Linking Ah receptor mediated effects of sediments and impacts on fish to key pollutants in the Yangtze Three Gorges Reservoir, China — A comprehensive perspective

Highlights

• Significant correlations between PAHs in sediment and in vitro/in vivo endpoints

• Low bioavailability of particle-bound pollutants

• Significant ethoxyresorufin-O-deethylase induction in fish at Chongqing city

• Results suggest rather poor condition of test fish species Pelteobagrus vachellii.

• Results suggest pivotal role of PAHs in observed ecotoxicological impacts.

Abstract

The Three Gorges Reservoir (TGR), created in consequence of the Yangtze River's impoundment by the Three Gorges Dam, faces numerous anthropogenic impacts that challenge its unique ecosystem. Organic pollutants, particularly aryl hydrocarbon receptor (AhR) agonists, have been widely detected in the Yangtze River, but only little research was yet done on AhR-mediated activities. Hence, in order to assess effects of organic pollution, with particular focus on AhR-mediated activities, several sites in the TGR area were examined applying the “triad approach”. It combines chemical analysis, in vitro, in vivo and in situ investigations to a holistic assessment. Sediments and the benthic fish species Pelteobagrus vachellii were sampled in 2011/2012, respectively, to identify relevant endpoints. Sediment was tested in vitro with the ethoxyresorufin-O-deethylase (EROD) induction assay, and in vivo with the Fish Embryo Toxicity Test and Sediment Contact Assay with Danio rerio. Activities of phase I (EROD) and phase II (glutathione-S-transferase) biotransformation enzymes, pollutant metabolites and histopathological alterations were studied in situ in P. vachellii. EROD induction was tested in vitro and in situ to evaluate possible relationships. Two sites, near Chongqing and Kaixian city, were identified as regional hot-spots and further investigated in 2013. The sediments induced in the in vitro/in vivo bioassays AhR-mediated activities and embryotoxic/teratogenic effects – particularly on the cardiovascular system. These endpoints could be significantly correlated to each other and respective chemical data. However, particle-bound pollutants showed only low bioavailability. The in situ investigations suggested a rather poor condition of P. vachellii, with histopathological alterations in liver and excretory kidney. Fish from Chongqing city exhibited significant hepatic EROD induction and obvious parasitic infestations. The polycyclic aromatic hydrocarbon (PAH) metabolite 1-hydroxypyrene was detected in bile of fish from all sites. All endpoints in combination with the chemical data suggest a pivotal role of PAHs in the observed ecotoxicological impacts.

Introduction

The Yangtze River basin is an elemental source for drinking water for millions of people and plays a significant role in freshwater fishery of the country (Liu and Cao, 1992, Liu et al., 2005, National Bureau of Statistics — China, 2004, Wong et al., 2007). One part of the upper Yangtze River section was impounded by the Three Gorges Dam (TGD) to form the Three Gorges Reservoir (TGR). The TGR stretches from Sandouping, Hubei Province, to the Jiangjin district of Chongqing Municipality, over a distance of 663 km (Fig. 1). Overpopulation of the TGR area and pollution, as a consequence of the impoundment, involve serious threats to the recently established vulnerable reservoirs ecosystem (Xinhua, 2007a, Xinhua, 2007b). Hence, this consequently also threatens an important source for drinking water and food for many people.

The population of Chongqing municipality already exceeded 29 million people in 2011, with Chongqing City (6–7 million in 2012) being the largest city at the TGR (BBC, 2012, National Bureau of Statistics — China, 2012). It is also one of the largest cities in western China and belongs to the fastest growing cities worldwide. Further, the increasing freight and shipping traffic on the TGR, due to the elevation of the water level, are intended to stimulate the regional economy (Xinhua, 2014). Progressive urbanization and industrialization will entail an increasing amount of wastewater in the urbanized regions. About 1000 Megatonnes (Mt) urban sewage (53%) and industrial wastewater (47%) were discharged annually in the TGR in the years 2004 to 2010 (Ministry of Environmental Protection – China, 2006–2012). In response the local governments adopted policies on emission reduction for the industry and improved the domestic wastewater treatment capacities from annually 515 Mt in 56 facilities in 2008, to 590 Mt in 71 facilities in 2010. However, both times domestic sewage contributed for about 98%, meaning that still 88 Mt domestic and 548 Mt industrial sewage in 2008, as well as 37 Mt and 307 Mt, respectively, in 2010 were discharged untreated (Ministry of Environmental Protection — China, 2010, Ministry of Environmental Protection — China, 2012, Wang et al., 2013). In addition, the submerged 13 cities, 140 towns and over 1300 villages with 1600 factories and abandoned mines (Smith, 2013) may release remaining contamination into the reservoir. Müller et al. (2008) already stressed the importance of growing wastewater fractions in the Yangtze River for the sections downstream of the TGD. They calculated that, in addition to rising levels of nutrients, dissolved organic carbon and heavy metals, 500 to 3500 kg industrial organic chemicals are discharged daily into the river. However, for the region upstream of the dam only little information on pollution and potential impacts is yet available, particularly for the recently established TGR (as reviewed recently by Floehr et al., 2013).

The Yangtze River's fish fauna is described as one of the richest worldwide, providing plentiful fish resources, but they showed a serious decline with a significantly reduced fishery yield in the past years (Chen et al., 2009, Fu et al., 2003). Further, the species composition shifted in its quantitative relationships and decreased in diversity (Chen et al., 2002a, Chen et al., 2002b, Chen et al., 2009). The impoundment of the TGR further threatened endemic and rare fish species, particularly those adaptable to the prior running water conditions, with demersal fishes like the darkbarbel catfish (Pelteobagrus vachellii) becoming more important. Yiming and Wilcove (2005) stated the most pervasive threats to Chinese vertebrate animals in general were overexploitation, habitat destruction and pollution, which contributed to the endangerment of 78%, 70% and 20%, respectively, of imperiled species. However, only few quantitative studies on Yangtze fishes are available, but a number of qualitative research demonstrates that aside habitat fragmentation and shrinkage, invasion of exotic species and resources overexploitation, also water pollution is one of main responsible factors for the registered depletion and structural changes of the fish resources (Chen et al., 2009). Industrial and communal sewage discharging toxicants into the environment are considered to trigger the destruction of spawning grounds, depletion of brood stocks, decrease in production and induction of mortality (Chen et al., 2004). Chen et al. (2009) warned of the increasing seriousness of pollution in the Yangtze River as consequence of proceeding industrialization and urged for a strengthened environmental monitoring and control of the water pollution to maintain a suitable fishery environment.

The impoundment also resulted in a reduction of the Yangtze Rivers flow velocity from prior 2 to 3 m s^− 1 in average of the upper and middle layer down to less than 0.05 to 1.5 m s^− 1 (Chen et al., 2005, Wang et al., 2009). This in turn affects the dilution of contamination from point sources, such as urban and industrial wastewater outlets, from where they have been swept away before, as well as the sedimentation rate of suspended particles and adhering contaminants. Thus, it can be expected that the pollution rather accumulates in the area of discharge from which they have been swept away before. Sediments can act as sink and source for pollutants, and thus play an important role for environmental assessment (Ahlf et al., 2002, Gerbersdorf et al., 2005, Hilscherova et al., 2007, Hollert et al., 2003, Li et al., 2014a, Li et al., 2014b, Wallis et al., 2014, Wölz et al., 2009). The Yangtze River is one of the largest sediment carriers in the world, and about 60 to 68% of the sediment materials that entered the TGR from upstream have been trapped annually since the operation of the dam, which account for annually 151 to 172 Mt between 2003 and 2008 during the impoundment (Hu et al., 2009, Yang et al., 2007).

A common way to monitor the pollution status of a water body is to determine the chemical concentrations in water and sediment with chemical–analytical methods. This approach however is insufficient. That way only selected compounds are in the scope of the quality assessment, and an even larger variety of toxic non-target parent compounds and metabolites is not considered. Moreover, aspects like bioavailability of the toxic compounds, their synergistic and antagonistic effects, as well as the metabolism in the organisms are not taken into account. Thus, in order to acknowledge the complex situation in the field a holistic assessment in environmental monitoring is demanded, that considers not only the concentrations of selected compounds, but integrates them with the toxic effects the environmental sample may induce (Brils, 2008, Heugens et al., 2001, Hollert et al., 2007, Hollert et al., 2009, Malaj et al., 2014, SedNet, 2004, Wernersson et al., 2015). One approved holistic assessment method is the triad approach by Chapman (1990), with in situ investigations of biomarkers in fish suggested as additional line of evidence (Chapman and Hollert, 2006). This approach integrates chemical analysis, in vitro/in vivo bioassays and in situ biomarkers for a comprehensive assessment of environmental conditions.

Organic pollutants, particularly aryl hydrocarbon receptor (AhR) agonists, appeared to play a crucial role in the Yangtze River in general (as reviewed by Floehr et al., 2013) and in the TGR in particular (Deyerling et al., 2014, Floehr et al., in press, Wang et al., 2009, Wang et al., 2013, Wolf et al., 2013). However, only limited research was done on biomarkers and bioassays, which determine cytochrome P450 (CYP) activity (Floehr et al., 2013) – a major enzyme family for the metabolism of lipophilic xenobiotics that plays a central role in detoxification mechanisms in organisms. The ethoxyresorufin-O-deethylase (EROD) belongs to the CYP1A enzyme subfamily, metabolizes PAHs, polychlorinated biphenyls (PCBs), dioxins and furans and is activated by the AhR. The EROD activity can be used in vitro to exemplarily determine the AhR-mediated dioxin-like activity of environmental samples – like water and sediment – (for review Eichbaum et al., 2014), or in organisms utilized as an in vivo and in situ biomarker (Behrens and Segner, 2005, Brack et al., 2005, Brinkmann et al., 2013, Hallare et al., 2011, Hilscherova et al., 2000, Whyte et al., 2000). EROD is a representative for phase I biotransformation enzymes, while glutathione S-transferase (GST) is a prominent representative for phase II biotransformation enzymes. It is also mediated inter alia by the AhR and can be utilized as a biomarker as well (Hayes and Pulford, 1995, Kammann et al., 2014, Schlenk et al., 2008, Sen and Kirikbakan, 2004). Both groups play an important role in organisms in the detoxification and excretion of xenobiotics (Lopez-Galindo et al., 2010, Reed, 1990, Sen and Semiz, 2007). As fish are in a particular sensitive state at early life stages, the in vivo Fish Embryo Toxicity Test (FET) (Braunbeck et al., 2005) and Sediment Contact Assay (SCA) with eggs of Danio rerio (Hollert et al., 2003) have proved to be suitable methods in ecotoxicological assessment. In particular, as the FET can serve as a substitute for the acute toxicity test with adult fish (OECD guideline 203) registering mortality, it also allows to record sublethal impacts that might affect the overall fitness of the fish (Braunbeck et al., 2005, Lammer et al., 2009, Nagel, 2002).

Due to the outlined conditions at the TGR – progressive urbanization and industrialization in addition to the submerged sites and the altered hydrological conditions – considerable environmental impacts were expected. Thus, the main objective of the present study was to evaluate the ecotoxicological status of the TGR, with focus on AhR-mediated activities, and to address potential environmental risks, in order to support further management strategies of the TGR and other Chinese water bodies. The triad approach with additional lines of evidence was applied in this region for the first time.

The specific aims of this study were to (i) identify relevant endpoints and (ii) a suitable monitoring fish species, (iii) to evaluate the ecotoxicological relevance of potentially contaminated sediment from several sites of the TGR, to (iv) assess possible links between effects determined via in vitro/in vivo bioassays and relevant impacts on fish from the field investigated in situ, and (v) to discuss them in context of key pollutants prior identified via chemical analysis (as described earlier by Floehr et al., in press). As additional lines of evidence AhR-mediated EROD induction were investigated in vitro and in situ in order to verify the ecological relevance of AhR-mediated modes of action.

To this end, sediment samples and fish were collected at several sites in the TGR in the administrative region of Chongqing municipality. Sediment extracts were tested (a) in vitro with the EROD induction assay with RTL-W1 cells for AhR-mediated dioxin-like activity and (b) in vivo with the FET. Further, (c) the bioavailable fraction of particle bound pollutants was tested with native (freeze-dried) sediments in vivo with the SCA. Both in vivo assays were performed with eggs of D. rerio to screen for embryotoxic and teratogenic impacts. To complement these studies, hepatic (d) EROD and (e) GST activities of the darkbarbel catfish (P. vachellii, Richardson 1846) were investigated in situ. Further, (f) bile of the fish was tested to identify a potential exposure to PAHs, and (g) histopathological alterations in liver and kidney were recorded in parallel. The results of the chemical analysis, as well as mutagenic and genotoxic impacts have been described before by Floehr et al. (in press).