Testing for the toxicity of chemicals with Tetrahymena pyriformis

A test was developed using Tetrahymena pyriformis in order to determine the toxicity of various chemicals. Pre-cultured T. pyriformis was exposed for 24 h at 30 degrees C to various concentrations of chemicals, and the number of T. pyriformis surviving were then counted. The concentration of the chemical, at which the proliferation of T. pyriformis was restricted to one-half of the blank test (EC50), was determined. The method, applied to 57 chemicals, demonstrated that it could be used to detect the chemicals at low concentrations rapidly and with ease. The EC50 values showed a good relationship with 48 h LC50 values for Himedaka (Orizias latipes), and could be explained on the basis of the partition coefficient between water and n-octanol.     

A Mixture Toxicity Study Employing Binary Combinations Of Dinitrobenzene And Trinitrobenzene Using The Bioluminescent Marine Bacterium Vibrio Harveyi As The Test Organism

A combination mixture study was conducted with two organic compounds, dinitrobenzene and trinitrobenzene, using the marine bioluminescent bacterium Vibrio harveyi . This assay employs the photogenic properties of this organism for determination of toxicity in a direct procedure. Evaluation of toxicity was performed using an additive index methodology, mixture toxicity index and graphic representations. Graphic representations employed were isobole plot and isobologram, both isopleths. The additive index procedure employed both chemicals at their estimated median effective concentration (EC 50 ) for determination of a toxicity value. Isopleths employed combinations of both chemicals, using an equitoxic concentration, at 20% intervals of their EC 50 . An additive index value for this mixture was statistically antagonistic. Mixture toxicity index suggests that these chemical in various combinations are antagonistic. Combinations for both isopleths were similar and exhibited additivity for three mixture values and statistical synergism for one mixture. Statistical analysis of combinations using the z test found some mixtures to be statistically different. This analysis, in part, supports findings for isopleths. These results suggest that different mixtures of the two chemicals may have different interaction outcomes.     

Combined toxicity effects of MTBE and pesticides measured with Vibrio fischeri and Daphnia magna bioassays

Methyl-tert-butyl ether (MTBE), a fuel oxygenate that is added to gasoline, commonly contaminates aquatic systems, many of which are already contaminated with pesticides. The toxic effects (EC(50) value) of several pure pesticides (Diuron, Linuron, Dichlofluanid, Sea nine, Irgarol and tributyltin (TBT)) were measured and compared with the EC(50) value of the pesticide mixed with MTBE, using the Vibrio fischeri and Daphnia magna acute toxicity assays. The interaction between chemicals was evaluated in terms of the effects of mixing on the EC(50) value (i.e. the concentration (mg/L) of a compound or mixture that is required to produce a 50% change in a toxic response parameter) and the time required to generate the toxic response. Presence of MTBE enhanced the EC(50) value of several pesticides (Diuron, Dichlofluanid, TBT and Linuron) and/or the toxic response manifested more rapidly than with pure pesticides. Toxicity enhancements were quite substantial in many cases. For example, the presence of MTBE increased the toxicity of Diuron by more than 50% when tested with the V. fischeri assay (5, 15 and 30 min exposure). Also, the toxic response manifested itself within 5 min whereas without the MTBE the same response arose in 30 min. Presence of MTBE increased the toxicity of Dichlofluanid by 30% when measured with the D. magna assay. Toxicities of only two pesticides (Sea nine and Irgarol) were not raised by the presence of MTBE.     

A Daphnia magna feeding bioassay as a cost effective and ecological relevant sublethal toxicity test for Environmental Risk Assessment of toxic effluents

Environmental Risk Assessment of chemical products and effluents within EC countries require the use of cost effective standardized toxicity tests that in most cases are restricted to acute responses to high doses. Thus, subtle ecological effects are underestimated. Here we propose a short-term one day Daphnia magna feeding inhibition test as a cost effective and ecological relevant sublethal bioassay. The sensitivity and reliability of the proposed bioassay was tested in the laboratory against standardized bacteria, algae growth, D. magna and fish acute toxicity test by using 16 chemical mixture x water type combinations that included four different water types fortified with four complex chemical mixtures. Water types included ASTM hard water and three selected effluents diluted 1/10 in water to mimic worse field situations that many overexploited arid river ecosystems suffer during summer months when effluents are discharged into them with little dilution. The results obtained denoted a greater sensitivity of the proposed feeding bioassay in 51 out of 65 tests performed with an average sensitivity 50 fold greater than that of the standardized tests. The greater differences were obtained for mixtures that included narcotic chemicals and the lowest differences for those containing pesticides. Furthermore, feeding responses to the studied contaminant mixtures behaved differently to increasing TOC content than those based on bioluminescent bacteria and algae. Increasing TOC coming from sewage treated effluents decrease toxicity to the latter bioassays but increased those of D. magna feeding bioassays. These results empathize the need to include additional bioassays to monitor more accurately and realistically the toxicity of effluents or surface waters dominated by effluent discharges, a quite common situation in America and Mediterranean arid regions.     

A rapid method for determining the toxicity of chemicals to activated sludge

This paper reports the development of a method to determine the toxicity of chemicals to activated sludge. In the method, activated sludge was exposed to various concentrations of test chemicals and the inhibition of [¹⁴C]glucose uptake was measured after 15 min. Data for the decrease in glucose uptake as a function of the log of the test chemical concentration was analyzed by a nonlinear regression model to determine the concentration of chemical inhibiting uptake by 50% (IC₅₀ value). In control experiments, glucose uptake in the absence of test chemicals was rapid, specific and totally dependent on the presence of metabolically-active activated sludge. However, uptake in the presence of inorganic and organic test chemicals showed significant, dose-dependent decreases as the concentration of test material increased. Inhibition of uptake was accurately described by the nonlinear regression model, and calculated IC₅₀ values for two chemicals, mercuric chloride and 3,5-dichlorophenol, agreed well with values reported in the literature to adversely affect wastewater treatment plant operation. Based on the results of our studies, inhibition of glucose uptake proved to be a rapid, accurate and reproducible method of determining the toxicity of chemicals to activated sludge.     

Adverse effects of organic arsenical compounds towards Vibrio fischeri bacteria

The most frequently encountered forms of organic arsenic, namely, dimethylarsinic acid, monomethylarsonic acid, arsenobetaine, arsenocholine and Roxarsone (4-hydroxy-3-nitrobenzene arsonic acid) were tested for toxicity either by using the Microtox bioassay, based on the rapid (within 15 min) fading of luminescence emitted by Vibrio fischeri marine bacteria, or by monitoring growth rate of the same bacteria for 3 days. Organic arsenic was generally found to be less toxic to these biological models than inorganic arsenic. In many cases, EC50 values for DMA, MMA or HNAA when using luminescence or growth inhibition assays could not be determined because of the low toxicity of the compounds. Nevertheless, results from the luminescence inhibition assay, which was found to be more sensitive than the growth inhibition assay, allowed to rank toxicity as follows: arsenate at pH 8>HNAA at pH 5>arsenate at pH 5>MMA at pH 5>HNAA at pH 8>DMA at pH 5. Arsenobetaine and monomethylarsonic acid were unexpectedly found to stimulate bacterial growth (hormesis effect). pH was found to have a strong influence on the observed toxicity as a consequence of the pH-induced changes in the chemical speciation of the tested molecules. In most cases it appeared that negatively charged forms were less toxic than the uncharged ones.     

In vitro toxicities of experimental jet fuel system ice-inhibiting agents.

One research emphasis within the Department of Defense has been to seek the replacement of operational compounds with alternatives that pose less potential risk to human and ecological systems. Alternatives to glycol ethers, such as diethylene glycol monomethyl ether (M-DE), were investigated for use as jet fuel system ice-inhibiting agents (FSIIs). This group of chemicals includes three derivatives of 1,3-dioxolane-4-methanol (M-1, M-2, and M-3) and a 1,3-dioxane (M-27). In addition, M-DE was evaluated as a reference compound. Our approach was to implement an in vitro test battery based on primary rat hepatocyte cultures to perform initial toxicity evaluations. Hepatocytes were exposed to experimental chemicals (0, 0.001, 0.01, 0.1, 1, 10 mM dosages) for periods up to 24 h. Samples were assayed for lactate dehydrogenase (LDH) release, MTT dye reduction activity, glutathione level, and rate of protein synthesis as indicators of toxicity. Of the compounds tested, M-1, especially at the 10-mM dose, appeared to be more potent than the other chemicals, as measured by these toxicity assays. M-DE, the current FSII, elicited little response in the toxicity assays. Although some variations in toxicity were observed at the 10-mM dose, the in vitro toxicities of the chemicals tested (except for M-1) were not considerably greater than that of M-DE.     

The combined toxic effects of nonpolar narcotic chemicals to Pseudokirchneriella subcapitata

This paper presents the toxicity data of 10 nonpolar narcotic chemicals on Pseudokirchneriella subcapitata (green algae) assessed by a new algal toxicity testing technique conducted under air-tight environment. Based on DO production, median effective concentration (EC50) varies from 1.73 mg/L (1-octanol) to 8,040 mg/L (2-propanol). The endpoint of algal growth rate reveals similar sensitivity as that from DO production. Compared to literature data, Pseudokirchneriella subcapitata and Nitrosomonas are apparently more sensitive to nonpolar narcotics than other organisms such as minnow, daphnia, and Tetrahymena pyriformis. Furthermore, good correlations between toxic effects observed from Pseudokirchneriella subcapitata and other aquatic organisms were found. Hence, algal toxicity test can be considered as a surrogate test for estimating the toxicity of nonpolar chemicals to fathead minnow, Microtox, activated sludge, Daphina magna, and Tetrahymena pyriformis. The combined effects of 13 binary mixtures of nonpolar chemicals were investigated using both additive-index method and isobologram analysis. Overall speaking, the joint actions between these chemicals are strictly additive. Model analyses indicate that these compounds act on identical reaction sites or receptors, which verify that these chemicals are of the same toxicity mechanism (narcosis).     

Estimating the toxicities of organic chemicals to bioluminescent bacteria and activated sludge

Toxicity assays based on bioluminescent bacteria have several advantages including a quick response and an easily measured signal. The Shk1 assay is a procedure for wastewater toxicity testing based on the bioluminescent bacterium Shk1. Using the Shk1 assay, the toxicity of 98 organic chemicals were measured and EC50 values were obtained. Quantitative structure-activity relationship (QSAR) models based on the logarithm of the octanol-water partition coefficient (log(Kow)) were developed for individual groups of organic chemicals with different functional groups. The correlation coefficients for different groups of organic compounds varied between 0.69 and 0.99. An overall QSAR model without discriminating the functional groups, which can be used for a quick estimate of the toxicities of organic chemicals, was also developed and model predictions were compared to experimental data. The model accuracy was found to be one order of magnitude from the observed values.     

The importance of light intensity in algal tests with coloured substances

Current algal test protocols do not distinguish between chemical toxicity and growth inhibition due to physical shading when coloured substances like dyes are evaluated. To eliminate the interfering physical effect, we investigated the importance of light intensity and culture volume in the algal growth inhibition tests with Desmodesmus subspicatus and three selected dyestuffs. A photon flux density above light saturation is needed to prevent inhibition of algal growth solely due to the light absorption by dyes. Furthermore, a smaller culture volume and moderate shaking of the test flasks is advantageous in that these conditions decrease the influence of the light absorption. At 120 microE s(-1) m(-2), the shading effect of three tested dyes could be eliminated, up to a concentration of 100 mg l(-1). In a mixture with a blue dyestuff, the model toxicant potassium dichromate showed nearly the same EC50 as if applied alone, indicating that the impact of the dye was negligible. Also a small culture volume decreases the inhibition of the average growth rate of Desmodesmus. Thus, by increasing the light intensity and optionally also reducing the culture volume, this method allows a more direct measure of a potential inherent chemical toxicity of coloured substances to algae. Additionally, high light intensities and lower culture volumes result in lower coefficients of variation, which improves the sensitivity of the test for the statistical calculation of toxicity data.     

Combined effect of copper, cadmium, and lead upon Cucumis sativus growth and bioaccumulation

Cucumis sativus (cucumber) was tested to assess an ecotoxicity in soils contaminated by the heavy metals copper (Cu), cadmium (Cd), and lead (Pb) separately and in combinations. The toxicity endpoint was plant growth, which was measured as shoot and root lengths after 5 day exposure. Sum of toxic unit (TU) at 50% inhibition for the mixture (EC50mix) was calculated from the dose (TU-based)-response relationships by the Trimmed Spearman-Karber method. Binary metal combinations of Cu+Cd, Cu+Pb, and Cd+Pb produced all three types of interactions; concentration additive (EC50mix=1TU), synergistic (EC50mix<1TU), and antagonistic (EC50mix>1TU) responses. Ternary combination of Cu+Cd+Pb produced an antagonistic response for the growth of Cucumis sativus. Bioaccumulations of Cu, Cd, and Pb were observed in Cucumis sativus and the bioaccumulation of one metal was influenced by the presence of other metals in metal mixtures. In general, antagonistic and/or synergistic responses reflected bioaccumulation patterns in some binary combinations, but the patterns in mixtures were not always consistent with toxicity data. This study indicated that TU approach appears to be a good model to estimate the combined effect of metals in plant systems, and mixture toxicity may be closely-related to the bioaccumulation pattern within plants. Combined effects of mixtures have to be taken into account to ecological risk assessment.     

Algae and crustaceans as indicators of bioactivity of industrial wastes

Freshwater (Selenastrum capricornutum) and marine (Skeletonema costatum) algae were exposed to liquid wastes from 10 industrial sites in laboratory bioassays. All wastes affected algal growth, either by stimulation only or by stimulation at low concentrations and inhibition at high concentrations. Generally, S. capricornutum and Sk. costatum responded similarly to each waste: SC20's (concentration that stimulated growth by 20%) were between 0.01 and 20.0% waste; EC50's (concentration that inhibited growth by 50%), between 5.1 and 85.5% waste. Since toxicity to S. capricornutum was usually lost by the sixth or seventh day of exposure in all wastes except one, it is recommended that algal tests be carried out for 4 days. Both algal species were more sensitive to the wastes than were Daphnia magna (freshwater) and Mysidopsis bahia (marine). Only three wastes were toxic to D. magna and two were toxic to M. bahia. SC20 and EC50 values are used to calculate the 7-day, 10-year flow rate of the receiving stream required for dilution of effluents to non-toxic concentrations.     

Nonionic surfactant effects on pentachlorophenol biodegradation

Several potential mechanisms of surfactant-induced inhibition of pentachlorophenol (PCP) biodegradation were tested using a pure bacterial culture of Sphingomonas chlorophenolicum sp. Strain RA2. PCP degradation, glucose degradation, and oxygen uptake during endogenous conditions and during glucose degradation were measured for batch systems in the presence of the nonionic surfactant Tergitol NP-10 (TNP10). TNP10 did not exert toxicity on RA2 as measured by dissolved oxygen uptake rates under endogenous conditions and glucose biodegradation rates. TNPIO reduced the substrate inhibition effect of PCP at high PCP concentrations, resulting in faster PCP degradation rates at higher concentrations of TNP10. Calculations of a micelle partition coefficient (Kmic) show that PCP degradation rates in the presence of surfactant can be explained by accounting for the amount of PCP available to the cell in the aqueous solution. A model is discussed based on these results where PCP is sequestered into micelles at high TNP10 concentrations to become less available to the bacterial cell and resulting in observed inhibition. Under substrate toxicity conditions, the same mechanism serves to increase the rate of PCP biodegradation by reducing aqueous PCP concentrations to less toxic levels.     

Measured concentrations of consumer product chemicals in California house dust: Implications for sources,exposure, and toxicity potential

Household dust is a reservoir of various consumer product chemicals. Thus, characterizing comprehensive chemical profiles of house dust may help improve our understanding of residential chemical exposure. We have previously developed a method for detecting a broad spectrum of chemicals in dust by applying a combination of target, suspect screening, and non-target methods with mass spectrometry preceded by liquid chromatography and gas chromatography. Building upon a previous study that detected 271 compounds in 38 dust samples, we presented concentrations of 144 compounds that were confirmed and quantified by standards in the same set of samples. Ten compounds were measured with median concentrations greater than 10 000 ng/g of dust: cis-hexadec-6-enoic acid, squalene, cholesterol, vitamin E, bis(2-ethylhexyl) phthalate, dioctyl terephthalate, linoleic acid, tricaprylin, tris(1-chloroisopropyl) phosphate, and oxybenzone. We also reviewed in vitro toxicity screening data to identify compounds that were not previously detected in indoor dust but have potential for adverse health effects. Among 119 newly detected compounds, 13 had endocrine-disrupting potential and 7 had neurotoxic potential. Toxicity screening data were not available for eight biocides, which may adversely affect health. Our results strive to provide more comprehensive chemical profiles of house dust and identified information gaps for future health studies.     

Xenobiotic organic compounds in leachates from ten Danish MSW landfills--chemical analysis and toxicity tests

A monitoring program comprising chemical analysis and biological toxicity testing of leachate samples from 10 Danish landfills (six engineered and four uncontrolled) revealed the presence of 55 different xenobiotic organic compounds (XOCs) and 10 degradation products of XOCs. The compounds belong to the following groups: BTEX, C3-benzenes, bicyclo compounds, napthalenes, chlorinated aliphatics, phenols (chloro-, methyl-, dimethyl, nonyl-), pesticides, and phthalates. Concentrations of single XOCs ranged from <0.1 to 2220 microg/L. A pesticide screening including 101 different compounds resulted in detection of 18 pesticides and three degradation products. The findings of degradation products of toluene, phenols, phthalates, pesticides, and nonylphenol ethoxylates show that degradation occurred inside the landfills. In biotests with bacteria and algae it was found that the non-volatile organic compounds were toxic as the samples only needed to be pre-concentrated from 1.3 to 9.4 times to give 50% inhibition of the test organisms. One of the ten samples proved to be genotoxic in the umuC test after 141 times pre-concentration. A major part of the organic chemicals causing toxicity remains unknown and it is recommended to combine chemical analyses and biotests in future monitoring programs.     

A comparison of microbial bioassays for the detection of aquatic toxicants

The toxicity of 35 test chemicals was analyzed using two microbial bioassay systems. The commercially available Microtox Toxicity Analyzer System™ and the two-organism procedure of Tchan were used to determine the concentration of test chemicals resulting in a 50% reduction in response (EC₅₀). Both of the tests employed a luminescent bacterium while the procedure of Tchan also utilized an alga. Results from the two microbial tests were compared with available data obtained with fish toxicity bioassays and with each other. The Microtox™ procedure was somewhat more sensitive than the Tchan bioassay in detecting most of the test chemicals and fish bioassays were generally more sensitive than either of the microbial tests. As a notable exception, photosynthesis-inhibiting herbicides were detected at remarkably lower concentrations with the procedure of Tchan than any of the other bioassays. Potential applications for these tests are discussed.     

Toxicity assessment of common xenobiotic compounds on municipal activated sludge: comparison between respirometry and Microtox

The toxicity of four xenobiotic compounds 3,5-dichlorophenol, formaldehyde, 4-nitrophenol and dichloromethane, representative of industrial wastewater contaminants was evaluated by a simple respirometric procedure set up on the basis of OECD Method 209 and by the Microtox bioassay. Very good reproducibility was observed for both methods, the variation coefficients being in the range of 2-10% for the respirometric procedure and 6-15% for Microtox, values that can be considered very good for a biological method. Comparison of EC(50) data obtained with the two methods shows that in both cases 3,5-dichlorophenol is more toxic than other compounds investigated and dichloromethane has a very low toxicity value. Intermediate EC(50) values were found for the two other chemicals, formaldehyde and 4-nitrophenol. Moreover, the Microtox EC(50) values are generally lower (except for dichloromethane) than the respirometric ones: these differences could be explained by the fact that the Microtox method uses a pure culture of marine species and, therefore, should not necessarily be expected to behave like a community of activated sludge bacteria. In conclusion, both methods can be usefully applied for toxicity detection in wastewater treatment plants but it is advisable to take into account that Microtox is more sensitive than respirometry in estimating the acute toxicity effect on the biomass operating in the plant.     

Rapid ecotoxicological bioassay using delayed fluorescence in the green alga Pseudokirchneriella subcapitata

A rapid and simple ecotoxicological bioassay allows quick estimation of the effects of Simazine (CAT) or 3,5-dichlorophenol (3,5-DCP) on the growth of the green alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum). The effects of a 15-min exposure to CAT or 3,5-DCP on delayed fluorescence (DF) in P. subcapitata were compared to the results of standard growth inhibition tests involving 72h of exposure to these chemicals at the same concentrations. Integrated DF intensity in the time period from 0.6 to 50s was found to correlate with algal growth inhibition as measured by the standard tests. In addition, the behaviour of DF in this time period exhibited characteristic kinetics indicative of the chemical being tested.     

Toxicity and phototoxicity of water-accommodated fraction obtained from Prestige fuel oil and Marine fuel oil evaluated by marine bioassays

Acute toxicity and phototoxicity of heavy fuel oil extracted directly from the sunken tanker Prestige in comparison to a standard Marine fuel oil were evaluated by obtaining the water-accommodated fraction (WAF) and using mussel Mytilus galloprovincialis and sea urchin Paracentrotus lividus embryogenesis bioassays, and copepod Acartia tonsa and fish Cyprinodon variegatus survival bioassays. Aromatic hydrocarbon (AH) levels in WAF were measured by gas chromatography. Prestige WAF was not phototoxic, its median effective concentrations (EC50) were 13% and 10% WAF for mussel and sea urchin respectively, and maximum lethal threshold concentrations (MLTC) were 12% and 50% for copepod and fish respectively. Marine WAF resulted phototoxic for mussel bioassay. EC50s of Marine WAF were 50% for sea urchin in both treatments and 20% for mussel under illumination. Undiluted Marine WAF only caused a 20% decrease in mussel normal larvae. Similar sensitivities were found among sea urchins, mussels and copepods, whilst fish were less sensitive. Unlike Marine WAF, Prestige WAF showed EC50 values at dilutions below 20%, and its toxicity was independent of lighting conditions. The differences in toxicity between both kinds of fuel could not be explained on the basis of total AH content.