Standard Toxicity Tests Research Articles

Towards more efficient testing strategies—Analyzing the efficiency of toxicity data requirements in relation to the criteria for classification and labelling

This contribution is based on the assumption that the aim of toxicity testing as required by chemicals legislation is to identify as many chemicals of concern to human health and the environment as possible, given a limited amount of resources allocated to testing. Based on this assumption we propose a method for the optimization of test systems for industrial chemicals, based on the calculation of efficiency ratios for tests and test systems. The efficiency ratio of a toxicity test depends on the monetary cost of performing the test and the probability that the test will identify a chemical of concern, as estimated by the rules for classification and warning labelling. Efficiency ratios are estimated based on the results of basic standardized toxicity testing for acute toxicity, subacute toxicity, irritation and sensitization of 1409 industrial chemicals notified in the European Union between 1994 and 2004. This careful evaluation of the regulatory consequences of testing indicated that many of these substances are classified based on short-term testing of acute toxicity, irritation and sensitization and that most of the substances classified due to a 28-day study were also classified based on short-term testing. These results indicate that, within the classification and labelling system, it is currently more efficient to perform short-term testing of a larger number of substances rather than to perform subacute toxicity studies on substances already tested for acute toxicity.

Characterizing the toxicity of pulsed selenium exposure to Daphnia magna

The acute toxicity of selenium (Se) to aquatic biota has been studied extensively for decades. However, most studies have used a constant concentration aqueous exposure of Se to an invertebrate species. Since constant concentration exposure of toxicants to invertebrates is unusual in the environment, episodic exposure or pulsed exposures may represent true risk to aquatic biota more accurately. This research was designed to characterize the toxicity effects of pulsed Se exposure to Daphnia magna. Selenium exposure was varied during a 21-d chronic toxicity test to examine the effects of exposure concentration, duration, and recovery on survival, growth, and reproduction of D. magna. While D. magna did not die during exposures, latent mortality was observed. Latent mortality increased with exposure concentration and duration. Hence, standard toxicity test using continuous exposures would underestimate Se toxicity. Risk assessment method using results of continuous exposure would underestimate risk of Se to biota. For double-pulse exposures, cumulative mortality on day 21 was higher when time interval between pulses was shorter. With the same total exposure time, continuous exposure caused higher toxicity than did pulsed exposures due to recovery and tolerance development in D. magna after earlier pulses. Growth and reproduction of surviving D. magna were not affected by pulsed Se exposure due to recovery of D. magna after removal of the pulses. Based on these results, risk assessment for Se should take latent effects and the effect of recovery in to account.

INCREASED SENSITIVITY AND VARIABILITY OF PHYTOTOXICITY RESPONSES IN ARCTIC SOILS TO A REFERENCE TOXICANT, BORIC ACID

Industrial and human activities in the Arctic regions may pose a risk to terrestrial Arctic ecosystem functions. One of the most common terrestrial toxicological end points, primary productivity, typically is assessed using a plant phytotoxicity test. Because of cryoturbation, a soil mixing process common in polar regions, we hypothesized that phytotoxicity test results in Arctic soils would be highly variable compared to other terrestrial ecosystems. The variability associated with phytotoxicity tests was evaluated using Environment Canada's standardized plant toxicity test in three cryoturbated soils from Canada's Arctic exposed to a reference toxicant, boric acid. Northern wheatgrass (Elymus lanceolatus) not only was more sensitive to toxicants in Arctic soils, its response to toxicants was more variable compared to that in temperate soils. The phytotoxicity of boric acid in cryosols was much greater than commonly reported in other soils, with a boric acid concentration of less than 150 microg/g soil needed to inhibit root and shoot growth by 20%. Large variability also was found in the phytotoxicity test results, with coefficients of variation for 10 samples ranging from 160 to 79%. The increased toxicity of boric acid in cryosols and variability in test response was not explained by soil properties. Based on our admittedly limited data set of three different Arctic soils, we recommend that more than 30 samples be taken from each control and potentially impacted area to accurately assess contaminant effects at sites in northern Canada. Such intensive sampling will insure that false-negative results for toxicant impacts in Arctic soils are minimized.

USING ACCELERATED LIFE TESTING PROCEDURES TO COMPARE THE RELATIVE SENSITIVITY OF RAINBOW TROUT AND THE FEDERALLY LISTED THREATENED BULL TROUT TO THREE COMMONLY USED RANGELAND HERBICIDES (PICLORAM, 2,4-D, AND CLOPYRALID)

We conducted 96-h static acute toxicity studies to evaluate the relative sensitivity of juveniles of the threatened bull trout (Salvelinus confluentus) and the standard cold-water surrogate rainbow trout (Onchorhyncus mykiss) to three rangeland herbicides commonly used for controlling invasive weeds in the northwestern United States. Relative species sensitivity was compared using three procedures: standard acute toxicity testing, fractional estimates of lethal concentrations, and accelerated life testing chronic estimation procedures. The acutely lethal concentrations (ALC) resulting in 50% mortality at 96 h (96-h ALC50s) were determined using linear regression and indicated that the three herbicides were toxic in the order of picloram acid > 2,4-D acid > clopyralid acid. The 96-h ALC50 values for rainbow trout were as follows: picloram, 41 mg/L; 2.4-D, 707 mg/L; and clopyralid, 700 mg/L. The 96-h ALC50 values for bull trout were as follows: picloram, 24 mg/L; 2.4-D, 398 mg/L; and clopyralid, 802 mg/L. Fractional estimates of safe concentrations, based on 5% of the 96-h ALC50, were conservative (overestimated toxicity) of regression-derived 96-h ALC5 values by an order of magnitude. Accelerated life testing procedures were used to estimate chronic lethal concentrations (CLC) resulting in 1% mortality at 30 d (30-d CLC1) for the three herbicides: picloram (1 mg/L rainbow trout, 5 mg/L bull trout), 2,4-D (56 mg/L rainbow trout, 84 mg/L bull trout), and clopyralid (477 mg/L rainbow trout; 552 mg/L bull trout). Collectively, the results indicated that the standard surrogate rainbow trout is similar in sensitivity to bull trout. Accelerated life testing procedures provided cost-effective, statistically defensible methods for estimating safe chronic concentrations (30-d CLC1s) of herbicides from acute toxicity data because they use statistical models based on the entire mortality:concentration:time data matrix.

Avoidance test with Enchytraeus albidus (Enchytraeidae): Effects of different exposure time and soil properties

Enchytraeids are ecologically relevant soil species and are commonly used in standardized toxicity tests. Their rapid reaction to a chemical exposure can be used as a toxicological measurement endpoint that assesses the avoidance behavior. The objectives of this investigation were to determine the effects of soil properties on the avoidance behavior of Enchytraeus albidus and to optimize the duration of avoidance test. The avoidance tests included (1) exposures in OECD artificial soil with standard or modified properties (pH, clay or peat content), and (2) exposures to copper chloride, cadmium chloride, and to the organic pesticides dimethoate and phenmedipham for different time periods. Results showed that alteration of OECD soil constituents significantly affected the avoidance behavior of enchytraeids, and that the 48-h exposure was the optimal duration of the test. Consideration of soil properties is important for selecting appropriate experimental design and interpreting the results of the enchytraeid avoidance test.

Effects of temperature and oxygen concentration in sediment toxicity testing

Joint effects of temperature and oxygen concentrations for the results of sediment toxicity tests were studied at 10 and 20 °C with 40% and 80% dissolved oxygen (DO) saturation. Growth, feeding rate, and reproduction of Lumbriculus variegatus (Oligochaete) and growth, emergence, and survival of Chironomus riparius (Diptera) were tested in a polluted and in a reference sediment. Both the feeding of L. variegatus and the emergence of C. riparius were significantly retarded at low temperature. Additionally, differences in the sex ratio of the emerged adults of C. riparius were observed. The oxygen concentration alone did not have any significant effect on the endpoints, but significant combined effects of polluted sediment and low DO were observed on the biomass of L. variegatus. The standard sediment toxicity tests might offer only limited data for risk assessment of contaminated sediments at sites where the actual conditions largely differ from the laboratory conditions.

Comparison of Bioluminescent Dinoflagellate (QwikLite) and Bacterial (Microtox) Rapid Bioassays for the Detection of Metal and Ammonia Toxicity

This study compared the sensitivity of two rapid toxicity tests, QwikLite and Microtox, to seven metals and ammonia. Both of these tests measure a reduction in light production from bioluminescent microorganisms (dinoflagellates and marine bacteria, respectively) as a means of toxicity detection and are simple and inexpensive to conduct compared to many standardized acute toxicity tests. For QwikLite tests, three marine dinoflagellate species (Lingulodinium polyedrum, Ceratocorys horrida, and Pyrocystis noctiluca) were separately evaluated following a 24-h exposure period. The marine bacterium, Vibrio fischeri, was used in the Microtox tests, in 15-min exposures to the same metal preparations as those used for the QwikLite tests. The QwikLite tests were generally one to two orders of magnitude more sensitive than the Microtox tests, as indicated by lower median effects concentrations (EC(50)). Both QwikLite and Microtox, however, resulted in similar toxicity rankings for the metals tested. The dinoflagellate species used in the QwikLite tests responded similarly for most compounds tested, with L. polyedrum appearing to be somewhat more sensitive than the other two species for most metals evaluated. QwikLite was also more comparable in sensitivity to several commonly used standardized toxicity tests. As with all toxicity tests, species selection for QwikLite should take into account study-specific factors, including the potential for sensitivity to confounding factors, such as ammonia.

Whole‐sediment toxicity identification evaluation tools for pyrethroid insecticides: II. Esterase addition

An esterase enzyme capable of catalyzing the hydrolysis of the ester bond common to all pyrethroid insecticides has been proposed as a toxicity identification evaluation procedure for the compounds when present in water samples. It appeared to show promise in previous applications to pore water and in one published application to bulk sediment. The present study was designed to provide the additional validation of the technique when applied to whole sediment, demonstrating its efficacy and specificity to pyrethroids. When added to the overlying water in a standard sediment toxicity test with the amphipod Hyalella azteca, the enzyme reduced toxicity of pyrethroid-spiked laboratory sediments. The technique had no effect on toxicity of DDT or chlorpyrifos, but it dramatically reduced the toxicity of cadmium, presumably by complexation of the cadmium with dissolved organic matter rather than by enzymatic activity. Esterase addition consistently reduced pyrethroid-related toxicity in 12 tests with field-collected sediments. The procedure, however, also results in substantial changes in water quality, provides an undesirable nutritional benefit to the test organisms exposed to the esterase, and achieves a substantial fraction of the protective capability simply by complexation of the pyrethroids with the esterase or the microbial growth that the esterase promotes. The esterase addition technique has potential for whole-sediment application, but further development of procedures and confirmation by independent lines of evidence are needed to reduce the potential for misinterpretation of results.

Acute silver toxicity in the euryhaline copepod Acartia tonsa: Influence of salinity and food

The euryhaline copepod Acartia tonsa was exposed to silver (AgNO(3)) in either the absence or the presence of food (diatom Thalassiosira weissflogii; 2 x 10(4) cells/ml). Standard static-renewal toxicity tests that included a fixed photoperiod of 16: 8 h light:dark and temperature (20 degrees C) were run in three different salinities (5, 15, and 30 ppt) together with measurements of pH, ions (Na(+), Cl(-), K(+), SO(4)(2-), Mg(2+), and Ca(2+)), alkalinity, dissolved organic carbon, and total and dissolved (0.45 microm) silver concentrations in the experimental media. In the absence of food, the 48-h EC50 (concentration causing effect to 50% of the individuals tested) values based on total and dissolved silver concentrations were 11.6, 87.2, and 163.2 microg Ag/L and 7.1, 79.2, and 154.6 microg Ag/L at salinities 5, 15, and 30 ppt, respectively. In the presence of food, they were 62.1, 98.5, and 238.4 microg Ag/L and 48.4, 52.3, and 190.9 microg Ag/L, respectively. In all experimental conditions, most of the toxic silver fraction was in the dissolved phase, regardless of salinity or the presence of food in the water. In either the absence or the presence of food, acute silver toxicity was salinity dependent, decreasing as salinity increased. Data indicate that changes in water chemistry can account for the differences in acute silver toxicity in the absence of food, but not in the presence of food, suggesting that A. tonsa requires extra energy to cope with the stressful conditions imposed by acute silver exposure and ionoregulatory requirements in low salinities. These findings indicate the need for incorporation of both salinity and food (organic carbon) in a future biotic ligand model (BLM) version for estuarine and marine conditions, which could be validated and calibrated using the euryhaline copepod A. tonsa.

Acute toxicities of trace metals and common xenobiotics to the marine copepod Tigriopus japonicus: Evaluation of its use as a benchmark species for routine ecotoxicity tests in Western Pacific coastal regions

Marine copepods have recently been recognized as important organisms in ecotoxicity testing for regulatory purposes. The harpacticoid copepod Tigriopus japonicus has a wide geographical distribution along the coast in the Western Pacific including Japan, South Korea, Taiwan, and Hong Kong. This study evaluated the acute toxicity sensitivity profile of Tigriopus japonicus against 12 common toxic substances including six endocrine disrupting chemicals (EDCs), three biocides and three trace metals. Through standard acute toxicity test procedures, toxicity endpoints LC(50), LC(10), and no observed effect concentration (NOEC) of each chemical were obtained. Although T. japonicus depicted different sensitivities towards different chemicals, a dose-response relationship was consistent in all cases. T. japonicus was particularly sensitive to most of the EDCs, but relatively less sensitive to molinate (a thiocarbate herbicide). Across all tested chemicals, tributyltin (TBT) was the most toxic to the copepod with the LC(50), LC(10), and NOEC of 0.05, 0.03, and 0.02 mg/L, respectively. A comparison made with available data on acute toxicities of these chemicals to other marine copepod species revealed that T. japonicus is generally more sensitive to EDCs and in particular to TBT. We, therefore, strongly advocate that T. japonicus shall be adopted as a benchmark marine species for routine ecotoxicity testing and ecotoxicological studies in Western Pacific coasts.

Acute toxicity of pesticides to the tropical freshwater shrimp Caridina laevis

To determine the potential risk of pesticides frequently used in Indonesia, a new toxicity test was developed using the indigenous freshwater shrimp Caridina laevis, which is representative of tropical ecosystems. The test species could easily be maintained in the laboratory. Acute toxicity tests with different insecticides, fungicides, and herbicides assessing adult survival after 24 and 96 h of static exposure showed low control mortality (<10%) and good reproducibility for diazinon. Juvenile C. laevis appeared approximately two to three times more sensitive to diazinon than adult ones. Compared to other species of freshwater crustacean commonly used in standard aquatic toxicity tests, C. laevis showed similar sensitivity for diazinon and lambda cyhalothrin, much greater sensitivity for endosulfan and paraquat and much lower sensitivity for carbofuran. It may be concluded that the acute toxicity test using C. laevis may be a suitable alternative for determining the potential risk of chemicals under tropical conditions.

Influence of organism age on metal toxicity to Daphnia magna

Aquatic organisms living in surface water experience contaminant exposure at different life stages. While some investigators have examined the influence of organism age on the toxicity of pollutants, the general assumption in toxicology has been that young organisms were more sensitive than older organisms. In fact, some standardized toxicity tests call for the use of organisms less than 24 h old. This research characterized the age sensitivity of the water flea Daphnia magna to copper, zinc, selenium, and arsenic. During 21-d toxicity tests, organisms were exposed to a single 12-h pulse of either 70 microg/L Cu, 750 microg/L Zn, 1000 microg/L Se, or 5000 microg/L As at different ages ranging from 3 h to 10 d old. Mortality and reproduction were compiled over 21 d. During the juvenile stage, mortality increased and cumulative reproduction decreased with age, respectively. However, mortality decreased and cumulative reproduction increased with age when organisms became adult. Peak sensitivity occurred in 4-d-old organisms exposed to Cu and Zn, while 2- to 3-d-old organisms were most sensitive to As and Se. Growth of D. magna over 21 d was not affected by the 12-h pulse of Cu, Zn, Se, or As given at any organism age. This indicates the recovery of the organisms after exposure termination.

Copper and zinc tolerance of two tropical microalgae after copper acclimation

Current toxicity tests with microalgae are often criticized as being overly sensitive to metals because algae are cultured in metal-deficient media. If such bioassays overestimate copper toxicity in surface waters, the relevance of water quality guidelines derived from these tests is questionable. In this study, the effect of acclimation to copper at environmentally relevant concentrations, on the sensitivity of the marine diatom Nitzschia closterium and the freshwater green alga Chlorella sp. to copper and zinc was examined. N. closterium was acclimated in culture medium containing 5 or 25 microg Cu L(-1) for 200 days, while Chlorella sp. was acclimated in medium containing 2 microg Cu L(-1) for 100 days. Changes in algal growth rates and copper and zinc tolerance were monitored using standard growth inhibition toxicity tests in minimal medium over 72 h. Neither of the two acclimated N. closterium cultures had increased zinc or copper tolerance compared with that of the nonacclimated algae, nor were there any changes in control growth rates. Similarly, no changes in copper tolerance or control growth rates were observed for the acclimated Chlorella sp. culture. This was supported by measurements of intracellular and extracellular copper which confirmed that there were no differences in copper accumulation in either acclimated or nonacclimated algae. These results suggest that these algae grown in standard culture media are generally no more sensitive than algae grown in a metal-enriched medium. This supports the continued use of current laboratory bioassays with microalgae, as part of a suite of tests for assessing metal bioavailability, for use in ecological risk assessments and for providing data for the derivation of water quality guidelines.

Toxic components leaching from tire rubber

Tire rubber is a complex mixture of a variety of chemicals, e.g., rubber polymers, carbon blacks, silicas, process and extender oils, vulcanization chemicals, and chemical antidegradents (Barbin and Rodgers, 1994). Leachates of tire rubber are toxic to a range of aquatic organisms; see for example the review by Evans (1997). Attempts made so far to identify the components causing the toxicity of rubber leachates have focused on chemical fractionation and characterization followed by toxicity testing of different fractions. Toxicity identification evaluations of leachates from tire plugs leached in deionized water have indicated that zinc was the main cause of toxicity to Ceriodaphnia dubia (Nelson et al., 1994). Microtox toxicity was caused by several organic compounds spread through a range of polarities (Anthony et al., 1995). A toxicity identification evaluation on leachates obtained from tire wear material leached in reconstituted water (pH 8, hardness 250 mg/L as CaC03), identified nonpolar organic compounds as the main cause of toxicity to Daphnia magna (Wik and Dave, 2006). In this previous study, we also found large differences in toxicity between different tires, which implies that the environmental impact of tire wear can be effectively reduced through rubber formulation if the most toxic components are identified. The aim of this study was to identify the components in tire rubber that are toxic to Daphnia magna, using a novel approach. Rubber formulations containing different additives were produced, and water leachates from the rubber samples were prepared and tested for toxicity using a standardized toxicity test. The main objective of this study was therefore to assess the toxicity to Daphnia magna of different rubber formulations in order to identify the most toxic tire leachate components.

Endpoint ‘floating leaves’ of Potamogeton natans: A new method to evaluate the development of macrophytes in pond mesocosms

One of the advantages of long-term mesocosm experiments as compared to short-term standard toxicity tests in the laboratory is the potential for detecting secondary effects due to the interaction of species and recovery with biomass of macrophytes being an important endpoint. However, generating biomass data by harvesting is often laborious, time-consuming, costly and restricted to the end of the experiment. Moreover, valuable information may get lost, in particular in single application studies, since maximal primary effects and secondary effects or recovery occur per se at different times. Potamogeton natans was used as an example in order to test whether number and area of floating leaves can be reliably measured and be used as intermediate and final end-points in mesocosm effect studies. Digital photos, which were taken of the water surface in the course of an indoor pond mesocosm study on herbicide effects, were subjected to image analysis. The results were compared to wet weight and ash-free dry weight of Potamogeton at the end of the herbicide study. Both number and area of floating leaves indicated the same herbicide effects as wet weight and ash-free dry weight of Potamogeton. Error introduced by the different work steps is small and can be further minimised by a number of method improvements. In indoor mesocosm studies, errors due to the perspective adjustment may be circumvented by taking the photos perpendicular to the water surface. Correction for lens aberration, identical light conditions and the use of fluorescence images are considered promising. Field applications are proposed.

Genetic impoverishment in laboratory cultures of the test organismChironomus riparius

Genetic diversity among laboratory and field populations of Chironomus riparius was investigated using microsatellite DNA analysis. Individuals of midge cultures reared in 10 different laboratories showed a clear reduction in the number of alleles per locus and in the level of heterozygosity compared to two natural populations sampled in southern Germany. To reconstruct the rate of genetic impoverishment under laboratory conditions, genetic diversity was monitored in a C. riparius laboratory population for 23 generations. Additionally, 11 populations originating from different laboratories were crossed, and genetic diversity was compared among pure, crossed, and natural populations. The results demonstrate that C. riparius strains used in standard toxicity tests exhibit low levels of genetic variability. In addition, our experiments show that refreshment of stocks with individuals from other laboratories is not sufficient to restore genetic variation. Potential consequences of genetic impoverishment and improvements for toxicological bioassays are discussed.

Daphnia magna responses to a vertebrate estrogen receptor agonist and an antagonist: A multigenerational study

Whereas ecological risk assessments rely on standardized aquatic toxicity tests to assess ecological hazards, these techniques have limited utility for endocrine-active compounds, including select pharmaceuticals. Due to structural similarity between of vertebrate estrogens and ecdysone, previous studies suggest that endocrine-active pharmaceuticals may interfere with invertebrate endocrine systems, while other investigations do not support these suggestions. We assessed effects of the pharmaceuticals 17 α-ethinylestradiol and faslodex, model therapeutics designed to interact with vertebrate estrogen receptors, on endocrine biomarkers and transgenerational life-history parameters of a model invertebrate, Daphnia magna. Identical studies were performed with 20-hydroxyecdysone and testosterone, which served as positive controls for ecdysteroid receptor agonism and antagonism, respectively. Results from this study at biochemical, individual and population levels suggest that a mammalian estrogen receptor agonist and antagonist did not act through the ecdysone receptor in D. magna. Acute-to-chronic ratios based on various chronic responses ranged from 2.59 to 5.18 for 17 α-ethinylestradiol and 1.29–12.9 for faslodex. Toxicity exerted by these therapeutics on D. magna likely resulted from non-endocrine-mediated responses. Mechanism-specific biomarkers, multigenerational designs and population growth models may be useful to assess organismal and population level responses to low-level exposures, which may serve to reduce uncertainty in future hazard assessments of invertebrate responses to endocrine-active pharmaceuticals in the environment.

Age and exposure duration as a factor influencing Cu and Zn toxicity toward Daphnia magna

Standardized toxicity tests are generally performed with juvenile test organisms, e.g., in Daphnia magna assays neonates <24 h old are used. The purpose of this research was to investigate the influence of a delayed exposure to Cu and Zn on population parameters and toxicity values derived from these endpoints. Juveniles (< 24 h; T0) and 7-d old daphnids (T7) were exposed for 21 and 14 d, respectively. For Cu, juveniles were significantly more sensitive than 7 d old organisms following acute (48 h) as well as chronic (14 d) exposure. After 14 d of exposure to 130 μg/L Cu, mortality was 80% and 10% in T0 and T7, respectively. Juveniles per surviving female at this concentration decreased by 78% and 14% compared to the control. 14 d-NOEC and LOEC values (based on juveniles per surviving female) were 75 and 90 μg/L Cu for T0 and both>130 μg/L for T7. For Zn, survival in T0 and T7 was similar. Although T7 organisms produced significantly more offspring, 14 d-NOEC and LOEC values were equal to those of T0, i.e., 80 and 115 μg/L, respectively. For Cu as well as for Zn effect concentrations based on 14 and 21 d exposure were similar (results from T0). It can be concluded that acute and chronic toxicity data obtained from juvenile D. magna are more sensitive or equally sensitive than obtained from 7 d old organisms. As differences are observed between the two metals extrapolations of these conclusions to other toxicants and other aquatic species cannot be made without further investigation.

Mysid crustaceans as standard models for the screening and testing of endocrine-disrupting chemicals

Investigative efforts into the potential endocrine-disrupting effects of chemicals have mainly concentrated on vertebrates, with significantly less attention paid to understanding potential endocrine disruption in the invertebrates. Given that invertebrates account for at least 95% of all known animal species and are critical to ecosystem structure and function, it remains essential to close this gap in knowledge and research. The lack of progress regarding endocrine disruption in invertebrates is largely due to: (1) our ignorance of mode-of-action, physiological control, and hormone structure and function in invertebrates; (2) lack of a standardized invertebrate assay; (3) the irrelevance to most invertebrates of the proposed activity-based biological indicators for endocrine disruptor (ED) exposure (androgen, estrogen, and thyroid); (4) limited field studies. Past and ongoing research efforts using the standard invertebrate toxicity test model, the mysid shrimp, have aimed at addressing some of these issues. The present review serves as an update to a previous publication on the use of mysids for the evaluation of EDs (Verslycke et al. 2004a). It summarizes recent investigative efforts that have significantly advanced our understanding of invertebrate-specific endocrine toxicity, population modeling, field studies, and transgeneration standard test development using the mysid model.

Characterization of the Action of Drug-Induced Stress Responses on the Immune System: Evaluation of Biomarkers for Drug-Induced Stress in Rats

Toxicological testing of compounds often is conducted at the maximum tolerated dose to identify potential target organs. Toxicities observed at these high doses may result in decreased body weight gain, food consumption and activity. These clinical signs are often associated with a generalized stress response. It has been known that stress may cause increased levels of corticosterone, which causes changes in circulating leukocyte profiles, decreases in thymus and spleen weights and changes in the microscopic structure of lymphoid organs. This makes it difficult to differentiate between stress-related changes and direct toxicity to the immune system in standard non-clinical toxicity testing in rats. In mice, MHC Class II expression was found to be a very sensitive biomarker of stress and maybe useful for the rat. Therefore, the objective of studies presented was to further characterize the effects of corticosterone and stressors on the immune system and identify potential biomarkers of stress in rats. Rats were treated with exogenous corticosterone (20 or 30 mg/kg BID) or ethanol (5 g/kg) for either 1 or 4 days. Restraint stress was also evaluated for a 3-day period. Blood and urine samples were collected during the treatment period for corticosterone measurements. At necropsy, blood samples for leukocyte differentials were collected. Spleen and thymus weights, cellularity, lymphocyte subpopulations and histopathology were also evaluated. Urine corticosterone levels were also investigated as a surrogate to measuring serum corticosterone. The results demonstrate that the pattern of responses to corticosterone or the stressors is different in mice and rats. Although, decreases in MHC Class II were found to be a sensitive indicator of stress in mice, only slight decreases were observed in rats with similar serum corticosterone AUC levels. Decreases in thymus weight were greater than spleen weight with corticosterone or ethanol or restraint stressor. No other single parameter or combination of parameters tested were obvious candidates as sensitive biomarkers of stress in rats. However, the good correlation between urine and serum corticosterone levels suggest that urine corticosterone may be a potential biomarker of stress induced changes to the immune response.