Effects of ozonated sewage effluents and pharmaceuticals in zebrafish (Danio rerio)

The aquatic environment is the main destination for many organic pollutants originating from human activity. Pharmaceuticals are significant contributors to this pollutant cocktail. One major route of pharmaceuticals into the aquatic environment is through discharges from sewage treatment plants (STPs) with inadequate pharmaceutical removal capacity. Adverse effects in aquatic organisms exposed to pharmaceuticals have been illustrated in a multitude of studies. Advanced sewage treatment technologies are therefore being evaluated as potential methods for improving pharmaceutical removal, for example whole-effluent ozonation. However, it remains important to assess how the ozonation treatment modulates the biological effect in aquatic organisms exposed to the STP effluent due to the formation of ozonation by-products (OBPs). This thesis aimed to investigate outcomes of ozonation treatment of STP effluents with a special focus on pharmaceuticals, by combining chemical analysis and in vivo testing using zebrafish (Danio rerio). Firstly the effects of an ozonated STP effluent on reproduction and behavior of adult zebrafish were evaluated at a Swedish STP (Knivsta municipal STP) with a full-scale ozonation step. Fish exposed over 21 days to the ozonated STP effluent spawned significantly more eggs than fish exposed to the nonozonated effluent and tap water. Vitellogenin induction (a biomarker for estrogenic pollutants) and a possible anxiety-related behavior were also observed in fish exposed to the ozonated STP effluent. Chemical analysis of the pre- and post ozonated STP effluent was used to screen for the presence of 105 pharmaceuticals, of which 24 could be detected. The average ozone removal efficiency of these pharmaceuticals was 77%. The thesis work then focused on how ozonation would affect zebrafish embryotoxicity of three pharmaceuticals detected in the Knivsta STP effluent (i.e. carbamazepine, diclofenac, and oxazepam). While embryotoxicity of diclofenac was eliminated, exposure to oxazepam and carbamazepine induced embryotoxic responses following ozonation, suggesting formation of OBPs. The thesis therefore proceeded to investigate the toxicities of isolated carbamazepine OBPs. It was revealed that two carbamazepine OBPs, BQM and BQD, were the drivers of ozonated carbamazepine embryotoxicity. The thesis concludes that the formation of specific pharmaceutical OBPs explains adverse biological outcomes of effluent ozonation. The results add valuable information for the continuing efforts to improve STP effluent treatment.