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.     

The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach

Within the frame of the 2nd Iberian Congress of Environmental Contamination and Toxicology (University of the Basque Country, Leioa, June 1998) a workshop was held about the use of biomarkers in marine pollution monitoring. Among others, the following biomarkers received special attention: metallothionein induction, acetylcholinesterase inhibition, cytochrome P450 system induction, imposex, lysosomal enlargement and lysosomal membrane destabilisation, and peroxisome proliferation. These biomarkers can be used to evaluate exposure to and effect of different contaminants (metals, organic xenobiotics and organometallic compounds) and they can be measured using different methodological approaches (biochemistry, cytochemistry, immunochemical methods based on the use of biotechnology). Before the application of a set of biomarkers in pollution monitoring programmes, well-defined protocols of Quality Assurance have to be established to allow adequate comparison of results. It is also necessary to include analysis of standard reference materials and to obtain detailed knowledge of basal values and seasonal variations of the biomarkers in various species, as well as to integrate the information obtained with the different biomarkers. Marine bivalve molluscs such as mussels are appropriate sentinel species for most of the biomarkers proposed except for the induction of the cytochrome P450 system, which should be measured in fish, and the degree of imposex, which is a biomarker of exposure to TBT specifically measured in certain gastropod molluscs. As a result of the workshop, a battery of biomarkers of contaminant exposure and effects are proposed that could be incorporated into programmes monitoring the quality of the coastal environment in the Iberian Peninsula. These measures would be undertaken in conjunction with chemical measures of contaminant burdens in selected sentinel species.     

Fate of steroid hormones and endocrine activities in swine manure disposal and treatment facilities

Manure may contain high concern endocrine-disrupting compounds (EDCs) such as steroid hormones, naturally produced by pigs, which are present at μg L⁻¹ levels. Manure may also contain other EDCs such as nonylphenols (NP), polycyclic aromatic hydrocarbons (PAHs) and dioxins. Thus, once manure is applied to the land as soil fertilizer these compounds may reach aquifers and consequently living organisms, inducing abnormal endocrine responses. In France, manure is generally stored in anaerobic tanks prior spreading on land; when nitrogen removal is requested, manure is treated by aerobic processes before spreading. However, little is known about the fate of hormones and multiple endocrine-disrupting activities in such manure disposal and treatment systems. Here, we determined the fate of hormones and diverse endocrine activities during manure storage and treatment by combining chemical analysis and in vitro quantification of estrogen (ER), aryl hydrocarbon (AhR), androgen (AR), pregnane-X (PXR) and peroxysome proliferator-activated γ (PPARγ) receptor-mediated activities. Our results show that manure contains large quantities of hormones and activates ER and AhR, two of the nuclear receptors studied. Most of these endocrine activities were found in the solid fraction of manure and appeared to be induced mainly by hormones and other unidentified pollutants. Hormones, ER and AhR activities found in manure were poorly removed during manure storage but were efficiently removed by aerobic treatment of manure.     

Characterizing the pollution produced by an industrial area: chemometric methods applied to the Lagoon of Venice

The industrial area of Porto Marghera discharges every year about 1.85 10(9) m(3) of waste waters in the Lagoon of Venice through its 142 discharge points, 17 of them being constantly active. The Anti-Pollution Department of Magistrato alle Acque, the Venice Water Authority, has been controlling these discharges for many years. The huge database built up during the last years could help the authorities in making choices regarding the water quality of the Venetian environment. The application of chemometric methods to the dataset obtained from chemical analyses of industrial waste water samples (almost 250, for each of them up to 57 chemical variables having been measured) is useful to answer fundamental questions related to the pollution generated by the industrial area: i) which are the main differences among the individual discharge points? ii) is there a temporal trend in global and punctual pollution? iii) which is the discharge point having the strongest relative impact on the waters? The results of the present work allow to 1) identify two different groups of discharge points, discriminated by the level of contamination and by the presence of different contaminants; 2) detect a relevant temporal trend in one of the main outfalls (the industrial and civil waste treatment plant); 3) set up a multivariate strategy to "measure" the relative modification induced on receiving lagoon waters by a single discharge. The application of such a "3-STEP multivariate analysis" to the present and future data of water quality could represent a relevant tool for monitoring industrial activities, providing at the same time a support in management decision processes.     

Quantitation of hepatitis A virus and enterovirus levels in the lagoon canals and Lido beach of Venice, Italy, using real-time RT-PCR

In order to assess the microbial water quality of the lagoon canals of Venice, Italy and nearby beach on Lido island, a study was conducted using real-time RT-PCR to enumerate levels of hepatitis A virus (HAV) and enteroviruses in these marine waters over a 3-year period from 2003 to 2005. A total of 17 sites (9 lagoon canal and 8 beach sites) were assayed. For the canals of the Venice Lagoon, 78% were positive for both HAV and enteroviruses, with levels ranging from 75 to 730 and 3 to 1,614 genome copies/L, respectively. At Lido beach, HAV was never detected, but enteroviruses were detected in all Lido beach samples at levels ranging from 2 to 71 genome copies/L. There was a statistically significant correlation between thermotolerant coliform densities and HAV levels (p=0.0002), but the relationship between thermotolerant coliform densities and enterovirus levels was not significant (p>0.05). Despite the fact that enteroviruses were detected at low levels in the surfzone at Lido beach, the risk for enteroviral infection (calculated using the beta-Poisson model) for recreational exposure from swimming, was in the range of 1.9 x 10(-3) - 6.1 x 10(-2), yielding a disease risk at or below the level (5% for gastroenteritis) deemed acceptable by European Guide standards.     

Determination of phenolic endocrine disrupting chemicals and acidic pharmaceuticals in surface water of the Pearl Rivers in South China by gas chromatography-negative chemical ionization-mass spectrometry

An analytical method for phenolic endocrine disrupting chemicals and acidic pharmaceuticals in river water was developed using gas chromatography mass spectrometry (GC-MS) coupled with negative chemical ionization (NCI) technique, and used for the determination of these compounds in the Pearl Rivers (Liuxi, Zhujiang and Shijing Rivers). Derivatization using pentafluorobenzoyl chloride (PFBOCl) and pentafluorobenzyl bromide (PFBBr) before GC-MS analysis were applied and optimized for phenolic compounds and acidic compounds, respectively. The target compounds were analyzed for river waters from the upstream to downstream of the Pearl Rivers. Phenolic compounds 4-tert-octylphenol (4-t-OP), 4-nonylphenol (4-NP), bisphenol-A (BPA), estrone (E1), estradiol (E2) and triclosan (TCS) were detected at trace or low levels in the water samples from Liuxi River and Zhujiang River. Diethylstilbestrol (DES) was not detected in the Pearl Rivers. The highest concentrations of the phenolic compounds were found in Shijing River, and they were 3150 ng/L for 4-t-OP, 11,300 ng/L for 4-NP, 1040 ng/L for BPA, 79 ng/L for E1, 7.7 ng/L for E2 and 355 ng/L for TCS, respectively. Only a few acidic pharmaceuticals were detected at low concentrations in water from Liuxi River and Zhujiang River, but the highest concentrations for the acidic pharmaceuticals were also found in Shijing River. The highest concentrations detected for clofibric acid, ibuprofen, gemfibrozil, naproxen, mefenamic acid and diclofenac were 17 ng/L, 685 ng/L, 19.8 ng/L, 125 ng/L, 24.6 ng/l and 150 ng/L, respectively. The results suggest Liuxi and Zhujiang Rivers are only slightly contaminated and can be used as drinking water sources, but Shijing River is heavily polluted by the wastewater from nearby towns.     

Mitochondrial membrane potential is a suitable candidate for assessing pollution toxicity in fish

Fish inhabiting polluted estuaries are highly exposed to severe stress characterized by an oxidant-antioxidant imbalance. The aim of the study was to explore the use of stress parameters such as adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, mitochondrial membrane potential (?ψm) and total protein expression patterns as biomarkers against oxidant exposures in hepatocytes of Mugil cephalus living in either a contaminated (Test; Ennore) or uncontaminated (Control; Kovalam) estuary. Earlier, the pollutant stress impact was determined through light and electron microscopy studies. The ATP/ADP ratio was measured using high performance liquid chromatography; ?ψm by fluorescent probe 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl benzimidazolcarbocyanine iodide (JC-1) dye and total protein expression patterns by protein profiling. The preponderance of stress impact was confirmed through microscopy studies that featured cytological alterations, disturbances in the surface morphology and in the cell organelles at the ultrastructural levels. Hepatocytes of test fish demonstrated a decrease in ATP and an increase in ADP and thereby alteration in ATP/ADP ratio (p < 0.05; 20.75%). A significant disturbance (p < 0.05; 26.57%) in ?ψm with a ratio of J-aggregates/JC-1 monomer of 1 was observed for test fish hepatocytes compared to control group with a J-aggregates/JC-1 monomer ratio of 1.5. Quantitative assessment of protein expression levels also revealed enhanced induction of both low and high molecular weight proteins in test fish hepatocytes. The findings highlight the use of these parameters as the highly sensitive biomarkers in response to contaminant exposure compared to the routinely used antioxidant and oxidant stress parameters in biomonitoring programs. Among the measured parameters, the determination of ?ψm may be suggested as a novel candidate as a biomarker because of its greater specificity and rapid quantitative risk assessment of pollutant exposures.     

Metal concentrations in digestive gland and mantle of Sepia officinalis from two coastal lagoons of Portugal

Concentrations of both essential (Fe, Cu, Zn) and non essential (Cd, Hg and Pb) metals were measured in the digestive gland and mantle of female cephalopods Sepia officinalis captured in two distinct lagoons in Portugal: Aveiro Lagoon, with a history of anthropogenic and industrial pollution, and Formosa Lagoon receiving urban effluents. We provide evidence for the following: (1) the digestive gland is the main target organ for both essential and non essential metals, frequently containing concentrations few orders of magnitude higher as compared to mantle; the sole exception from this was the Hg that is equally distributed in the two tissues; (2) unexpectedly, the higher levels of metals were found in animals captured in the less polluted lagoon, except for Cd whose bioavailability in Aveiro lagoon might be related to industrial sources, while the influence of Cd speciation in local pray composition should not be ruled out (3) size influenced metal concentration in different way: smaller individuals accumulated significantly more Cu, while Hg concentrations showed the opposite trend; (4) Cd is positively correlated to Zn and Cu in digestive gland of specimens collected in spring in Aveiro Lagoon, and no relationship was found in Formosa Lagoon; (5) the molar ratios Cd:Zn and Cd:Cu in digestive gland increased with body weight in specimens from Aveiro area, both ratios becoming particularly higher in older individuals. Metal-specific accumulation patterns in both mantle and digestive gland at the two sites are discussed in the light of their toxicological implications.     

Introduction of the land snail Eobania vermiculata as a bioindicator organism of terrestrial pollution using a battery of biomarkers

The present study aimed to enrich the group of sentinel organisms of terrestrial pollution biomonitoring, by investigating the efficacy of the land snail Eobania vermiculata. For this reason, a package of biomarkers was performed on land snails E. vermiculata collected from polluted areas in the field or treated with heavy metals in the laboratory. The biomarkers used were neutral red lysosomal retention assay of the haemocytes, acetylcholinesterase activity in the digestive gland and the haemolymph, and metallothionein content of the digestive gland. Moreover, the morphometric changes in the lysosomal system and the morphometric alterations of the neutral lipids were also investigated. In addition, the content of cadmium, lead and copper was evaluated in the digestive gland of the snails. The results revealed appreciable alterations in the biomarker values both in field- and laboratory-conditions, accompanied by significant correlations among the biomarkers. Therefore, this exploratory study suggests the utility of E. vermiculata as a sentinel organism for biomonitoring the biologic impact of terrestrial pollution, and supports the package's efficacy of the selected biomarkers.     

Chemical contaminants, health indicators, and reproductive biomarker responses in fish from rivers in the Southeastern United States

Largemouth bass (Micropterus salmoides) and common carp (Cyprinus carpio) were collected from 13 sites located in the Mobile (MRB), Apalachicola-Flint-Chattahoochee (ARB), Savannah (SRB), and Pee Dee (PRB) River Basins to document spatial trends in accumulative chemical contaminants, health indicators, and reproductive biomarkers. Organochlorine residues, 2,3,7,8-tetrachlorodibenzo-p-dioxin-like activity (TCDD-EQ), and elemental contaminants were measured in composite samples of whole fish, grouped by species and gender, from each site. Mercury (Hg) and polychlorinated biphenyls (PCBs) were the primary contaminants of concern. Concentrations of Hg in bass samples from all basins exceeded toxicity thresholds for piscivorous mammals (>0.1 microg/g ww), juvenile and adult fish (>0.2 microg/g ww), and piscivorous birds (>0.3 microg/g ww). Total PCB concentrations in samples from the MRB, ARB, and PRB were >480 ng/g ww and may be a risk to piscivorous wildlife. Selenium concentrations also exceeded toxicity thresholds (>0.75 microg/g ww) in MRB and ARB fish. Concentrations of other formerly used (total chlordanes, dieldrin, endrin, aldrin, mirex, and hexachlorobenzene) and currently used (pentachlorobenzene, pentachloroanisole, dacthal, endosulfan, gamma-hexachlorocyclohexane, and methoxychlor) organochlorine residues were generally low or did not exceed toxicity thresholds for fish and piscivorous wildlife. TCDD-EQs exceeded wildlife dietary guidelines (>5 pg/g ww) in MRB and PRB fish. Hepatic ethoxyresorufin O-deethylase (EROD) activity was generally greatest in MRB bass and carp. Altered fish health indicators and reproductive biomarker were noted in individual fish, but mean responses were similar among basins. The field necropsy and histopathological examination determined that MRB fish were generally in poorer health than those from the other basins, primarily due to parasitic infestations. Tumors were found in few fish (n=5; 0.01%); ovarian tumors of smooth muscle origin were found in two ARB carp from the same site. Intersex gonads were identified in 47 male bass (42%) representing 12 sites and may indicate exposure to potential endocrine disrupting compounds. Comparatively high vitellogenin concentrations (>0.35 mg/mL) in male fish from the MRB, SRB, and PRB indicate exposure to estrogenic or anti-androgenic chemicals.     

Evaluating environmental impact of STPs situated on streams in the Czech Republic: an integrated approach to biomonitoring the aquatic environment

Sewage water treatment plants (STPs) are frequently associated with the release of xenobiotics and, consequently, with biological responses of fish to these substances. The impact of three STPs situated on small streams was assessed in 2009. Brown trout (Salmo trutta fario L.), captured upstream and downstream of these STPs, were used as biomonitors. The concentrations of 39 organic pollutants (PCBs, OCPs, PBDEs, HBCDs, and MCs), and the biological responses related to oxidative stress (lipid peroxidation and carbonyl protein), and antioxidant responses (superoxide dismutase, glutathione peroxidase and glutathione reductase) were measured. Through chemometrics of these parameters, three groups with 97.62% of the total accumulated variance were distinguished. Integration of the assessed biomarkers using the IBR index, ranked environment impact on sites as: DS Pacov > DS Prachatice > DS Brloh > US Pacov > US Prachatice > US Brloh (most to least affected). STPs are a major source of xenobiotic pollution in streams of the Czech Republic. The combined use of chemical analysis and biological responses is necessary to validate the efficacy of a battery of biomarkers chosen to detect environmental stress due to pollution.     

Pesticides in the atmosphere of the Mississippi River Valley, part I--rain

Lysosomal responses are widely accepted cellular effect biomarkers of general stress. Up to now, these biomarkers have been analysed by means of conventional techniques based on enzyme histochemical methods, where lysosomal enzymes such as acid phosphatase and beta-glucuronidase (beta-GUS) have been employed as markers of lysosomes. The aim of the present work was to develop more advanced and sensitive methods based on the use of polyclonal antibodies to measure lysosomal enzymes in different sentinel organisms. For this purpose, we have studied the cross-reactivity of two commercial polyclonal antibodies against the lysosomal enzymes acid phosphatase and beta-GUS with molluscan digestive gland by means of immunoblotting and immunohistochemistry. The antibody against acid phosphatase cross-reacted specifically with the lysosomal fraction of the digestive gland, while unspecific immunoreaction occurred with digestive gland whole homogenates and tissue sections. The antibody against beta-GUS cross-reacted specifically with digestive gland whole homogenates and tissue sections. The cross-reactivity of this antibody was tested also in crab hepatopancreas and mullet liver where the same successful results were obtained. The second aim of the present study was to test if the immuno-based approach was sensitive enough to detect lysosomal alterations provoked by contaminants. For this purpose two experiments were carried out with mussels treated with cadmium in two ways: in vivo treatment by injection and in vitro treatment using digestive gland explants. Afterwards immunoblotting studies with the antibody against beta-GUS were applied and immunoreactive bands were quantified by means of a gel analysis programme. We found that beta-GUS protein levels were higher in treated mussels when compared with controls in either in vivo or in vitro treatments. All these data suggest that the polyclonal antibody against beta-GUS is adequate to be used in immuno-based approaches to detect contaminant-induced lysosomal alterations.     

Importance of organic matter lability for monomethylmercury production in sulfate-rich marine sediments

Sediment cores were collected from two shallow sites in the Venice Lagoon, Italy, in order to study the lability of organic matter and the methylation rate of inorganic Hg(II). Measurements were made of concentrations of total Hg and monomethylmercury (MMHg), Hg(II) methylation rates, concentrations of total organic carbon and total nitrogen in the sediments, and dissolved sulfate, sulfide, and alkalinity in sedimentary pore waters. A positive linear relationship was detected between the specific Hg(II) methylation rate constant and the fraction of total Hg comprised of MMHg (%MMHg/Hg), indicating that short-term Hg(II) methylation rate reflects a long-term accumulation of MMHg in sediment. In addition, the %MMHg/Hg and specific Hg(II) methylation rate constant in sediment increased with decreasing ratios of total organic carbon to total nitrogen (C/N), whereas concentrations of dissolved sulfate, sulfide, and alkalinity in pore water remained constant. This result suggests that the Hg(II) methylation rate was affected by lability of organic matter. In particular, surface sediments, which contained large fractions of fresh algal organic material (C/N = 5.8-7.8), showed higher Hg(II) methylation rates than did deeper sediments (C/N > 10). Our results indicate that the C/N ratio can be used as a proxy for the lability of organic matter that influences Hg(II) methylation rate in sulfate-rich marine sediments.     

Mercury in sediments and Nassarius reticulatus (Gastropoda Prosobranchia) in the southern Venice Lagoon

The southern basin of the Venice Lagoon has been the focus of fewer studies concerning contamination from heavy metals than the northern and central basins. A recent increase in urban waste waters from Chioggia town, as well as dockyards, shipping and fishing activities, affect this part of the lagoon. The aim of this study was to investigate the total mercury (THg) incidence in sediments and Nassarius reticulatus gastropods in order to assess its distribution and evaluate the level of contamination. THg concentration measured in bottom sediments ranged between 0.1 and 3.4 mg/kg d. wt. The enrichment factor (EF) showed high values (avg. 30, max 49) near the dockyards of Chioggia; the lowest (avg. 9, max 17) were found in the coastal marine sediments near the port entrance of the southern basin. THg in marine scavenger gastropods accumulated in N. reticulatus with concentrations falling within the range of 0.3-1.3 mg/kg d. wt. A positive correlation was found between THg concentration in sediments and in N. reticulatus in all sites, excluding the dockyards. A first local cause for mercury pollution might be attributed to the antifouling paints used in great quantity in the recent past near the town of Chioggia. Moreover, fine suspended sediments associated with tidal flushing are suggested as possibly being the vehicle for pollutant dispersal from the Marghera industrial area to the whole of Venice's lagoon.     

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.     

Occurrence of endocrine disrupting compounds, pharmaceuticals, and personal care products in the Han River (Seoul, South Korea)

The occurrence of 31 selected endocrine disrupting compounds (EDCs) and pharmaceuticals and personal care products (PPCPs) in Korean surface waters was investigated. The area was selected since there is a lack of information in the Seoul area on the suspected contamination of rivers by micropollutants, although over 99% of drinking water is produced from surface waters in this area that has a population of approximately 15 million inhabitants. Samples were collected from upstream/downstream and effluent-dominated creeks along the Han River, Seoul (South Korea) and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Most target compounds were detected in both the Han River samples (63%) and the effluent-dominated creek samples (79%). Iopromide, atenolol, TCPP, TECP, musk ketone, naproxen, DEET, carbamazepine, caffeine, and benzophenone were frequently detected in both river and creek samples, although the mean concentrations in effluent-dominated creek samples (102 ng/L-3745 ng/L) were significantly higher than those in river samples (56 ng/L-1013 ng/L). However, the steroid hormones 17β-estradiol, 17α-ethynylestradiol, progesterone, and testosterone, were not detected (< 1 ng/L) in both the river and creek samples. Numerous target compounds (15) were found to be positively correlated (over 0.8) to the conventional water quality parameters (chemical oxygen demand, biochemical oxygen demand, dissolved organic carbon, and ultraviolet absorbance). Results of this study provide increasing evidence that certain EDCs and PPCPs commonly occur in the Han River as the result of wastewater outfalls.     

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.