Evaluation of wastewater reclamation technologies based on in vitro and in vivo bioassays.

When municipal secondary effluent is used as the main supplementation water source for surface water bodies, its potential adverse ecological effects should not be neglected. The objective of this work was to investigate the effectiveness of several technologies, i.e. combination of coagulation and sand filtration (CSF), ultraviolet (UV) irradiation, chlorination, ozonation, ultrafiltration (UF) and reverse osmosis filtration (RO), on the removal of acute ecotoxicity, genotoxicity and retinoic acid receptor (RAR) agonist activity from the municipal secondary effluent. The effects of treated effluents on the development of Japanese medaka (Oryzias latipes) embryos were also evaluated. The secondary effluent exhibited a mutagenic effect on Salmonella typhimurium strain TA 1535/pSK1002, acute invertebrate toxicity to Daphnia magna, and weak RAR alpha activity. RO and ozonation demonstrated remarkable removals of the genotoxic effect, acute toxicity and RAR activity from secondary effluent, while chlorination could elevate both genotoxicity and acute toxicity. CSF, UV, UF, chlorination as well as RO could decrease the 4-day mortality of medaka embryos and accordingly increase the hatching success rate, comparing with the secondary effluent. Ozonation at 4 mg/l and higher doses, however, elicited significantly higher 4-day mortality, leading to the reduction of the hatching success rate.

Contamination with retinoic acid receptor agonists in two rivers in the Kinki region of Japan.

This study was conducted to investigate the agonistic activity against human retinoic acid receptor (RAR) alpha in the Lake Biwa-Yodo River and the Ina River in the Kinki region of Japan. To accomplish this, a yeast two-hybrid assay was used to elucidate the spatial and temporal variations and potential sources of RARalpha agonist contamination in the river basins. RARalpha agonistic activity was commonly detected in the surface water samples collected along two rivers at different periods, with maximum all-trans retinoic acid (atRA) equivalents of 47.6 ng-atRA/L and 23.5 ng-atRA/L being observed in Lake Biwa-Yodo River and Ina River, respectively. The results indicated that RARalpha agonists are always present and widespread in the rivers. Comparative investigation of RARalpha and estrogen receptor alpha agonistic activities at 20 stations along each river revealed that the spatial variation pattern of RARalpha agonist contamination was entirely different from that of the estrogenic compound contamination. This suggests that the effluent from municipal wastewater treatment plants, a primary source of estrogenic compounds, seemed not to be the cause of RARalpha agonist contamination in the rivers. Fractionation using high performance liquid chromatography (HPLC) directed by the bioassay found two bioactive fractions from river water samples, suggesting the presence of at least two RARalpha agonists in the rivers. Although a trial conducted to identify RARalpha agonists in the major bioactive fraction was not completed as part of this study, comparison of retention times in HPLC analysis and quantification with liquid chromatography-mass spectrometry analysis revealed that the major causative contaminants responsible for the RARalpha agonistic activity were not RAs (natural RAR ligands) and 4-oxo-RAs, while 4-oxo-RAs were identified as the major RAR agonists in sewage in Beijing, China. These findings suggest that there are unknown RARalpha agonists with high activity in the rivers.

Identification of retinoic acid receptor agonists in sewage treatment plants.

Retinoic acid receptor (RAR) agonists are speculated to be one possible cause for the widely observed frog deformities in North America, although little is known about the specific RAR agonists in aquatic environments. We identified the specific RAR egonists in sewage treatment plants (STPs) and receiving rivers using an RAR yeast two-hybrid bioassay. Water samples were extracted by solid-phase extract cartridges, which were successively eluted by hexane, ethyl acetate, and methanol for bioassay. Among the three fractions, the ethyl acetate fraction showed the highest RAR agonistic activities. The bioassay-derived activity, expressed as all-trans-retinoic acid (all-transRA) equivalents (ATRA-EQ) were 10.9 +/- 2.2 and 1.7 +/- 1.0 ng/L in the STP influents and effluents, respectively, while the ATRA-EQs were as high as 7.1 and 8.3 ng/L in the two rivers receiving STP effluents. Following a two-step fractionation using high-performance liquid chromatography and ultra-performance liquid chromatography (UPLC) directed by the bioassay, two bioactive fractions were obtained from Gaobeidian STP influent and all-trans-4-oxo-RA (4.7-10.4 ng/L in influents, < 0.2-0.9 ng/L in effluents) and 13-cis-4-oxo-RA (2.3-7.1 ng/L in influents, < 0.4-1.1 ng/L in effluents) were identified in these fractions with UPLC-MS/MS. The EC50 for all-trans-4-oxo-RA or 13-cis-4-oxo-RA relative to that of all-trans-RA in exhibiting RARalpha agonistic activity was calculated to be 3.87 and 0.46, respectively.