Sublethal Toxicity Tests Research Articles

Toxicity of 1,4-dichlorobenzene in sediments to juvenile polychaete worms

Investigation of sediment contamination associated with a marine sewage outfall in Victoria (BC, Canada) found elevated concentrations of 1,4-dichlorobenzene (1,4-DCB). Juvenile polychaete worm ( Neanthes) growth was significantly reduced at or near the outfall, roughly corresponding to elevated 1,4-DCB concentrations. There are few data on 1,4-DCB toxicity to marine organisms and no published literature on its toxicity to benthic marine organisms. To determine whether reduced polychaete growth (measured as dry weight) was due to 1,4-DCB exposure, a laboratory investigation was conducted. Uncontaminated marine sediment was spiked with 1,4-DCB and juvenile Neanthes were exposed in 20-d sublethal toxicity tests. There were no adverse effects on survival at any test concentration; mean survival was 80–100%. Statistically significant decreases in average dry weight only occurred at the highest 1,4-DCB concentration (19,900 μg/kg, dry weight); this represented a 1,4-DCB concentration more than 10 times higher than previously measured at the outfall (1710 μg/kg, dry weight). There were no adverse effects on survival or dry weight at the range of concentrations previously measured in sediments from the vicinity of the outfall.

Swimming and ammonia toxicity in salmonids: the effect of sub lethal ammonia exposure on the swimming performance of coho salmon and the acute toxicity of ammonia in swimming and resting rainbow trout

This study tested the hypothesis that swimming exacerbates ammonia toxicity in fish. Both sub-lethal and acute toxicity testing was conducted in a swim tunnel on swimming and resting coho salmon and rainbow trout, respectively. The sub lethal tests on coho salmon also considered the compartmentalization of ammonia within the fish. Coho salmon showed a significant linear decrease in U crit both with increasing water ammonia (0, 0.02, 0.04 and 0.08 mg per l NH 3) and increasing plasma ammonia. Data collected included plasma pH and ammonia, muscle pH and ammonia and muscle membrane potential. Based on results found in these experiments it was concluded that the reduction in swimming performance was due to both metabolic challenges as well as depolarization of white muscle. Acute toxicity testing on swimming and resting rainbow trout revealed that swimming at (60% U crit or approximately 2.2 body lengths/s) decreased the LC 50 level from 207±21.99 mg N per l in resting fish to 32.38±10.81. The LC 50 for resting fish was significantly higher than that for swimming fish. The acute value set forth by the US EPA at the same pH is 36.1 mg N per l and may not protect swimming fish. In addition the effect of water hardness on ammonia toxicity was considered. It was found that increased water calcium ameliorates ammonia toxicity in fish living in high pH water.

Monitoring Sublethal Toxicity in Effluent Under the Metal Mining EEM Program

Abstract The second national application of environmental effects monitoring (EEM) in Canada will be under the amended Metal Mining Effluent Regulations (MMER). Under the EEM program, sublethal toxicity testing will be included in a suite of complementary tools to assess whether fish populations, fish habitat, and use of the fisheries resource, are protected in water bodies receiving mining effluent. The rationale for including sublethal toxicity tests was provided by an extensive literature review during the Aquatic Effects Technology Evaluation (AETE) program that showed 84% agreement between sublethal toxicity test results and observed impacts on receiving water. In addition, laboratory testing during the AETE program identified tests which would be the most cost effective and efficient in assessing mining effluent toxicity. Based on the findings of the AETE program, the multistakeholder EEM Metal Mining Working Group and its Toxicology Subgroup selected tests on fish, invertebrates, algae and an aquatic plant species, and developed consensus recommendations on the minimum requirements for sublethal toxicity testing and technical guidance on how to implement the recommended monitoring. The rationale for method selection and application of test results, and general testing requirements are discussed.

A Rapid Respiratory Toxicity Test Using Caenorhabditis elegans with an Oxygen Electrode System.

We describe a novel approach to evaluating the respiratory toxicity of chemicals in the free-living nematode Caenorhabditis elegans. Using DOX-96KT, a general purpose, multi-channel dissolved oxygen (DO) measuring system, we measured the DO concentration in culture media containing C. elegans exposed to chemicals to assay for respiratory toxicity. The current value, which is an index of the dissolved oxygen concentration in culture media, was measured every 10 sec for 30 min at 24°C. We focused on the respiration levels of the exposed worms between 500 and 1800 sec. This method produces results that are similar to the computer tracking system measuring behavioral toxicity. Since it can do multiple dilution series tests at a given time, it is useful for concentration-activity correlation studies. This novel technique is not only an alternative to the computer tracking system for measuring behavioral toxicity but also a rapid sublethal toxicity test for chemical hazard assessment.

Burrowing responses of the short-neck clam Ruditapes philippinarum to sediment contaminants

The burrowing responses of a common tropical bivalve, the short-neck clam Ruditapes philippinarum, to cadmium (Cd)-spiked sediment, variations of sediment grain size and natural sediments collected from 15 locations in Hong Kong's inshore waters were investigated through a series of laboratory tests. Results showed that the burrowing response exhibited a negative relationship with an increase in Cd concentration in the spiked sediments. The level of Cd was also found to be directly proportional to the percent mortality of the clam. However, there was no significant difference ( p>0.05) in the time elapsed for the clam to burrow into sediments with different grain size composition. The elapsed time for 50% of the test clams to burrow into the sediment (ET 50) over a period of 48 h was calculated for the sediment samples collected from the 15 locations. Results of ANOVA showed significant difference ( p<0.05) among the sediment samples. Tukey's multiple comparison test revealed two groups of sediments: group 1 containing 3 sediment samples collected from Victoria Harbour and group 2 containing 12 samples obtained from other coastal areas of Hong Kong. The ET 50 value for group 1 sediments was found to be greater than 2880 min whilst the ET 50 for group 2 sediments had a mean of 173.9 min. Agglomerative classification of the sediment samples, based on metal content (As, Cd, Cr, Cu, Ni, Pb, Zn), also showed two similar groups, suggesting that the ET 50 values were correlated with the metal level of the sediment samples. Group 1 sediments collected from Victoria Harbour had greater concentrations of Cd, Cr, Cu, Ni and Zn as compared to that in group 2. The present study demonstrated that high metal content in the sediment tends to inhibit the burrowing responses of the clam and that there is potential to develop the burrowing responses of R. philippinarum as a sublethal sediment toxicity test.

In Vitro Development of Parthenogenetic Eggs: A Fast Ecotoxicity Test with Daphnia magna?

In the present study, the authors investigated and defined development stages of Daphnia magna eggs that could be potential endpoints for sublethal toxicity tests with in vitro parthenogenetic egg cultures. Such an “egg test” could be a cost-effective alternative to the classic 21-day test with D. magna. Three main stages of embryonic development were considered: release of the egg external membrane, release of the internal membrane, and separation of the caudal spine. The first embryonic stage was attained approximately 30 h after transfer of eggs from ovaries to the brood chamber (considered as time zero), the second stage at 48 h, and the third stage at 68 h. Embryonic development was considered completed with the caudal spine separation. Thereafter, juveniles were able to swim in the water column. Egg mortality, duration of each egg stage, egg diameter, and egg abnormalities were investigated as potential endpoints. In vitro tests were carried out with several toxicants (DBS, 3,4-DCA, cadmium, and copper) and with acid mine drainage, sensitivity generally being higher than with the accepted chronic 21-day test with D. magna.

The garden snail (Helix aspersa) as a bioindicator of organophosphorus exposure: Effects of dimethoate on survival, growth, and acetylcholinesterase activity

AbstractThe garden snail (Helix aspersa) is currently used as bioindicator of metallic pollution. Our objective was to extend its use to organic chemicals by studying the effects and tissue concentrations of the organophosphorus pesticide dimethoate follo wing dietary uptake. After exposure for four weeks to increasing doses of pesticide in the diet, the median lethal concentration (LC50) was determined to be 3,700 μg/g food. Clinical signs indicated a no‐observed‐effect concentration of 100 μg/g and a lowest‐observed‐effect concentration of 250 μg/g. The growth parameters were decreased with increasing exposure to the pesticide. The median effective concentration (EC50), which was evaluated based on both shell diameter and dry weight inhibitions, was 665 and 424 μg/g, respectively, and the EC10 was 180 and 145 μg/g, respectively. Accumulation in the viscera was related to the amount of dimethoate in the food. The bioconcentration factors were low (&gt;6 × 10−3). Acetylcholinesterase (AChE) activity was strongly decreased (80% from 250 μg/g). In conclusion, we demonstrated that the species H. aspersa could be a useful sentinel organism for organophosphorus contamination surveys. Among the effects measured, the inhibition of AChE activities and clinical signs were the most sensitive, followed by the growth parameters. These results confirm the suitability of the garden snail for development of sublethal toxicity tests using primary consumers and aboveground organisms.

Sea urchin fertilization assay: An evaluation of assumptions related to sample salinity adjustment and use of natural and synthetic marine waters for testing

Most industrial effluents discharged into the marine coastal environment are freshwater in nature and therefore require manipulation prior to testing with marine organisms. The sea urchin fertilization test is a common marine bioassay used for routine environmental monitoring, investigative evaluations, and/or regulatory testing of effluents and sediment pore waters. The existing Canadian and U.S. Environmental Protection Agencies test procedures using sea urchin (and sand dollar) gametes allow for sample salinity adjustment using either brine or dry salts. Moreover, these procedures also allow for the use of either natural or synthetic marine water for culturing/holding test organisms and for full-scale testing. At present, it is unclear to what extent these variables affect test results for whole effluents. The test methods simply state that there are no data available and that the use of artificial dry sea salts should be considered provisional. We conducted a series of concurrent experiments aimed at comparing the two different treatments of sample salinity adjustment and the use of natural versus synthetic seawater in order to test these assumptions and evaluate effects on the estimated end points generated by the sea urchin fertilization sublethal toxicity test. Results from these experiments indicated that there is no significant difference in test end points when dry salts or brine are used for sample salinity adjustment. Similarly, results obtained from parallel (split-sample) industrial effluent tests with natural and artificial seawater suggest that both dilution waters produce similar test results. However, data obtained from concurrent tests with the reference toxicant, copper sulfate, showed higher variability and greater sensitivity when using natural seawater as control/dilution water.

The Evaluation of Pulp and Paper Mill Effluents Using Biological Assessment

The effect of pulp and paper mill effluents on the environment should be understood accurately, because the quantity of water discharged from a mill is very large. Current effluent regulations are based on biological oxygen demand (BOD) or chemical oxygen demand (COD), but these measures do not reflect the actual environmental effect of effluent.Bioassay are already used to determine the effect of effluent discharge on the environment in several foreign countries, and even in Japan it is expected that a bioassay will be introduced in the future. The type of bioassay used in nearly every country is the sublethal toxicity test, using fish, water frea, or green algae, although test conditions are slightly different in each country.The bioassay is not only for effluent monitaring ; it is also an effective procedure for toxicity identification evaluation (TIE) and toxicity reduction evaluation (TRE). By using TRE method, we would be able to further decrease the environmental effect. Although a total effluent free (TEF) system is the ideal, that is difficult to realize right away.We should aim at sustainable development while balancing considerations of risk and benefit.

The garden snail (Helix aspersa) as a bioindicator of organophosphorus exposure: effects of dimethoate on survival, growth, and acetylcholinesterase activity.

The garden snail (Helix aspersa) is currently used as bioindicator of metallic pollution. Our objective was to extend its use to organic chemicals by studying the effects and tissue concentrations of the organophosphorus pesticide dimethoate following dietary uptake. After exposure for four weeks to increasing doses of pesticide in the diet, the median lethal concentration (LC50) was determined to be 3,700 microg/g food. Clinical signs indicated a no-observed-effect concentration of 100 microg/g and a lowest-observed-effect concentration of 250 microg/g. The growth parameters were decreased with increasing exposure to the pesticide. The median effective concentration (EC50), which was evaluated based on both shell diameter and dry weight inhibitions, was 665 and 424 microg/g, respectively, and the EC10 was 180 and 145 microg/g, respectively. Accumulation in the viscera was related to the amount of dimethoate in the food. The bioconcentration factors were low (>6 x 10(-3)). Acetylcholinesterase (AChE) activity was strongly decreased (80% from 250 microg/g). In conclusion, we demonstrated that the species H. aspersa could be a useful sentinel organism for organophosphorus contamination surveys. Among the effects measured, the inhibition of AChE activities and clinical signs were the most sensitive, followed by the growth parameters. These results confirm the suitability of the garden snail for development of sublethal toxicity tests using primary consumers and aboveground organisms.

Qualification of spontaneous undirected locomotor behavior of fish for sublethal toxicity testing. Part II. Variability of measurement parameters under toxicant‐induced stress

AbstractSpontaneous locomotor behavior of semiadult zebra fish (Brachydanio rerio) was recorded under sublethal short‐term exposure to the anionic technical surfactant, linear alkylbenzene sulfonate (C10–13‐LAS) and cadmium in single compound tests using an automated video‐monitoring and object‐tracing system. Vertical position and swimming velocity in the horizontal and vertical directions were used as behavioral measurement parameters. Data were analyzed by different statistical methods. In pairwise comparisons, consistent, statistically significant, and toxicant‐induced alterations of locomotor behavior were observed only for test concentrations, which also caused aspectoric symptoms of intoxication. This comparatively low sensitivity of the behavioral indication criteria was related to high variation in the measurement parameters and corresponding high, minimum detectable, statistically significant, and toxicant‐induced deviations. In contrast, results obtained by regression analysis showed significant trends in locomotor activity over the range of toxicant concentrations tested. Thus, our findings support the inappropriateness of no observed effect concentrations and the lowest observed effect concentrations as summary measures of toxicity and indicate that the regression analysis approach is superior to the analysis of variance approach.

Qualification of spontaneous undirected locomotor behavior of fish for sublethal toxicity testing. Part I. Variability of measurement parameters under general test conditions

AbstractAn automated, personal computer–based video‐processing, object‐recognition, and object‐tracing system was used to record and analyze undirected spontaneous locomotor behavior of small groups of undisturbed semiadult zebra fish (Brachydanio rerio) in laboratory tanks. The primary data provided by the monitoring system were the individually assigned, time‐stamped coordinates of the fish in two‐dimensional projection. Secondary parameters (position, velocity of movement in the horizontal and the vertical direction, and temporal intraindividual and interindividual association) were calculated. The computed parameters offered a multidimensional description of spontaneous undirected swimming behavior of the fish and proved to be largely independent of water temperature, length, weight, and sex ratio of the zebra fish within the standardized range, but varied significantly with the feeding regime, time of day, number of fish per tank, and batch. Statistical characteristics of the behavioral parameters confirmed them as being appropriate for parametric statistical analyses.

生物試験による紙パルプ工場排水の環境影響評価

We have initiated a variety of laboratory studies to assess the environmental impacts of pulp and paper mill effluents in Japan. Here we present the outline of lethal and sublethal short-term toxicity tests employed for the assessment and some results obtained by testing 17 whole mill effluents.

The swimming behaviour of Brachionus calyciflorus (rotifer) under toxic stress. I. The use of automated trajectometry for determining sublethal effects of chemicals

Changes in the locomotory behaviour of the freshwater rotifer Brachionus calyciflorus were used as sublethal indicators of toxic stress. To that end, the swimming behaviour of this rotifer was analysed using an automated tracking system. The swimming speed (temporal factor), the swimming sinuosity (spatial factor), and the periods of swimming were measured and the influence of four chemicals, each representing a distinct chemical class (copper, pentachlorophenol, lindane and 3,4-dichloroaniline), on the rotifer's swimming characteristics were examined. The three test parameters exhibit different sensitivities depending on the chemical tested. The 2-h EC 50s obtained with the behavioural test were of the same order of magnitude as the 24-h LC 50s resulting from conventional acute toxicity tests with the same test species. This potential use of behavioural test criteria for sublethal toxicity testing with rotifers is briefly discussed.

Life history characteristics of the freshwater ostracod Cyprinotus incongruens and their application to toxicity testing

: The biology of ostracod crustaceans suggests that this group could be a useful model for sediment toxicity testing. We examined life history characteristics of the freshwater ostracod Cyprinotus incongruens in the laboratory (25°C), then used length after 10 days as a response variable in a sediment toxicity test. Life history results indicated that both fecundity (0-64 total eggs per female), and egg development time (1->157 days), were highly variable. In contrast, juvenile development time showed less variation (7-16 days), and thus body length after 10 days was chosen as a sublethal bioassay character. A bioassay experiment using sediments isolated from mine-impacted cobble streams indicated that C. incongruens had a higher sensitivity to variation in sediment quality than the cladoceran Ceriodaphnia dubia. Surprisingly, the results indicated no correspondence between concentration of metals and toxicity in either C. incongruens or C. dubia. Overall our results indicate that ostracods are a good candidate taxon for sublethal toxicity tests of contaminated sediments.

Tissue distribution and histopathological effects of dietary methylmercury in benthic grubby Myoxocephalus aenaeus

There is a need to test deterministic models predicting the behavior and effects of chemicals on aquatic systems by conducting experiments with more than one trophic step at a time. This approach requires the set-up of an experimental food chain in pounds or mesocosm facilities which can be used for dietary uptake studies and assessment of sublethal stress induced by contaminated food. In the course of our current research program at the INRS marine mesocosms facilities, a model benthic food chain including inter- and sub-tidal species such as the mussel (Mytilus edulis), clam (Mya arenaria), starfish (Leptasterias polaris), polychaete (Nereis virens), amphipod (Gammarus sp.), gastropod (Buccinum undatum), and fishes (Pleuronectec americanus, Myoxocephalus aenaeus), is used for testing food uptake models and for the development of sublethal toxicity tests which could be used in the environmental assessment of coastal and estuarine waters. Among these test organisms, the grubby (M. aenaeus) is a small coastal fish (12-15 cm) characterized by a broad head. The grubby is tolerant of water temperature and salinity variations and lives on a wide variety of bottom strata at low depths. The grubby is carnivorous and consumes a wide variety of molluscs and the young of many speciesmore » of fish. Because of its size, its estuarine and coastal distribution and its large spectrum of prey, this species was seen as an ideal fish to fit into our experimental food chain model. This paper reports a preliminary experiment designed to measure distribution of mercury in tissues and to test the response of various histopathological and biochemical stress indicators in grubby exposed to dietary contamination by methylmercury (MeHg) for a 20-day exposure period. MeHg was chosen because it is rapidly bioaccumulated by most living organisms and its toxicity has been studied for decades in numerous aquatic ecosystems. 17 refs., 2 tabs.« less

Effect of pH and ammonia on survival and growth of the early larval stages of Penaeus monodon Fabricius

Lethal toxicity tests of ammonia at different pH levels (7, 7.5, 8, and 8.5) and its effect on survival and growth of the early larval stages of Penaeus monodon were determined. An increase in ammonia toxicity when the water pH increased was revealed in 96 h toxicity tests. Estimated LT 50 decreased from 101.09 to 25.16 h for protozoea exposed to 8 ppm ammonia, from 115.79 to 11.26 h for mysis exposed to 24 ppm, and from 51.41 to 22.58 h for PL exposed to 52 ppm ammonia with increase in pH levels. The effect of 3 and 6 ppm ammonia levels at pH levels of 7.0, 7.5, 8.0 and 8.5 on the survival and growth of P. monodon larvae and postlarvae was also investigated in a 16-day sublethal toxicity test. Results indicated that ammonia at 3 and 6 ppm affects both survival and growth of shrimp. Survival was decreased by 27% in 3 ppm and by 48% in 6 ppm ammonia, while growth was reduced by 4.4% in 3 ppm and by 6.5% in 6 ppm ammonia. Increasing pH of the rearing water resulted in significantly lower survival in protozoea, mysis, and postlarval stages. No interactive effect of pH and ammonia was detected.

A short-exposure, sublethal, sediment toxicity test using the marine bivalveMulinia lateralis: Statistical design and comparative sensitivity

Over the last 10 years a great deal of research effort has concentrated on determining the effects of contaminated sediments on aquatic organisms. For marine systems, this effort has emphasized acute sediment toxicity tests using amphipods, although a variety of other end points and species have been used. Another candidate species for marine, solid-phase, sublethal sediment toxicity testing is the bivalve Mulinia lateralis. Useful attributes of this euryhaline bivalve include a wide geographic distribution (along the North American Atlantic coast from Prince Edward Island to the Gulf of Mexico), easy lab culture, and amenability to toxicity testing applications (end points are mortality and growth [milligrams per organism dry weight]). Detailed in this paper are organism selection and culture, establishment of statistical design, and an estimate of organism mortality and sublethal response variability. Results of Mulinia lateralis toxicity tests with 65 contaminated sediments from eight sites are reported, as well as results of comparative toxicity tests using two amphipod species, Ampelisca abdita and Eohaustorius estu-arius. Analysis of statistical power indicates treatment weight and survival responses that are 25% different from the site control responses can be detected with a probability of 95%. Results of comparative toxicity tests illustrate that although Mulinia lateralis and amphipod acute end points are relatively similar in sensitivity, utilization of the Mulinia lateralis sublethal growth end point greatly increases test sensitivity. This paper describes a new marine sediment toxicity test that complements the existing suite of marine sediment toxicity assessment techniques.

The effect of 2‐(Thiocyanomethylthio)benzothiazole on juvenile coho salmon (Oncorhynchus Kisutch): Sublethal toxicity testing

AbstractThis study examined the sublethal toxicity of 2‐(thiocyanomethylthio)benzothiazole (TCMTB), a wood preservative used in British Columbia's lumber industry, to coho salmon, Oncorhynchus kisutch. Swimming performance, oxygen consumption, and blood lactate levels were used as indicators of sublethal TCMTB toxicity. Assessment of swimming performance consisted of subjecting preexposed (48 h) fish to a series of increasing velocity increments within an annular swim chamber. An automated respirometer apparatus was used to measure oxygen consumption rates. Blood lactate levels were assayed upon termination of the respirometer trials. Reductions in critical swimming speed and elevations in blood lactate occurred in a concentration‐dependent manner with thresholds of 10 and 7.5 μg/L, respectively. A similar concentration‐dependent response was not observed with respect to oxygen consumption rates.

Sublethal effects of zinc and municipal effluents on larvae of the red abalone Haliotis rufescens

Experiments were conducted to develop a sensitive sublethal toxicity test protocol to determine the toxicity of municipal wastewater effluents to larvae of the red abalone Haliotis rufescens. In multiple tests, fertilized abalone embryos were exposed for 48 h to dilutions of a reference toxicant, zinc sulfate, and to dilutions of primary-and secondary-treated effluents. The resulting veliger larvae were examined microscopically for larval shell abnormalities. In a longer flowthrough experiment, abalone were exposed for the entire larval phase, from the two-cell stage through metamorphosis, to compare zinc effects on metamorphosis with zinc effects on short-term larval shell development. Dissolved oxygen, pH, salinity and temperature were measured daily in test solutions, and zinc concentrations were verified by chemical analysis. “No observed effect concentrations” (NOECs) for zinc were 39±2.1 μg l-1 in three 48 h exposures, and 19 μg l-1 for the 9 d exposure through metamorphosis. Median effect concentrations (EC50s) were 68±6.9 μg l-1 in 48 h tests and 50 μg l-1 in the 9 d test. Abalone larvae were affected at lower concentrations of primary than of secondary effluent.