An automated overlying water-renewal system for sediment toxicity studies

An automated water-renewal toxicity test system is described for exposing benthic invertebrates to whole sediments. The system will intermittently deliver laboratory or on-site water for overlying water replacement in sediment exposures. A range of cycle rates can be used to produce different volume additions of overlying water per day to exposure chambers. The system can be used with six different treatments and eight replicates per treatment producing 48 exposure chambers. Three formulated sediments with variable organic carbon (1.5%, 7.5%) and sand (14%, 63%) content were prepared to test the system exposing amphipods, Hyalella azteca and midges, Chironomus tentans in 10 day whole sediment tests. Intermittent water flow was used with a 90 min cycle time to create two volume additions of laboratory water per 24 h in exposure chambers (180 ml sediment, 320 ml water). Overlying water quality conditions, and survival and growth of both species were consistent and within acceptable limits for the testing requirements of the U.S. EPA guidelines for sediments with freshwater invertebrates.     

Influence of storage time on toxicity of freshwater sediments to benthic macroinvertebrates

Guidance concerning recommended storage times for sediments to be used in toxicity tests generally has not been based upon systematically collected experimental data. The objective of this study was to better define the effects of storage time on toxicity of a series of freshwater sediments. Sixteen sediments with varying types of contaminants were collected, homogenized and stored at 4 degrees C in 1 liter aliquots, which were periodically tested for toxicity to the amphipod Hyalella azteca and the midge Chironomus tentans after storage times of up to 101 weeks. The sediments ranged from non-toxic to extremely toxic (100% mortality) in 10-day assays, with several of the samples displaying an intermediate degree of toxicity (e.g. partial mortality, reduced growth). Biological responses in most of the samples did not vary with time relative to their statistical relationship to control values; samples identified initially as toxic (or non-toxic) tended to remain toxic (or non-toxic) regardless of when they were tested. The variations that were observed in biological responses over time generally were not systematic; that is, there were no apparent trends in samples becoming more (or less) toxic in the 10-day assays. This suggests that the source of at least some of the temporal changes in toxicity were due to inherent biological variability of the assays used to assess the sediments, rather than the effects of storage. In C. tentans tests with the least toxic sediments, among-replicate variability tended to be greater in initial assays than in tests with samples that had been stored for some period of time. This may have been due to the presence of indigenous competitive or predatory organisms that did not survive during prolonged storage.     

Direct comparison of amphipod sensitivities to dredged sediments from Spanish ports

The sensitivity of the benthic amphipod species Ampelisca brevicornis and Corophium volutator to dredged sediments was compared through simultaneous testing on the standard 10 days sediment toxicity test. The results of mortality obtained for 22 harbor sediments sampled at several Spanish ports were studied together with the physico-chemical characteristics of the samples to obtain the incidence of toxicity in terms of dredged material categories and to identify possible differences in the amphipod mortality results when using one or another test species. The results showed a similar incidence of toxicity for medium-high and highly contaminated sediments for both amphipod species, similar to that obtained through the comparison of the chemical concentrations measured in sediments with the single limit values used in Spain for dredged material characterization and management. On the contrary, C. volutator presented a higher mortality and a higher incidence of toxicity when exposed to low and medium-low contaminated sediments, which may have been caused by the lower sensitivity of A. brevicornis when exposed to sediments from its natural environment. When compared to other amphipod species used for whole sediment toxicity assessment, both amphipod species used in this study reported slightly higher sensitivities although these differences could have been associated to the different set of chemical compounds considered when characterizing the sediment samples. In this sense, the amphipod mortality results were better predicted through the use of mean quotients than just by comparing the measured chemical concentrations with the single limit values used in Spain, which indicates that the toxic response of both species was caused by the cocktail of contaminants present in the sediments. Finally, the correlation analysis identified a higher association between A. brevicornis mortality and the metallic contaminants while C. volutator was more correlated with the organic micro-pollutants. Despite these differences, the results indicate that Ampelisca brevicornis can be used as test organism for dredged material characterization when enough individuals of other recommended species such as Corophium volutator are not available.     

Identifying primary stressors impacting macroinvertebrates in the Salinas River (California, USA): relative effects of pesticides and suspended particles

Laboratory dose-response experiments with organophosphate and pyrethroid pesticides, and dose-response experiments with increasing particle loads were used to determine which of these stressors were likely responsible for the toxicity and macroinvertebrate impacts previously observed in the Salinas River. Experiments were conducted with the amphipod Hyalella azteca, the baetid mayfly Procloeon sp., and the midge Chironomus dilutus (Shobanov, formerly Chironomus tentans). The results indicate the primary stressor impacting H. azteca was pesticides, including chlorpyrifos and permethrin. The mayfly Procloeon sp. was sensitive to chlorpyrifos and permethrin within the range of concentrations of these pesticides measured in the river. Chironomus dilutus were sensitive to chlorpyrifos within the ranges of concentrations measured in the river. None of the species tested were affected by turbidity as high as 1000 NTUs. The current study shows that pesticides are more important acute stressors of macroinvertebrates than suspended sediments in the Salinas River.     

In situ exposures using caged organisms: a multi-compartment approach to detect aquatic toxicity and bioaccumulation

An in situ toxicity and bioaccumulation assessment approach is described to assess stressor exposure and effects in surface waters (low and high flow), the sediment-water interface, surficial sediments and pore waters (including groundwater upwellings). This approach can be used for exposing species, representing major functional and taxonomic groups. Pimephales promelas, Daphnia magna, Ceriodaphnia dubia, Hyalella azteca, Hyalella sp., Chironomus tentans, Lumbriculus variegatus, Hydra attenuatta, Hexagenia sp. and Baetis tibialis were successfully used to measure effects on survival, growth, feeding, and/or uptake. Stressors identified included chemical toxicants, suspended solids, photo-induced toxicity, indigenous predators, and flow. Responses varied between laboratory and in situ exposures in many cases and were attributed to differing exposure dynamics and sample-processing artifacts. These in situ exposure approaches provide unique assessment information that is complementary to traditional laboratory-based toxicity and bioaccumulation testing and reduce the uncertainties of extrapolating from the laboratory to field responses.     

Cyst-based toxicity tests XVI--sensitivity comparison of the solid phase Heterocypris incongruens microbiotest with the Hyalella azteca and Chironomus riparius contact assays on freshwater sediments from Peninsula Harbour (Ontario,Canada)

In view of the complexity and costs of "traditional" whole sediment assays, a "culture/maintenance free" direct contact microbiotest has been developed with the freshwater ostracod Heterocypris incongruens. The new Toxkit assay (named Ostracodtoxkit) has been applied to 33 sediment samples from Peninsula Harbour, located in Lake Superior of the Great Lakes water basin in Ontario, Canada. The microbiotest was applied in parallel to direct contact tests with the amphipod Hyalella azteca and the midge larva Chironomus riparius, to compare its relative sensitivity with that of the two "conventional" assays. The study was undertaken in the framework of remediation action plans for specific areas of concern, to enable decision making by the Canadian authorities for the restoration of impacted aquatic environments. Most sediments were found non-toxic (<20% mortality) to both the conventional test species and the ostracod. For the large majority of samples, a very good correspondence was found between the two crustacean test species for the intensity of the toxic signal "mortality", as reflected by a 0.71 (p<0.05) correlation coefficient. Growth inhibition, which is determined in the ostracod microbiotest as a sublethal effect criterion, allowed the earmarking of some sediment samples, which were apparently more toxic for the amphipod than to the ostracod. For 20% of the samples, substantially higher mortality scores were noted with the ostracod assay than with the midge larvae tests and the overall correlation coefficient between these two tests was lower (r=0.60,p<0.05). The results obtained in the present study corroborate those of previous research on sediments collected from various rivers in Flanders, Belgium, and confirm the potential of the new ostracod microbiotest as a reliable and sensitive low cost alternative for traditional whole sediment assays.     

Sediment toxicity testing using large water-sediment ratios: an alternative to water renewal

Deterioration of overlying water quality during toxicity tests with benthic invertebrates is a serious problem with some sediments. One solution is periodic renewal of overlying water. However, this is either labour intensive or requires construction and maintenance of special equipment. Furthermore, water renewal has the potential for flushing toxic chemicals out of the test chamber and establishes nonequilibrium conditions between the water and sediment. An alternative is testing under static conditions using atypical test vessels (e.g. Imhoff settling cones) with a large water volume (1 l) overlaying a much smaller sediment volume (e.g. 15 ml). This results in dramatic improvement of overlying water quality compared to standard static toxicity tests. Compared to water renewal, the test method is much simpler, all toxic substances leached from the sediment are retained in the test vessel, and contaminant concentrations in water and sediment have more time to equilibrate. Chronic sediment toxicity tests (10-28 days) have been conducted successfully under these conditions with Chironomus riparius, Hexagenia sp., Hyalella azteca and Tubifex tubifex.     

Sediment toxicity and stormwater runoff in a contaminated receiving system: consideration of different bioassays in the laboratory and field.

Several field and laboratory assays were employed below an urban storm sewer outfall to define the relationship between stormwater runoff and contaminant effects. Specifically, two bioassays that measure feeding rate as a toxicological endpoint were employed in the field and in the laboratory, along with bioassays measuring survival and growth of test organisms. In 7 to 10 d in situ exposures, amphipod leaf disc processing, growth and survival were monitored. Different exposure scenarios were investigated by varying the mesh size (74 microns or 250 microns mesh) and method of deployment (water column, sediment surface, or containing sediment) of in situ exposure chambers. Hyalella azteca, Daphnia magna, and Pimephales promelas survival were monitored in 48 h in situ exposures. Feeding inhibition was investigated via enzyme inhibition of H. azteca and D. magna and via leaf disc processing measurements of the detritivore H. azteca. Additionally, we investigated the extent of phototoxicity at this site via field exposures in sun and shade and laboratory exposures with and without UV light. The measurement of detritivore leaf disc processing, and thus its usefulness as an endpoint, was hindered by individual variability in the amount of leaf consumed and by leaf weight gain during the summer field exposures. For D. magna, enzyme inhibition measured in a laboratory exposure did not reveal the toxicity observed in field exposures. For H. azteca, enzyme inhibition measured in the laboratory indicated toxicity similar to that observed in short term chronic in situ exposures. Enzyme inhibition also did not detect differences in toxicity due to variations in flow conditions. There were no statistically significant effects of any exposure on P. promelas survival or H. azteca growth, and there were no statistically significant effects due to mesh size or sun exposure. Survival of H. azteca was the most sensitive and the least variable endpoint. Effects on survival were noted in the same treatments over short-term chronic exposures in the laboratory and in situ. Significant differences in survival were noted due to the method of deployment under low flow conditions. In situ chambers containing sediment resulted in greater mortality in the 10 d low flow in situ experiments. Under high flow conditions, significant reductions in survival and leaf disc processing were noted under all methods of deployment at the two impacted sites over a 7 d exposure. Also under high flow conditions, significantly greater mortality of H. azteca was reported at the downstream field site when sediment was included in the chamber at deployment. These results suggest that significant toxicity at this site is due to accumulation of contaminants in the sediment and the mobilization of these contaminants during a storm event. In situ exposures detected toxicity not observed in laboratory exposures. These results suggest that a combination of laboratory and field bioassays is most useful in defining field effects.     

Integrated assessment of the impacts of agricultural drainwater in the Salinas River (California,USA)

The Salinas River is the largest of the three rivers that drain into the Monterey Bay National Marine Sanctuary in central California. Large areas of this watershed are cultivated year-round in row crops and previous laboratory studies have demonstrated that acute toxicity of agricultural drainwater to Ceriodaphnia dubia is caused by the organophosphate (OP) pesticides chlorpyrifos and diazinon. In the current study, we used a combination of ecotoxicologic tools to investigate incidence of chemical contamination and toxicity in waters and sediments in the river downstream of a previously uncharacterized agricultural drainage creek system. Water column toxicity was investigated using a cladoceran C. dubia while sediment toxicity was investigated using an amphipod Hyalella azteca. Ecological impacts of drainwater were investigated using bioassessments of macroinvertebrate community structure. The results indicated that Salinas River water downstream of the agricultural drain is acutely toxic to Ceriodaphnia, and toxicity to this species was highly correlated with combined toxic units (TUs) of chlorpyrifos and diazinon. Laboratory tests were used to demonstrate that sediments in this system were acutely toxic to H. azteca, which is a resident genus. Macroinvertebrate community structure was moderately impacted downstream of the agricultural drain input. While the lowest macroinvertebrate abundances were measured at the station demonstrating the greatest water column and sediment toxicity and the highest concentrations of pesticides, macroinvertebrate metrics were more significantly correlated with bank vegetation cover than any other variable. Results of this study suggest that pesticide pollution is the likely cause of laboratory-measured toxicity in the Salinas River samples and that this factor may interact with other factors to impact the macroinvertebrate community in the system.     

Photo-induced toxicity of PAHs to Hyalella azteca and Chironomus tentans: effects of mixtures and behavior

In the aquatic environment, polycyclic aromatic hydrocarbon (PAH) contamination can result from several anthropogenic sources such as petroleum runoff, industrial processes, and petroleum spills. When ultraviolet light (UV) is present at sufficient intensity, the acute toxicity of some PAHs to aquatic biota is greatly enhanced. This photo-induced toxicity of PAHs is directly influenced by the amount of PAH and by the level of UV intensity present in the aquatic environment. Thus, behavioral responses and habits that affect an aquatic organism's exposure to UV as well as exposure to PAHs can influence the extent to which damage due to photo-induced toxicity occurs. Experiments demonstrated the effects of photo-induced toxicity of anthracene and fluoranthene on the survival of two benthic macroinvertebrates, the midge Chironomus tentans and the freshwater amphipod Hyalella azteca. This study further investigated the survival and behavior of the test organisms in different substrates (no substrate, a sand monolayer, leaf discs, and sediment) with and without UV. The free-swimming, epibenthic H. azteca avoided the effects of photo-induced toxicity of PAHs to some extent by hiding in leaves when this substrate was available. Results emphasize the importance of organisms' behavior in affecting the photo-induced toxicity of PAHs in the aquatic environment.     

Nickel partitioning in formulated and natural freshwater sediments

A natural, field-collected sediment high in organic carbon (OC) and low in acid-volatile sulfide (AVS) was used to evaluate nickel (Ni) complexation to organic matter (OM) over a range of pH under anoxic conditions. It was found that OM strongly influenced Ni partitioning and that Ni complexation to OM was significantly influenced by pH, with complexation increasing with increasing pH (from pH 6 to 8). Using an equilibrium partitioning approach incorporating both [SEM(Ni)]-[AVS] and OC content, lethal and non-lethal toxicity test endpoints were calculated (predicted) and compared to observed toxicity test results using the amphipod, Hyalella azteca, exposed to four Ni-spiked natural sediments varying in OC and AVS content. Generally, lethal and non-lethal toxicity test endpoints were reasonably predictable in low AVS sediments. Due to the apparent lack of equilibrium between dissolved pore-water Ni and the pure Ni sulfide (likely the result of additional dissolved metal binding ligands), and the possible competition of liberated Fe2+ with Ni2+ for binding sites on organic matter, toxicity predictions (based on sediment OC and AVS content) overestimated the combined protective effects of AVS and OC in the sediments containing mid to high (27.87-44.05 micromol/g d.w.) AVS concentrations. Overall, it was found that equilibrium partitioning-based sediment quality guidelines can be improved through the incorporation of nickel complexation to sedimentary OM (in addition to AVS), although further research is required to fully describe nickel-OM interaction.     

Waterborne and sediment toxicity of fluoxetine to select organisms

Ecological risk assessments of pharmaceuticals are currently difficult because little-to-no aquatic hazard and exposure information exists in the peer-reviewed literature for most therapeutics. Recently several studies have identified fluoxetine, a widely prescribed antidepressant, in municipal effluents. To evaluate the potential aquatic toxicity of fluoxetine, single species laboratory toxicity tests were performed to assess hazard to aquatic biota. Average LC(50) values for Ceriodaphnia dubia, Daphnia magna, and Pimephales promelas were 0.756 (234 microg/l), 2.65 (820 microg/l), and 2.28 microM (705 microg/l), respectively. Pseudokirchneriella subcapitata growth and C. dubia fecundity were decreased by 0.044 (14 microg/l) and 0.72 microM (223 microg/l) fluoxetine treatments, respectively. Oryias latipes survival was not affected by fluoxteine exposure up to a concentration of 28.9 microM (8.9 mg/l). An LC(50) of 15.2 mg/kg was estimated for Chironomus tentans. Hyalella azteca survival was not affected up to 43 mg/kg fluoxetine sediment exposure. Growth lowest observed effect concentrations for C. tentans and H. azteca were 1.3 and 5.6 mg/kg, respectively. Our findings indicate that lowest measured fluoxetine effect levels are an order of magnitude higher than highest reported municipal effluent concentrations.     

Metal bioavailability and toxicity through a sediment core.

Sediment cores from Richard Lake near Sudbury, Ontario, were sectioned and analyzed for total metal content, plus metal bioavailability and toxicity to Hyalella azteca (after equilibration with oxygenated overlying water). Strong and similar sediment profiles were observed for Cd, Co, Cu and Ni in the sediment. However, these differed from metal bioavailability profiles (bioaccumulation by Hyalella and metals in overlying water). Bioavailability profiles for Cu also differed from those for Cd, Co or Ni. The deepest sediment layers, deposited prior to industrial development, were non-toxic. Sediment toxicity was attributed to Ni dissolution into overlying water. Moreover, differential bioavailability of Ni in surface and deeper sediment layers was observed. This can affect the interpretation of toxicity data for sediments collected by different methods (e.g. core vs. grab samples). Based on Pb-210 dating and trends in Ni in the core, chronic toxicity of surface sediments from Richard Lake might approach non-toxic levels in about 15 years.     

Toxicity of copper in an oxic stream sediment receiving aquaculture effluent.

Sediments were collected from a stream (upstream, outfall and downstream) receiving copper laden catfish pond effluent to assess toxicity to non-target biota. No significant reduction in Hyalella azteca survival or growth (10 d), or Typha latifolia germination and root and shoot growth (7 d) were observed after exposure to upstream and outfall sediments. A significant reduction in H. azteca survival was observed after exposure to the downstream sediment sample; however, no reduction in T. latifolia germination or seedling growth was detected. Bulk sediment copper concentrations in the upstream, outfall and downstream samples were 29, 31, and 25 mg Cu/kg dry weight, respectively. Interstitial water (IW) concentrations ranged from 0.053 to 0.14 mg Cu/l with 10 d IW toxicity units > or = 0.7. Outfall samples were amended with additional concentrations of copper sulfate so that bulk sediment measured concentrations in the amended samples were 172, 663, 1245, and 1515 mg Cu/kg dry weight. Survival was the most sensitive endpoint examined with respect to H. azteca with a no observed effects concentration (NOEC) and lowest observed effects concentration (LOEC) of 1245 and 1515 mg Cu/kg, respectively. NOEC and LOEC for T. latifolia root growth were 663 and 1245 mg Cu/kg, respectively. IW copper concentrations were > or = 0.86 mg Cu/l with H. azteca intersitial water toxicity unit (IWTU) concentrations > or = 1.2. Sequential extraction qualitatively revealed the carbonate and iron oxide fractions which accounted for a majority of the copper binding. In this instance, the copper which was applied to catfish ponds does not appear to be adversely impacting the receiving stream system.     

Applying adult emergence as an endpoint in a post-exposure laboratory test using two midge species (Diptera: Chironomidae)

Several approaches have been used to evaluate biological impairment in aquatic ecosystems which can be categorized as either laboratory or field. In the recent years, the laboratory toxicity test approach has been extended to field exposures where ambient factors are allowed to influence the test response. Field exposures of laboratory test organisms require method modifications. In this paper, a novel in situ method is described which measures growth, survival and emergence of sediment inhabiting insects (Diptera: Chironomidae) that are used in standardized laboratory toxicity testing. Two standard chironomid species (Chironomus riparius and Chironomus tentans) were used to test the suitability of the approach and to compare the performance of the species. The larvae were transferred to the laboratory for emergence after 7 days in situ exposure which was compared to laboratory responses. Growth, survival and emergence were significantly lower in the in situ pre-exposure than in the laboratory. Also, emergence success was significantly lower in one reference sediment (LMR) than in the other test sediments in both in situ and the laboratory treatment. These lower response levels likely resulted from sediment characteristics and artifacts related to the exposure in the in situ chamber. Feeding and water quality within the exposure chamber appear to be factors that may differ markedly from the laboratory exposure and may affect organism responses. C. riparius developed (growth, emergence time) faster than C. tentans in all treatments, otherwise the species responded similarly. C. riparius may be a better alternative for the chronic in situ exposures because of shorter exposure times and reduced feeding requirements.     

Effects of low dissolved oxygen on organisms used in freshwater sediment toxicity tests

Minimum dissolved oxygen requirements are part of standard guidelines for toxicity testing of freshwater sediments with several benthic invertebrates, but the data underlying these requirements are somewhat sparse. We exposed three common test organisms to ranges of dissolved oxygen concentrations to determine their responses in 10-d exposures, relative to published guidelines for sediment toxicity tests. The oligochaete, Lumbriculus variegatus, showed 100% survival in all exposures down to the lowest concentration tested, 0.7 mg O(2)l(-1). Midge (Chironomus dilutus) larva showed a more pronounced response; while survival was less than 90% only below 1.0mg O(2)l(-1), the biomass endpoint showed EC(50), EC(20), and EC(10) values of 1.00 (0.91-1.10), 1.41 (1.16-1.71), and 1.67 (1.25-2.24) mg O(2)l(-1). The amphipod, Hyalella azteca, showed no adverse effects at concentrations as low as 2.12 mg O(2)l(-1). The combination of these data with other literature data suggest that DO minima in current North American 10-d sediment test guidelines are reasonable.     

Occurrence and potential toxicity of pyrethroids and other insecticides in bed sediments of urban streams in central Texas

Despite heavy insecticide usage in urban areas, only a few studies have investigated the impact of current-use insecticides on benthic invertebrates in urban streams. The objective of this study was to measure the presence and concentration of current-use pesticides in sediments of residential streams in central Texas. Additionally, toxicity of these sediments to Hyalella azteca was evaluated. Sediment samples were collected from several sites in urban streams over the course of a year, of which, 66% had greater than one toxic unit (TU) of insecticide. Bifenthrin was the greatest contributor accounting for 65% of the TUs, and sediment toxicity to H. azteca correlated with the magnitude of total insecticides and bifenthrin TUs. The results of this study further raise concerns over the environmental consequences posed by many current-use insecticides, especially pyrethroids, in urban settings.     

Toxicity of sediment cores from Yangtze River estuary to zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos

Toxicity evaluation is an important segment in sediment quality monitoring in order to protect aquatic organisms and human health. The purpose of this study is to assess the toxicity of sediments from three sediment cores in Yangtze River Estuary, China, using the zebrafish (Danio rerio) embryo tests. Fertilized zebrafish eggs were exposed to both whole sediments and sediment organic extracts prepared from collected sediments, in order to provide a comprehensive and realistic insight into the bioavailable toxicity potential of the sediments. As end points, development parameters (mortality, hatching rate, and abnormality) in the developing embryos were recorded during the 96-h exposure. The results showed that some samples increased mortality, inhibited the hatching of embryos, and induced morphological abnormalities. The embryonic toxicities presented serrated changes and irregular distribution with depth, which may be related to hydrodynamic effect and unstable environmental input. However, lethal and sub-lethal effects were more significant at the sub-surface sediments (10～40 cm), which indicated that the pollution is more serious in recent decades.     

Lac Dufault sediment core trace metal distribution, bioavailability and toxicity to Hyalella azteca

To determine changes in metal distribution, bioavailability and toxicity with sediment depth, two 20-cm-long replicate cores were collected from a lake historically subjected to the influence of metal mining and smelting activity. The vertical distribution of Pb, Cd and Cu in sediment was similar for all three metals, with the surface layers showing enrichment and the deeper (pre-industrial) layers showing lower concentrations. Toxicity of each sediment core section was determined in laboratory tests with the freshwater amphipod Hyalella azteca. Bioavailable metal in each sediment slice was estimated from metal concentrations in overlying water in these toxicity tests and, for Cd, also from metal bioaccumulation. The profile for Cd in tissue was comparable to Cd in sediment and overlying water, but relative Cd bioavailability from sediment increased with sediment depth. Survival increased with increasing sediment depth, suggesting that surface sediments were probably less or non-toxic before industrialization.     

Toxicity to freshwater organisms from oils and oil spill chemical treatments in laboratory microcosms

Toxicity and temporal changes in toxicity of freshwater-marsh-microcosms containing South Louisiana Crude (SLC) or diesel fuel and treated with a cleaner or dispersant, were investigated using Chironomus tentans, Daphnia pulex, and Oryzias latipes. Bioassays used microcosm water (for D. pulex and O. latipes) or soil slurry (for C. tentans) taken 1,7, 31, and 186 days after treatment. SLC was less toxic than diesel, chemical additives enhanced oil toxicity, the dispersant was more toxic than the cleaner, and toxicities were greatly reduced by day 186. Toxicities were higher in the bioassay with the benthic species than in those with the two water-column species. A separate experiment showed that C. tentans' sensitivity was intermediate to that of Tubifex tubifex and Hyallela azteca. Freshwater organisms, especially benthic invertebrates, thus appear seriously effected by oil under the worst-case-scenario of our microcosms. Moreover, the cleaner and dispersant tested were poor response options under those conditions.