Submitochondrial Particle Research Articles

Exploring the binding pocket of quinone/inhibitors in mitochondrial respiratory complex I by chemical biology approaches.

ATP: adenosine triphosphate; BODIPY: boron dipyrromethene; complex I: proton-translocating NADH-quinone oxidoreductase; DIBO: dibenzocyclooctyne; EM: electron microscopy; FeS: iron-sulfur; FMN: flavin adenine mononucleotide; LDT: ligand-directed tosylate; NADH: nicotinamide adenine dinucleotide; ROS: reactive oxygen species; SMP: submitochondrial particle; TAMRA: 6-carboxy-N,N,N',N'-tetramethylrhodamine; THF: tetrahydrofuran; TMH: transmembrane helix.

320 - Differential Protein Acetylation Mediates Mitochondrial Localization and Pentameric Aggregation of SOD2 in the Murine Heart of SOD2-Tg

Cardiac-specific overexpression of SOD2 (mitochondrial manganese superoxide dismutase, mouse model of SOD2-tg) induces supernormal and physiologically hypertrophic phenotypes in the mouse heart. However, the reductive stress imposed by SOD2 overexpression also results in protein aggregation of SOD2 pentamer and differential acetylation of SOD2 in the compartments of mitochondrial matrix, mitochondrial inner membrane, and cytosol. We studied SOD2 acetylation in the matrix, submitochondrial particle (SMP), and cytosol from the SOD2-tg mouse heart. LC/MS/MS analysis revealed that K68, K75, K89, K114, K122, K130, K132, K134, and K154 residues of the cytosolic SOD2 were acetylated. LC/MS/MS analysis further indicated that K68, K122, and K130 of SOD2 in SMP and K68, K122, K 130, K202 of matrix SOD2 were acetylated. LC/MS/MS also detected a signaling peptide, 12QLAPVLGYLGSR23, in the SMP SOD2. The results implicated that mitochondrial localization of SOD2 resulted in specific deacetylation of K75, K89, K114, K132, K134, K154 residues, and site-specific acetylation of K202 residue in the matrix. In vitro acetylation of matrix SOD2 with acetic anhydride de-aggregated pentameric SOD2, restored the profile of hyperacetylation observed in cytosolic SOD2, and decreased the enzymatic activity of matrix SOD2. As indicated by the 3D structure of SOD2, specific acetylation of K89, K134, and K154 detected in cytosolic SOD2 induces unfolding tertiary structure and breaking salt bridges for stabilizing the quaternary structure. Stable isotope labeling of both wild type and SOD2-tg matrix proteins followed by LC/MS analysis indicated significant up-regulation of stress-induced mitochondrial hsp70 (GRP75) and down-regulation of presequence protease (PREP), thus supporting increased reductive stress, compromised mitochondrial localization, and weakening correct folding of the nascent SOD2 in the SOD2-tg murine heart. In conclusion, hyperacetylation maintains the unfolded cytosolic SOD2 and mediates importation of SOD2 across the mitochondrial membrane. Activation of the matrix SOD2 is subsequently initiated by deacetylation that further induces SOD2 misfolding for pentameric aggregation.

Mitochondrial Potassium Channels in Dictyostelium Discoideum

Mitochondria are crucial not only in energy metabolism but also in regulation of cell senescence and apoptosis. The strict control of inner mitochondrial membrane permeability and selective ion transport is essential for mitochondria functioning. Potassium ions homeostasis is an important process for mitochondrial optimal functioning. Potassium channels such as ATP-regulated, large conductance calcium activated and voltage dependent channels were observed in inner mitochondrial membrane in various mammalian tissues. Recently, we have identified potassium channels in inner mitochondrial membrane of potato Solanum tuberosum and Acanthamoeba castellanii. Currently we characterize mitochondrial potassium channels from one of Dictyostelium species. It is commonly used as a model organism to study cell differentiation, metabolism and programmed cell death. Preliminary experiments are focused on biophysical and pharmacological characterization of mitochondrial ion channels. Purified inner mitochondrial membranes (submitochondrial particles) were reconstituted into planar lipid bilayer. To form model membranes asolectin from soybean mixture of phospholipids was used. We observed two types of potassium selective ion channels in submitochondrial particle samples: a large- and small-conductance channels. Experiments were performed both in gradient solution 50/150 mM KCl (cis-trans) and in symmetrical solution 150/150 mM KCl at voltages from −50 to 50 mV. Regulation of the channel activity by divalent cations such as Ca2+ and Mg2+ was explored. Additionally, interaction of the ATP with mitochondrial potassium channels was characterized. The knowledge on mitochondrial ion channels may contribute to understanding molecular mechanism of Dictyostelium discoideum functioning.Thiswork was supportedby Polish Mitochondrial Network. MitoNet.pl

Effect of storage temperature on the activity of submitochondrial particles

The submitochondrial particle (SMP) assay employs processed mammalian mitchondria to assess the toxicity of chemical contaminants in aqueous solutions. Particles and associated reagents are commercially available to support two individual procedures, the electron transfer (ETr) and reverse electron transfer (RET) assays. The objective of the present study was to assess the effect of storage temperature on SMP activity. One RET and one ETr assay were conducted with sodium dodecylsulfate on each of two vials of particles stored at -20 and -80 degrees C at periodic intervals over a six-month span. Results demonstrated that SMP could remain active in either assay through six months of storage at either temperature. However, there were isolated vials of particles stored at -20 degrees C that exhibited unacceptable reductions in activity for both the ETr and the RET assays that were not related to storage duration. These results were used to develop guidance in assessing the acceptability of particle activity in SMP assays.

Toxicity of landfill leachate to sea urchin development with a focus on ammonia

Sea urchin gametes and embryos serve as a model system to evaluate toxicity in the marine environment. In this study, the toxicity of complex chemical mixtures in leachate samples to sea urchin development was examined with a focus on ammonia, which was the main contaminant of concern in most samples. Two rapid tests, the submitochondrial particle function and bacterial luminescence tests, were also used. Ammonia is highly toxic to sea urchin embryos with an EC50 of 1.3 mg l(-1) for the embryos of the Australian sea urchin Heliocidaris tuberculata. Leachate ammonia levels were well above these EC50 concentrations. To assess the contribution of ammonia to leachate toxicity in sea urchin development, we compared the predicted toxic units (PTU) and observed toxic units (OTU) for ammonia for each sample. The PTU/OTU comparison revealed that the sensitivity of the sea urchin embryos to ammonia were altered (enhanced or decreased) by other chemicals in the leachates. This result emphasises the need for parallel chemical analyses and a suite bioassays for evaluating the toxicity of complex and variable chemical mixtures.

Carrier-mediated urea transport across the mitochondrial membrane of an elasmobranch (Raja erinacea) and a teleost (Oncorhynchus mykiss) fish

In osmoregulating teleost fish, urea is a minor nitrogen excretory product, whereas in osmoconforming marine elasmobranchs it serves as the major tissue organic solute and is retained at relatively high concentrations ( approximately 400 mmol/l). We tested the hypothesis that urea transport across liver mitochondria is carrier mediated in both teleost and elasmobranch fishes. Intact liver mitochondria in rainbow trout (Oncorhynchus mykiss) demonstrated two components of urea uptake, a linear component at high concentrations and a phloretin-sensitive saturable component [Michaelis constant (K(m)) = 0.58 mmol/l; maximal velocity (V(max)) = 0.12 mumol.h(-1).mg protein(-1)] at lower urea concentrations (<5 mmol/l). Similarly, analysis of urea uptake in mitochondria from the little skate (Raja erinacea) revealed a phloretin-sensitive saturable transport (K(m) = 0.34 mmol/l; V(max) = 0.054 mumol.h(-1).mg protein(-1)) at low urea concentrations (<5 mmol/l). Surprisingly, urea transport in skate, but not trout, was sensitive to a variety of classic ionophores and respiration inhibitors, suggesting cation sensitivity. Hence, urea transport was measured in the reverse direction using submitochondrial particles in skate. Transport kinetics, inhibitor response, and pH sensitivity were very similar in skate submitochondrial particle submitochondrial particles (K(m) = 0.65 mmol/l, V(max) = 0.058 mumol.h(-1).mg protein(-1)) relative to intact mitochondria. We conclude that urea influx and efflux in skate mitochondria is dependent, in part, on a bidirectional proton-sensitive mechanism similar to bacterial urea transporters and reminiscent of their ancestral origins. Rapid equilibration of urea across the mitochondrial membrane may be vital for cell osmoregulation (elasmobranch) or nitrogen waste excretion (teleost).

Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex

The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The quinol oxidase (Q(o)) site in this complex oxidizes a hydroquinone (quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the "Rieske" iron-sulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a "Q-cycle" mechanism, which generates proton motive force for ATP synthesis. Since semiquinone intermediates of quinol oxidation are generally highly reactive, one of the key questions in this field is: how does the Q(o) site oxidize quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Q(o) site semiquinone (or quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O(2) reduction; 2) the Q(o) site semiquinone is highly stabilized making it unreactive toward oxygen; and 3) the Q(o) site catalyzes a quantum mechanically coupled two-electron/two-proton transfer without a semiquinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-cycle and superoxide production is essentially identical, consistent with model 1 but requiring modifications to models 2 and 3.

Study on the toxicity of phenolic and phenoxy herbicides using the submitochondrial particle assay

A simple and rapid in vitro toxicological assay, utilizing submitochondrial particles (SMP), has been used to evaluate the toxic effects of fifteen herbicides belonging to the phenol and phenoxyalkanoic acid chemical classes. The SMP assay allows the quantitative evaluation of the toxicity of compounds with different mechanisms of action: uncouplers, inhibitors of the enzyme complexes involved in reverse electron transfer and in oxidative phosphorylation and chemicals that alter the membrane structure. The two groups of herbicides showed different levels of toxicity. For phenol derivatives, EC 50 values ranged from 0.16 μM (ioxynil) to 6.7 μM (2,4-dinitrophenol), whereas for phenoxy herbicides EC 50 values ranged from 21 μM (2,4,5-trichlorophenoxyacetic acid, 2,4,5-T) to 110 μM (4-chloro-2-methylphenoxyacetic acid, MCPA). On the average, the toxicity of phenolic compounds is greater than that of phenoxyalkanoic acids by two orders of magnitude. Quantitative structure–activity relationships (QSAR) were developed between EC 50 values and various molecular descriptors. The results suggest the existence of different mechanisms of action for the two classes of compounds. The findings obtained for phenolic herbicides are consistent with a protonophoric uncoupling mechanism, whereas for phenoxy herbicides a non-specific mode of action at membrane level can be hypothesized.

Oxidatively damaged proteins of heart mitochondrial electron transport complexes.

Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I-V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.

Repeatability of the Submitochondrial Particle Assay

A study assessing the intralaboratory precision of the in vitro submitochondrial particle (SMP) electron transfer (ETr) and reverse electron transfer (RET) assays was undertaken using the standard reference toxicants, pentachlorophenol (PCP), sodium dodecyl sulfate (SDS), and zinc sulfate 7-hydrate (ZnSO 47H 2O). One to three trials of each assay were manually conducted daily for at least 5 days with each toxicant using commercially available sources of particles and reagents. Composite coefficients of variation (CVs) for the ETr assay ranged from 20.6% for ZnSO 4 to 29.3% for PCP (n≥15). Composite CVs for the RET assay ranged from 6.5% for SDS to 16.5% for PCP (n≥15). Comparison of intralaboratory results with in-house and published data demonstrates that the precision of both of these SMP assays is comparable to that of the more common in vivo, whole-organism bacterial, invertebrate, and fish toxicity tests.

Comparisons of human-cell-based and submitochondrial particle bioassay responses to the MEIC compounds in reference to human toxicity data

Toxicity results from submitochondrial particle (SMP) bioassays were compared to results from multiple human-cell-based assays to evaluate the SMP tests’ ability to indicate cellular toxicity. Correlation analyses between cell-based and SMP responses were conducted on a series of diverse chemicals of human toxicologic interest chosen in the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) study and suggest a high degree of ordering. The r 2 correlation coefficient obtained when comparing the log-transformed SMP results to the average cellular response for all compounds was 0.75 ( n=42). When specific mitochondrial inhibitors (to which SMP arc extremely sensitive) and toxic metals (which SMP modeled poorly) were removed, the correlation improved to 0.91 ( n=34). When the SMP assay of each individual cell-based assay was compared to the average toxic response of all the cell-based assays for these 34 compounds, the SMP r 2 was greater than the median r 2 of the cell-based assays, indicating its ability to predict cell-based toxic responses with a high degree of accuracy. Comparisons of the SMP data to the human toxicity data are similar to the cell line assays, where removal of the specific mitochondrial toxicants and metals greatly improves the relationship.

Differential effects of magnesium on the hydrolysis of ADP and ATP in human term placenta: Effect of substrates and potassium

This study evaluates the effect of Mg 2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K +, and different oxidizable substrates. Mg 2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K +, succinate and 1 mM Mg 2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg 2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg 2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM

Assessment of chloroaniline toxicity by the submitochondrial particle assay

The effects on mitochondrial respiration of 15 chloroanilines were recorded by using the in vitro response of submitochondrial particles (SMP) from beef heart mitochondria. The bioassay procedure for SMP is based on the process of reverse electron transfer, which can be negatively affected by inhibitors of electron transport, by uncouplers, and by chemicals that impair membrane integrity. The EC50 values, determined for the tested chloroanilines, indicate a general tendency of increasing toxicity with increasing chlorine substitution. In order to validate the results obtained and to evaluate the capability of the SMP assay to reproduce the toxic effects of the examined compounds on different freshwater species, the EC50 values were compared with literature data from other biological assays regarding both in vitro systems and whole organisms. A good correlation was found in particular with two widely used testing systems, the Microtox and the Tetrahymena assays. In addition, quantitative structure-activity relationships (QSARs) were established between the EC50 values and various molecular descriptors for hydrophobic, steric, and electronic interactions. The results obtained were utilized to elucidate the mechanism of toxic action of chloroanilines, which are commonly reported to act by the polar narcosis mode of action. Moreover, they confirmed that the SMP assay can be a useful tool for studying the toxicity of chemicals that act nonspecifically by impairing membrane structure and functions.

The in vitro evaluation of the toxicities of three related herbicide formulations containing ester derivatives of 2,4,5-T and 2,4-D using sub-mitochondrial particles

The aim of this investigation was to determine the contribution made by the different components of herbicide formulations to the overall toxicity of the formulations. Three related herbicide formulations were chosen. The first, Agent Orange, consisted only of the butyl esters of 2,4,5-T and 2,4-D. The second was Agent Orange diluted with diesel fuel and the third formulation tested was a tree and blackberry killer, which consisted of the butyl ester of 2,4,5-T, the ethyl ester of 2,4-D, diesel fuel and two surfactants. The potential toxic effects of these three formulations were evaluated by determining their inhibitory effects on the oxidative functions of submitochondrial particles prepared from beef heart mitochondria. The effective concentration that caused a 50% inhibition of the activities of the submitochondrial particles was determined for all three formulations. When the toxicity of the individual components of these formulations was evaluated, it was established that the so-called ‘inert’ components i.e. diesel fuel and surfactants contributed approximately 50% of the overall toxicity of the complete formulations. Hence the results confirm the importance of evaluating the toxicity of complete formulations, rather than only focussing on the active components. While cellular and sub-cellular assays cannot account for pharmacokinetic and pharmacodynamic changes that may affect the toxicity of xenobiotics, the sub-mitochondrial particle test is useful as an initial screening assay.

Groundwater toxicity assessment using bioassay, chemical, and toxicity identification evaluation analyses

Contaminated groundwater from a pesticide production facility was assessed using whole-organism and in-vitro bioassays to determine the potential for these tests to replace or augment traditional chemical analyses being employed at this site. The groundwater, which is contaminated with a variety of pesticides (organochlorine, organophosphorus, and carbamate insecticides) and pesticide adjuvants (spreading agents, emulsifiers, etc.) was sampled from an activated-carbon-based on-site remediation system before, during, and after the cleanup procedure. Toxicity was evaluated at each point using whole-organism (96-h acute Pimephales promelas, chronic 3-brood Ceriodaphnia dubia) and in-vitro [submitochondrial particle (SMP) and Microtox®] bioassays. Chemical indicator parameters [pentachlorophenol, gasoline-range organics (GROs), and benzene, toluene, ethylbenzene, and xylenes (BTEX)] were also measured to compare with bioassay responses. 50% effective concentrations (EC 50 ) s from the different tests indicate that the whole-organism assays were slightly more sensitive than the in-vitro assays: the order of sensitivity was C. dubia > P. promelas > SMP > Microtox®, but responses were quite similar. All assays showed decreasing toxicity as cleanup progressed, mimicking the concomitant decrease in chemical concentration parameters. A toxicity identification evaluation (TIE) analysis, conducted using the in-vitro bioassays, identified pentachlorophenol as the major contributor to toxicity in the samples with additional contributions likely from other less-prevalent compounds. Results suggest that these in-vitro tests can be credible alternatives to whole-organism bioassays for monitoring remediation of system effluents and that they can complement chemical analyses with useful effects-based toxicity data.

Fluoxetine interacts with the lipid bilayer of the inner membrane in isolated rat brain mitochondria, inhibiting electron transport and F1F0-ATPase activity.

The effects of fluoxetine on the oxidative phosphorylation of mitochondria isolated from rat brain and on the kinetic properties of submitochondrial particle F1F0-ATPase were evaluated. The state 3 respiration rate supported by pyruvate + malate, succinate, or ascorbate + tetramethyl-p-phenylenediamine (TMPD) was substantially decreased by fluoxetine. The IC50 for pyruvate + malate oxidation was approximately 0.15 mM and the pattern of inhibition was the typical one of the electron-transport inhibitors, in that the drug inhibited both ADP- and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-stimulated respirations and the former inhibition was not released by the uncoupler. Fluoxetine also decreased the activity of submitochondrial particle F1F0-ATPase (IC50 approximately 0.08 mM) even though K0.5 and activity of Triton X-100 solubilized enzyme were not changed substantially. As a consequence of these effects, fluoxetine decreased the rate of ATP synthesis and depressed the phosphorylation potential of mitochondria. Incubation of mitochondria or submitochondrial particles with fluoxetine under the conditions of respiration or F1F0-ATPase assays, respectively, caused a dose-dependent enhancement of 1-anilino-8-naphthalene sulfonate (ANS) fluorescence. These results show that fluoxetine indirectly and nonspecifically affects electron transport and F1F0)-ATPase activity inhibiting oxidative phosphorylation in isolated rat brain mitochondria. They suggest, in addition, that these effects are mediated by the drug interference with the physical state of lipid bilayer of inner mitochondrial membrane.

Comparison of toxicities and mechanism of action ofn‐alkanols in the submitochondrial particle and theVibrio fischeribioluminescence (Microtox®) bioassay

AbstractMany studies on the toxicity ofn‐alkanols have been conducted and quantitative structure‐activity relationships (QSARs) established comparing bioassay toxicity data to carbon number. The results typically indicate increasing toxicity with increasingn‐alkanol chain length, but often higher homologues are not assessed, where toxicity no longer increases with carbon number—a phenomenon commonly called the cutoff effect. The mode of toxic action of these compounds has been designated narcosis I; however, the specific mechanism is unknown and widely debated. This study compares results for two commonly used bioassays, the submitochondrial particle (SMP) and the Microtox® bioassay, for the homologous series ofn‐alkanols ranging from methanol (C1‐OH) to stearyl alcohol (C18‐OH). Analysis of dose‐response curve slopes indicates that the SMP assay exhibits a general mechanism of toxicity, whereas the Microtox assay exhibits this general mechanism to short‐chain alkanols, but subsequently switches to specific interaction with the higher alkanols. This specific interaction is likely competitive inhibition of the bacterial luciferase. Comparison of the toxicities in these assays with octanol/water partition coeffients (Kow), the results of whole‐organism tests, and a bacterial luciferase assay further substantiate this claim and indicate that the SMP is a better model of toxicity in whole organisms.

Comparison of in vitro submitochondrial particle and Microtox® assays for determining the toxicity of organotin compounds

AbstractThe toxicity of 14 organotin compounds was investigated by means of two short‐term in vitro bioassays, the submitochondrial particle (SMP) test and the Microtox® test. The first bioassay makes use of SMPs and is based on the effects of toxicants on reverse electron transport, which is induced by adenosine triphosphate and succinate at the first site level of the respiratory chain. Microtox is a well known test system that uses marine luminescent bacteria and quantifies toxicity by measuring the reduction of luminescence caused by toxic chemicals. Very good agreement was observed between the median effective concentration (EC50) values determined for organotin compounds by means of the two bioassays. Toxicity depended on both the number and kind of organic substituents bound to the tin atom. It followed the order triorganotins &gt; diorganotins ≈ tetraorganotins &gt; monoorganotins. Within each series, butyltin and phenyltin compounds exhibited the highest toxicity. Microtox and SMP EC50 values were successfully correlated with toxicity data for aquatic organisms, demonstrating the usefulness of these bioassays as prescreening or complementary tools for monitoring aquatic toxicity. Moreover, to investigate the suitability of the two assays in providing information on the mechanism of toxic action of organotins, EC50 values were correlated with various physicochemical and structural parameters of the tested compounds. The results obtained showed that these parameters are poor descriptors of organotin toxicity; in particular, the poor correlations found between EC50 values and log Kow could be ascribed to the fact that different modes of action govern the biological activity of mono‐, di‐, tri‐, and tetraorganotin compounds, respectively.

Use of various acute, sublethal and early life-stage tests to evaluate the toxicity of refinery effluents

The toxicities of effluents from three Ontario, Canada, refineries were assessed with microbes, plants, invertebrates, and fish. Acute toxicity was assessed by the Microtox™ test, an assay based on electron transport activity in submitochondrial particles, and Daphnia magna (water flea) and Oncorhynchus mykiss (rainbow trout, 96-h exposure) bioassays. Short-term toxic effects were measured with bioassays of growth and survival of Pimephales promelas (fathead minnow) larvae; survival and reproduction of Ceriodaphnia dubia (water flea); growth of Selenastrum capricornutum (alga); growth of Lemna minor (aquatic plant); germination of Lactuca sativa (nonaquatic plant); survival, growth, and maturation of Panagrellus redivivus (nematode); and genotoxicity in the SOS-Chromotest. Only the Microtox test and the submitochondrial particle test detected acute toxicity in the effluent samples. Reduced survival and sublethal responses were caused by some effluents, but not all effluents were toxic, and none caused a response in all of the tests applied. The results suggest that the effluent treatment systems used at Ontario refineries have largely eliminated acute toxicity to the organisms in our test battery. Although reduced survival and sublethal effects were detected in some of the effluents, the effects were minor. Some of the tests provided evidence, albeit weak, of variations in the responses of the test organisms to a temporal series of effluent samples. Not unexpectedly, there were also minor differences in the responses of the tests to effluents from the three refineries. The fathead minnow test seems to be a sensitive indicator of the sublethal toxicity of Ontario refinery effluents.

USE OF VARIOUS ACUTE, SUBLETHAL AND EARLY LIFE-STAGE TESTS TO EVALUATE THE TOXICITY OF REFINERY EFFLUENTS

The toxicities of effluents from three Ontario, Canada, refineries were assessed with microbes, plants, invertebrates, and fish. Acute toxicity was assessed by the Microtox™ test, an assay based on electron transport activity in submitochondrial particles, and Daphnia magna (water flea) and Oncorhynchus mykiss (rainbow trout, 96-h exposure) bioassays. Short-term toxic effects were measured with bioassays of growth and survival of Pimephales promelas (fathead minnow) larvae; survival and reproduction of Ceriodaphnia dubia (water flea); growth of Selenastrum capricornutum (alga); growth of Lemna minor (aquatic plant); germination of Lactuca sativa (nonaquatic plant); survival, growth, and maturation of Panagrellus redivivus (nematode); and genotoxicity in the SOS-Chromotest. Only the Microtox test and the submitochondrial particle test detected acute toxicity in the effluent samples. Reduced survival and sublethal responses were caused by some effluents, but not all effluents were toxic, and none caused a response in all of the tests applied. The results suggest that the effluent treatment systems used at Ontario refineries have largely eliminated acute toxicity to the organisms in our test battery. Although reduced survival and sublethal effects were detected in some of the effluents, the effects were minor. Some of the tests provided evidence, albeit weak, of variations in the responses of the test organisms to a temporal series of effluent samples. Not unexpectedly, there were also minor differences in the responses of the tests to effluents from the three refineries. The fathead minnow test seems to be a sensitive indicator of the sublethal toxicity of Ontario refinery effluents.