Mixed Function Oxygenase Induction Research Articles

In Vivo and In Vitro Mixed-Function Oxygenase Activity and Vitellogenin Induction in Fish and in Fish and Rat Liver Cells by Stilbenes Isolated from Scotch Pine (Pinus sylvestris)

Many types of pulp and paper mill effluents have the ability to induce mixed-function oxygenase (MFO) activity and vitellogenin (VTG) protein in exposed male fish. The search for the compounds responsible for MFO induction has led to several classes of compounds, among them retene and stilbenes. The objective of this study was to investigate the biological activities of candidate stilbene compounds. Three stilbenes, 3,5-dihydroxystilbene (pinosylvin; P1), 3-hydroxy-5-methoxystilbene (P2), and 3,5-dimethoxystilbene (P3), were extracted from Scotch pine (Pinus sylvestris) and purified to evaluate their ability to induce MFO activity in vitro using ethoxyresorufin-O-deethylase (EROD) activity in a rat hepatoma cell line (H4IIE). As these compounds may be chlorinated during pulp bleaching, chlorination of P2 was undertaken, producing di- and trichlorinated isomers (Cl-P2), which were also tested. Compounds were tested for EROD-inducing ability in vivo by exposing juvenile rainbow tout (Oncorhynchus mykiss) to waterborne concentrations (0.010 to 1.0 mg/L) for 4 days. Compounds were also tested for their ability to induce VTG in trout primary liver cells in vitro. The stilbenes were weak inducers of EROD and VTG. H4IIE EROD was induced by all four compounds, with the most potent induction by P3, followed by P1, the Cl-P2 mixture, and then P2. Induction for all four stilbenes was from 3.13 × 10⁻³ to 3.57 × 10⁻⁴ as potent as retene and about 1.11 × 10⁻⁵ to 1.20 × 10⁻⁶ as potent as TCDD. Juvenile rainbow trout did not show EROD induction after exposures to P1, P2, or the Cl-P2 mixture, whereas P3 caused activity fourfold above that of controls. P1, P3, and Cl-P2 all weakly induced VTG in rainbow trout hepatocytes. The most potent inducer of VTG was Cl-P2, followed by P3 and P1. The results show the ability of wood-derived stilbenes to cause weak MFO induction in fish and in rat liver cells and to weakly induce vitellogenin in fish liver cells.

The Possible Role of Reduced Sulfur Compounds in the Toxic Properties of Pulp and Paper Mill Effluents

AbstractA comparative analysis of different bioassays was carried out using as an example black liquor solutions, simulating wastewaters of the Kraft process for the digestion of cellulose. Alongside the analysis of products of lipid peroxidation (LPO) in blood plasma and the liver of mice in vivo, the most sensitive assays were based on the analysis of Ceriodaphnia reproduction, the analysis of LPO intensity in vitro, the mixed function oxygenase (MFO) assay in fish liver, and the population growth test of Tetrahymena pyriformis. The toxicological analysis of pulp and paper mill effluents at different stages of their formation and treatment has demonstrated two relationships, as follows:(i) a correlation between toxicity and the presence of reductive substances (as titrated by hydrogen peroxide in the presence of a catalyst), and(ii) a correlation between the content of these reductive substances in wastewater and the reduced sulfur compounds formed during cellulose digestion.Using the MFO induction assay and the LPO intensity assay in the model test system has demonstrated that the addition of hydrogen peroxide into solutions of black liquor results in their detoxification. It is concluded that the reduced sulfur compounds play a significant role in the toxicity of pulp and paper mill effluents.

MFO induction potential of fish species native to the Swan-Canning Estuary, Western Australia.

The use of mixed function oxygenase (MFO) enzymes as a potential biomarker of pollution in the Swan-Canning Estuary was investigated in four indigenous fish species, Perth herring (Nematalosa vlaminghi), sea mullet (Mugil cephalus), yellowtail trumpeter (Amniataba caudavittata), and black bream (Acanthopagrus butcheri). Each experimental fish was intraperitoneally injected with 10 micrograms/kg of 3,3',4,4',5-pentachlorobiphenyl per kilogram of fish using corn oil as a carrier. Controls were injected with 1 mL corn oil per kilo. Induction of the hepatic ethoxyresorufin-O-deethylase (EROD) activity was measured 10 days post injection. Perth herring did not survive the experimental period due to the poor ability of this fish to endure captivity and handling. Yellowtail trumpeter demonstrated a low EROD induction response (2-fold relative to controls), while sea mullet displayed a moderate EROD induction potential (3.5-fold relative to controls). Because of their lower EROD induction potential, yellowtail trumpeter and sea mullet were not considered the most suitable bioindicator species for Western Australian estuarine waters. Black bream has desirable population traits and had a relatively high MFO induction potential response (9-fold relative to controls). From the results it was concluded that, when using EROD induction as a biomarker, black bream is a suitable fish to use as a bioindicator species for investigations into the health of the Swan-Canning Estuary.

Pink snapper (Pagrus auratus) as a bioindicator of aquatic. Environmental health in Western Australia.

Pink snapper (Pagrus auratus), an endemic Western Australian fish species, was tested for its potential as a bioindicator of aquatic environmental health. Mixed function oxygenase (MFO) induction (as a biomarker of exposure), and sorbitol dehydrogenase (SDH) activity (as a biomarker of liver damage) were of special interest to the study as these biochemical tools have not been validated for any Western Australian fish species. Juvenile pink snapper were injected intraperitoneally (i.p.) with 0, 10, 100, 500, and 1000 micrograms PCB-126 per kilogram. Fish were sacrificed 10 days postinjection, and livers and blood samples were collected for MFO and SDH analysis, respectively. Doses of 10 and 100 micrograms PCB-126 per kilogram caused the highest MFO induction, while doses of 0 and 1000 micrograms PCB-126 per kilogram did not result in higher MFO activity relative to carrier-injected control fish. SDH activities were not significantly different among treatments indicating that hepatocellular damage was not responsible for the reduced MFO activity at the highest dose. The result of the study demonstrates that pink snapper may potentially be used as a bioindicator species in Western Australia when MFO is used as a biomarker.

Isolation of MFO inducers from tissues of white suckers caged in bleached kraft mill effluent

White sucker ( Catostomus commersoni) caged for 3 days in a bleached kraft mill effluent (BKME) stream had elevated mixed function oxygenase (MFO) activities 15–90 fold those of fish caged in a reference stream. Liver composites of male and female fish were ground and extracted with dichloromethane (DCM), methanol or 50% DCM/methanol, and tested for MFO activity in rat hepatoma cells (H4IIE). There was no difference in the potency of H4IIE EROD induction among the three solvents, so DCM extracts were split into 31 fractions using reverse phase high pressure liquid chromatography (HPLC). H4IIE MFO activity was elevated in several fractions, with three early peaks and several later peaks of induction, indicating several classes or compounds causing MFO induction were present in the fish livers. Polychlorinated dibenzo- p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and chlorinated diphenylethers (CDPEs) were detected in several late-eluting fractions, but concentrations were not high enough for these compounds to be solely responsible for the observed induction. Induction by liver extracts decreased as cell exposure times increased (24, 48 or 72 h), suggesting that some inducers were more easily metabolized and eliminated from the H4IIE cells. In contrast, 2,3,7,8-tetrachlorodibenzo- p-dioxin (2378-TCDD) had similar potency over 24, 48 and 72 h, as it was relatively resistant to metabolism.

Enhanced biological treatment of bleached kraft mill effluents — II. Reduction of mixed function oxygenase (MFO) induction in fish

Biological treatment studies with bleached kraft mill effluents (BKME) assessed the removal of hepatic MFO induction potency towards fish. Bench-scale activated sludge, facultative stabilization basin, and aerated stabilization basin processes were compared under conservative conditions of elevated sludge age and hydraulic retention times as well as moderate temperatures for the treatment of bleachery effluents from a modernized bleached kraft mill using oxygen delignification and 60% chlorine dioxide substitution. Optimized treatment for the removal of hard-to-degrade organics also consistently removed more than 85–90% of effluent-induced MFO activity of fish, based on laboratory bioassays. Follow-up mill-site pilot scale testing with an activated sludge treatment process in extended-aeration mode also demonstrated >90% reductions in the MFO induction potential of whole mill effluent. Characterization of various in-plant process waters sampled at the mill indicated that the softwood-line bleach plant was a major contributor (>70%) to the MFO induction potential of untreated and biologically-treated BKME.

MFO Induction of three Australian fish species

The use of mixed function oxygenase (MFO) enzymes as a biomarker of pollution in Port Phillip Bay was investigated in three indigenous fish species, sand flathead (Plathycephalus bassensis), bluethroat wrasse (Notalabrus tetricus), and sixspine leatherjacket (Meuschenia freycineti). Investigation of the relative MFO induction potential was performed by intraperitoneally injecting each experimental fish species with 100 μg/kg of 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) using corn oil as a carrier. Hepatic induction was fluorimetrically measured by the activity of 7-ethoxyresorufin O-deethylase (EROD) activity 10 days post-injection. Of the three species tested, the sand flathead was selected as the most suitable indicator species for its high MFO induction potential and desirable characteristics related to its population biology. Bluethroat wrasse appeared relatively uninducible by PCB 126, while sixspine leatherjacket demonstrated a medium induction potential but was unsuitable as a biomonitoring tool because of its scarcity and distribution limited to reef beds. ©2000 John Wiley & Sons, Inc. Environ Toxicol 15: 1–7, 2000

Kinetics of mixed function oxygenase induction and retene excretion in retene-exposed rainbow trout (Oncorhynchus mykiss).

The polycyclic aromatic hydrocarbon 7-isopropyl-1-methylphenanthrene (retene) induces mixed function oxygenase (MFO) activity of fish. Bile levels of retene and its metabolite(s) were measured in relation to exposure time, exposure concentration, and induction of MFO activity. Synchronous fluorescence spectrometry provided a rapid means of measuring the amount of retene present in the bile of exposed fish, whereas conventional fluorescence spectrometry was used to quantify the amount of retene metabolites. Based on bile analysis, increased retene exposure resulted in an increased uptake of retene and a curvilinear increase in hepatic MFO activity. Retene was present in the bile within 6 h of initial exposure. However, retene metabolite(s) only appeared in the bile after MFO induction had occurred, 12 h after exposure had commenced, suggesting that MFO activity is required for metabolism. Transfer of fish to clean water after 48 h of exposure resulted in a rapid decrease in the presence of retene and its metabolite(s) in the bile, with a calculated half-life of about 14 h. In vitro additions of retene directly to the ethoxyresorufin O-deethylase assay demonstrated that retene is capable of acting as a competitive inhibitor. Thus, retene contamination of postmitochondrial supernatant (S9 fraction) could result in false-negative results in the MFO assay. The MFO activity in extrahepatic tissues (gills, heart, and kidney) was not significantly induced with retene exposure. Thus, the major site of retene metabolism seems to be in the liver. These results confirm that retene is rapidly taken up, metabolized, and excreted by rainbow trout, and that retene metabolism and excretion are linked to hepatic MFO induction.

Developing Biological Endpoints for Defining Virtual Elimination: a Case Study for PCDDs and PCDFs

Abstract Defining virtual elimination has created considerable debate. A traditional approach has been to use chemically defined detection limits or levels of quantification that are determined using the best currently available methodologies. Ever increasing improvements in analytical techniques could lead to corresponding pressure to reduce the targets for virtual elimination. The current Toxic Substances Management Policy in Canada recognizes this and clearly states that it is not the intent of virtual elimination to have a moving target or to chase down the last molecule of the chemical of concern. Although it may be possible to reduce a chemical to less than some extremely sensitive detection limit, the chemical may or may not exert biological effects at that level. The chemically defined detection limits may be much lower than background levels in the environment, making it an unrealistic target. Conversely biological responses may result from trace levels of a compound that are not detectable in effluents or selected compartments of the environment (i.e., water) using current chemical techniques. Alternatively, an effect-based approach can establish biologically meaningful endpoints to defining virtual elimination. Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are used in this study as an example to evaluate the advantages and limitations of several possible approaches of using biological endpoints to determine the presence of these compounds in the environment and ultimately define virtual elimination. A review of the biological responses to PCDD/PCDFs is included to demonstrate the importance of selecting appropriate biological endpoints. Mixed function oxygenase (MFO) induction, although not recommended at this point, is used as an example of a possible sensitive endpoint that could potentially be used to detect exposure of biota to these chemicals. Three different approaches are explored: (1) measuring MFO induction in a sentinel species in the environment; (2) testing environmental extracts for MFO induction in cell lines; and (3) using biological endpoints (MFO induction) to define chemical targets for virtual elimination. While the use of biological end-points is the most desirable approach to defining virtual elimination, there are significant knowledge gaps which limit our selection and application of this approach.

Natural Recovery Reduces Impact of the 1970 Arrow Oil Spill

ABSTRACT In 1970 the tanker Arrow ran aground releasing 2,000 m3 of Bunker C crude oil along 300 km of Nova Scotia's coastline, of which only 10% was subjected to cleanup, the rest was left to degrade naturally. Sediment and interstitial water collected in 1993 and 1997 from Black Duck Cove in Chedabucto Bay, a representative untreated site, showed that the remaining residual oil has undergone substantial biodegradation. The environmental significance of this intrinsic remediation process was assessed with a battery of microscale biotests: CYP1A and mixed function oxygenase induction in winter flounder, Amphipod Survival, Echinoid Fertilization, Grass Shrimp Embryo-Larval Toxicity, Microtox® Solid-Phase and 100% Tests. While much oil remains in the sediment (426–12,744 ppm), results of the biotests show that it is of low toxicity and habitat recovery is evident from the level of benthic diversity.

The Use of Semipermeable Membrane Devices to Concentrate Chemicals in Oil Refinery Effluent on the Mackenzie River

To concentrate natural and refinery-derived inducers of mixed function oxygenase (MFO), semipermeable membrane devices (SPMDs) were deployed for 11-12 days in Norman Wells refinery effluent and upstream and downstream on the Mackenzie River, Northwest Territories, Canada. SPMDs, which are layflat polyethylene membrane tubes containing a thin film of purified triolein, absorb freely dissolved neutral organic chemicals that diffuse through the polyethylene membrane. Fish liver cells ( Poeciliopsis lucida hepatoma; PLHC-1) were dosed with SPMD extracts; then MFO activity was determined. SPMDs from the effluent contained potent MFO inducers, equivalent to 4830-8700 pg 2, 3, 7, 8-tetrachlorodibenzo- p -dioxin (TCDD)/g SPMD. SPMDs from the site 0.5 km downstream of the refinery outfall also induced MFO, but downstream extracts were about 1/40 as potent as those from effluent-exposed SPMDs. Comparing sites on the Mackenzie River, extracts of SPMDs from downstream of the refinery outfall were over 20 times as potent as those from upstream SPMDs. Polyaromatic hydrocarbons (PAHs) and C1- to C3-substituted PAHs were abundant in extracts of SPMDs from the effluent and, to a lesser degree, in extracts of SPMDs from 0.5 km downstream. In effluent-exposed and downstream SPMDs, concentrations of parent PAHs were lower than those of C1-substituted PAHs. The highest concentrations were those of C2- and C3-substituted PAHs. It is unknown which compounds in the SPMDs caused the MFO induction, but there was a very strong correlation between the concentration of total C3-phenanthrene/anthracene in the SPMD extracts and MFO-induction potency. The study shows both the use of SPMDs as concentrators of neutral organic compounds in harsh environments (such as high temperature effluents and fast-flowing rivers) and the utility and sensitivity of the fish liver cell line for ranking MFO-inducing potencies of environmental SPMD extracts. Key words: semipermeable membrane devices (SPMDs), mixed function oxygenase (MFO), Ethoxyresorufin-O-deethylase (EROD), oil refinery effluent, Mackenzie River, Norman Wells, cell line, PLHC-1, polyaromatic hydrocarbons (PAHs)

Chronic retene exposure causes sustained induction of CYP1A activity and protein in rainbow trout (Oncorhynchus mykiss)

AbstractThe removal of persistent chlorinated organic compounds from pulp mill effluents has not eliminated mixed function oxygenase (MFO) induction by these effluents. Therefore, continuous MFO induction downstream of pulp mills may be due to exposure to more labile compounds, such as retene (an alkyl‐substituted phenanthrene), which typically cause transient induction after a single brief exposure. Because fish are exposed continuously to pulp mill effluents, we have tested, and rejected, the null hypothesis that continuous exposure of fish to retene does not cause sustained MFO induction. Rainbow trout exposed continuously to retene, a component of some pulp mill effluents and sediments downstream of pulp mills, showed concentration‐dependent increases in hepatic ethoxyresorufin‐O‐deethylase (EROD) activity. The increase in EROD activity was sustained over 32 d of continuous exposure, but it diminished to background levels within 4 d after transfer to clean water. The enzymatic response was confirmed by measuring changes in the content of immunodetectable cytochrome P4501A (CYP1A) protein. These data support a role for labile, nonhalogenated compounds in chronic effects of pulp mill effluents on fish.

Identification of the lampricide 3‐trifluoromethyl‐4‐nitrophenol as an agonist for the rainbow trout estrogen receptor

AbstractLampricide formulations containing 3 ‐trifluoromethyl‐4‐nitrophenol (TFM) have been associated with induction of hepatic mixed function oxygenase (MFO) activity and altered levels of circulating steroids in fish. Bioassay‐directed chemical fractionations have shown MFO induction to be associated with impurities in TFM field formulations. A similar toxicity identification/evaluation approach coupled to competitive binding to rainbow trout estrogen receptors (ER) was used to identify compounds associated with estrogenic responses in fish. In contrast to MFO induction, nearly all binding to the ER was associated with fractions containing TFM, TFM isomers, andp‐chlorophenol.p‐Chlorophenol did not show any affinity for the receptor when tested separately. Diphenyl ether impurities identified in the fractions causing MFO induction as well as analogues to dibenzo‐p‐dioxins suspected in these fractions also showed no affinity for the receptor when tested individually. Relative to estradiol, TFM demonstrated an affinity of 5.03 × 10−5, compared to 2.47 × 10−4forp‐nonylphenol, a reported estradiol agonist. Vitellogenin induction in primary cultures of rainbow trout hepatocytes indicated that TFM acts as an estradiol agonist.

Source and identity of compounds in a thermomechanical pulp mill effluent inducing hepatic mixed-function oxygenase activity in fish

The source and identity of two mixed-function oxygenase (MFO)-inducing substances present in the primary-treated effluent of a thermomechanical pulp (TMP) mill producing newsprint was determined. The source was pinpointed by exposing rainbow trout (Oncorhynchus mykiss) to various process effluents sampled throughout the mill. Exposure concentrations were based on the flow of these process streams in relation to the final effluent flow. Contaminated TMP steam condensates were identified as the major process source of MFO-inducing substances. Using conventional extraction and fractionation procedures, an MFO-inducing fraction was isolated. The major gas chromatographic peaks in this fraction were identified by gas chromatography/mass spectrometry as juvabione, dehydrojuvabione, and manool, all naturally occurring extractives in balsam fix (Abies balsamea). These substances were extracted and isolated from balsam fir and TMP condensates. Trout exposed to juvabione and dehydrojuvabione responded by exhibiting significant hepatic MFO inductions. No MFO induction was observed for manool. Secondary treatment in an activated sludge system effectively eliminated the MFO-inducing potential of the combined mill effluent consistent with a corresponding 90% reduction of both juvabione and dehydrojuvabione.

A comparison of the potential of primary- and secondary-treated pulp and paper mill effluents to induce mixed function oxygenase (MFO) activity in fish

Primary- and secondary-treated effluents from 13 mills representative of the kraft, chemi-thermomechanical and thermomechanical pulping sectors were tested for their potential to induce mixed function oxygenase (MFO) enzyme activity in rainbow trout ( Oncorhynchus mykiss). Fish were exposed in the laboratory to 1, 5 and 10% (v/v) concentrations of effluents collected at the inlet and the outfall of secondary treatment plants. After 4 days of exposure, hepatic MFO enzyme activities were measured by the ethoxyresorufin-O-deethylase (EROD) assay. Primary-treated effluent from chemi-thermomechanical, thermomechanical and bleached kraft mills showed potential for MFO induction in fish at concentrations of 10% or lower. The MFO-inducing potential of these effluents could not be linked to any specific operating variable such as type of pulping, wood furnish and pulp bleaching or brightening. The ability of the secondary-treated effluents to cause MFO induction was significantly lower or absent at the concentrations tested.

MFO induction in fish by spent cooking liquors from kraft pulp mills

AbstractWithin bleached kraft mills, the main sources of compounds inducing the mixed function oxygenase (MFO) activities of fish are waste cooking liquor, black liquor, and effluents from the first stages of the bleaching process. We measured the potency for MFO induction in rainbow trout (Oncorhynchus mykiss) of black liquors from pulp mills using kraft pulping and either softwood or hardwood furnish. Concentrations causing induction ranged from 0.0032 to 0.2%(v/v), and below critical thresholds, concentration‐response relationships were linear. Above threshold concentrations, activity declined, and lethality occurred at concentrations greater than 0.1% (v/v) black liquor during 4‐d exposures. Hardwood pulping generated black liquor that was less potent than that from softwood, but much of the difference was due to a higher water content; hardwood black liquor was more dilute as indicated by normalizing results to dissolved organic carbon. Compounds inducing MFO activity may be natural wood extractives associated with wood resins, or compounds created by the digestion of lignin. A high potency of black liquor from alcohol pulping supports the hypothesis of a natural compound because alcohol extraction does not chemically alter lignin to the same extent as kraft pulping; inducing compounds can also be extracted from wood chips with dichloromethane. The high potency of black liquor compared to bleaching or extraction‐stage effluents suggests that control of black liquor losses may be very important in limiting exposure of fish to inducing compounds.

Mixed Function Oxygenase Induction in Pre- and Post-Spawn Herring ( Clupea pallasi) by Petroleum Hydrocarbons

We compared the uptake of water-accommodated fractions of Alaska North Slope crude oil in pre- and post-spawn herring ( Clupea pallasi) and the resulting induction of mixed function oxygenase activity. Both groups of herring accumulated significant quantities of polycyclic aromatic hydrocarbons in muscle tissue. Pre-spawn herring accumulated higher concentrations of hydrocarbons and retained them longer. Induction of mixed function oxygenase activity in pre-spawn fish was one-third to one-half that in post-spawn fish. Inability of pre-spawn herring to induce mixed function oxygenases at as high a level as post-spawn fish may explain higher tissue concentrations and retention of hydrocarbons in the pre-spawn animals.

環境 ＩＶ 紙パルプ排水の生理学的な生物影響の評価方法について

MFOs (mixed function oxygenases) are a family of enzymes induced when organisms are exposed to a variety of xenobiotics. MFO assay has been utilized to evaluate the toxic potency of mill effluents. It has also been demonstrated that, particularly, pulp and paper mill effluents induce MFOs of various organisms.We have recently developed a method to detect the induction of MFOs using chick embryos as a test organism. In this report, we describe the effect of total effluents of pulp and paper mills in Japan on the MFOs induction. The mill effluents were directly injected into chick embryos. The chick embryos were incubated for 48 hours, after which microsomal fractions of liver were prepared for the EROD (7-ethoxyresorufin-O-deethylase) assay.Pulp and paper mill effluents induced the MFOs up to 1.6-fold compared with control embryos. Similarly, approximately 1.7-fold induction was observed for the EROD activity in carps exposed to effluents at mill sites. These values are significantly lower than those obtained in other countries, suggesting that the pulp and paper mill effluents in Japan may not contain a significant amount of toxic chemicals or the inducers for the MFOs.

A bleached-kraft mill effluent fraction causing induction of a fish mixed-function oxygenase enzyme

Pulp mill effluents contain a myriad of chemicals that have the potential to cause deleterious effects on aquatic biota in receiving waters. Some of these chemicals evoke an acute lethal response of exposed biota while others evoke sublethal responses. One such sublethal response is the induction of mixed-function oxygenases (MFO) in fish, specifically the CYP1A1 enzyme ethoxy-resorufin-o-deethylase (EROD). Compounds causing MFO induction include congeners of polychlorinated biphenyls (PCBs), dioxins, furans, and polycyclic aromatic hydrocarbons (PAHs). The authors followed the partitioning of the inducing chemicals in pulp mill effluent fractions by Toxicity Identification Evaluation (TIE), or bioassay-driven chemical analysis. This procedure was eventually modified to a more direct technique involving centrifugation, filtration, cleanup procedures, and C{sub 18} solid-phase adsorption. The extracts from the fractionation of two pulp mill effluents after secondary treatment were tested for EROD-inducing activity in a 4-d rainbow trout bioassay. The methanol extracts of particulates/colloids showed significant inducing capacity in Mill A effluent but not in Mill B effluent. The C{sub 18} methanol extracts induced activity from both effluents, with extracts from Mill A causing the greatest response. The particulate/colloidal extract (Mill A) was used as the source material for chemicals which caused EROD induction. The fraction wasmore » purified by solid-phase extraction techniques and reverse-phase high-performance liquid chromatography. The majority of the EROD activity was found in the moderately nonpolar region of the chromatogram (K{sub ow} = 4.6 to 5.1).« less

Measuring the potency of pulp mill effluents for induction of hepatic mixed-function oxygenase activity in fish.

A bioassay protocol was optimized for measuring the potency of effluents or waterborne chemicals for inducing mixed-function oxygenase (MFO) activity of rainbow trout (Oncorhynchus mykiss). Measurements of ethoxyresorufin O-deethylase (EROD) can be made with an established endpoint assay using large volumes of reagents and tissue. However, a new kinetic microplate assay offers significant savings in time, reagents, and sample volumes. Data are distributed lognormally and must be log transformed before statistical analyses. EROD activity increases with exposure time to pulp mill effluent, and a 4-d exposure provides a near-maximal response. Optimum fish size conforms to standard practices in fish toxicology; loading rates should not exceed 1 g of fish per liter of test solution per day. Feed should be withheld from test fish 48 h before testing to reduce the variance of measured activity, and anaesthetizing fish with MS-222 does not affect their response to MFO inducers. Pulp mill effluents do not lose their potency during 2-3 wk of exposure at temperatures ranging from -20 to 13 degrees C, whether stored in plastic or glass. Steel containers were associated with slight losses in potency. Bioassays of MFO induction in fish exposed to liquid effluents are practical and conform to standard practice for testing the lethality of waterbone chemicals. The results are sufficiently precise that differences among means based on live fish per treatment can be discriminated statistically when activity changes by threefold or more.