Microbial Evaluation and Validation of Biomarkers for Environmental Contaminants

Abstract

Aquatic environment is the ultimate sink for most environmental contaminants. There is a serious need for water quality monitoring and to validate the sensitive biomarkers for aquatic pollution. The main focus of this study was to evaluate the microbial diversity of a water reservoir and validation of biomarkers for environmental contaminants BPA and herbicide 2,4-D. Water quality and bacterial diversity from Rawal Lake was investigated for a period of eight months. Rawal Lake in Islamabad Pakistan is an artificial reservoir having several reports of pollution from adjacent areas. Grab water samples were collected from surface layer of ten various locations of the lake to study the water quality characteristics. The physicochemical parameters exceeded WHO guideline values for drinking water at few locations. Bacterial strains were isolated and template DNA was prepared following 16S rRNA analysis and sequencing. The sequences of nearest relative microbes were identified by BLAST and used as reference sequences for phylogenetic analysis. Various phylotypes of microbes including Firmicutes, Teobacteria and Proteobacteria were predominant. Bisphenol A (BPA) is a xenobiotic compound known for its endocrine disruptive effects, originating from polycarbonate plastics in large quantities. Besides, 2,4-D herbicide is one of the most widely used halogenated agricultural chemicals around the world and also a well-known endocrine disrupting chemical (EDC). These chemicals were tested for the biological effects by differential expression of genes including, Cytochrome P4501A1 (CYP1A), Aromatase B (Aro-B), heat shock proteins (HSP70) and Glutathione peroxidase (GPX) using quantitative real time polymerase chain reaction (Q-PCR) assay BPA concentration of 100 μg/L caused significant induction of Aro-B and HSP70 genes after xiv exposure of 72 hpf. Similarly, mRNA levels of CYP1A gene increased about two fold while GPX expression was found to be almost unchanged under similar exposure. Furthermore, BPA in aqueous solution was subjected to direct UV photolysis and tested for its residual effects. The results indicated significant effect of UV treatment on mRNA levels of tested genes and disappearance of biological activity of BPA with reduced mortality of zebrafish embryos. The study employed TiO2 based photocatalytic degradation of 2,4-D in aqueous solution. The residual biological activity was determined by transcriptome analysis of selected genes in zebrafish embryos. Statistically significant induction of CYP1A gene was observed for 2,4-D exposure, whereas HSP70 and Aro-B expression was significantly lowered after its UV/TiO2 photocatalytic degradation. Adult goldfish liver cell culture technique was used to determine the transcriptomic alterations of selected genes due to BPA and 2,4-D exposure in vitro. The results showed significant down regulation of insuline like growth factor (IGF-1) gene expression in 100 and 50 μg/L BPA and 2,4-D exposure respectively. While goldfish vitellogenin (VTG) gene expression demonstrated altered expression. This work provides an important insight into predominant microbial evaluation and validity of few biomarker genes in fish affected by BPA and 2,4-D herbicide. Aro-B, HSP70 and CYP1A gene expression showed significant alteration in exposed zebrafish embryos. The efficacy of UV based degradation of these chemicals has also been tested biologically. The findings might be helpful to create awareness about pollutant loading to lakes, rivers and other aquatic ecosystems and to devise promising techniques to remediate the problem.