The evaluation of estrogenic activities and aryl hydrocarbon receptor (AhR) agonists in water from Three Gorges Reservoir (TGR) China was conducted by in vitro bioassays combined with SPMD-based virtual organisms (VO). VOs were deployed at seven sites in the Three Gorges Reservoir (TGR), China for two periods in 2009. The estrogenic activity was assessed using a rapid yeast estrogen bioassay, based on the expression of a green fluorescent reporter protein (yEGFP). The AhR activity was detected employing rat hepatoma cell line (H4IIE). The results indicate that AhR agonists distributed widely in water of TGR and almost homogenously distributed in most area of TGR. Weak antiestrogenic activities were also found homogenously distributed in water of TGR. Further studies are needed to determine the identities of these estrogenic compounds and AhR agonists and their potential adverse effects on wild biota in TGR.
The Three Gorges Dam (TGD) constructed on the Yangtze River in China is the largest hydro-electricity project in the world and created the Three Gorges Reservoir (TGR) with an area of 1080 km2. The closing of the TGD has resulted in drastic environmental alterations especially obvious changes in dominant fish species. Over the last decades, a decline in fish catches has been reported in the Yangtze River and fish have tended to be smaller and younger. Caught fish often exhibit diseases with increased frequencies of occurrence of malformations [
The results from conventional chemical analysis of priority pollutants [
Risk assessment for endocrine disrupters or dioxin-like compounds in surface water is difficult mainly because these compounds are usually low in concentrations, as well as the effects of many endpoints typically falling below the detection limit. In this case, necessary enrichment measures are demanded to improve the detection limit. The SPMD-based passive sampler which is also referred to be virtual organism (VO) imitates the exposure of animals in water and has been used to estimate time-integrated averages of dissolved contaminants [
The objective of the present study was to determine ecotoxicological effect in water of TGR by using two in vitro bioassays indicative of two specific endpoints―AhR- and ER-mediated activities combined with SPMD- based VO sampling. AhR agonistic potentials were determined by the micro-EROD bioassay based on exposure of H4IIE rat hepatoma cells. Presence of ER (ant) agonists was determined by application of a yeast estrogen bioassay expressing a green fluorescent reporter protein upon exposure to estrogens.
The SPMD-based VO samplers were prepared in the manner described in our previous paper [
The sampling sites spanned the whole reservoir from the upstream Chongqing to the great dam Maoping, covering more than 600 km long. The sampling sites were MP (Maoping), GJB (Guojiaba), BD1 (Badong), BD2 (Badong), WZ (Wanzhou), CS (Changshou), CQ (Chongqing) (
the river bank. The VOs for the determination of blanks were prepared together with the other VOs for sampling. VO blanks were transported to the sampling sites also in clean airtight jars, not deployed. After retrieval, the VOs were transported to the laboratory in the corresponding glass jars in the darkness and were kept in a freezer at −28˚C until processing.
All organic solvents were of picograde quality and were obtained from LGC standards (Wesel, Germany). The extraction method was according to our previous study [
The induction of EROD activity was measured upon exposure of rat hepatoma cell line (H4IIE) to samples. The cell culture and EROD bioassay were performed according to the procedure described in our previous paper [
The yeast assay used in this study is based on a cytosensor expressing the human estrogen receptor (hERα) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. The bioassay was performed according to the procedure described in our previous paper [
To test the antagonistic properties of the extracts, the 1-μL amounts of the extract stock solutions were co- exposed with 1-μL of E2 solution giving about a half-maximal response. RU5866 (ToCRIS, USA) which is a strong pure synthetic antagonist was used as reference standard in the study. Solvent blank and control samples containing only E2 or E2 plus RU were included in each experiment.
Each VO sample was measured at least three times and each sample concentration was tested in quadruplicate wells. The results of the standards were fitted and plotted by SigmaPlot (V.8.02, four parameter logistic function). The levels of AhR agonists in samples were obtained by comparing EROD activities to the TCDD concentration-response curve created using the standard solution on the same plate and expressed as TCDD equivalents (TEQ) in pg TCDD g−1 lipid (triolein). The limit of detection (LOD) was 2 pg TCDD g−1 lipid. A result is valid when the EROD activity falls in the linear part of the TCDD standard curve.
Similarly, the E2 equivalence (E2EQ) of samples was generated based on the E2 dose-response curve. The EC50 values of E2 dose-response curve were 1 nM which was in line with those reported previously [
ANOVA and t test were used to analyze difference among samples. Data were analyzed using the students t-test and the results expressed as mean ± SD of three independent replicates.
In this study, sample extracts were tested in the EROD assay without acid silica gel clean up, which destroys all but persistent compounds such as PCDDs/Fs. However, most organic chemicals could be metabolized by cultured cells after 72 h incubation [
The sediment load in the Yangtze River is 500 Mt (1 Mt = 1,000,000 t) year−1 [
and WZ [
Our previous study [
More persistent AhR agonists accounted for 18% to 37% of the total AhR agonists in sediments which is slightly higher than the ratio in water in the present study, indicating a little more of persistent AhR agonists settled in sediment. The majority of high affinity AhR ligands include halogenated aromatic hydrocarbons (organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDD/Fs), etc.) and polycyclic aromatic hydrocarbons (PAHs). However, our previous study [
No estrogenic activity was found in all of the VO samples and VO blanks (data not shown). In order to clarify if antagonistic effects in these VO extracts may have masked an estrogenic effect, sample extracts were co- exposed with 1 nM E2 in the estrogen bioassay and checked for antagonism. RU58668 is a synthetic compound with antiestrogenic activity in breast cells which was proven to be useful to treat patients with breast cancer. The antiestrogenic property of the compound was also tested by coadministration of 1 nM E2. The pure antiestrogen did not show any agonistic activity (data not shown), but an inhibition of the response of more than 50% caused by E2 that gave about a half-maximal response (1 nM) was observed at 1 mM (
Our previous study detected weak estrogenic activities in sediments from WZ to GJB areas [
sedimentation effect in the reservoir. Since nearly half of trapped suspended solids in TGR settled in the most industrialized and urbanized areas CS and WZ in the TGR [
The virtual organism sampling combined with in vitro bioassays provides an effective tool for ecological risk assessment for environmentally relevant water borne pollutants in surface water. The results indicate that AhR agonists distributed widely in water of TGR and almost homogenously distributed in most area of TGR. Weak antiestrogenic activities were also found homogenously distributed in water of TGR. Many antiestrogenic compounds may be more water soluble and could move forward with water flow, while estrogenic compounds may easily absorbed on suspended particles and settle down in sediment through natural setting process. The finding of antiestrogenic and EROD activities in TGR provides possible weight-of-evidence of potential ecotoxicological causes for the declines in fish populations which have been observed during the past decades in TGR. Further studies are needed to determine the identities of the estrogenic compounds and the unidentified AhR agonists and their potential adverse effects on wild biota in TGR.
This project was funded by the German Ministry of Education and Research (BMBF 02WT1132). The modified yeast strain was a gift from Toine Bovee (RIKILT, Institute of Food Safety, Wageningen University and Research Center, The Netherlands).
Jingxian Wang,Bernhard Henkelmann,Toine F. H. Bovee,Gerd Pfister,Liang Zhang,Karl-Werner Schramm, (2016) Evaluation of Estrogenic Receptor (ER) and Aryl Hydrocarbon Receptor (AhR) Agonists in Three Gorges Reservoir, China Using SPMD-Based Virtual Organisms. Journal of Environmental Protection,07,323-330. doi: 10.4236/jep.2016.73028