A Test Battery Approach for the Ecotoxicological Evaluation of Estuarine Sediments

The purpose of this study was to evaluate the overall sensitivity and applicability of a number of bioassays representing multiple trophic levels, for the preliminary ecotoxicological screening (Tier I) of estuarine sediments. Chemical analyses were conducted on sediments from all sampling sites to assist in interpreting results. As sediment is an inherently complex, heterogeneous geological matrix, the toxicity associated with different exposure routes (solid, porewater and elutriate phases) was also assessed. A stimulatory response was detected following exposure of some sediment phases to both the Microtox and algal bioassays. Of the bioassays and endpoints employed in this study, the algal test was the most responsive to both elutriates and porewaters. Salinity controls, which corresponded to the salinity of the neat porewater samples, were found to have significant effects on the growth of the algae. To our knowledge, this is the first report of the inclusion of a salinity control in algal toxicity tests, the results of which emphasise the importance of incorporating appropriate controls in experimental design. While differential responses were observed, the site characterised as the most polluted on the basis of chemical analysis was consistently ranked the most toxic with all test species and all test phases. In terms of identifying appropriate Tier I screening tests for sediments, this study demonstrated both the Microtox and algal bioassays to be more sensitive than the bacterial enzyme assays and the invertebrate lethality assay employing Artemia salina. The findings of this study highlight that salinity effects and geophysical properties need to be taken into account when interpreting the results of the bioassays.     

Use of protozoan communities to assess the ecotoxicological hazard of contaminated sediments

Protozoan communities developed on artificial substrates were used in a series of in situ and laboratory tests to evaluate the toxic potential of harbor sediments contaminated with polychlorinated biphenyl (PCB). Colonization dynamics in polluted and clean harbors were compared. Laboratory tests were community bioassays using standard techniques to produce sediment elutriate. Results of the in situ colonization and the community tests measuring structural changes (e.g., “decolonization”) were similar. In general, sediments from the contaminated harbor caused significant (p ? 0.05) reductions in the number of taxa, in total protozoan abundance, and in phototroph abundance; however, the abundance of heterotrophic species increased in some in situ tests. Process-level parameters (e.g., respiration; island-epicenter colonization rates) were more sensitive than measurements of community structure. Phototrophs were more sensitive to sediment elutriate than were other trophic types. The information provided by this series of protozoan community tests is more complex than that provided by single-species bioassays. Although community tests may provide more information on the effects of sediment contamination on actual ecosystems than tests based on single species, they require careful interpretation to avoid misleading conclusions.     

Toxic effects of zinc from trout farm sediments on ATP, protein, and hemoglobin concentrations of Limnodrilus hoffmeisteri

Zinc (Zn) is a nutritionally essential metal, and deficiency results in severe health consequences to aquatic organisms. In this study toxicity data for Limnodrilus hoffmeisteri produced by Zn in systems using three natural sediments (trout farms: El Oyamel, El Truchón, and El Potrero) are presented. Hemoglobin, adenosine triphosphate (ATP), and protein concentrations were measured in L. hoffmeisteri exposed to spiked sediments, as indicators of exposure. Physicochemical characteristics of water and sediments were also considered. Zn concentrations were measured in water and sediment. El Oyamel, El Truchón, and El Potrero pond sediments did not have similar physicochemical characteristics. Zn concentrations of water obtained from the rustic ponds were near 0.4575 mg/L; however, this metal was always found to be higher in the sediments (0.0271-0.9754 mg/kg). The bioassay with worms demonstrated that pond sediments from El Oyamel, El Potrero, and El Truchón produced toxicity since ATP and protein concentrations were low compared to controls (organisms without metal). All spiked sediments had a significant reduction effect on ATP, protein, and hemoglobin concentrations. This investigation clearly shows that sediments of El Truchón, El Oyamel, and El Potrero possess toxicity potential. These results suggest the usefulness of these bioassays to evaluate the toxicity of sediments polluted with heavy metals.     

Use of four microbial tests to assess the ecotoxicological hazard of contaminated sediments

Three single-species bioassays (Microtox, Selenastrum capricornutum, and Panagrellus redivivus) and a test using microbial communities developed on artificial substrates were used in a series of in situ and laboratory tests evaluating the ecotoxicological hazard of contaminated sediments at two sites on Lake Michigan: Waukegan (Illinois) Harbor and The Chicago Area Confined Disposal Facility Study. In the single-species tests, exposure to elutriates of contaminated sediments significantly inhibited bacterial luminescence, algal photosynthesis, and nematode survival and growth at polluted stations, while elutriates from control stations did not. The battery of three tests is a promising screening tool for in-place pollutants. Protozoan species richness and protozoan phototroph abundance were inhibited by elutriates from contaminated sites, but the abundance of heterotrophic protozoans was enhanced by sediment elutriates from some stations. Microbial community photosynthesis was significantly inhibited by most sediment elutriates, while community respiration was often stimulated; thus, functional responses paralleled the structural changes. Overall, the results of the microbial community tests were consistent with expected patterns of toxicity at the two sites on Lake Michigan. In general, single-species test results agreed with the community bioassays. Although community tests may be more realistic than single-species bioassays in predicting the impact of sediment contamination on actual ecosystems, caution must be exercised in interpreting the results.     

TOXIC EFFECTS OF ZINC FROM TROUT FARM SEDIMENTS ON ATP,PROTEIN, AND HEMOGLOBIN CONCENTRATIONS OF LIMNODRILUS HOFFMEISTERI

Zinc (Zn) is a nutritionally essential metal, and deficiency results in severe health consequences to aquatic organisms. In this study toxicity data for Limnodrilus hoffmeisteri produced by Zn in systems using three natural sediments (trout farms: El Oyamel, El Truchon, and El Potrero) are presented. Hemoglobin, adenosine triphosphate (ATP), and protein concentrations were measured in L. hoffmeisteri exposed to spiked sediments, as indicators of exposure. Physicochemical characteristics of water and sediments were also considered. Zn concentrations were measured in water and sediment. El Oyamel, El Truchon, and El Potrero pond sediments did not have similar physicochemical characteristics. Zn concentrations of water obtained from the rustic ponds were near 0.4575 mg/ L; however, this metal was always found to be higher in the sediments (0.0271-0.9754 mg/kg). The bioassay with worms demonstrated that pond sediments from El Oyamel, El Potrero, and El Truchon produced toxicity since ATP and protein concentrations were low compared to controls (organisms without metal). All spiked sediments had a significant reduction effect on ATP, protein, and hemoglobin concentrations. This investigation clearly shows that sediments of El Truchon, El Oyamel, and El Potrero possess toxicity potential. These results suggest the usefulness of these bioassays to evaluate the toxicity of sediments polluted with heavy metals.     

The effects of elutriates from PAH and heavy metal polluted sediments on Crassostrea gigas (Thunberg) embryogenesis,larval growth and bio-accumulation by the larvae of pollutants from sedimentary origin

The release, bio-availability and toxicity of contaminants, when sediments are resuspended have been examined, studying concurrently their effects on the embryogenesis and on the larval growth of the Crassostrea gigas larvae and their bio-accumulation in those organisms. Three characteristic sediments have been selected (one contaminated by PAHs, a second by heavy metals and the last by the both pollutants). The organisms were directly exposed to elutriates obtained from each sediment or fed on algae (Isochrysis galbana) contaminated with the same elutriates. The elutriates used in this study show contamination levels similar to those observed in some polluted coastal and estuary environments. The larval growth test has appeared to be more sensitive that the embryotoxicity test. The biological effects and the contaminant bio-accumulation were more pronounced when larvae were directly exposed to different elutriates. In the case of PAHs, the contamination of algae was sufficient to lead to effect on the larval growth of the Crassostrea gigas. In all cases, a fraction of contaminants adsorbed on suspended particles was bio-available and accumulated by the larvae. This study has shown that resuspending polluted sediments constitutes a threat to pelagic organisms and than the C. gigas larval growth may be proposed as a test to protect the most sensitive areas.     

Using an Integrated Approach to Assess the Sediment Quality of an Mediterranean Lagoon,the Bizerte Lagoon (Tunisia)

The present study investigates the quality of surface sediments from the Bizerte lagoon (North Tunisia) using an integrated approach including chemical contaminant analysis, bioassays and sediment quality guidelines (SQGs). Sediment samples were collected at 9 sites and analyzed for eight heavy metals (Hg, Cd, Cr, Cu, Pb, Zn, Ni, Fe and Mn). PAHs, PCBs, OCPs were measured previously in the same sediment samples. Our results indicated that the highest concentrations of metals were found near urban areas due to the municipial and industrial wastewater discharges. Sediment pollution assessment was carried out using geoaccumulation index and enrichment factor, which indicate a widespread pollution by Cd, Pb, Ni and Zn in the studied sediments. For bioassays, aqueous and organic extracts were used to assess toxicity and genotoxicity in sediments by using Microtox^® and SOS Chromotest, respectively. Toxicity levels were compared to metallic and organic pollutants contents. Our results highlight differences in the pattern of responses between the different assays and show no correlation with all the studied contaminants, emphasizing the influence of other contaminants not analyzed in the present study. Based on SQGs, the results of toxicity assessment indicated that adverse effects caused by Ni and Zn would be expected frequently. Nickel was found to have the highest predicted acute toxicity, followed by Zn, Pb, Cd, Cu and Cr. There was no significant relationship between sediment toxicity calculated from heavy metal concentrations (SQG approach) and those measured with bioassays. These findings support the use of integrated approachs for evaluating the environmental risks of sediments.     

A novel bioassay approach: Direct application of the toxi-chromotest and the SOS chromotest to sediments

Routine testing of sediments or suspended sediments for toxicant or genotoxicants by the bioassay route often involves time-consuming and expensive organic extraction procedures. In most instances these extraction procedures are more time-consuming and costly than the bioassays that will be used on these extracts. A direct sediment test procedure (DSTP) was developed to alleviate the problem and thus returning bioassays to one of their original roles, i.e., short, quick, screening tests to identify priority samples for more intensive chemical analysis. The DSTP was developed in conjunction with the Toxi-Chromotest and SOS Chromotest kits. The SOS Chromotest can be used with or without S9. Our results presented in this paper indicate the sensitivity of DSTP and cost effectiveness compared to some commonly used sediment extraction procedures.     

Integrated measures of ambient toxicity and fish community diversity in Chesapeake Bay tributaries

The goals of this study were to assess the results of a suite of sediment and water column bioassays in the framework of a toxicological risk ranking model and evaluate correlations of model output with fish community metrics. The test sites were located in four tributaries of Chesapeake Bay that are impacted on by industrial, urban and agricultural land use (Curtis Creek, Rock Creek, Fishing Bay and Wicomico River). The mortality, reproduction and growth rates in the water column assays indicated low-level chemical contamination impacts in Curtis Creek and Rock Creek. The results from the Wicomico River and Fishing Bay did not indicate contaminant impacts, but some borderline effects were seen. The sediment bioassays demonstrated greater toxicological responses than the water column assays. The sediments in the Curtis Creek and Rock Creek sites were contaminated with heavy metals and PAHs. The heavy metal concentrations were an order of magnitude lower in the Fishing Bay sediments and below detection in the Wicomico River sediments, except for zinc. The acid-volatile sulphides:simultaneously extractable metals (AVS:SEM) ratios were below 1 in all cases. All four systems had detectable petroleum hydrocarbon contamination. Organic contaminants were below detection for all analyses in the Wicomico River and Fishing Bay samples. The risk ranking model ranked Curtis Creek as the most toxicologically impacted site, followed by Rock Creek, Fishing Bay and Wicomico River, which were essentially equal. The diversity index for fish communities sampled by bottom trawl was significantly correlated with the toxicological risk scores for sediment. The toxicological results indicate sediment contamination effects on the deep water fish community in Curtis Creek and indicate that contaminant impacts are not likely to be a contributing factor to disturbed fish communities in Fishing Bay     

A test battery approach to the ecotoxicological evaluation of cadmium and copper employing a battery of marine bioassays

Heavy metals are ubiquitous contaminants of the marine environment and can accumulate and persist in sediments. The toxicity of metal contaminants in sediments to organisms is dependent on the bioavailability of the metals in both the water and sediment phases and the sensitivity of the organism to the metal exposure. This study investigated the effects of two metal contaminants of concern (CdCl₂ and CuCl₂) on a battery of marine bioassays employed for sediment assessment. Cadmium, a known carcinogen and widespread marine pollutant, was found to be the least toxic of the two assayed metals in all in vivo tests. However, CdCl₂ was found to be more toxic to the fish cell lines PLHC-1 and RTG-2 than CuCl₂. Tisbe battagliai was the most sensitive species to both metals and the Microtox^® and cell lines were the least sensitive (cadmium was found to be three orders of magnitude less toxic to Vibrio fischeri than to T. battagliai). The sensitivity of Tetraselmis suecica to the two metals varied greatly. Marine microalgae are among the organisms that can tolerate higher levels of cadmium. This hypothesis is demonstrated in this study where it was not possible to derive an EC₅₀ value for CdCl₂ and the marine prasinophyte, T. suecica. Conversely, CuCl₂ was observed to be highly toxic to the marine alga, EC₅₀ of 1.19 mg l^?1. The genotoxic effect of Cu on the marine phytoplankton was evaluated using the Comet assay. Copper concentrations ranging from 0.25 to 2.50 mg l^?1 were used to evaluate the effects. DNA damage was measured as percent number of comets and normal cells. There was no significant DNA damage observed at any concentration of CuCl₂ tested and no correlation with growth inhibition and genetic damage was found.     

Bioassay assessment of impacts of tar sands extraction operations

This report describes the application of ten bioassays (battery of tests approach) to waters and sediments collected from the tar sands area of northern Alberta, Canada. In this study there were three main goals: (1) to establish the presence and sources of toxicants in these northern waters and sediments, (2) to determine if their presence is related to sediment composition, and (3) to establish which of the various sediment extracting procedures used was most efficient in producing responses in the bioassays used. Results of these investigations indicated that there were two obvious sources of toxicants/genotoxicants, the tar sands extracting area and streams passing through tar sands or oil shales and at least two unknown sources. Based on the techniques used in this study, there does not appear to be a strong sediment structure–toxicant response relationship. Extraction procedure results were variable and are discussed in detail in the report. © by John Wiley & Sons, Inc.     

Comparison of the Qwiklite algal bioluminescence test with marine algal growth rate inhibition bioassays

Although marine algal bioassays based on growth rate inhibition over 72-96 h have been widely used to assess the toxicity of contaminants in waters and sediments, changes in pH over the test duration can lead to changes in contaminant speciation and consequently an under- or over-estimation of toxicity. In addition, high cell densities are used in order to obtain a detectable response, further reducing the tests' environmental relevance in marine waters. There is a need for rapid acute tests with ecologically relevant test endpoints that may be used as surrogates for longer-term chronic tests. This study compares the sensitivity and reproducibility of a rapid marine dinoflagellate (Pyrocystis lunula) bioluminescence test (QwikLite) with standard algal growth rate bioassays (Nitzschia closterium and Entomoneis c.f. punctulata) using ammonia and several antifouling agents (tributyltin [TBT], copper, and diuron) as reference toxicants. QwikLite was of similar sensitivity to ammonia as standard algal growth rate tests, but was less sensitive to copper, diuron and TBT, with 24-h EC50 values of 10 +/- 1.1 mg N/L, 0.128 +/- 0.021 mg Cu/L, 19 +/- 13 mg diuron/L, and 0.226 +/- 0.028 mg TBT/L. Inter-test precision using different batches of P. lunula was generally acceptable. On the basis of NOEC values, QwikLite was more sensitive to copper and ammonia at 25 degrees C than at 21 degrees C. QwikLite shows promise as a rapid, inexpensive screening test for acute toxicity of contaminants in marine environments.     

Interlaboratory precision study of a whole sediment toxicity test with the bioluminescent bacterium Vibrio fischeri

The reproducibility of sediment toxicity bioassays using the Microtox® solid-phase test (SPT) with the luminescent bacterium Vibrio fischeri was estimated in an interlaboratory precision study. A preliminary study, with five labs testing six solid-phase samples, was used to evaluate proposed method modifications. As a result, it was recommended that the SPT protocol be revised to include whole sample testing with subsequent wet-weight to dry-weight correction, and the use of NaNO₃, rather than NaCl, as the diluent for freshwater sediment samples. The revised protocol was then examined in a definitive precision study, with 18 laboratories each testing eight samples. Coefficients of variation for the eight samples ranged from 35.8 to 78.0%. One possible source of error is the separate moisture content determination performed by each laboratory in order to calculate the wet-weight to dry-weight correction. Out of a total of 143 bioassays performed by the 18 labs, only two results (1.4%) exceeded the critical value of the interlaboratory consistency statistic h. With coefficients of variation comparable to other interlaboratory precision studies, and an extremely low number of results exceeding the critical value of h, it is concluded that the V. fischeri SPT has an acceptable level of precision and can be developed as a standardized test method. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 339–345, 1999     

Toxicity evaluation of water samples collected near a hospital waste landfill through bioassays of genotoxicity piscine micronucleus test and comet assay in fish Astyanax and ecotoxicity Vibrio fischeri and Daphnia magna

In this study, we analyzed samples of water from a river and a lake located near a hospital waste landfill with respect to physico-chemical parameters and conducted bioassays of ecotoxicity using Vibrio fischeri and Daphnia magna, which are species commonly used to evaluate the water toxicity. We also evaluated damage to the genetic material of fish (Astyanax sp. B) that were exposed (96 h) to water from these two sites that were located near the tank ditch, using the alkaline comet assay and the piscine micronucleus test. Parameters including aluminum, manganese, biochemical oxygen demand, sulfide, conductivity, phenol, total coliforms and Escherichia coli counts, were above acceptable levels that have been established in environmental legislation. However, the toxicity bioassays that we carried out in Vibrio fischeri and Daphnia magna and the piscine micronucleus test in fish showed no immediate risk due to acute effects. Based on the results of the comet assay, however, it was possible to detect damage to genetic material in fish that were acutely exposed in the laboratory to water samples from the river and lake that are located near the trench septic tank. Thus, our results suggest that tests beyond those usually employed to test water toxicity, such as the comet assay we used in the fish, are required to assess the toxicity of water with greater accuracy.     

Battery of screening tests approach applied to sediment extracts

This paper reports the application of a battery of microbiological and toxicant screening tests to sediments collected from the Port Hope area. The sediments were extracted using Milli-Q water and solvents. The solvent extracts indicated that the whole sampling area has been impacted by toxic chemicals. The Daphnia magna test was the most sensitive screening test in Milli-Q water extracts. Microbiological studies indicated that the sampling area was, and is continuing to be, impacted by fecal pollution.     

Predicting toxicity in marine sediment in Taranto Gulf (Ionian Sea,Southern Italy) using Sediment Quality Guidelines and a battery bioassay

The goal of this study was to assess coastal marine pollution in the Mar Piccolo and Taranto Gulf (Ionian Sea, Southern Italy) by combining chemical and toxicological data in order to compare and integrate both approaches. Pollutants levels, traditionally, have limited ability to predict adverse effects on living resources. Moreover, in order to provide information on the ecological impact of sediment contamination on aquatic biota Numerical Sediment Quality Guidelines (SQGs) and sediment toxicity bioassays were carefully recommended. In this study ERL (effect range low)/ERM (effect range medium value) and TEL (threshold effect level)/PEL (probable effect level) guidelines have been used. Bioassays were performed with two species of amphipods Gammarus aequicauda and Corophium insidiosum, one species of isopod Idotea baltica and bivalve Mytilus galloprovincialis larvae. The TEL/PEL analysis suggested that, especially for stations 1 and 2, sediments in Mar Piccolo should contain acutely toxic concentrations of metals. In particular Hg content, in station 1, was about 17 times PEL value. 96 h LC₅₀ and 48 h EC₅₀ values were estimated for cadmium, copper and mercury in these species using the static acute toxicity test. M. galloprovincialis larvae was more sensitive than other species to all the reference toxicants tested (EC₅₀ determined for cadmium copper and mercury were of 0.59, 0.11 and 0.01 mg/l respectively). Significant differences in sensitivity of species tested to all reference toxicants (ANOVA p < 0.001) were recorded. Bioassays with these species allowed to estimated sediment toxicity from the different studied sites. On the basis of results obtained a good agreement was reported between chemical data and response of the biological endpoints tested.     

Ecotoxicity testing of heavy metals using methods of sediment microbiology

Current measures of microbe-mediated biogeochemical processes in sediments were examined for their potential use as indicators of heavy metal ecotoxicity in both river sediments and bacterial cultures. Assays were carried out with HgCl₂, CuSO₄, and 3CdSO₄ · 8H₂O added to sediment samples and bacterial cell suspensions at concentrations ranging from 0.1 to 10 mM and 0.1 μM to 1 mM, respectively. Chemoautotrophic CO₂ fixation by Elbe River sediment microbiota was most sensitive to Hg²⁺ and Cd²⁺, but not to Cu²⁺. Among the estimates of heterotrophic productivity, incorporation of leucine into cellular protein showed clearer dose responses than incorporation of thymidine into bacterial DNA. Thymidine incorporation was highly resistant to and even stimulated by metal ions, particularly in starved and anaerobic cultures of a test strain of Vibrio anguillarum. Similar metal ion induced “overshoot” responses beyond the levels of untreated controls were noted for mineralization of ¹⁴C-glucose by V. anguillarum and, in the case of Cd²⁺, also in sediment. As a less complex measure of microbial respiratory activity, succinate dehydrogenase (SDH) showed normal dose responses without stimulatory effects, as long as bacterial cell homogenates were assayed. Despite this result, it is concluded that levels of SDH in natural sediment microbiota are inevitably affected by metal-induced processes of selection and enzyme synthesis, and would thus fail to provide an appropriate measure of metal ecotoxicity. The final conclusion is that current parameters of microbial production and activity often reveal dose responses that do not fulfill basic requirements of ecotoxicity testing in metal-polluted sediments. © 1993 John Wiley & Sons, Inc.     

Precautions for routine use of INT-reductase activity for measuring biological activities in soil and sediments

To evaluate toxicity of xenobiotics on microflora of soils and sediments, reduction of tetrazolium dye [2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT) or 2,3,5 triphenyltetrazolium chloride (TTC)] by dehydrogenase activities is commonly used, but many questions are posed about the biological relevance of this test. In this work, INT was chosen to evaluate the influence of some experimental factors on reductase activity. INT adsorption to soil, concentration of INT, and sterilization methods were tested on different soils and sediments to determine bioavailability of INT, saturation concentration, and intensity of chemical reduction. INT adsorption shown dramatic variations vs type of soil, from 0 to 100%, and was particularly linked with cation exchange capacity. Formaldehyde seems to be the best method with irradiation to determine abiotic reduction, particularly with sediments. However, it must be stressed that the tetrazolium reduction in microbial systems can be carried out by other enzymatic systems than malate dehydrogenase. INT is an excellent substrate of membrane hydrogenases found in numerous anaerobic bacteria. This was observed with experiments conducted with Rhodobacter capsulatus in our laboratory. © by John Wiley & Sons, Inc.     

Standardization of bulb and root sample sizes for the Allium cepa test

Although the Allium cepa test has been widely used to identify potentially cytotoxic and genotoxic pollutants in aquatic environments, variable non-standardized choices have been made regarding the number of plant bulbs and roots analyzed. We propose numbers for bulbs and roots per bulb when comparing the frequencies of micronuclei, mitotic anomalies and mitotic index with this test. Roots that had been treated with aqueous solutions, such as water samples collected in August 2007 from the Paraíba do Sul River at the Brazilian cities of Tremembé and Aparecida; negative and positive controls were used for bioassays. The presence of pollutants in the river water had been presumed based on our previous cytological data and an official report by the S?o Paulo State Environmental Agency (Brazil) on presence of fecal contaminants (Tremembé and Aparecida) and elevated dissolved aluminium (Aparecida) in the water under study. The sampling of ten bulbs and five roots per bulb was found adequate for comparative studies to evaluate with the A. cepa test the potential damage inflicted by pollutants in aquatic environments. Furthermore, even one bulb and one root per bulb was sufficient in discerning this damage, thereby shortening the time required to attain a statistically confident comparative evaluation. However, to allow for the use of statistical programs based on the evaluation of average values, and to avoid criticism based on genetic variability, we propose that three bulbs and three roots per bulb be considered as standard sample sizes for the A. cepa test.     

A rapid and simple toxicity assay based on growth rate inhibition of Pseudomonas fluorescens

A simple and rapid test, based on growth rate inhibition (GRI) of Pseudomonas fluorescens, is described for toxicity screening and assessment. The test involves challenging standardized suspensions [0.2 optical density (OD) at 600 nm] of exponentially growing cells with various concentrations of a toxicant at 33°C and monitoring changes in the growth rate by OD measurements after 0.5, 1.0, 1.5, and 2.0 h. The toxicity end point is measured as the effective concentration of a test sample that causes 50% inhibition of growth rate (EC₅₀) during each designated time period. Several metal salt solutions, organic compounds, and complex effluents were screened using this test and data compared with those obtained using the Microtox® assay. Results indicated that the P. fluorescens GRI test could be used as a valuable part of a battery of short-term bioassays for toxicity screening and assessment.