Bioavailability of PAHs in the Biobio river (Chile): MFO activity and biliary fluorescence in juvenile Oncorhynchus mykiss.

Cytochrome P450-dependent monooxygenase (MFO) activity and levels of bile PAH metabolites were measured in fish (Oncorhynchus mykiss) exposed to Biobio river (Chile) water. Experiments were performed simultaneously in both the field and laboratory to investigate whether the river water contained notable bioavailable PAH fractions. The field experiment was conducted using fish caged for 41 days at La Mochita (Biobio river mouth) whereas the laboratory experiment involved a 21-day exposure treatment with water collected in the same area. Induction of hepatic MFO, assayed by benzo(a)pyrene monooxygenase activity (BaPMO), and levels of PAH metabolites in the bile were measured in groups of eight (field experiment) and four specimens (laboratory experiment) sampled after 5, 10, 21 and 41 days of exposure. BaPMO was induced by a factor of 23 and 25 in fish caged for 21 and 41 days, respectively. Likewise, a significant BaPMO induction was found in the fish group experimentally exposed to river water for 21 days. Biliary fluorescence measurements, recorded by fixed wavelength fluorescence (FF) and synchronous fluorescence spectroscopy (SFS), revealed significantly high levels of PAH metabolites in bile samples of fish caged for 21 and 41 days. A positive relationship was found between BaPMO activity and biliary PAH metabolites. This study provides evidences that (i) Biobio river water contains a significant bioavailable PAH fraction in terms of marked effects on fish and (ii) the biological indicators MFO activity and bile PAH metabolites represent an excellent screening methodology to assess PAH exposure.     

Mixed-function oxidase-specific activity in wild and caged speckled sanddabs Citharichthys stigmaeus in Elkhorn Slough, Moss Landing Harbor and nearshore Monterey Bay, California

The goal of this study was to characterize the spatial, seasonal and annual hepatic activities of mixed-function oxidase (MFO) in the speckled sanddab Citharichthys stigmaeus, the most common fish in the Moss Landing area. In addition, techniques to monitor MFO activities in caged speckled sanddabs were developed and tested. Once the relationship between MFO activities in caged and wild fish populations is determined, caged fish could be used to monitor potential hydrocarbon impacts at Moss Landing, or other marine sites. During each of the spatial, seasonal and annual sediment samplings conducted in 1985-1987 as part of a separate hydrocarbon variability study at Moss Landing, 12 wild speckled sanddabs were collected from Moss Landing Harbor, Elkhorn Slough and nearshore Monterey Bay sites. In addition, four locations were chosen for a 14-day field caged fish experiment. The caged fish experiments successfully demonstrated the feasibility of using caged sanddabs as indicators of hydrocarbon exposure. The major source of variability in hepatic aryl hydrocarbon hydroxylase activity in wild speckled sanddabs from the Moss Landing area is due to seasonal rather than site differences. Significant relationships between caged fish MFO response and sediment hydrocarbon concentrations were found. It is possible that caged fish could be used in place of costly sediment sampling and analysis, and provide a more direct method to assess biological impacts. Many of the caging techniques demonstrated in this study can easily be transferred to other benthic flatfish, and other marine and freshwater environments.     

Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-TCDD equivalents (TEQs) in sediment from the Hyeongsan River, Korea

Sediment, pore water and water samples from the Hyeongsan River, Korea were analyzed for several classes of halogenated aromatic hydrocarbons (HAHs) and their dioxin-like activities were evaluated using the in vitro H4IIE-luc bioassay. Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) were detected in sediments from all six sampling locations with mean concentrations of 2.8 x 10(2) pg/g, 190 pg/g, and 61.4 ng/g, dw, respectively. Polycyclic aromatic hydrocarbons (PAHs) were predominated by 4-6 ring compounds with concentrations in the range of 5.30-7680 ng/g, dw. Chemical profiles of target analytes in sediment and water samples revealed that there was a gradient of concentrations along the river from upstream to downstream, which suggested that the primary source was a wastewater reservoir adjacent to a sewage treatment plant (STP). TEQs derived by summing the product of concentrations of individual congeners by their respective relative potencies (REPs or TEFs) ranged from 4.3 x 10(-1) to 1.1 x 10(3) pg/g, dw. Raw Soxhlet extracts from all six sampling locations induced significant dioxin-like responses in the H4IIE-luc bioassay. TCDD-EQs derived from H4IIE bioassay ranged from 7 x 10(-3) to 1.5 x 10(3) pg/g, dw, which were significantly correlated with TEQs (r2 = 0.994, p < 0.05). Among the three Florisil fractions tested, PCDD/Fs in fraction (F2) induced the greatest magnitude of response (range: 24-83%-TCDD-max.) in the H4IIE-luc assay. Comparison of the TEQ and TCDD-EQ suggested little non-additive interaction between fractions and AhR-active and inactive compounds. Concentrations of individual congeners as well as TEQs and TCDD-EQs suggest inputs from the industrial center waste stream in the Hyeongsan River.     

A laboratory exposure procedure for screening pulp and paper mill effluents for the potential of causing increased mixed function oxidase activity in fish

To better understand the relationships between pulp manufacturing processes and mixed function oxidase (MFO) enzyme induction in fish, a practical and standardized exposure procedure is required. This study was undertaken to develop a laboratory-based exposure procedure to quantify the relative MFO induction potencies of different types of pulp and paper mill effluents. One major consideration in developing the procedure was to ensure that the protocol was practical so that tests could be performed in a short time, with small volumes of effluents and using simple experimental conditions. A series of concentration-response and time-course experiments were conducted to find the minimum time and effluent concentration which could distinguish the ability of different effluents to cause significant MFO induction in rainbow trout in the laboratory. Experiments were also conducted to determine the effects of biotic and abiotic factors such as loading density, fish size and feeding regime. This study showed that the exposure of rainbow trout in the laboratory to 10% concentration of secondary-treated effluent for 96 h caused significant increases in hepatic MFO activity. The magnitude of MFO induction was comparable to other field and laboratory observations. While fish size, loading density and feeding regime were found to affect the test results, consistent responses within a laboratory using this protocol are possible, provided that these factors are standardized. Therefore, the short-term exposure approach described in this paper could be a relevant tool for assessing the ability of different types of pulp and paper mill effluents to cause MFO induction in fish.     

Effect of long term exposure to petroleum on mixed function oxygenases in fish: Further support for use of the enzyme system in biological monitoring

A number of long term, low level petroleum hydrocarbon exposures were carried out from 1978 to 1981 in order to further evaluate use of the mixed function oxygenase (MFO) system in biological monitoring. The studies involved exposure, at ambient temperatures, of fish from three separate families including (a) spawning codfish ($\text{Gadus morhua}$), (b) post-spawned sculpins ($\text{Myoxocephalus octodecemspinosus}$), and (c) gonadally maturing flounder ($\text{Pseudopleuronectes americanus}$). MFO activity remained elevated approximately four-fold in the liver, and three-fold in the gills of codfish exposed for four months to water concentrations of 0.30–0.60 ppm petroleum, at ?1-1°C. Similarly, liver enzyme activity remained elevated approximately three-fold in sculpins exposed to very low levels (0.15–0.20 ppm) of petroleum for three months, at 4–12°C. Results indicate that several years may be required before hydrocarbon concentrations are reduced in petroleum contaminated sediments, such as those heavily impacted by oil spills, to non-inducing threshold levels. Oil contaminated sediment was, even after a year of weathering, quite effective in inducing a high level of MFO activity (seven-fold) in flounder, at 11-6°C. A number of factors that might detract from use of the mixed function oxygenase system in biological monitoring were not realized in these long term studies with petroleum hydrocarbons.     

The Mutagenic Activity of Particulate Organic Matter Collected with a Dilution Sampler at Coal-Fired Power Plants

The Deep Creek Lake Study of 1983 provided an opportunity to obtain emission samples from coal-fired power plants with a dilution sampler for mutagenicity testing. Stack and ambient samples of particulate matter were collected with a dilution sampler at three coal-fired power plants in West Virginia. Samples were sequentially extracted with cyclohexane (CX), dichloromethane (DCM) and acetone (ACE) and tested for mutagenicity in the Ames Salmonella/microsome assay using TA98 (-S9). For the stack samples, the CX, DCM and ACE fractions constituted 1.0, 0.7 and 98.1 percent of the total extractable organic material (EOM), respectively, compared to 28.5, 7.4 and 64.1 percent for the ambient samples. In contrast, the mutagenic activity of the organic fractions was concentrated in the CX and DCM fractions.

The cyclohexane- and dichloromethane-soluble fractions of the stack samples from all locations exhibited mutagenicity when tested in the plate incorporation assay. No significant response was observed with the acetone fraction. When tested with Kado's modification of the preincubation assay, the acetone-soluble fraction did exhibit mutagenic activity comparable to that of the other fractions when expressed in units of revertants per milligram of particular matter. Chemical analyses of one of the acetone-soluble fractions indicated that half of the mass was sulfuric acid while the remainder consisted of C, H and O. More than 30 peaks were detected in the high pressure liquid chromatogram of this fraction.

Although little mutagenic activity was detected in the polar ACE fraction of the diluted stack emissions samples with this single bioassay, in view of the large mass of this fraction, further investigation of the chemical composition and genotoxic activity of this fraction would be prudent.     

Effect of a base-catalyzed dechlorination process on the genotoxicity of PCB-contaminated soil

We evaluated the genotoxicity of dichloromethane (DCM) extracts of PCB-contaminated soil before and after the soil had been treated by a base-catalyzed dechlorination process. The treatment process involves heating a mixture of the soil, polyethylene glycol (or hydrocarbons with boiling points of 310–387°C), and sodium hydroxide to 250–350°C. Dechlorination reduced by >99% the PCB concentration of the soil, which was initially 2,200 ppm. The DCM extracts of both control and treated soils were not mutagenic in strain TA100 of Salmonella, but they were mutagenic in strain TA98. Based on results in strain TA98, the base-catalyzed dechlorination process reduced the mutagenic potency of the soil by approximately one-half. The DCM extracts of the soils before and after treatment were equally genotoxic in a prophage-induction assay in . , which detects some chlorinated organic carcinogens that are not detected by the Salmonella mutagenicity assay. These results suggest that treatment of PCB-contaminated soil by base-catalyzed dechlorination reduced the mutagenicity of the soil slightly.     

Correlation between bioassay-derived P4501A1 induction activity and chemical analysis of clam (Laternula elliptica) extracts from McMurdo Sound, Antarctica

Variable levels of halogenated aromatic hydrocarbons were measured in clams (Laternula elliptica) collected from McMurdo Sound, Antarctica. Clams collected in and near Winter Quarters Bay contained high levels of organochlorine compounds, particularly polychlorinated biphenyls (PCBs). A strong gradient has been documented in Winter Quarters Bay that been linked to human activities at McMurdo Station. The activity of clam extracts as inducers of P4501A1-dependent ethoxyresorufin O-deethylase (EROD) activity was determined using in vitro bioassays utilizing rat hepatoma H4IIE cells. The extracts which exhibited the highest induction activities were those derived from clams collected in contaminated areas. Additionally, there was an excellent linear correlation between induced EROD activity versus total PCB levels (r²=0.96). The complimentary nature of both the analytical and bioanalytical data confirms the utility of the latter assay and provides a method for estimating the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents in extracts from marine biota.     

In vitro EROD induction equivalency factors for the 10 PAHs generally monitored in risk assessment studies in The Netherlands

The ethoxy resorufin dealkylase (EROD) inducing potency of 10 polycyclic aromatic hydrocarbons (PAHs) is measured in the H4IIE in vitro bioassay and the results are compared to those reported in literature. The selected PAHs varied considerably in their potency to induce EROD activity. Anthracene (Ant) and phenanthrene (Phe) showed consistently no response. Naphthalene (Nap) showed no or a very weak response on EROD activity. Fluoranthene (Fla) and benzo[g,h,i]perylene (BghiP) showed weak responses at the highest doses. The other PAHs, including indeno[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), benzo[a]pyrene (BaP), chrysene (Chr) and benzo[k]fluoranthene (BkF), showed full bell shaped dose-response curves. BaP EROD induction equivalency factors (BaP-1EF) were calculated and increased in the order Ant approximately Phe < Fla < Nap < BghiP < IP < BaA < BaP < Chr < BkF. Comparison of BaP-IEFs based on 50% effect concentration (EC50) or lowest effect concentration (LEC), yielded a significant relationship between both methods described by the equation log(BaPIEF(EC50) = 0.55 x log(BaPIEF(LEC)) + 0.07 (r2 = 0.913). BaP-IEFs as derived from our measurements and as reported in literature and measured in other in vitro assays deviated up to a factor of 17 among the different studies, but the potency rankings were comparable. For the PAH mixture as on average present in the human diet an overall tetrachlorodibenzo-p-dioxin (TCDD)-IEF of 1 x 10(-4) was estimated. The total PAH based TCDD induction equivalents (IEQ) intake then was calculated 300 pg/day, which is approximately 2 times higher then the PHAH based TCDD-EQ intake reported for humans.     

Microsomal monooxygenase activity in Tilapia (Oreochromis mossambicus) exposed to a bleached kraft mill effluent using different exposure systems.

Bleached kraft pulp and paper mill effluents (BKMEs) are known to have adverse effects on aquatic organisms. One of the effects of BKMEs is its ability to induce cytochrome P4501A activity in exposed fish. 7-Ethoxyresorufin O-deethylase (EROD) activity is the most common biomarker used to measure the mixed-function monooxygenase activity. In this study, Tilapia were exposed to BKMEs using different exposure systems and their hepatic EROD activity, as well as liver/somatic index (LSI), were determined. In the Phase I study, Tilapia treated with betaNF and a whole (100%) BKME using a static, non-renewal system exhibited statistically significant EROD induction, but LSI values were not altered. In the Phase II study, fish were either caged in the mill's fishpond with the whole effluent passing through or cultured in tanks receiving 100% of the BKME continuously using a flow-through system in the laboratory. Their EROD activities were then compared with the non-exposed fish (control). The EROD activities in both groups of fish were elevated significantly with the greatest induction being observed in the field-exposed group. The LSI values in all of the field-exposed fish were significantly greater than the control Tilapia. The EROD assay was sensitive in detecting biological changes in fish exposed to the BKME. Further studies are warranted to better understand the impacts of BKMEs on aquatic organisms in Taiwan.     

The changes in common carp blood after short-term zinc exposure

Blood zinc level, hematological parameters and blood cell morphology were evaluated in common carp immediately after 3 h exposure to 20 mg dm(-3) of zinc (Zn0), and in 24, 48 and 96 hours after the end of it (Zn24, Zn48, Zn96). Blood zinc level in the non-exposed fish was 8 mg dm(-3), reached a maximum of 20 mg dm(-3) in Zn48, while it dropped to 9 mg dm(-3) in Zn96. Zinc caused a stress reaction in fish indicated by an increase in hematocrit value in Zn0, and elevated plasma glucose level and trombocytosis which persisted until the end of the experiment. Zinc-exposed fish showed an increased frequency of abnormal erythrocytes, and a compensatory release of immature erythrocytes to the blood stream. In zinc-treated fish, leukocyte count initially increased and subsequently decreased significantly below the control level due to a drop in lymphocyte number. Lymphocyte viability was reduced, and abnormal lymphocytes appeared. A decreased count of juvenile neutrophiles, and reduced phagocyte activity also occurred. The results indicate possible zinc-induced disturbances in both specific and non-specific immune mechanisms.     

The chemical and biogenotoxic characterization of organic xenobiotics in aquatic sediment materials 1. The application and comparison of chemically non-specific and biogenotoxic methods

The aim of this work was to evaluate the Ames assay and mixed function oxidase (MFO)-Induct Test used in parallel with chemical group tests (ECD fingerprint and PAH estimation) for the characterization of the organic pollution of water sediment materials. Sediment materials were collected from “clean” and relatively heavily polluted locations in the Middle Adriatic Sea, and from some locations in continental Croatia polluted with wastewaters from different enterprises. Characterization of the organic extracts of the sediment materials investigated was performed chemically using UV spectrofluorometry for the determination polyaromatic hydrocarbons (PAH) and gas chromatography for the determination of volatile EC detector sensitive materials. Genotoxic analysis of the extracts was performed using the MFO-Induct Test and mutagenicity testing using the Standard Plate Incorporation Test as described by Maron and Ames with Salmonella typhimurium TA 98. Measurement of the BaPMO enzyme activity in the livers of carp treated i.p. with total extracts of the sediment investigated confirmed that the methanol extracts generally contained more inducing matter than the petroleum ether extracts. Ames assay showed that for all the samples following the elimination of the sulfur, there was an increase in the number of revertants in comparison to the control number, which indicates that the samples contained mutagenic substances. The larger doses of extracts generally demonstrated cytotoxicity, as evidenced by a reduced number of spontaneous revertants in the Salmonella/Microsome Test. Investigation of the correlation of the chemical parameters with the biological parameter showed that the induction of BaPMO exhibited a statistically significant correlation with the level of the ECD fingerprint of the petroleum ether sediment extract.     

Receptor-mediated in vitro bioassay for characterization of Ah-R-active compounds and activities in sediment from Korea

Sediment extracts of stream sediments, collected from inland areas of Lake Shihwa (LSI) and Masan Bay (MBI), were screened for their abilities to induce aryl hydrocarbon receptor (Ah-R) mediated gene expression in vitro. Cell viability assay was also performed to examine cytotoxic effects on the Ah-R-mediated activities of sediment samples. Over 80% (30 out of 36) of sediment raw extracts (REs) induced significant Ah-R-mediated activities in the H4IIE-luc cell bioassays. Ah-R-mediated activities of sediment REs from LSI locations (mean=58%-TCDD-max; n=21) were greater than those of sediment REs from MBI locations (mean=35%-TCDD-max; n=15), in general. Seven (mean+/-SD=100+/-14%-TCDD-max) of 21 sediment REs from LSI showed Ah-R-mediated activities comparable to that (set to, 100%-TCDD-max) elicited by 1240 pM TCDD. Whereas, in MBI, only two REs from M1 (93%-TCDD-max) and M9 (82%-TCDD-max) showed significantly great responses that comparable to maximum response of TCDD standard curve. Sample potencies relative to the TCDD standard (TCDD-EQs) were estimated based on full dose-response characteristics of REs and TCDD-EQs were found to be 14-868 pg TCDD/g, dw and 17-275 pg TCDD/g, dw, in LSI and MBI, respectively. A range of TCDD-EQ20-80 of samples, based on multiple estimates of relative potency (REP20-80), did not vary greatly (2-4-fold) in the H4IIE-luc bioassays, which indicated relatively low degree of uncertainties in point estimates of REP for sediment REs examined. Acid-treatment of REs samples improved quantitative biological responses of samples followed by decreases in cytotoxicity identified by MTT cell viability assays.     

Melting and incineration plants of municipal waste. Chemical and biochemical diagnosis of thermal processing samples (emission,residues)

Control of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) in emissions and thermal residues from incinerators has been a cause of public concern for more than one decade. Recently, several studies showed that other persistent organic pollutants (POPs) such as coplanar polychlorinated biphenyls (co-PCBs) also have dioxin-like activity and are released from incinerators. Therefore, the present study was aimed at making a risk assessment about dioxin-like activity in extracts of thermal waste residues (e.g. combustion gas; fly ash, slag) from incineration and melting processes in Germany and Japan. For this purpose, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs), coplanar polychlorinated biphenyls (co-PCBs), polychlorinated naphthalenes (PCNs) and polyaromatic hydrocarbons (PAHs) were analyzed by chemical analysis. Additionally, 2, 3, 7, 8-TCDD equivalents (EROD-TEQs) were determined by in vitro Micro-EROD bioassay using rat H4IIE hepatoma cells. EROD-TEQs could be correlated to I-TEQ values (from PCDD/Fs/co-PCBs) analyzed by chemical analysis resulting in a maximal sixfold higher estimate. Our study indicates minor influences of co-PCBs, PAHs and PCNs to the sum of dioxin-like toxicity in the extracts of thermal waste residues as determined here. Furthermore, we showed that the levels of dioxins and co-PCBs contained in slag from melting processes and bottom ashes from incineration processes were lower by 1-2 orders of magnitude than that in fly ash.     

Responses of European eel (Anguilla anguilla L.) circulating phagocytes to an in situ closed pulp mill effluent exposure and its association with organ-specific peroxidative damage

The effect of bleached kraft pulp mill effluent (BKPME) persistent compounds on phagocyte activities and its organ-specific influence in gill, kidney and liver was studied in European eel (Anguilla anguilla L.). Eels were caged and plunged at 3 different sites-50 m (site 1), 100 m (site 2) and 2,000 m (site 3) away from the end of the closed BKPME discharging channel for 8 and 48 h. Gill, head kidney and peritoneum phagocytes oxidative burst activity (OBA) was measured by the nitroblue tetrazolium reduction assay whereas lipid peroxidation (LPO) in eel gill, kidney and liver was measured by thiobarbituric acid reaction. A significant gill OBA induction was found at 8h on site 2, and on sites 1, 2 and 3 at 48 h exposure. However, in head kidney and peritoneal exudate phagocytes, OBA induction was significant only at sites 2 and 3 after 48 h exposure. In those particular sites, a significant increase in gill, kidney and liver LPO was measured that is assumed to result from OBA induction. Considering OBA and LPO, gill is the most affected tissue compared to kidney and liver. Gill vulnerability towards peroxidative damage was demonstrated at 8h on site 2 and at 48 h on sites 1, 2 and 3, whereas in kidney was observed at sites 2 and 3 only at 48 h. Liver LPO increased at site 2 only after 48 h exposure. Our results demonstrate that the OBA activation pattern in gill and kidney is associated with the induced peroxidative damage extent in those organ, together with water pollution the exposure route, resulting from previous BKPME effluent sediment contamination which may affect the activation pattern of circulating fish phagocytes.     

Monitoring contaminants from oil production at sea by measuring gill EROD activity in Atlantic cod (Gadus morhua)

An ex vivo gill EROD assay was applied in Atlantic cod (Gadus morhua) as a biomarker for waterborne CYP1A-inducing compounds derived from oil production at sea. Exposure to nominal concentrations of 1 ppm or 10 ppm North Sea crude oil in a static water system for 24 h caused a concentration-dependent gill EROD induction. Further, exposure of cod for 14 days to environmentally relevant concentrations of produced water (PW, diluted 1:200 or 1:1000) from a platform in the North Sea using a flow-through system resulted in a concentration-dependent induction of gill EROD. Crude oil (0.2 ppm) from the same oil field also proved to induce EROD. Finally, gill EROD activity in cod caged for 6 weeks at 500-10 000 m from two platforms outside Norway was measured. The activities in these fish were very low and did not differ from those in fish caged at reference sites.     

Selenium and other trace metals in fish inhabiting a fly ash stream: implications for regulatory tissue thresholds

Bluegill sunfish (Lepomis macrochirus) and caddis flies (Trichoptera: Hydropsychidae) were collected from a stream receiving fly ash discharge and nearby reference streams to determine tissue levels of selenium (Se) and other metals, and compare these levels to published "no effect" thresholds. Stingy Run samples contained elevated levels of several metals. Mean Se concentrations in bullhead minnow whole body, bluegill whole body, bluegill ovary, and testes tissues were 44.6, 17.3, 32.5, and 37.1 microg/g (dry wt), respectively. These levels were 2-3 times higher than proposed toxic thresholds for fish whole body (7.9 microg/g) and ovary (17 microg/g). Although monitoring indicated a persistent bluegill population, some reproductive impairment may have occurred. Tissue residue data should be treated with caution because feral fish may accumulate several metals. In Stingy Run, persistence of a bluegill population may be explained by antagonistic interactions with other metals that were also elevated in the fish.     

Impact of cypermethrin on stream fish populations under field-use in biotech-soybean production

The impact of biotech-soybean technology on freshwater ecosystems is being evaluated in the Rolling Pampas region, Argentina. The effect of cypermethrin, the main soybean insecticide, on low-order temperate-stream fish populations was investigated for two consecutive crop cycles under field-use conditions in biotech-soybean production. Cypermethrin was unable to induce mortality or behavioral effects on any of the fish species resident in a first-order stream across a crop field (pulsed acute exposure scenario) sprayed according to conventional practices. No spatially or temporally dependent effects were observed on population parameters (size-class structure, abundance, survival, sex and immature/mature ratio, condition factor) of resident or caged Cnesterodon decemmaculatus after spraying or rainfall events, not even one year after, at the beginning of the next crop cycle. Although cypermethrin was "very highly toxic" to C. decemmaculatus in laboratory water (96h-LC(50)=0.43microg/l), its toxicity was reduced in filtered (78%) and unfiltered (92%) stream water. Changes in LC(50) values were mainly correlated with the OC content of each water fraction (r(2)=0.99; p<0.01; n=9), showing that both DOC and TOC contributed proportionally to toxicity reduction. Protective effects of stream water (12-fold reduction LC(50) values) explained the lack of effects on fish populations in the field, despite cypermethrin water concentrations after spraying reached values comparable with the 96h-LC(50). Therefore, cypermethrin under field-use conditions in transgenic-soybean production represents a low risk of acute exposure for fish populations inhabiting low-order temperate-streams rich in TOC. The relationship between LC(50) and TOC could be a convenient way to improve risk estimation based on laboratory toxicity testing.     

AhR-active compounds in sediments of the Haihe and Dagu Rivers, China

Total concentrations of compounds that can cause activation of the aryl hydrocarbon receptor (AhR) in extracts of river sediments from various locations in the Haihe and Dagu Rivers, Tianjin, China were determined by use of the in vitro H4IIE-luc cell line. AhR-active compounds were isolated from sediments by Soxhlet extraction, and the crude extracts were fractionated using a Florisil column into three Fractions. The response of samples was compared to the TCDD standard and expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TCDD-EQs). Significant dioxin-like activity was observed in each sample. The TCDD-EQs in crude extracts of sediments (SCEs), as determined with the bioassay were 2-4 times greater than the sum of the TCDD-EQs of the eluent from fractions separated with a Florisil column. The results also showed that Fractions 2 and 3 contained most of the AhR-mediated activity. The results obtained by using the bioassay were compared with those of previous measurements of the concentrations of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) and the structurally-similar AhR-active polychlorinated biphenyls (PCBs). It was determined that sediments from the Dagu River contained greater concentrations of TCDD-EQ than did sediments from the Haihe River except at Jingangqiao (location R3), which is associated with industrial activities in the adjacent densely populated area. The concentrations of TCDD-EQs, based on the EC20 and the relative potency ranges, of SCEs ranged from 330 to 930 and 1200 to 13,900 pg TCDD-EQ g(-1) dry wt in Haihe and Dagu Rivers, respectively.     

The suitability of gastropods as biomarkers

The measurement and induction of mixed function oxyigenases (MFOs) of limnetic gastropods were investigated to estimate their suitability as biomarkers. A determination of MFO activities was performed through the measurement of ethoxyresorufin-O-dealkylase (EROD), pentoxy-(PROD) and benzoxyresorufin-O-dealkylase-activity (BROD). Optimal measuring conditions of these activities were investigated in preparations of the digestive gland of 4 species of limnetic gastropods. Results indicate that MFO-activity inhibiting substances are accumulating in the microsomal pellet, the fraction commonly used for the measurement of MFO-activities. Therefore, the fraction used for induction studies was the postmitochondrial supernatant (PMS). EROD, PROD, and BROD activity ofPlanorbis planorbis andPlanorbis carinatus were measured after a treatment with Aroclor 1254 for 1–17 days. Maximal induction of EROD and PROD were 6 and 10 times the value in the control group ofP. carinatus, respectively induction inP. planorbis was lower. BROD-activity could not been measured inP. planorbis. InP. carinatus, BROD-activity increased to a maximum of 2 times after traetment with Aroclor 1254. The level of induction of EROD-activity is comparable to results described in analog studies with fish.