Metal distribution and metallothionein in loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles

The first aim of our study was to determine the concentrations of selected trace elements (Zn, Cu, Fe, Mn, Cd and Pb) in tissues of green turtles from Tortuguero National Park on the North Caribbean coast of Costa Rica and of loggerheads from the Mediterranean Sea. Zn, Cu, Fe, Mn and Cd were present at detectable concentrations in all samples and showed clear organotropism, whereas Pb was not always over the detection limit and did not show any particular tissue distribution. The two species presented significant differences: Cu and Cd in liver and kidney of Chelonia mydas were significantly higher with respect to the concentrations found in Caretta caretta. The second and major goal of our study was to evaluate hepatic and renal metallothionein (MT) as a biomarker of environmental metal exposure. The present paper is the first to describe and quantify MT in kidney and liver of loggerhead turtles and in kidney of green turtles. MT concentrations were higher in green than in loggerhead turtles. In addition, positive correlations were found between Cu and Cd concentrations and Cu-MT and Cd-MT in liver and kidney in both species, suggesting a pivotal role of MT in metal storage and detoxification. The quantification of metals and MT in liver and kidney may be a valid biomarker of metal exposure in the aquatic environment to assess the health of marine sea turtles as long as accurate analytical methods are adopted.     

Physiological and biochemical markers in the process of resistance and/or tolerance of heavy metals in the abandoned mining area of Sidi Kamber,Skikda, Algeria

This paper reports the biomonitoring of metallic pollution in the mine of Sidi Kamber Skikda town, to determine the resistance and/or tolerance to Zn, Pb, Cd and Cu in metalliferous plants. During the period of March–May, 2015, soil and plants samples were collected from three different stations in the study area. The total heavy metal fraction was determined by ICP/MS, whereas biochemical markers were determined by spectrophotometric techniques. The results obtained showed high levels of heavy metals in soil and plants compared to international standards. The translocation factor revealed that the studied plants can transfer and/or accumulate metals in their roots or their aerial parts as a function of characteristics of each metal, soil contents and physiological role in the plant. The contents of chlorophyll, proline, total sugars and total proteins are directly influenced by changes in bioavailable contents of heavy metals. It has been found that the plants studied and biomarkers are better suited for phytoremediation and biomonitoring of heavy metals pollution.     

Bioaccumulation of heavy metals by the aquatic plants Potamogeton pectinatus L. and Potamogeton malaianus Miq. and their potential use for contamination indicators and in wastewater treatment

The concentrations of heavy metals in the leaves of two aquatic plants Potamogeton pectinatus L. and Potamogeton malaianus Miq., and the corresponding water and sediment samples from the Donghe River in Jishou City of Hunan Province, China were studied to investigate metal contamination from the intensive industrial activities in the surrounding area. Results showed that the concentrations of heavy metals in the sediments, especially Cd, Mn and Pb, were much higher than the eco-toxic threshold values developed by the U. S. Environmental Protection Agency. Between the two plant species, P. pectinatus showed the higher capacity in metal accumulation. The highest concentrations of Cd, Pb, Cu, Zn and Mn were found in the leaves of P. pectinatus, reaching 596, 318, 62.4, 6590 and 16,000 mg kg(-1) (DW), respectively. Significantly positive relationships were observed among the concentrations of Zn, Cu and Mn in the leaves of both aquatic plants and those in water, indicating the potential use of the two plants for pollution monitoring of these metals. In addition, a laboratory experiment was conducted to investigate the ability of P. pectinatus and P. malaianus to remove heavy metals from contaminated river water. The average removal efficiencies by P. pectinatus and P. malaianus for Cd, Pb, Mn, Zn and Cu from the spiked Donghe River water were 92%, 79%, 86%, 67% and 70%, respectively. The results indicated that P. pectinatus and P. malaianus had high capabilities to remove heavy metals directly from the contaminated water. The potential use of these plants in wastewater treatment is worth further exploration.     

Seasonal concentrations of cadmium and zinc in native pasture plants: consequences for grazing animals

Aboveground concentrations of Cd and Zn in various grazing plants at three stages of the growing season are reported. The plants were collected at a natural habitat exposed to extensive atmospheric deposition of heavy metals from other parts of Europe. Concentrations of both metals varied considerably among species, also among morphologically similar species growing in the same soil. The two metals correlated in the material as a whole, but the concentration variations were most pronounced for Cd. There were also seasonal variations (generally a concentration reduction during spring). Transfer of metals from twigs to leaves seemed to differ between 'high' and 'low' metal accumulators. The inter-species and seasonal variations in plant metal concentrations complicate assessments of metal exposure to grazing animals through diet. By using diet information from a study conducted in an area similar to the present one, we roughly estimated a daily intake of 1-2 mg Cd for moose (Alces alces) in the autumn. Among the plant species investigated, Populus and Salix species were by far the most important Cd contributors. In areas where high Cd accumulators grow more widely, the daily Cd intake by moose could be as high as 7 mg or more.     

Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site

Contamination of heavy metals represents one of the most pressing threats to water and soil resources as well as human health. Phytoremediation can be potentially used to remediate metal-contaminated sites. This study evaluated the potential of 36plants (17species) growing on a contaminated site in North Florida. Plants and the associated soil samples were collected and analyzed for total metal concentrations. While total soil Pb, Cu, and Zn concentrations varied from 90 to 4100, 20 to 990, and 195 to 2200mg kg(-1), those in the plants ranged from 2.0 to 1183, 6.0 to 460, and 17 to 598mg kg(-1), respectively. None of the plants were suitable for phytoextraction because no hyperaccumulator was identified. However, plants with a high bioconcentration factor (BCF, metal concentration ratio of plant roots to soil) and low translocation factor (TF, metal concentration ratio of plant shoots to roots) have the potential for phytostabilization. Among the plants, Phyla nodiflora was the most efficient in accumulating Cu and Zn in its shoots (TF=12 and 6.3) while Gentiana pennelliana was most suitable for phytostabilization of sites contaminated with Pb, Cu and Zn (BCF=11, 22 and 2.6). Plant uptake of the three metals was highly correlated, whereas translocation of Pb was negatively correlated with Cu and Zn though translocation of Cu and Zn were correlated. Our study showed that native plant species growing on contaminated sites may have the potential for phytoremediation.     

Heavy metal pollution status in surface sediments of the coastal Bohai Bay

Bohai Bay, the second largest bay of Bohai Sea, largely due to the huge amount of pollutants discharged into it annually and its geohydrologic condition, is considered to be one of the most polluted marine areas in China. To slow down, halt and finally reverse the environmental deterioration of Bohai Sea, some researchers have proposed to connect it with Jiaozhou Bay in the western coast of Southern Yellow Sea by digging an interbasin canal through Shandong Peninsula. In order to assess the heavy metal pollution and provide background information for such a large geoengineering scheme, surface sediments from 42 stations covering both riverine and marine regions of the northwestern coast of Bohai Bay were analyzed for heavy metal content and fractionation (Cd, Cr, Cu, Ni, Pb and Zn). Three empirically derived sediment quality guidelines were used to assess the pollution extent of these metals. The studied metals had low mobility except for Cd at all stations and Zn at some riverine stations. Although a high mobility of Cd was observed, it could hardly cause a bad effect on the environment owing to its low total concentrations. Anthropogenic influence on the accumulation of studied heavy metals in sediments of Bohai Bay was obvious, but their contents were relatively lower to date comparing with some other marine coastal areas that receive important anthropogenic inputs. Taking as a whole, surface sediments of northwestern Bohai Bay had a 21% probability of toxicity based on the mean effects range-median quotient.     

Biomonitoring of mercury in polluted coastal area using transplanted mussels

The Kastela Bay is heavily polluted with inorganic mercury originated from direct discharges from the chlor-alkali plant, which operated in the period from 1950 to 1990. Even though the plant was closed 15 years ago, elevated levels of total mercury are still evident in surface sediments of the bay. In order to assess the availability of remobilized mercury to marine organisms, cultured mussels (M. galloprovincialis) were transplanted from pristine area to Kastela Bay, in the period from September 2000 to March 2001. Mussel samples were collected for the analysis of THg and MeHg in whole soft tissue, gills and digestive gland. Surface sediments and suspended matter were collected for the analysis of THg. Digestive gland was the target organ for the accumulation of THg, while concentrations of MeHg were similar in all analyzed tissues. The percentage of MeHg in mussel tissues (4-27%) was characteristic for the areas contaminated with inorganic mercury. A significant negative correlation was observed between the THg concentration in the tissues and the percentage of MeHg. Concentrations of THg in mussel tissues, which were decreasing from the source of contamination in an anticlockwise direction towards the exit of the bay, were significantly positively correlated to THg content in sediment and suspended particles. Spatial distribution of mercury species (THg and MeHg) in different environmental compartments was in accordance with the prevailing circulation in the bay. Data obtained through 6 months of biomonitoring experiment indicated that digestive gland was more sensitive indicator of THg concentrations in the environment than the whole organism or gills. As for MeHg, all tissues were equally suitable as biomonitors of MeHg concentrations in the environment.     

The use of Micropogonias furnieri and Mugil liza as bioindicators of heavy metals pollution in La Plata river estuary, Argentina

This paper presents, the concentrations of total Hg, Cd and Zn in edible muscle tissue, and in the liver of the fish Micropogonias furnieri and Mugil liza from the Samborombon Bay, La Plata river estuary, Argentina. The highest accumulation of the three metals was observed in liver samples, whereas in muscle tissue concentrations they were always lower, values even lower than the detection limit of the analytical method used. A marked relationship between metal contents of the studied species and their trophic and ecological habits was observed. In all cases, metal levels found in edible muscle tissue were lower than the international standards of reference for organisms directed to human consumption. Finally, the validity of using these two species as bioindicators of heavy metal pollution in the assessed ecosystem was discussed.     

Heavy metal fluxes at the sediment-water interface of three coastal ecosystems from south-west of the Iberian Peninsula

Concentrations of the heavy metals Cr, Cu, Fe and Mn were measured in sediments and porewater samples collected in three coastal ecosystems southwest of the Iberian Peninsula: the Odiel and Barbate River Salt Marshes and the Bay of Cadiz. Both the sediment and the porewater metal concentrations in the Odiel River Salt Marshes are higher than the values found in the Bay of Cadiz and Barbate River Salt Marsh, particularly for copper, a metal associated with mining activity. In porewater, the profiles were not the same as those in the solid phase and reflect the different behaviours of the elements in relation to the redox conditions. The heavy metals Cr and Cu show a typical enrichment in the porewater of the oxic zone. The heavy metals Mn and Fe show an increase in the porewater at the depths where the maximum nitrate and phosphate concentrations occur, respectively. Significant differences between background levels for each heavy metal in the various studied zones exist. Iron and Cu showed larger background levels in the Odiel River Salt Marshes than those in the Cadiz Bay and the Barbate River Salt Marshes. In the Bay of Cadiz the background levels are also high, particularly for Cr. At the Odiel River Salt Marshes the diffusive flux of Cu is high (1.3-230.1 microg cm(-2) year(-1)), which suggests that the Odiel River Salt Marshes are subject to strong contamination by Cu, which is presumably introduced to the sediment in particulate form. In the Bay of Cadiz, Cr is the only metal with positive diffusive flux (2.15 microg cm(-2) year(-1)). It is higher than those obtained in other coastal ecosystems including the Odiel River Salt Marshes. The positive diffusive flux of Cr has been associated with the input of this metal by the naval industry and the manufacturing of car and aircraft components.     

Trace elements in fruiting bodies of ectomycorrhizal fungi growing in Scots pine (Pinus sylvestris L.) stands in Poland

The trace metal contents in fruiting bodies of ectomycorrhizal (ECM) fungi, symbiotic partners of Scots pine, were studied on three sites situated in west-central Poland. Elements were determined by atomic absorption spectrometry in 123 samples of 16 species. The study explored the differences in metal accumulation in relation to site, fungal species, age and part of the fruiting body and results were related to metal content in soil and plant material (roots and needles). Soil analysis revealed that results were obtained under environmental conditions not subject to strong anthropogenic pressure. Median metal concentrations did not differ disparately between sites, although the concentrations of each of the tested metals in the individual species varied to a large extent. Extremely high levels of Al with a large bioconcentration factor (BCF) were found in sporocarps of Thelephora terrestris. The spread between the highest and the lowest concentration (max/min) was very wide in Al, Cd and Pb and these elements may be considered to be absorbed preferentially by fruiting bodies of some species whereas Fe, Mn and Zn, with relatively low values of max/min, are normally absorbed by the majority of fungi. There was no clear relationship between caps and stipes in metal content. However, a tendency to higher metal concentration in the caps was observed. The metal content in young and older fruiting bodies of five different fungi was species dependent. In order to estimate the degree of accumulation of each element by plant and mushrooms, bioconcentration factors (BCFs) were calculated. In plant material (roots and needles), highest values of BCFs were noted for essential metals, like Zn and Mn. Lead showed a definite exclusion pattern (BCF below 1). In fruiting bodies of tested fungi, especially in Amanita muscaria, cadmium was the most intensively accumulated metal. Lead was excluded by plants but was accumulated or excluded by fungi depending on the species. The significance of mycological observation and quantification of the metal content in monitoring of the forest ecosystem is discussed.     

Congener-specific profile and toxicity assessment of PCBs in green turtles (Chelonia mydas) from the Hawaiian Islands.

Chemical pollution may play a role in the etiology of fibropapillomatosis in green turtles (Chelonia mydas). In this preliminary study, polychlorinated biphenyls (PCBs) were measured in the livers and adipose fats of green turtles collected after they were stranded on Oahu Island, Hawaii in 1992-1993. Average concentrations of total PCBs were 45-58 ng/g dry weight and 73-665 ng/g in the liver and adipose tissues, respectively. Hexachlorobiphenyls were predominant homologues, PCBs 153 and 138 were dominant congeners in all the turtle tissues. Among the most toxic coplanar congeners, in the order of abundance, were PCB 77 > 126 > 169. Estimated toxic equivalents (TEQs) of PCBs to 2,3,7,8-tetrachlorodibenzo-p-dioxin were 8-15 pg/g in the livers and 13-48 pg/g in the adipose tissues. PCB 126 contributed 85-91% of the total TEQs.     

Heavy metal and pesticide content in commonly prescribed individual raw Chinese Herbal Medicines

Heavy metal and pesticide contamination has previously been reported in Chinese Herbal Medicines (CHMs), in some cases at potentially toxic levels. This study was conducted to determine general patterns and toxicological significance of heavy metal and pesticide contamination in a broad sample of raw CHMs. Three-hundred-thirty-four samples representing 126 species of CHMs were collected throughout China and examined for arsenic, cadmium, chromium, lead, and mercury. Of the total, 294 samples representing 112 species were also tested for 162 pesticides. At least 1 metal was detected in all 334 samples (100%) and 115 samples (34%) had detectable levels of all metals. Forty-two different pesticides were detected in 108 samples (36.7%), with 1 to 9 pesticides per sample. Contaminant levels were compared to toxicological reference values in the context of different exposure scenarios. According to a likely scenario of CHM consumption, only 3 samples (1%) with heavy metals and 14 samples (5%) with pesticides were found with concentrations that could contribute to elevated background levels of contaminant exposure. According to the most conservative scenario of CHM consumption, 231 samples (69%) with heavy metals and 81 samples (28%) with pesticides had contaminants that could contribute to elevated levels of exposure. Wild collected plants had higher contaminant levels than cultivated samples. Cadmium, chromium, lead, and chlorpyrifos contamination showed weak correlations with geographic location. Based on our assumptions of the likely mode of consumption of raw CHMs, the vast majority (95%) of the 334 samples in this study contained levels of heavy metals or pesticides that would be of negligible concern. However, given the number of samples with detectable contaminants and the range between the more likely and more conservative scenarios of contaminant exposure, more research and monitoring of heavy metals (especially cadmium and chromium) and pesticide residues (especially chlorpyrifos) in raw CHMs are advised.     

The Posidonia oceanica marine sedimentary record: A Holocene archive of heavy metal pollution

The study of a Posidonia oceanica mat (a peat-like marine sediment) core has provided a record of changes in heavy metal abundances (Fe, Mn, Ni, Cr, Cu, Pb, Cd, Zn, As and Al) since the Mid-Holocene (last 4470 yr) in Portlligat Bay (NW Mediterranean). Metal contents were determined in P. oceanica. Both, the concentration records and the results of principal components analysis showed that metal pollution in the studied bay started ca. 2800 yr BP and steadily increased until present. The increase in Fe, Cu, Pb, Cd, Zn and As concentrations since ca. 2800 yr BP and in particular during Greek (ca. 2680-2465 cal BP) and Roman (ca. 2150-1740 cal BP) times shows an early anthropogenic pollution rise in the bay, which might be associated with large- and short-scale cultural and technological development. In the last ca. 1000 yr the concentrations of heavy metals, mainly derived from anthropogenic activities, have significantly increased (e.g. from ~ 15 to 47 μg g^− 1 for Pb, ~ 23 to 95 μg g^− 1 for Zn and ~ 8 to 228 μg g^− 1 for As). Our study demonstrates for the first time the uniqueness of P. oceanica meadows as long-term archives of abundances, patterns, and trends of heavy metals during the Late Holocene in Mediterranean coastal ecosystems.     

A five-year study on the heavy-metal pollution of Guanabara Bay sediments (Rio de Janeiro, Brazil) and evaluation of the metal bioavailability by means of geochemical speciation

Surface sediments of the Guanabara Bay (Rio de Janeiro, Brazil) were analyzed by a sequential extraction procedure for Cd, Cr, Cu, Pb, Zn, Mn and Fe, determining their distribution among five geochemical phases and in the nitric acid extractable phase. Bioavailable phases and non-bioavailable phases have been determined in six transects in the bay to define the significant level of pollution due to sediment metal contamination. A multiple correlation showed limited responsibility of Mn and Fe oxides and humic acids in the adsorption process, which allowed discrimination among the different processes and suggested the strong influence of the hydrogen sulfide present in the highly reduced bay bottom environment. The authors suggest the need to avoid disturbing bottom sediment by dredging or by artificial bottom aeration which could result in a rapid worsening of the environment due to the accelerated formation of more soluble oxygenated metal compounds making the toxic metals much more available to the benthic fauna and to the bay biota in general.     

Heavy metals in aquatic plants,clams, and sediments from the Chesapeake Bay,U.S.A. implications for waterfowl

Dry weight concentrations of cadmium, lead, copper and zinc were measured in clams, submerged aquatic macrophytes and sediments from several locations in the Chesapeake Bay region. Clam species included Macoma balthica, Mya arenaria, and Rangia cuneata; plant taxa included Najas spp., Potamogeton spp., Ruppia maritima, Vallisneria americana, and Zostera marina. Cadmium and lead concentrations were generally greater in whole plants than in soft tissues of clams, while the reverse was true for zinc; no marked trend was observed in copper concentrations. These results suggest that the changes occurring in the food habits of some Chesapeake Bay waterfowl towards increased clam utilization as a result of declining submerged plant abundances is not increasing ingestion of the nonessential metals cadmium and lead. Highest lead, copper and zinc concentrations were observed in sediments from the Patapsco River, which is probably due to the proximity of Baltimore Harbor. Highest cadmium concentrations were observed in sediments in the Port Tobacco River, a tributary of the Potomac River. These observations were generally consistent with high concentrations of the respective metal(s) in biota from these locations. Concentrations of the four metals in sediments were highly correlated to organic matter content.     

Biomonitoring of mercury in the Kastela Bay using transplanted mussels

Kastela Bay, located on the central part of east Adriatic coast, is heavily contaminated by mercury. The main source of contamination was a chlor-alkali plant, which was operating for 40 years since 1949. Previous studies showed that mercury concentrated in the sediment close to the plant is being dispersed throughout the bay by remobilization processes. Subsequent remobilization of mercury buried in the sediment may be harmful to various marine organisms. In order to assess the availability of remobilised mercury to marine organisms and to assess the health risk of consumption of shellfish species from the bay, a monitoring programme was initiated in September 1997. One thousand six hundred specimens of mussels (Mytilus galloprotincialis) of the same size and age, collected in a very clean area, were transferred to four stations in the bay 1.5 m above the sea bottom. Every month during the summer period and every second month in the winter period, 50 mussels were taken from each station for the analysis of mercury and monomethylmercury (MeHg) content. The results obtained during 1 year of biomonitoring suggest that the equilibrium concentration in transplanted mussels was established in a relative short period of time. The digestive gland is preferential organ for the accumulation of total mercury. Seasonal variation of mercury content both in the whole soft tissue, and organs (gills and digestive gland) was observed at all stations. However, methyl mercury is more concentrated in soft tissues than in digestive gland and gills, despite the fact that it is absorbed through these organs. The mercury levels in tissues were below accepted limits for human consumption. Obtained results showed that the implantation of mussels in an area containing sediment contaminated by mercury may be a good monitoring tool for the assessment of the availability of remobilised mercury to marine organisms.     

Heavy Metals In Water,Suspended Particles,Sediments And Aquatic Plants Of Habbaniya Lake,Iraq

Seasonal samples were taken from four selected stations on the Habbaniya lake, middle of Iraq during 1997, to study six heavy metals (Cu, Cd, Pb, Ni, Mn, and Zn), in water suspended particles, sediments and aquatic plants. Five plant species were collected, represented the most dominant in the lake, namely Myriophyllum verticillatum, Potamogeton crispus, P. pectinatus, Ceratophyllum demersum and Vallisnaria spiralis . Zn had the highest concentration among the studied metals in filtered water and suspended particles with an average of 4.08 w g/l and 26088 w g/g, respectively. Whereas, Mn had the highest in the sediments and all studied aquatic plants, which ranged between 213.3- 2027 w g/g and 100-1725 w g/g, respectively, Meanwhile, Cd had the lowest concentration in all studied samples.     

北京地区8种园林树木的叶片和枝条重金属含量比较

为了解不同园林树木重金属的吸收情况,以北京市陶然亭公园、中科院植物园、紫竹院公园、马甸公园、皇城根遗址公园和营城建都滨水绿道为样地,采用ICP光谱仪测定了8种园林树木叶片、一年生枝条以及相应土壤中重金属Cd、Pb、Cu的含量,并对叶片与一年生枝条中重金属含量以及树木与土壤重金属含量进行了相关性分析。结果表明,不同树种重金属含量有显著差异且因重金属种类而异;叶片和枝条中Cd含量排序为:紫薇金银木太平花锦带珍珠梅紫丁香木槿砂地柏,Pb含量排序为:锦带木槿太平花金银木紫丁香紫薇砂地柏珍珠梅,Cu含量排序为:金银木紫薇紫丁香珍珠梅木槿太平花锦带砂地柏;树木不同器官中重金属含量也不同,叶片与枝条中Pb含量具有显著相关性;树木叶片和一年生枝条的Cd、Pb、Cu重金属既来源于土壤,也来源于大气污染。8种园林树木中,对Cd、Pb、Cu吸收量最大的分别是紫薇、锦带和金银木;紫薇和金银木同时对Cd和Cu、太平花同时对Cd和Pb有较强的吸收力,而砂地柏吸收力较弱。本研究对园林绿地选择富集重金属能力强的树种有参考价值。     

Lead,iron, chromium and zinc in road runoff at Pullman,Washington

The inputs of iron, lead, chromium and zinc to a 0.6 acre road surface were investigated. The selection of a viaduct in Pullman, Washington as a study area permitted the exclusion of metal input from non-road surfaces. Dust and precipitation metal deposition rates were measured at the viaduct as well as at a control location. It was found that the primary source of metals at the road surface was the operation of motor vehicles. For three separate storm events, runoff samples were collected, flow was measured and runoff samples were analyzed for total solids, total metals and dissolved-colloidal metals. Less than 5 percent of each metal in the runoff samples existed in the dissolved-colloidal fraction. Mean concentrations of total metals in road runoff were 1.86, 89, 0.139 and 19.1 mg/l for Pb, Fe, Cr, and Zn, respectively. A mathematical model was developed allowing the estimation of metal loadings for a storm event typical of the area, given the total runoff volume for the particular storm event. It was also found that a residual amount of metals remains on the road surface after each storm.     

A field study on serum vitellogenin levels in male Reeves' pond turtles (Chinemys reevesii) from estrogen-contaminated sites and a reference site

To ascertain whether wild male turtles were influenced by environmental estrogens, we examined serum vitellogenin (VTG) levels of male Reeves' pond turtles (Chinemys reevesii) collected from four study sites (A-D) in Kyoto, Japan. Sites A-C, which were impacted by domestic or industrial wastewater and effluents from sewage treatment plants, were chosen as contaminated sites, and site D was intended as a reference site. This contaminated/reference site characterization was confirmed by measuring estrogenic activities of the water samples collected at each site for over a year. Serum VTG levels in the turtles were quantified by an enzyme-linked immunosorbent assay established previously. Estrogenic activities of the water samples were measured using a previously validated yeast two-hybrid assay and expressed as the estradiol-17beta equivalent. Estrogenic activity was observed at high levels at sites A-C, but was almost undetected at site D throughout the sampling period: the mean and range were 0.74 (<0.07-2.1), 0.52 (0.17-1.6), 1.7 (<0.07-7.3), and 0.07 (<0.07-0.62) ng/l at sites A-D, respectively. Significant differences were found only in site D versus sites A, B, and C. Therefore, site D and sites A-C were confirmed to be a reference site and contaminated ones, respectively. Overall, 320 male turtles were captured and examined. The majority of the turtles showed normal VTG values (0.10-0.74 microg/ml). Although only five turtles from sites A-C showed unusually high VTG values (1.1-5.9 microg/ml, nearly one order of magnitude higher than normal values but much lower than values in adult females), there was no significant difference in the incidence of these high values between sites A-C and site D. Moreover, among the five turtles, one turtle was captured again 2 months later, but its VTG value dropped to the normal level. The unusually high VTG values may therefore be transient elevation caused by incidental and/or individually specific agents. Excluding the unusually high values, the mean serum VTG (accompanied with the range) was 0.22 (0.10-0.74), 0.27 (0.11-0.62), 0.27 (0.17-0.68), and 0.23 (0.10-0.57) microg/ml at sites A-D, respectively. There were no significant differences in the mean VTG values among sites A-D. Although it should be clarified how and why the unusually high VTG values occurred at sites A-C, our results suggested that wild male C. reevesii turtles would not be significantly affected by xenobiotic estrogens at environmentally relevant levels in terms of serum VTG elevation.