Elsevier

Water Research

Volume 131, 15 March 2018, Pages 33-44
Water Research

An integrated approach with the zebrafish model for biomonitoring of municipal wastewater effluent and receiving waters

https://doi.org/10.1016/j.watres.2017.12.017Get rights and content

Highlights

  • Zebrafish toxicogenomics, targeted genes and morphology for biomonitoring.

  • Effluent exposure deregulated 631 genes at 0.5× and 1.0× original concentration.

  • Genes linked to 18 molecular functions, 10 organ-systems and 11 signaling pathways.

  • Effluent exposure affected nervous, hepatic, and vascular-myeloid systems.

  • Integrated effect-based biomonitoring tools for effluent and receiving waters.

Abstract

Comprehensive monitoring of water pollution is challenging. With the increasing amount and types of anthropogenic compounds being released into water, there are rising concerns of undetected toxicity. This is especially true for municipal wastewater effluents that are discharged to surface waters. This study was designed to integrate zebrafish toxicogenomics, targeted gene expression, and morphological analyses, for toxicity evaluation of effluent discharged from two previously characterized wastewater treatment plants (WWTPs) in Pima County, Arizona, and their receiving surface water. Zebrafish embryos were exposed to organic extracts from the WWTP1 effluent that were reconstituted to represent 1× and 0.5× of the original concentration. Microarray analyses identified deregulated gene probes that mapped to 1666, 779, and 631 unique human homologs in the 1×, 0.5×, and the intersection of both groups, respectively. These were associated with 18 cellular and molecular functions ranging from cell cycle to metabolism and are involved in the development and function of 10 organ systems including nervous, cardiovascular, haematological, reproductive, and hepatic systems. Superpathway of cholesterol biosynthesis, retinoic acid receptor activation, glucocorticoid receptor and prolactin signaling were among the top 11 perturbed canonical pathways. Real-time quantitative PCR validated the expression changes of 12 selected genes. These genes were then tested on zebrafish embryos exposed to the reconstituted extract of water sampled downstream of WWTP1 and another nearby WWTP2. The expression of several targeted genes were significantly affected by the WWTP effluents and some of the downstream receiving waters. Morphological analyses using four transgenic zebrafish lines revealed potential toxicity associated with nervous, hepatic, endothelial-vascular and myeloid systems. This study demonstrated how information can be obtained using adverse outcome pathway framework to derive biological effect-based monitoring tools. This integrated approach using zebrafish can supplement analytical chemistry to provide more comprehensive monitoring of discharged effluents and their receiving waters.

Introduction

The monitoring of water quality is challenging due to the numerous natural and anthropogenic chemicals found in the water bodies. With the increasing amount and types of anthropogenic compounds being introduced into water, in addition to transformation products formed during water treatment, it is not feasible to analytically detect and quantify every chemical contaminant in water (Merel et al., 2015). Moreover, even if individual chemicals are detectable, multiple chemicals may interact to induce toxicity in aquatic organisms via mechanisms such antagonism, additivity, or synergism (Adebambo et al., 2015, Carvalho et al., 2014). Therefore, biomonitoring is recommended as a complementary approach to assess the potential toxicity of water (Altenburger et al., 2015, Gerbersdorf et al., 2015, Escher et al., 2014, Leusch and Snyder, 2015).

Fish are the most commonly used vertebrates for biomonitoring of water quality. Acute tests in adult fish (OECD, 1992) and fish embryo tests (OECD, 2013) based on mortality rate and gross morphological changes have been used to assess toxicity of aquatic contaminants in the laboratory and field. However, gross morphological changes can be difficult to quantify or lack adequate sensitivity. Transgenic fish with fluorescent proteins expressed in specific tissues can facilitate the visualization (Kim et al., 2013) and enable the quantitative measurement of changes in these tissues, even when no gross morphological changes can be observed in wild-type fish (Chen et al., 2010, Fetter et al., 2014, Kim et al., 2013, Ng and Gong, 2013). Yet, current application of transgenic fish in biomonitoring is still limited, especially transgenic fish with specific compound-inducible reporter gene, partly because they lack sensitivity to detect the specific compounds at environmentally-relevant concentrations (Lee et al., 2015b). Expression analysis of targeted genes that are known to be impacted by specific compounds is also used; for example, biomarker genes for oxidative stress, estrogenic, and androgenic compounds were used to evaluate whether water downstream from a WWTP discharge could change the expression of these genes in fish (Jasinska et al., 2015).

Toxicogenomic approaches that capture genome-wide expression changes induced by contaminants can provide insight on: (1) the molecular mechanism of toxicity; (2) candidate biomarkers for exposure to toxicants; and (3) inference of the potential effects that have not yet reflected as morphological changes (Chen et al., 2012, Naciff and Daston, 2004). For instance, certain transcriptome profiles were identifiable in rats (Hamadeh et al., 2002), in zebrafish embryos (Yang et al., 2007), and in whole adult zebrafish (Lam et al., 2008) exposed to certain compounds. Moreover, transcriptomic profiling of fish, such as largescale suckers (Christiansen et al., 2014), fathead minnows (Sellin Jeffries et al., 2012), and zebrafish (Bluhm et al., 2014, Kosmehl et al., 2012), exposed to environmental samples with unknown chemical composition can reveal affected biology and provide information on the exposure conditions or presence of potential classes of contaminants. However, challenges still remain in distinguishing between responses induced by specific classes of contaminants and the more universal detoxification mechanisms (Bluhm et al., 2014). Moreover, molecular changes are influenced more by the bioavailability of the compounds in sample mixture, which is determined by sample processing and exposure methods, than the original composition of the mixture (Kosmehl et al., 2012). Therefore, the transcriptomic changes detected are only molecular changes that may or may not be directly driven by the compound(s) being studied. Nevertheless, candidate biomarkers identified from toxicogenomic studies can be used as effect-based monitoring tools in future studies or facilitate the development of environmental risk assessment that is based on the adverse outcome pathway (AOP) framework by providing evidence for ‘molecular initiating events’ or ‘cellular key events’ in the toxicity pathways that lead to ‘organ key events’ and adverse outcome at the organism and population levels (Ankley et al., 2010, Lee et al., 2015a).

The Santa Cruz River in Pima County, Arizona, USA is an effluent-dependent stream receiving effluent from two WWTPs. Previous studies have been performed using chemical analyses, in vitro bioassays, and expression analysis of target genes for glucocorticoid, results of which have raised concern on the potential toxicity of the effluent, especially the glucocorticoid activity and estrogenic activity (Chen et al., 2016, Dong et al., 2015, Jia et al., 2015). Yet, it is not known whether other pathways or expression of other genes can also be affected in vivo by this effluent-dependent stream. Given the availability of transgenic lines, genomic platforms, and the advantages of zebrafish as a model (Scholz et al., 2008), the current study aimed to integrate zebrafish approaches involving microarray-based toxicogenomics, targeted gene expression, and transgenic embryos to assess the potential toxicity of the effluent from the two WWTPs and the receiving effluent-dependent segment of the Santa Cruz River.

Section snippets

Sample collection and extraction

Effluent samples were collected from two WWTPs located in Pima County, Arizona, USA which receive predominantly municipal sewage from over half a million domestic residents. At the time of sample collection, WWTP1 used a trickling filter treatment system with a capacity of 1.80 m3/s. WWTP2 is located approximately 6 km downstream to WWTP1 along the Santa Cruz River, which uses an activated sludge treatment system and has a treatment capacity of 1.64 m3/s. Both facilities include disinfection in

Embryo toxicity testing of effluent from WWTP1

A 5-day embryo toxicity test was performed using WWTP1 effluent extract that has been reconstituted to 0.2× to 5× of the original concentration. Before hatching, no difference was observed in the number of embryos with coagulation (<= 2). Embryos of all groups showed normal tail detachment, somite development, eye development, blood circulation and spontaneous movement at 24 hpf and 48 hpf. Heartbeat rate was counted at 48 hpf and there was a notable reduction of 5–13% in 0.5× and higher

Conclusion

The present study demonstrates how molecular initiating events, cellular and organ level key events within AOP framework can be obtained or inferred using the zebrafish embryo exposed to an effluent and further applied for monitoring of the receiving waters. Although no adverse outcome was detected using a short-term toxicity test in this study, similar approaches would have been employed to obtain relevant and practical information if a more toxic effluent that causes malformation of embryos

Acknowledgement

We thank Darcy VanDervort for her help in water sampling and sample preparation. This study is supported by a grant from the Singapore National Research Foundation under its Environmental & Water Technologies Strategic Research Programme and administered by Public Utilities Board (PUB), Singapore. The zebrafish microarrays were supported by a special funding from Agilent Technologies. We thank the NUS Environmental Research Institute (NERI) for their laboratory and administrative support.

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