Effect assessment of reclaimed waters and carbamazepine exposure on the thyroid axis of Xenopus laevis: Gene expression modifications.

Reclaimed water (RW) obtained from wastewater treatment plants (WWTP) is used for irrigation, groundwater recharge, among other potential uses. Although most pollutants are removed, traces of them are frequently found, which can affect organisms and alter the environment. The presence of a myriad of contaminants in RW makes it a complex mixture with very diverse effects and interactions. A previous study, in which tadpoles were exposed to RW and RW spiked with Carbamazepine (CBZ), presented slight thyroid gland stimulation, as suggested by the development acceleration of tadpoles and histological findings in the gland provoked by RW, regardless of the CBZ concentration. To complement this study, the present work analysed the putative molecular working mechanism by selecting six genes coding for the thyroid-stimulating hormone (TSHß), thyroid hormone metabolising enzymes (DIO2, DIO3), thyroid receptors (THRA, THRB), and a thyroid hormone-induced DNA binding protein (Kfl9). Transcriptional activity was studied by Real-Time PCR (RT-PCR) in brains, hind limbs, and tails on exposure days 1, 7, and 21. No significant differences were observed between treatments for each time point, but slight alterations were noted when the time response was analysed. The obtained results indicate that the effects of RW or RW spiked with CBZ are negligible for the genes analysed during the selected exposure periods.

Use of a novel battery of bioassays for the biological characterisation of hazardous wastes.

Four toxicity bioassays were used for the biological characterisation of nine hazardous wastes and extracts. This evaluation included conventional and novel bioassays, and combined in vivo and in vitro tests in order to facilitate the effect characterisation. This test battery incorporated different relevant taxonomic groups for the aquatic compartment and covered several ecotoxicological endpoints. The toxicity bioassays used for this characterisation were the acute immobilisation daphnia test, an acute toxicity test with larvae of Xenopus laevis, an in vitro test with the fish cell line RTG-2 comprising endpoints for cellular defence and viability, and finally the DR-CALUX assay to detect dioxin-like compounds. The aim of this study is to contribute to the development of a cost-effective battery of toxicity tests for the acute screening of hazardous and toxic wastes for the aquatic compartment. For this objective, the correlations between toxicity data derived from all bioassay were studied using a multivariate analysis, including the Principal Component Analysis. The results showed that Daphnia and Xenopus were effective assays to detect toxicity and they could be incorporated to a screening test battery. On the other hand, the toxicity results with the in vitro test RTG-2 showed that this test could be a good alternative to in vivo tests, demonstrating an acceptable sensitivity for toxicity detection and contributing other advantages as reducing assays cost and animal testing. Finally DR-CALUX test implemented the tests-batteries in the screening of hazardous wastes when there is a suspicious that dioxin-like compounds are presented in the samples.