Determination of estrogens and estrogenic activity in wastewater effluent by chemical analysis and the bioluminescent yeast assay

A scheme of bioassay-directed analysis has been developed which combines a yeast assay screening for estrogenic activity with a liquid chromatographic-mass spectrometric (LC-MS/MS) chemical analysis, chromatographic fractionation, solid phase extraction and freeze-drying. The test scheme was applied on effluent samples collected from a municipal sewage treatment plant. The aim was to determine the substances responsible for main portion of the estrogenic activity in the samples and to compare the efficiency of different procedures for isolation and concentration of estogenicity. LC-MS/MS analyses were used for the quantification of 17beta-estradiol, estrone, estriol and 17alpha-ethinylestradiol, and the measured concentrations compared with the activities found in the yeast assay. Following conversion of the concentrations measured by LC-MS/MS to 17beta-estradiol equivalents it was concluded that freeze-drying, solid phase extraction and the chemical analysis gave comparable activities. Since estrone was the major estrogen in the effluent, this estrogen was also the major contributor to the estrogenic activity in the effluent. The estrogenic activity was equivalent to 4-7 ng/L of 17beta-estradiol. The yeast assay results from the tests of the chromatographic fractions showed that the major activity resides in the fraction where estrone, 17beta-estradiol and 17alpha-ethinylestradiol eluted. The activity of this fraction was substantially higher than the activity of the original wastewater sample. The reason for this could in part be explained by an inhibition of activity occurring in the original water sample.     

Survey of contamination of estrogenic chemicals in Japanese and Korean coastal waters using the wild grey mullet (Mugil cephalus)

We monitored the contamination by environmental estrogens (EEs) of coastal areas in Korea and Japan using the wild grey mullet. The grey mullet were collected from Ansan, Jeju, Yeosu, Tongyeong, and Busan in Korea and Nagasaki, Omuta, and Fukuoka in Japan. Contamination by EEs was determined by measuring vitellogenin (VTG) levels in serum and identifying gonadal abnormalities histologically (i.e., testis-ova). In four sites in Korea (Ansan, Yeosu, Tongyeong, and Busan) and two sites in Japan (Nagasaki and Fukuoka), serum VTG in immature and male grey mullet was detected at levels greater than 1.0 μg/ml, which is considered to be an abnormal level. Although, testis-ova were observed in some individuals collected in Ansan, Tongyeong, and Busan in Korea and Omuta in Japan, there was no correlation between individuals with testis-ova and individuals with abnormal levels of VTG. Furthermore, in Japan, serum VTG levels of fish collected from Nagasaki and Fukuoka were also greater than 1.0 μg/ml. Although individuals with testis-ova were found in Omuta, these fish expressed normal levels of serum VTG. Our results suggest that the grey mullets living in these coastal areas are influenced by EEs in the environment. Furthermore, it appears that the production of VTG and the occurrence of testis-ova are caused by different mechanisms.     

Use of native species Crenicichla menezesi (Ariidae) as a model for in situ evaluation of genotoxicity in surface water

The aim of this work was to analyze the genotoxicity in surface water from the Pitimbú River (Natal, Brazil) using a neotropical species Crenicichla menezesi as bioindicator for in situ evaluation. The genotoxicity was analyzed using the micronucleus (MN) and comet assays in erythrocytes. The animals were collected from five sites of the river. A significant increase of MN frequency and comet class L1 to L4 were observed only in specimens collected from three sampling sites (SS3, SS4 and SS5) located into urban areas. These same sites also presented high metals concentration. Correlation of DNA damage and concentrations of Cu (Spearman r = 0.8080), Cr (Spearman r = 0.9772), and Ni (Spearman r = 0.9909) were observed. These data indicate the presence of genotoxic agents. Moreover the C. menezesi species showed to be a good indicator for prompt analyses since this species presents sedentary and carnivore habits.     

Endocrine disrupting, haematological and biochemical effects of polybrominated diphenyl ethers in a terrestrial songbird, the European starling (Sturnus vulgaris)

We exposed female European starlings (Sturnus vulgaris) during a 6 month period to environmentally relevant concentrations of a pentabromodiphenyl ether (Penta-BDE) mixture (about 1740 μg/kg body weight) through subcutaneous implants, and examined endocrine disruptive, haematological and biochemical effects. To investigate endocrine disrupting effects of PBDEs, testosterone, estradiol and thyroid hormones (T₃ and T₄) were measured in the plasma. We found no significant differences between the control and exposed groups for any of the hormones. However, 14 days after implantation, T₃ concentrations tended to be lower in the exposed group compared to the control group. For albumin, our results suggest that short term exposure of PBDEs may result in a negative effect on albumin, while the opposite was observed on a longer term (6 months after implantation). However, from the statistical analysis, effects on albumin disappeared when excluding females that laid eggs. These results demonstrate that observed effects during the breeding season should be interpreted carefully when the breeding status of females is unknown. There were no significant differences found between the control and exposed groups for the other biochemical parameters (total protein, uric acid, cholesterol, aspartate transaminase, total antioxidative capacity, high-density lipoproteins and creatine kinase) and haematological parameters (amount of red blood cells, haemoglobin concentration, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin). All together, our results suggest that European starlings, similar to other passerine species, may be less sensitive to the effects of organohalogenated pollutants, such as PBDEs, than other bird species.     

Evaluation of wastewater nitrogen transformation in a natural wetland (Ulaanbaatar, Mongolia) using dual-isotope analysis of nitrate

The Tuul River, which provides water for the daily needs of many residents of Ulaanbaatar, Mongolia, has been increasingly polluted by wastewater from the city's sewage treatment plant. Information on water movement and the transformation of water-borne materials is required to alleviate the deterioration of water quality. We conducted a synoptic survey of general water movement, water quality including inorganic nitrogen concentrations, and isotopic composition of nitrogen (δ¹⁵N-NO₃⁻, δ¹⁸O-NO₃⁻, and δ¹⁵N-NH₄⁺) and water (δ¹⁸O-H₂O) in a wetland area that receives wastewater before it enters the Tuul River. We sampled surface water, groundwater, and spring water along the two major water routes in the wetland that flow from the drain of the sewage treatment plant to the Tuul River: a continuous tributary and a discontinuous tributary. The continuous tributary had high ammonium (NH₄⁺) concentrations and nearly stable δ¹⁵N-NH₄⁺, δ¹⁵N-NO₃⁻, and δ¹⁸O-NO₃⁻ concentrations throughout its length, indicating that nitrogen transformation (i.e., nitrification and denitrification) during transit was small. In contrast, NH₄⁺ concentrations decreased along the discontinuous tributary and nitrate (NO₃⁻) concentrations were low at many points. Values of δ¹⁵N-NH₄⁺, δ¹⁵N-NO₃⁻, and δ¹⁸O-NO₃⁻ increased with flow along the discontinuous route. Our results indicate that nitrification and denitrification contribute to nitrogen removal in the wetland area along the discontinuous tributary with slow water transport. Differences in hydrological pathways and the velocity of wastewater transport through the wetland area greatly affect the extent of nitrogen removal.     

Chronic exposure to sublethal hexavalent chromium affects organ histopathology and serum cortisol profile of a teleost, Channa punctatus (Bloch)

Effects of chronic exposures (one and two months) to sublethal doses of hexavalent chromium (2 and 4 mg/L potassium dichromate) on organ histopathology and serum cortisol profile were investigated and their overall impact on growth and behavior of a teleost fish, Channa punctatus was elucidated. Histopathological lesions were distinct in the vital organs gill, kidney and liver. The gill lamellae became lifted, fused, and showed oedema. Hyperplasia and hypertrophy of lamellar epithelial cells were distinct with desquamation. Hypertrophy of epithelial cells of renal tubules and reduction in tubular lumens were observed in the trunk kidney. The atrophy of the head kidney interrenal cells and decreased serum cortisol level indicated exhaustion of interrenal activity. Hepatocyte vacuolization and shrinkage, nuclear pyknosis and increase of sinusoidal spaces were observed in the liver. Abnormal behavioral patterns and reduced growth rate were also noticed in the exposed fish. The chronic hexavalent chromium exposure thus by affecting histopathology of gill, kidney (including interrenal tissue) and liver could impair the vital functions of respiration, excretion, metabolic regulation and maintenance of stress homeostasis which in the long-run may pose serious threat to fish health and affect their population.     

Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta

Hepatotoxic microcystins (MCs) produced by cyanobacteria are known to accumulate in gastropods following grazing of toxic cyanobacteria and/or absorption of MCs dissolved in water, with adverse effects on life history traits demonstrated in the laboratory. In the field, such effects may vary depending on species, according to their relative sensitivity and ecology. The aims of this study were to i) establish how various intensities of MC-producing cyanobacteria proliferations alter the structure of gastropod community and ii) compare MC tissue concentration in gastropods in the field with those obtained in our previous laboratory experiments on the prosobranch Potamopyrgus antipodarum and the pulmonate Lymnaea stagnalis. We explored these questions through a one-year field study at three stations at Grand-Lieu Lake (France) affected by different intensities of cyanobacteria proliferations. A survey of the community structure and MC content of both cyanobacteria and gastropods was associated with a caging experiment involving P. antipodarum and L. stagnalis. In total, 2592 gastropods belonging to 7 prosobranch and 16 pulmonate species were collected. However, distribution among the stations was unequal with 62% vs 2% of gastropods sampled respectively at the stations with the lowest vs highest concentrations of MC. Irrespective of the station, pulmonates were always more diverse, more abundant and occurred at higher frequencies than prosobranchs. Only the pulmonate Physa acuta occurred at all stations, with abundance and MC tissue concentration (≤ 4.32 µg g DW^− 1) depending on the degrees of MC-producing cyanobacteria proliferations in the stations; therefore, P. acuta is proposed as a potential sentinel species. The caging experiment demonstrated a higher MC accumulation in L. stagnalis (≤ 0.36 µg g DW^− 1 for 71% of individuals) than in P. antipodarum (≤ 0.02 µg g DW^− 1 for 12%), corroborating previous laboratory observations. Results are discussed in terms of differential gastropod sensitivity and MC transfer through the food web.     

Bioaugmentation with the nicotine-degrading bacterium Pseudomonas sp. HF-1 in a sequencing batch reactor treating tobacco wastewater: Degradation study and analysis of its mechanisms

The highly effective nicotine-degrading bacterium Pseudomonas sp. HF-1 was augmented in an SBR system that is used to treat tobacco wastewater. Compared to the non-bioaugmented (non-BA) system, the bioaugmented (BA) system exhibited considerably stronger pollution disposal abilities, with 100% nicotine degradation and more than 84% chemical oxygen demand (COD) removal within 12 h. Nicotine degradation had a significant effect on COD removal in SBRs (r = 0.928, p < 0.01). The mechanisms of bioaugmentation were systematically investigated using a combination of polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) and a toxicity assay (protein carbonyl (PC) and DNA-protein crosslinking (DPC)). DGGE fingerprint profiles showed that the number of bands and the Shannon-Wiener index decreased at a nicotine load of 250 mg/L compared to a 40-130 mg/L nicotine load in the non-BA system. However, a stepwise increase in the Shannon-Wiener index was found during all periods in the BA system. A comparison of sequences excised from DGGE gels demonstrated significant differences in the dominant microbial species between the two SBRs. This result suggested that bioaugmentation of strain HF-1 could select cooperators for treating complicated tobacco wastewater. The PC content and the DPC coefficient increased significantly at levels higher than 80 mg/L in the non-BA system; nevertheless, no increase was observed in the BA system during the stepwise nicotine load. This indicated that bioaugmentation of strain HF-1 resulted in the maintenance of high treatment activity by minimizing the nicotine toxicity for other microbes in the BA system. In conclusion, the rapid nicotine degradation of strain HF-1 performed a vital function in SBR by influencing the microbial community structure, dynamics and activity of the activated sludge system.     

Simultaneous determination of microcystin contaminations in various vertebrates (fish, turtle, duck and water bird) from a large eutrophic Chinese lake, Lake Taihu, with toxic Microcystis blooms

This is the first to conduct simultaneous determination of microcystin (MC) contaminations in multi-groups of vertebrates (fish, turtle, duck and water bird) from Lake Taihu with Microcystis blooms. MCs (-RR, -YR, -LR) in Microcystis scum was 328 μg g^− 1 DW. MCs reached 235 μg g^− 1 DW in intestinal contents of phytoplanktivorous silver carp, but never exceeded 0.1 μg g^− 1 DW in intestinal contents of other animals. The highest MC content in liver of fish was in Carassius auratus (150 ng g^− 1 DW), followed by silver carp and Culter ilishaeformis, whereas the lowest was in common carp (3 ng g^− 1 DW). In livers of turtle, duck and water bird, MC content ranged from 18 to 30 ng g^− 1 DW. High MC level was found in the gonad, egg yolk and egg white of Nycticorax nycticorax and Anas platyrhynchos, suggesting the potential effect of MCs on water bird and duck embryos. High MC contents were identified for the first time in the spleens of N. nycticorax and A. platyrhynchos (6.850 and 9.462 ng g^− 1 DW, respectively), indicating a different organotropism of MCs in birds. Lakes with deaths of turtles or water birds in the literatures had a considerably higher MC content in both cyanobacteria and wildlife than Lake Taihu, indicating that toxicity of cyanobacteria may determine accumulation level of MCs and consequently fates of aquatic wildlife.     

Formation, modeling and validation of trihalomethanes (THM) in Malaysian drinking water: a case study in the districts of Tampin, Negeri Sembilan and Sabak Bernam, Selangor, Malaysia

A modeling procedure that predicts trihalomethane (THM) formation from field sampling at the treatment plant and along its distribution system using Tampin district, Negeri Sembilan and Sabak Bernam district, Selangor as sources of data were studied and developed. Using Pearson method of correlation, the organic matter measured as TOC showed a positive correlation with formation of THM (r=0.380,P=0.0001 for Tampin and r=0.478,P=0.0001 for Sabak Bernam). Similar positive correlation was also obtained for pH in both districts with Tampin (r=0.362,P=0.0010) and Sabak Bernam (r=0.215,P=0.0010). Chlorine dosage was also found to have low correlation with formation of THM for the two districts with Tampin (r=0.233,P=0.0230) and Sabak Bernam (r=0.505,P=0.0001). Distance from treatment plant was found to have correlation with formation of THM for Tampin district with r=0.353 and P=0.0010. Other parameters such as turbidity, ammonia, temperature and residue chlorine were found to have no correlation with formation of THM. Linear and non-linear models were developed for these two districts. The results obtained were validated using three different sets of field data obtained from own source and district of Seremban (Pantai and Sg. Terip), Negeri Sembilan. Validation results indicated that there was significant difference in the predictive and determined values of THM when two sets of data from districts of Seremban were used with an exception of field data of Sg. Terip for non-linear model developed for district of Tampin. It was found that a non-linear model is slightly better than linear model in terms of percentage prediction errors. The models developed were site specific and the predictive capabilities in the distribution systems vary with different environmental conditions.     

Assessment of soil and groundwater impacts by treated urban wastewater reuse. A case study: application in a golf course (Girona, Spain)

Starting in July 2000, treated wastewater of urban origin has been used for the "Serres de Pals" golf course irrigation (Girona, Spain). To evaluate if the soil and the aquifer underneath are affected by the utilization of this type of water, samples have been taken along a period of several months from the wastewater treatment plant, the stabilization lagoon, groundwater and soil profiles. Analyses have been performed for total coliforms and aerobic bacteria, soil water pressure and soil water content as well as chemical analyses of the irrigation water, aquifer and water of the vadose zone. Soil profiles taken at several times during the study indicate the absence of coliforms except for a short period during summer. In the vadose zone an increase of more than 1000 mg kg(-1) of NaO(2) in the top 60 cm of soil was observed while Cl(-) concentration in the aquifer reached up to 1200 mg l(-1) ten months after starting the irrigation.     

Speciation of lead, copper, zinc and antimony in water draining a shooting range—Time dependant metal accumulation and biomarker responses in brown trout (Salmo trutta L.)

The speciation of Pb, Cu, Zn and Sb in a shooting range run-off stream were studied during a period of 23 days. In addition, metal accumulation in gills and liver, red blood cell ALA-D activity, hepatic metallothionine (Cd/Zn-MT) and oxidative stress index (GSSG/ tGSH levels) in brown trout (Salmo trutta L.) exposed to the stream were investigated. Fish, contained in cages, were exposed and sampled after 0, 2, 4, 7, 9, 11 and 23 days of exposure. Trace metals in the water were fractionated in situ according to size (nominal molecular mass) and charge properties. During the experimental period an episode with higher runoff occurred resulting in increased levels of metals in the stream. Pb and Cu were mainly found as high molecular mass species, while Zn and Sb were mostly present as low molecular mass species. Pb, Cu and Sb accumulated on gills, in addition to Al origination from natural sources in the catchment. Pb, Cu and Sb were also detected at elevated concentration in the liver. Blood glucose and plasma Na and Cl levels were significantly altered during the exposure period, and are attributed to elevated concentrations of Pb, Cu and Al. A significant suppression of ALA-D was detected after 11 days. Significant differences were detected in Cd/Zn-MT and oxidative stress (tGSH/GSSG) responses at Day 4. For Pb the results show a clear link between the HMM (high molecular mass) positively charged Pb species, followed by accumulation on gills and liver and a suppression in ALA-D. Thus, high flow episodes can remobilise metals from the catchment, inducing stress to aquatic organisms.     

Effects of laying order and experimentally increased egg production on organic pollutants in eggs of a terrestrial songbird species, the great tit (Parus major)

In this study, concentrations and profiles of organic pollutants were investigated in a passerine species with a large clutch size, the great tit (Parus major). In the first clutches, mean egg concentrations decreased significantly in relation to the laying order from 3025 ± 416 ng/g lw to 2267 ± 386 ng/g lw for sum PCBs and from 989 ± 339 ng/g lw to 695 ± 320 ng/g lw for sum DDTs. Sum PBDE concentrations also decreased in relation to the laying order from 68 ± 10 ng/g lw to 53 ± 11 ng/g lw, but not significantly. Although laying order effects were found, variation in concentrations within clutches was smaller than among clutches. To further investigate the impact of laying large numbers of eggs on levels and profiles of organic pollutants, initiation of replacement clutches was experimentally induced. Mean sum PCB and sum PBDE concentrations were significantly lower in eggs of replacement clutches compared to first clutches. In addition, first clutches had a higher contribution of the higher chlorinated and more persistent PCB congeners, CB 170, 180 and 183, and a lower contribution of CB 52, 95 and 149 compared to replacement clutches. Because of the differences in concentrations and profiles between the first and replacement clutches, the combined use of eggs from both the first and replacement clutches for monitoring purposes is not recommended. In conclusion, we suggest that, due to the larger variation among clutches compared to the variation within clutches, one randomly collected great tit egg from a first clutch is useful as a biomonitoring tool for organic pollutants. To our knowledge, this is the first study in which the impact of an experimentally increased clutch size on the levels and profiles of contaminants in eggs has been investigated.