Hepatic metallothionein level and mixed function oxidase activity in fingerling rainbow trout (Oncorhynchus mykiss) after acute exposure to pulp and paper mill effluents

Hepatic metallothionein (MT) levels and mixed function oxidase (MFO) activity (7-ethoxyresorufin-o-deethylase or EROD) were measured in fingerling rainbow trout (Oncorhynchus mykiss) exposed to sublethal concentrations of 12 pulp and paper effluents, after completion of 96 h static acute lethality assays. Barring one primary-treated effluent where MFO levels were significantly depressed and two secondary-treated effluents where no significant MFO induction were observed, all other effluents triggered significant induction of MT and EROD, regardless of mill process/treatment or of effluent lethality and chemical characteristics. MT and EROD inductions were significant, however, at higher concentrations for secondary-treated effluents than for primary-treated ones. Lethal (96 h LC₅₀s) to sublethal (MT and EROD lowest observable effect concentrations) ratios were variable and indicated that significant biochemical effects were present at effluent concentrations that were roughly 4–33 (MT) and 3–59 (EROD) times lower than the LC₃₀. Enzyme induction ranged from 1.3 to 2.5-fold for MT and from 1.3 to 9.4-fold for EROD compared to controls. Limited chemical data available suggest that there were indeed classes of compounds present capable of inducing MT or EROD. Observed patterns of MT/MFO responses also suggest that contaminant interactions may have interfered with induction for some of the effluents studied. Refinements of this combined (sub)lethal bioassay procedure are envisaged to determine whether it can provide an efficient means of detecting hazardous chemicals in industrial wastewaters.     

Endocrine alteration and other biochemical responses in juvenile turbot exposed to the Prestige fuel oil

Juvenile turbot (Scophthalmus maximus) were exposed to different concentrations of the Prestige fuel oil through the diet. The effects on hepatic biotransformation enzymes, namely, 7-ethoxyresorufin O-deethylase (EROD), catalase, and phase II activities - UDP-glucuronosyltransferases (UGT) and cytosolic sulfotransferases (SULT) -, were monitored after 42-day exposure. Additionally, potential alterations on the endocrine system of juvenile turbot were assessed by measuring circulating levels of testosterone and 17beta-estradiol in plasma, together with gonadal P450 aromatase activity and the glucuronidation of testosterone by liver microsomal fractions. Fluorescent aromatic compounds (FACs) in bile were determined as an indicator of exposure to polycyclic aromatic hydrocarbons (PAHs). EROD activity increased in a dose dependent manner, and a positive correlation between levels of FACs in bile and EROD activity was observed. A significant increase in UGT activity was observed in fish exposed to medium and high fuel doses, whereas SULT cytosolic activity was not responsive to exposure. Exposure to the Prestige fuel oil sharply reduced circulating levels of testosterone in plasma. The obtained results suggest the ability of the Prestige fuel oil to alter hepatic biotransformation enzymes and to disrupt endogenous hormone levels in juvenile turbot, with unknown consequences in terms of sexual differentiation and reproduction.     

原代培养细胞法测试水中类二噁英物质

采用固相萃取方法对北方某水厂水源水和各处理工艺出水进行富集,对各提取物采用草鱼原代培养肝细胞的7-乙氧基异吩唑酮-脱乙基酶(EROD)体外测试方法进行了类二口恶英物质的检验。结果表明,该水厂水源水和各处理工艺出水中,类二噁英物质浓度相当于0.20～0.50pg/L的2,3,7,8-TCDD(2,3,7,8-四氯代二苯并二噁英)当量,出水TCDD当量远远低于美国环保局饮用水二噁英类化合物含量标准(30pg/L)。研究表明,水样的固相萃取前处理方法结合原代培养细胞EROD分析,可以快速、有效地筛选和定量分析水样中未知的具有EROD效应的物质及其总体效应。     

The checkered puffer (Spheroides testudineus) and its helminths as bioindicators of chemical pollution in Yucatan coastal lagoons

The suitability of using helminth communities as bioindicators of environmental quality of the Yucatan coastal lagoons status was tested on the checkered puffer (Spheroides testudineus) in four coastal lagoons along the Yucatan coast. The concentration of chemical pollutants in sediments, water quality parameters, helminth infracommunity characteristics, as well as fish physiological biomarkers, including EROD (7-ethoxyresorufin-O-deethylase) and catalase activities, were measured. Results from sediment analyses demonstrated the presence of hydrocarbons, organochlorine pesticides and polychlorinated biphenyls at varying concentrations, some of which exceeded the Probability Effect Level (PEL). Significant negative associations among organochlorine pesticides, infracommunity characteristics and fish physiological responses were observed in most of the lagoons. Results suggest that EROD activity and parasite infracommunity characteristics could be useful tools to evaluate the effects of chemical pollutants on the fish host and in the environment. Importantly, certain parasites appear to influence biomarker measurements, indicating that parasites should be considered in ecotoxicological studies.     

An assessment of estrogenic organic contaminants in Canadian wastewaters

A suite of 30 primarily estrogenic organic wastewater contaminants was measured in several influent/effluent wastewater samples from four municipal wastewater treatment plants and effluents from one bleached kraft pulp mill (BKME) using an ultra-trace analytical method based on gas chromatography-high resolution mass spectroscopy (GC-HRMS). In vitro recombinant yeast assay detection of the estrogenic equivalent (EEq) on whole and solid phase extracted (SPE) and fractionated wastewater was also performed. 19-norethindrone was the most frequently detected and abundant (26-224 ng/L) of all the synthetic estrogens/progesterones in the influent samples. 17alpha-ethinylestradiol was the more frequently detected synthetic estrogen/progesterone in the effluents occurring at or below 5 ng/L with some sporadic occurrences of up to 178 ng/L. The greatest levels of steroidal estrogens in municipal effluents were E1>E2>E3 which were all <20 ng/L. Nonylphenol and di(2-ethylhexyl) phthalate were found to be the highest non-steroidal synthetic compounds surveyed in both municipal influent and effluent samples, both occurring at 6-7 microg/L in municipal effluents. BKME contained relatively large amounts of the plant sterol stigmasterol (4 microg/L) but low amounts of fecal sterols, and steroidal estrogens (E2 only at 6 ng/L) when compared to the municipal effluents. In vitro EEq in the wastewater surveyed ranged from 9-106 ng E2/L and ranked from municipal influent>municipal effluent approximately BKME, with most of the estrogenicity fractionating in the 100% methanol SPE fraction followed by a secondary amount in the diethyl ether (for municipal) or methyl-tert butyl ether (for BKME) SPE fractions. Most correlations between chemical and in vitro estrogenic equivalency were weak (p>0.05 in most cases). Unexpected inverse correlations between in vitro estrogenic activity and concentrations of the estrogenic contaminant bisphenol A were found which likely contributed to the weakness of these correlations. A modified toxicity identification and evaluation procedure was continued with the SPE extracts from the more potent 100% methanol SPE fractions of municipal effluent. High performance liquid chromatography band elution retention times, based on in vitro estrogen detection, indicated that steroidal estrogens such as E2 were responsible for most of the estrogenicity of the samples. Subsequent collection and GC-MS analysis of active bands did not confirm the presence of steroidal estrogens, but expanded the possibility of phthalate esters (i.e. dibutyl phthalate) and natural sterols (i.e. beta-sitosterol) contributing to the overall estrogenic load.     

Biomonitoring of the genotoxic potential (micronucleus assay) and detoxifying activity (EROD induction) in the River Dadou (France), using the amphibian Xenopus laevis

Within the framework of a general survey of the water quality of the river Dadou (Tarn, France), different physico-chemical parameters were measured and an inventory of the fish population was made along the water course, around the Rassisse dam. With the aim of monitoring the potential genotoxic effects and the detoxifying activities induced in organisms exposed to the river water, two in vivo bioassays were performed in laboratory experiments, using larvae of the amphibian Xenopus laevis. The first was the micronucleus test, using red blood cells, and the second the assay of ethoxyresorufin-O-deethylase (EROD) induction in the liver of exposed animals. Eight water samples were taken from the river and at outlet points from the two major industrial activities of the studied section of the water course: a spar-fluor mine and a water treatment plant. Genotoxic impact and EROD induction were measured in the larvae. The effluent of the filter-washing process from the water treatment plant was found to be particularly genotoxic, even after dilution in pure reconstituted water, but no particular genotoxicity was found, either in Dadou river water, or in the effluents from the mine. On the other hand, most of the water samples tested produced a clear induction of EROD activity compared to the level of enzymatic activity found in the liver of larvae reared in the river water sampled upstream of the industrial activities. These results were interpreted taking into account (i) the high concentrations of pollutants (fluorine and manganese) measured in the river water, (ii) the very low population levels inventoried in the downstream section of the river and (iii) the possible interactions between the substances present in the river water, particularly the classical EROD inducers PAHs and PCBs.     

Trace metal accumulation in Clarias gariepinus (Teugels) exposed to sublethal levels of tannery effluent

The concentration and distribution of Cr, Cu, Pb and Zn among the tissues of a freshwater fish, Clarias gariepinus exposed to combined (composite) tannery effluent was investigated at two sublethal concentrations (2 and 6%) in static bioassay for 8 weeks. The distribution of the four metals in fish was of the order of Pb > Cr > Cu > Zn and the accumulation was found to be dose- and time-dependent. The metal levels in the liver were significantly higher (P > 0.05) than other tissues. This was followed by the gill and the gut. Relatively low accumulation of these metals was found in muscle tissue.     

Avoidance of bleached kraft mill effluent by pre-exposed Coregonus albula L.

The ability of vendace (Coregonus albula L.) to avoid different concentrations of bleached kraft mill effluent (BKME) was studied in the laboratory with fish pre-exposed to BKME for 1 week and non-pre-exposed fish. Pre-exposure to BKME in sublethal concentrations reduced the avoidance of BKME by vendace. Throughout the range of exposure concentrations (0.13–4.50 v/v%), pre-exposed fish tended to be observed less in the clean water than non-pre-exposed fish or even to prefer contaminated water. In field conditions, changes in behaviour might lead to a chain reaction: the fish come into contact with concentrations of BKME lower than the threshold level for avoidance and their threshold level increases gradually due to this exposure, which makes exposure to higher concentrations possible and weakens the ability to return to clean water. Desensitization caused by pre-exposure and long distances between pronounced BKME concentration differences in the field together makes it improbable that the vendace can show avoidance responses in the field. Analyses of the various waste-water samples support the conclusion that the most toxic components in BKME, resin acids and chlorophenols, are of minor importance to the avoidance reaction.     

Sparus aurata L. liver EROD and GST activities, plasma cortisol, lactate, glucose and erythrocytic nuclear anomalies following short-term exposure either to 17beta-estradiol (E2) or E2 combined with 4-nonylphenol

Immature Sparus aurata L. (gilthead seabream) were exposed to 17beta-estradiol (E(2)) 4000 ng/l and to the same E(2) concentration mixed with 50,000 ng/l 4-nonylphenol (E(2) + NP) during 4, 8, 12 and 16 h. E(2) availability and E(2) plasma level variations were assessed. Liver biotransformation capacity was measured as ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST) activities. Plasma cortisol, lactate and glucose were also determined. Genotoxicity was assessed through erythrocytic nuclear anomalies (ENA) frequency. Liver EROD activity significantly decreased during the whole experiment for both treatments, with the exception of 16 h exposure to E(2). Liver GST activity was significantly increased after 8 and 12 h of exposure either to E(2) or E(2) + NP. An endocrine disruption expressed as plasma cortisol decrease was observed after 16 h exposure under both tested conditions, concomitantly with a plasma lactate increase. No genotoxic responses, measured as ENA frequency, were detected. Analyzing the E(2) water concentration in aquaria without fish it was demonstrated an intense and fast E(2) loss, considerably reducing its availability to fish. In the presence of fish, E(2) water levels were drastically reduced after 4 h exposure, being this reduction more pronounced in E(2) aquarium when compared to E(2) + NP aquarium. In addition, it was demonstrated a rapid E(2) uptake from the water since the highest E(2) plasma concentrations were observed after 4 h exposure, followed by a continuous decrease, which became more pronounced between 8 and 12 h of exposure. Furthermore, during the first 8 h exposure to E(2) and E(2) + NP, seabream plasma E(2) concentrations were higher than the initial water exposure concentration. Comparing the E(2) plasma levels in both seabream-exposed groups, it was clear that its concentration is always higher in E(2) + NP-treated fish. Despite the previous results, no significant differences were found in the measured responses between E(2) and E(2) + NP.     

Endocrine disruption in wild populations of chub (Leuciscus cephalus) in contaminated French streams

The aim of this study was to assess endocrine disruptive effects in wild population of fish in five French rivers selected to represent different pollution contexts at two seasons (summer and fall). For that purpose, a panel of biometrical parameters (length, weight, and gonado-somatic index: GSI) and biochemical (ethoxyresorufin-O-deethylase: EROD, vitellogenin: VTG, and brain aromatase) and histological biomarkers (gonads histology) were used in chub (Leuciscus cephalus), a common cyprinid fish species. In fish from the reference site, EROD activity and VTG levels were low at the two seasons. Brain aromatase activities (AAs) were similar to other species and increased with increasing GSI and gonad maturation. Among the four contaminated sites, the Jalle d'Eysines River was the most impacted site. At this site, fish were exposed to estrogenic substances as demonstrated by the VTG induction in males and the arrest of development of the gonads that led to lower brain AA compared to fish from the reference site. In fish from other contaminated sites, EROD activity was induced as compared to fish from the reference site and some males had elevated concentrations of VTG. Moreover, the presence of aromatase-inhibiting compounds was demonstrated in the sediments of three contaminated sites, even if the precise nature of contaminants is not known. This study provides new data concerning endocrine disruption in wild fish populations inhabiting French rivers and demonstrates that measurements of in vivo and in vitro aromatase could be used as biomarkers of endocrine disruption in field studies.     

Multiscale approach of fish responses to different types of environmental contaminations: a case study

In order to study the responses of wild fish to environmental contaminations, different sites (two references and three contaminated) were sampled across the Walloon hydrographical network (southern Belgium). The status of fish communities was characterized according to an index of biotic integrity (IBI). Furthermore, population structure, reproductive parameters and biochemical assays were performed on chub (Leuciscus cephalus) as sentinel species. The results showed impaired responses in fish sampled downstream paper mill effluents (BKME), in a polychlorinated biphenyls (PCB) contaminated site, and in a river receiving pesticides (i.e. atrazine and hexachlorocyclohexane). High level of hepatic 7-ethoxyresorufin-Odeethylase (EROD) activity were evidence of direct toxicant exposure, while population structures showed unbalanced size-frequency distributions. Moreover, in the pesticide contaminated site, a higher proportion of atretic oocytes was found in females and abnormally high plasma alkali-labile phosphorus (ALP) values were noticed in males, compared to reference sites, suggesting that chubs were exposed to estrogenic compounds. IBI scores from both sites were also lower than in reference sites. On the other hand, no particular response was found in hepatic glutathione-S-transferase (GST) activities, hepatic heat-shock protein HSP70 concentrations and plasma sex steroids (estradiol-17beta, 11-ketotestosterone and testosterone) in chubs from any sites. By using a multiparametric approach, a better understanding of response mechanisms to pollution in fish may be achieved in order to improve the ecological status of river ecosystems.     

Comparative study of the estrogenic responses of mirror carp (Cyprinus carpio) exposed to treated municipal sewage effluent (Lisbon) during two periods in different seasons

To assess the estrogenic potency of the treated domestic sewage effluent from a sewage treatment plant (STP) located in Lisbon (Chelas), 60 mirror carp (Cyprinus carpio) were exposed to different concentrations of the sewage effluent (0%, 25%, 50% and 100%) for two periods of 28 days in two different seasons (winter/spring). Vitellogenin induction in males was used as a biomarker of exposure to xenoestrogens. At the end of the experiment, blood samples were taken for vitellogenin analysis and the fish were sacrificed and dissected. Gonad samples were taken for histological evaluation of the sewage effects. The results showed an increase in vitellogenin induction in exposed fish, both males and females, depending on the different dilutions of the sewage effluent. In comparison with controls, the gonadosomatic index decreased significantly (P < 0.05) in fish exposed to 100% treated effluent. Although statistically not significant, the hepatosomatic index (HSI) was high in all exposed fish. Histological abnormalities in fish gonads were evaluated and related to the different percentages of sewage effluent. Seasonal variations found in estrogenic responses were attributed to weather influences on sewage dilution.     

A video-based movement analysis system to quantify behavioral stress responses of fish

Behavioral alterations can be measured as endpoints for sublethal toxicity, and serve as a tool for environmental risk assessment and analysis of toxicological impact. Numerous technical and biological factors have made sublethal effects on fish behavior difficult to quantify. In order to investigate stress- and contaminant-induced behavioral alterations, a video analysis system was designed by our laboratory. With this system up to 12 fish may be individually housed in 20 L exposure arenas and automatically videotaped at multiple and discrete intervals during an experimental period. Analog video data can then digitized, converted into x,y coordinates, and finally transformed into relevant behavioral endpoints using software designed for tracking fish movement combined with specific algorithms. These endpoints include velocity, total distance traveled, angular change, percent movement, space utilization, and fractal dimension (path complexity). Data from fish exposed to a reference toxicant, MS222, and simulation experiments, are presented to exemplify alterations in fish behavior associated with exposure, and accuracy and precision, respectively. The system provides flexibility to analyze any observed movement behavior, is remotely controlled, and can be transportable. These movement analyses can be used to identify characteristic behavioral responses to a variety of environmentally-relevant stressors, and assist in risk assessment and the development of more sensitive lowest observable effect level and no observable effect level for sentinel species.     

River pollution by anticholinesterase agents

The effects of effluent discharged into the Blue River, near its confluence with the Missouri River in Kansas City, Missouri, by a manufacturer of organophosphate and carbamate pesticides were investigated. Since these pesticides act as nerve poisons by inhibiting the neurotransmitter modulating enzyme acetylcholinesterase (AChE) in the nervous system, poisoning of fishes was diagnosed by measurement of brain-AChE in fishes collected from the Missouri River upstream and downstream from the mouth of the Blue River. Other fish were exposed to diluted effluent in glass jars and their brain-AChE measured to determine combined poisoning potential of compounds present. Fishes immediately downstream repeatedly had lower brain-AChE activity than fishes upstream, and fish exposed to diluted effluent had lower brain-AChE activity than unexposed fish. Chemical analyses showed substantial amounts of AChE-inhibiting pesticides in the effluent relative to their toxicities. These data indicate the effluent is a contributing factor in the reduced brain-AChE activity of Missouri River fishes, and that brain-AChE measurement in fishes is a sensitive and reliable indicator of such pollution.     

Effects of chemical pollution on the activities of hepatic xenobiotic metabolizing enzymes in fish from the River Rh?ne

Polychlorobiphenyl (PCBs) levels and hepatic xenobiotic metabolizing enzyme activities were measured in fish from three locations of the River Rh?ne to study the consequences of a constant loading of PCBs from a PCB incineration plant. Our results show that levels of PCBs and enzyme activities were higher in fish living downstream from the plant than in fish from two locations upstream, suggesting enzyme induction by PCBs (known to be potent inducers in laboratory conditions). Enzyme activities were studied in spring and autumn in three species: nase (Chondrostoma nasus), roach (Rutilus rutilus) and grayling (Thymallus thymallus). Induction was observed for three cytochrome P-450-dependent monooxygenase activities (MO), i.e. 7-ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD). There was a close correlation between EROD and AHH activities (for all species). Glutathione S-transferase activities were also shown to be related to the PCB levels. Conversely, cytochrome P-450 content and benzphetamine N-demethylase activity were not "PCB level-dependent". This study clearly demonstrates a close relationship between PCB contamination and MO activities in fish from the field and thus clearly emphasizes the interest in MO as a monitoring tool for estimating water quality.     

Comparison of six different sewage treatment processes—Reduction of estrogenic substances and effects on gene expression in exposed male fish

Treated sewage effluents often contain a mixture of estrogenic compounds in low concentrations. The total combined activity of these, however, may be sufficiently high to affect the reproduction of aquatic vertebrates. The introduction of advanced treatment technologies has been suggested as a way to remove micro-contaminants, including estrogenic substances. In this study, one municipal influent was treated with six different processes in parallel on a semi-large scale in order to assess their potential to reduce substances that could contribute to estrogenic effects in male fish. The effluent from a conventional, activated sludge treatment line was compared to a similarly treated effluent with a final sand-filtering step. The addition of ozonation (15 g O₃/m³), a moving bed biofilm reactor (MBBR) or both in combination was also evaluated. There was also a separate treatment line that was based on a membrane bioreactor. A small battery of hepatic estrogen-responsive genes was measured in the exposed fish using quantitative PCR. Concentrations of steroid estrogens and estrogenic phenols in the effluents were measured by GC-ECNI-MS. The ozonated effluents were the only tested effluents for which all measured biological effects in exposed fish were removed. Chemical data suggested that the MBBR technology was equally effective in removing the analyzed estrogens; however, elevated expression of estrogen-responsive genes suggested that some estrogenic substances were still present in the effluent. The membrane bioreactor removed most of the measured estrogens and it reduced the induction of the estrogen-responsive genes. However, fish exposed to this effluent had significantly enlarged livers. Given that the same influent was treated in parallel with a broad set of technologies and that the chemical analyses were combined with an in vivo assessment of estrogenic responses, this study provides valuable input into the assessment of advanced treatment processes for removing estrogenic substances.     

Mutagenic activity of fish and sediments in the Sheep River, Alberta

Using the Ames Salmonella/mammalian microsome assay the mutagenic activity of fish and benthic sediments from a section of the Sheep River. Alberta receiving a chlorinated sewage effluent were investigated. The benthic sediments gave no indication of increased mutagenic activity, however fish collected from the chlorinated sewage plume showed a significant increase in mutagenic activity. The results are discussed with respect to the effects of a chlorinated sewage effluent on the lotic ecosystem in this section of the river.     

The influence of the high and low molecular weight fractions of a bleach kraft mill effluent on the microbial activity of activated sludge

The high molecular weight (HMW) fraction (> 1000 Da) of bleached kraft mill effluent (BKME) is the principal source of adsorbable organic halide (AOX), colour and chemical oxygen demand in treated effluent. The influence of HMW material on the microbial activity of activated sludge microorganisms was studied in a series of batch growth experiments using fractionated effluent. The HMW material in the untreated effluent comprised 80% of the AOX but only 20% of the soluble COD. Multivalent metal ions had a greater affinity for this HMW fraction. The addition of HMW material to the LMW fraction had a stimulatory effect on microbial activity as measured by increases in biomass and substrate utilization. Chemical characterization and subsequent growth experiments revealed that nitrogen, which was deficient in the LMW effluent, was primarily associated with the HMW material. The association of nitrogen with the HMW fraction may have an important role on the behaviour of nitrogen within a biological treatment system and in the receiving environment.