON and its effects on adult fish were examined. A recombinant yeast estrogen screen (rYES) was used as an in vitro assay to determine effects on the activation of estrogen receptor-regulated genes. Zebrafish were exposed to ZON in a 42 day reproduction experiment enabling assessment of reproductive as well as physiological and morphological parameters. In a third step, the estrogenic potency of ZON was studied in a life-cycle experiment. This study was designed to investigate possible effects of continuous long-term exposure including a subsequent depuration period as well as possible transgenerational effects of F0 exposure on F1 generation. The offspring of the F0 generation that was exposed to ZON for 21 days was raised in a 140 day exposure experiment from embryo to adult. In a subsequent 42 day reproduction experiment, spawning groups of the F1 generation were either exposed to ZON (after growing up in clean water) or clean water (after growing up under ZON exposure) and effects on developmental, reproductive as well as physiological parameters were assessed.
Exposure to ZON had no effects on embryonic development up to 2000 ng/L, but a positive correlation between the body length and ZON concentration of up to 100 ng/L suggests a growth promoting effect for zebrafish exposed during 30 days. Although ZON revealed a moderate estrogenic potency in vitro (rYES), it exhibited a comparably strong effect on induction of VTG (1000 ng/L) and reproduction (100 ng/L) in vivo during 21 days exposure. It was further demonstrated that ZON influenced growth (1000 ng/L), sexual differentiation (320 ng/L), reproduction (1000 ng/L), and VTG levels (1000 ng/L) in zebrafish exposed from fertilization to day 140. Furthermore, ZON revealed a possible transgenerational effect on growth caused by short-term exposure of F0 and F1 generation each for 21 days to 1000 ng/L.
In the framework of this project, relevant data of the ecotoxicological relevance of ZON were collected. The estrogenic potential of ZON was confirmed in vitro and in vivo and detrimental effects on zebrafish reproduction were demonstrated. This indicates that ZON may have an effect on fish populations in the aquatic environment. Effects were observed at concentrations near the maximum measured values in surface waters (approximately double) and below the maximum values measured in effluents (approximately half). Even though to date there is not much data published presenting ZON concentrations in the aquatic environment and effects on different (native) fish species, this project demonstrated that ZON has the potential to pose a threat to fish in the aquatic environment.
In a second research project, the embryo toxicity and genotoxicity of biofilm and sediment from the catchment area of a STP was investigated. An embryo toxicity test with zebrafish and the comet assay (single cell gel electrophoresis) with primary cells isolated from the embryos were applied to investigate native biofilm for the first time. Biofilms were sampled from different sections of the sewage system and sediments were taken from the rive Wyna directly at, upstream and downstream of the STP discharge Mittleres Wynental (Switzerland). The aim of this study was to verify the applicability of the aforementioned test system to investigate native biofilm and to evaluate the toxicity of the different sampling sites.
Clear differences in the embryo toxicity and genotoxicity of the biofilm from different sewage system sections as well as from the sediment sampling sites were found. Significant genotoxicity was determined in all biofilm and sediment samples. Temporal variability in toxicity were observed in some of the biofilm and sediment samples. Based on the results of this study it can be suggested that biofilm implemented in ecotoxicological bioassays such as the embryo toxicity test and comet assay with zebrafish can be a useful tool to assess (waste) water quality.