Ecological effects testing under the Toxic Substances Control Act: Acrylamide

Analysis of acrylamide aquatic toxicity data submitted under section 4 of the Toxic Substances Control Act revealed that for acute toxicity studies with bluegill, fathead minnow, and rainbow trout, concentrations of acrylamide necessary to produce 50% mortality decreased by 50-70% as a function of increasing exposure from 1 to 4 days. Analysis of acute toxicity data for Mysidopsis bahia suggested that increases in mortality with increased exposure to acrylamide might also be observed in saltwater organisms. Ratios of acute (4-day) LC₅₀ values to chronic (28-day) maximum acceptable toxic concentrations for this saltwater invertebrate were 26 for parent and offspring survival, 115 for female dry weights, and 975 for male dry weights. These ratios illustrate that long-term acrylamide exposure to sensitive life stages of M. bahia produced adverse effects on reproduction and growth at acrylamide concentrations significantly lower than those suggested by acute LC₅₀ or EC₅₀ values.     

Relative sensitivity of algae,bacteria, invertebrates,and fish to phenol: Analysis of 234 tests conducted for more than 149 species

An analysis was conducted to estimate the relative sensitivities of algae, bacteria, fish, and invertebrates to one chemical—phenol. Results from 234 studies to estimate the toxicity (EC₅₀ or LC₅₀ value) of phenol to 1 rotifer, 3 algal species, 4 leech species, 9 worm species, 12 fish species, 13 water mite species, 20 bivalve species, 22 crustacean species, greater than 26 bacterial species, and 39 insect species were analyzed to estimate relative, not absolute, sensitivities within and between species and between groups of species. EC50 values from conducting ten 5-min Microtox® tests were very similar as were EC50 values from conducting two activated sludge respiration inhibition tests. Three-fold and thirty-fold differences were observed for two 120-min Motility and two Oxygen-depletion tests, respectively. Most of the fish within-species variability could be accounted for by differences in test temperatures. Most of the invertebrate within-species variability could be accounted for by lab-to-lab or test-to-test variability. Differences between bacterial tests to measure five physiological endpoints (bioluminescence, respiration, growth, dehydrogenase, motility) were estimated. Differences between the EC50 values for the two tests to measure bioluminescence were significant. However, none of the mean EC50 values from tests to measure five physiological end-points appeared to be significantly different. Mean LC50 values from conducting fish tests were compared; only the rainbow trout and fathead minnow were significantly different. A comparison of relative species sensitivity suggested that bluegills, daphnids, fathead minnows, featherbacks, giant gourami grass shrimp, guppies, P. phosphoreum in the 5-min Microtox® test or rainbow trout were among the more sensitive species to the acute effects of phenol. A comparison of groups of species suggested that caddisfly or dragonfly larvae, crustaceans, daphnids, fish, shrimp and water fleas were among the more sensitive species groups to the acute effects of phenol.     

Toxicity of the synthetic pyrethroids,permethrin and AC 222, 705 and their accumulation in early life stages of fathead minnows and snails

Early life stages of fathead minnows (Pimephales promelas) were exposed to permethrin and AC 222, 705 and snails (Helisoma trivolvis) were exposed to permethrin in continuous flow-through exposures for approximately 30 days. Saturated solutions of each pesticide were used to avoid the use of solvents.Survival of newly hatched larvae and early juveniles was found to be the most sensitive measure of effect on fathead minnows of both pesticides. AC 222, 705 was approximately 20 times more toxic to fathead minnows than permethrin by the end of the test period. Based on the chronic limits, as defined in these tests, the predicted chronic no-effect concentrations for fathead minnows were between 0.66 and 1.4 μ/l for permethrin and 0.03 and 0.07 μ/l for AC 222, 705. The no-effect concentration for permethrin and snails was ≥ 0.33 μg/l. The chronic values for these compounds were approximately one-sixteenth and one-fourth of the corresponding 96-h LC₅₀ values, respectively, for fathead minnows in Lake Superior water.The mean bioconcentration factors (BCFs) for permethrin were 2800 for fathead minnows and 800 for snails. The mean BCF for AC 222, 705 and fathead minnows was 4000. Residue concentrations for both pesticides increased with increased water concentrations.     

Acute toxicity of tralopyril,capsaicin and triphenylborane pyridine to marine invertebrates

A need for environmentally acceptable alternative antifouling (AF) biocides has arisen through restrictions in the use of many common biocides in the European Union through the Biocidal Product Regulation (Regulation EU No. 528/2012). Three such alternatives are triphenylborane pyridine (TPBP), tralopyril and capsaicin. This study aims at extending the available information on the toxicity of these three emerging AF biocides to key marine invertebrates. Here we investigate the toxicity of tralopyril and capsaicin to the early life stages of the mussel Mytilus galloprovincialis and the sea urchin Paracentrotus lividus and also of tralopyril, capsaicin and TPBP to the early life stages of the copepod Tisbe battagliai. The EC₅₀ that causes abnormal development of mussel’s D-veliger larvae and impairs the growth of sea urchin pluteus larvae are respectively 3.1 and 3.0 μg/L for tralopyril and 3,868 and 5,248 μg/L for capsaicin. Regarding the copepod T. battagliai, the LC₅₀ was 0.9 μg/L for tralopyril, 1,252 μg/L for capsaicin and 14 μg/L for TPBP. The results obtained for the three substances are compared to a reference AF biocide, tributyltin (TBT), and their ecological risk evaluated. These compounds pose a lower environmental risk than TBT but still, our results suggest that tralopyril and TPBP may represent a considerable threat to the ecosystems.     

Effects of copper on development of the fathead minnow,Pimephales promelas Rafinesque

Embryos of the fathead minnow, Pimephales promelas Rafinesque, were exposed to total copper concentrations (Cu_T) of 0.6, 61, 113, 204, 338 and 621 μg/l from 5 to 10 h post-fertilization through 2 days post-hatch. A decrease in hatching time was observed with increasing total copper concentration but there was no decrease in embryonic developmental rate. Therefore, embryos hatched at earlier stages of development. Significant (P ≤ 0.05) declines in percent survival and percent total hatch were observed at 621 μg/l Cu_T) but not at 338 μg/l Cu_T or lower concentrations. The percentage of embryos with abnormalities was greater at 338 and 621 μg/l Cu_T than at 204 μg/l Cu_T and lower concentrations.Individuals exposed to copper during early development were then exposed to the same test concentrations for 28 days post-hatch. Survivors at 113 through 338 μg/l Cu_T were at earlier stages of development than were control fish. The percentage of fish surviving decreased with increasing copper concentration over the range 61 through 204 μg/l Cu_T. The percentage of fish surviving at 204 μg/l Cu_T was not significantly different from that at 338 μg/l Cu_T, and there were no survivors at 621 μg/l Cu_T. Surviving larvae at all copper concentrations from 61 through 621 μg/l Cu_T showed decreased length, weight and coefficient of condition compared to controls. The percentage of larvae with abnormalities increased significantly with increasing copper concentration. The calculated 96-h LC₅₀ (larval stage) was 250 μg/l Cu_T and the 28-day LC₅₀ (larval stage) was approximately 123 μg/l Cu_T.     

Role of glucuronide formation in the selective toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) for the sea lamprey: comparative aspects of TFM uptake and conjugation in sea lamprey and rainbow trout

This report is concerned with the comparative aspects of disposition and conjugation of 3-trifluoromethyl-4-nitrophenol (TFM), a selective sea lamprey larvicide in sea lamprey (Petromyzon marinus) and rainbow trout (Salmo gairdneri). When exposed to identical water concentrations of TFM, sea lamprey ammocoetes exhibited a higher rate of accumulation and an increased steady-state level of TFM compared to trout. Regardless of the life stage of sea lamprey or size of trout studied, sea lamprey revealed a higher concentration of free TFM in blood and tissues. The higher concentration of TFM in lamprey blood was demonstrated at a variety of water concentrations of TFM. In vitro glucuronyl transferase assays demonstrated that the sea lamprey had a lower rate of formation of TFM glucuronide when compared to trout. In vivo, sea lamprey exhibited a much higher ratio of free to conjugated TFM than trout. Pretreatment of sea lamprey and trout with salicylamide, an inhibitor of glucuronyl transferase, shifted the LC₅₀ for trout from 9.7 mg/liter to 3.6 mg/liter and did not alter the LC₅₀ for sea lamprey.     

The Toxicity of Acetylenic Alcohols to the Fathead Minnow,Pimephales Promelas: Narcosis and Proelectrophile Activation

1. The 96-h LC₅₀ values for 16 acetylenic alcohols in the fathead minnow (Pimephales promelas) were determined using continuous-flow diluters. The measured LC₅₀ values for seven tertiary propargylic alcohols agreed closely with the QSAR predictions based upon data for other organic non-electrolytes acting by a narcosis mechanism.

2. Four primary and four secondary propargylic alcohols were 7 to 4600 times more toxic than the respective narcotic toxicity estimated by QSAR. Metabolic activation to electrophilic α,β-unsaturated propargylic aldehydes or ketones is proposed to account for the increased toxicity.

3. 3-Butyn-1-o1 and 4-pentyn-2-ol, primary and secondary homopropargylic alcohols, were 320 and 160, respectively, times more toxic than predicted. In this case an activation step involving biotransformation to an allenic electrophile intermediate was proposed.     

Development toxicity of functionalized single-walled carbon nanotubes on rare minnow embryos and larvae

Abstract

Carbon nanotubes (CNTs) are widely used in industrial and commercial applications, but few studies systematically evaluate their developmental toxicity on aquatic organism. Using rare minnow (Gobiocypris rarus) at early life stages as experimental models, developmental toxicity of functionalized single-walled CNTs (SWCNTs) was investigated following exposure to 0–320?mg/L for 144?h. Results revealed that significantly increased in mortality and malformation was only observed after hatching. Decreased body length, heart rate and swimming speed provide a concentration-dependent manner on larvae; values of 144?h LC₅₀ and EC₅₀ were 140.8 and 109.8?mg/L, respectively. Antioxidant enzyme activities (superoxide dismutase, catalase and glutathione S-transferase) and antioxidant enzyme related mRNA expressions were significant changed; cell apoptosis activities (caspase-3, -8, -9) and cell apoptosis related mRNA expressions were significant up-regulated; reactive oxygen species and DNA damage were significantly induced when the concentration of SWCNTs above 100?mg/L. Fluorescence and electron microscopy sliced observation show that SWCNTs were well dispersed in larvae within 0.5?h, eventually cleared from the larvae at 144?h. This is the first study to define uptake kinetics and to focus on behavioral consequences, physiological changes and mRNA expression following SWCNTs exposure in the early life stages of fish. The results obtained in the present study demonstrated that functionalized SWCNTs have the potential to affect aquatic life when released into the aquatic environment and reached high concentration. In the increasing economical context of SWCNTs, complementary studies must be undertaken, especially including mechanistic and environmental investigations.     

The relative sensitivity of microtox®, daphnid,rainbow trout,and fathead minnow acute lethality tests

Relative sensitivity and correlations between the Microtox® test and three commonly used acute lethality bioassays (i.e., rainbow trout, fathead minnow, Daphnia) are reviewed and discussed. All relevant data available for comparison were separated and evaluated based on chemical groupings. Generally, Microtox was more sensitive than or as sensitive as the acute lethality tests for pure individual organics, but was less sensitive to most inorganics. Microtox was not as sensitive as acute lethality tests to effluents or leachates with a high component of insecticides, herbicides, inorganics, pharmaceuticals or textiles, or highly lipophilic contaminants. As the complexity and toxicity of industrial effluents increased, the correlations and sensitivity of Microtox increased, with a corresponding decrease in data variability. Additionally, limitations of the available data are that (1) comparisons of relative sensitivity varied with the compounds and organisms tested; (2) there was a lack of standardization in approach and a failure to provide adequate details on the origin of the cited toxicity data, which confused and obscured comparisons; and (3) published reviews commonly failed to identify fish species and test details for cited data.     

The joint toxicity of pesticide tank-mixes to rainbow trout

The joint toxicity to rainbow trout fingerlings (Salmo gairdneri Richardson) of eleven tank-mixes composed of pairs of six fungicides and herbicides (prochloraz, fenpropimorph, diclofop-methyl, tridemorph, benzoylprop-ethyl and propiconazole) was measured using 96 h LC₅₀ tests. The toxicity of the mixtures ranged from being half of that expected on the basis of additive toxicity of the components, to less than 1.4 times the expected value. In view of the degree of experimental error inherent in the LC₅₀ determinations, these data provide no evidence for the existence of synergistic (i.e. more-than-additive) toxicity of the respective tank-mixes.     

Window of sensitivity for the estrogenic effects of ethinylestradiol in early life-stages of fathead minnow,Pimephales promelas

Sexual differentiation in fish occurs after hatching during early life-stages and is believed to be a time when the gonad has a heightened sensitivity to disruption by chemicals that mimic hormones. In this study fathead minnows (Pimephales promelas) were exposed to an environmentally relevant concentration of ethinylestradiol (EE2) for short intervals in fish early life-stages and vitellogenic and gonadal responses were measured at 30 and 100 dph (sexual maturity), respectively. All EE2 exposure regimes induced vitellogenin (VTG) synthesis and disruption in duct development (a feminization) in males, with a window of enhanced sensitivity between 10 and 15 dph (where 60% of the males had feminized ducts). There was an altered pattern in sex cell development in males (inhibition of spermatogenesis) in the solvent controls (ethanol 0.1 ml/l) and all EE2 treatments when compared with the dilution water controls. Furthermore, fewer spermatozoa were observed in the testis of males exposed to EE2 from 15 to 20 dph and fertilized eggs (<24 h post-fertilization)-20 dph, compared with both the solvent and dilution water controls. These data show that short exposures of embryos/very early life-stage fathead minnows to an environmentally relevant concentration of EE2 lead to alterations in gonadal development that potentially have reproductive consequences and thus population level effects.     

Estimation of toxicity to marine species with structure-activity models developed to estimate toxicity to freshwater fish

Structure-activity models which were developed to estimate toxicity of chemicals to freshwater fish were tested for use with an estuarine fish (Cyprinodon variegatus) and mysids (Mysidopsis bahia). Significant linear and polynomial relationships that correlated well existed between reported 96-h LC₅₀ values for each marine species and log P (log octanol/water partition coefficient). Good linear relationships were obtained when the 96-h LC₅₀ values for C. variegatus and M. bahia were regressed on water solubility (μmol/l). These models were compared to models developed for freshwater fish using log P and log S. Models using log P to estimate acute toxicity for two freshwater fish produced 96-h LC₅₀ values similar to those measured for C. variegatus and M. bahia, whereas, those models developed with water solubility produced 96-h LC₅₀ values similar to those for C. variegatus, but not for M. bahia. The data indicated that models developed with log P for freshwater fish can be used to estimate toxicity to C. variegatus for a minimum of 58% of the chemicals, whereas models using water solubility estimated toxicity to C. variegatus for a minimum of 77% of the chemicals within an order of magnitude for screening purposes. The calculated 96-h LC₅₀ values were compared to the measured values for each marine species and those measured for Pimephales promelas (fathead minnow) and Poecilia reticulata (guppy). Tests indicated generally that calculated 96-h LC₅₀ values were overestimates of the measured 96-h LC₅₀ values when models for freshwater fish were used to estimate toxicity to each marine species. More data are required for marine species to determine if highly significant relationships between marine and freshwater fish exist with comparisons using larger sample sizes.     

Species-related differences in the inhibition of brain acetylcholinesterase by paraoxon and malaoxon

Interspecies comparisons indicate that fish are relatively more resistant to acute intoxication with parathion and paraoxon than are rodents. In contrast, fish are more sensitive to malathion and malaoxon. The following investigation was designed to determine if species-related differences in the sensitivity of brain acetylcholinesterase (AChE) to inhibition by paraoxon and malaoxon could contribute to the interspecies differences in toxicity. Brain AChE activity was significantly greater in fathead minnows and rainbow trout than in rats and mice. The fathead minnow and rainbow trout IC50 values for paraoxon were 228- to 1879-fold greater than the corresponding values for rat and mouse. Similarly, the Ki (bimolecular inhibition constant) was 159- to 1663-fold greater in rodents than in fish, which reflected both a higher KA (association constant) and kp (phosphorylation constant) in rodents. The rodent IC50 values for malaoxon were 30-80% that of the fish IC50, and the Ki was 30-50% greater in rodents than in fish. These data suggest that the greater sensitivity of rodent brain AChE to inhibition by paraoxon may contribute to the greater toxicity of parathion and paraoxon in rodents than in fish. In contrast, the lack of correlation between the inhibition of brain AChE by malaoxon and species-related differences in acute I D50 suggests that other factors, such as the limited carboxylesterase activity in fish, may be responsible for this species selectivity.     

Hydrolysis of permethrin,a pyrethroid insecticide,by rainbow trout and mouse tissues in vitro: A comparative study

Permethrin, a pyrethroid insecticide, is highly toxic to fish relative to its toxicity to mammals and the role of permethrin metabolism in this differential toxicity has not been investigated. A previous study, however, has shown that little hydrolysis of permethrin occurs in vivo in rainbow trout in contrast to mammals where ester hydrolysis is a major detoxification reaction. In the present study, the rates of permethrin hydrolysis in rainbow trout and mouse tissues in vitro were estimated. Mouse liver, kidney, and plasma incubated at 37°C, hydrolyzed trans-[¹⁴C]permethrin approximately 166, 38, and 59 times faster, respectively, than the same rainbow trout tissues incubated at 12°C. At an incubation temperature of 37°C trout liver microsomes hydrolyzed trans-permethrin approximately 45 times slower than mouse liver microsomes. When the total capacity of trout and mouse tissues to hydrolyze trans-permethrin was compared on a whole body basis mice hydrolyzed trans-permethrin 184 times faster than rainbow trout. The results suggest that rainbow trout tissues have a much lower capacity than mouse tissues to hydrolyze permethrin, and this may explain the relative absence of permethrin hydrolysis products in permethrin-exposed raibow trout. It is impossible that the high toxicity of permethrin to rainbow trout is in part related to a low capacity to hydrolyze permethrin.     

Effect of hydroxymethylfurfural (HMF) on mortality of artificially reared honey bee larvae (<Emphasis Type="Italic">Apis mellifera carnica</Emphasis>)

Hydroxymethylfurfural (HMF) is a heat-formed, acid-catalyzed contaminant of sugar syrups, which find their way into honey bee feeding. As HMF was noted to be toxic to adult honey bees, we investigated the toxicity of HMF towards larvae. Therefore we exposed artificially reared larvae to a chronic HMF intoxication over 6 days using 6 different concentrations (5, 50, 750, 5000, 7500 and 10,000 ppm) and a control. The mortality was assessed from day 2 to day 7 (d7) and on day 22 (d22). Concentrations ranging from 5 to 750 ppm HMF did not show any influence on larval or pupal mortality compared to controls (p > 0.05; Kaplan–Meier analysis). Concentrations of 7500 ppm or higher caused a larval mortality of 100 %. An experimental LC₅₀ of 4280 ppm (d7) and 2424 ppm (d22) was determined. The calculated LD₅₀ was 778 µg HMF per larva on d7 and 441 µg HMF on d22. Additionally, we exposed adult honey bees to high concentrations of HMF to compare the mortality to the results from larvae. On d7 larvae are much more sensitive against HMF than adult honey bees after 6 days of feeding. However, on d22 after emergence adults show a lower LC₅₀, which indicates a higher sensitivity than larvae. As toxicity of HMF against honey bees is a function of time and concentration, our results indicate that HMF in supplemental food will probably not cause great brood losses. Yet sublethal effects might decrease fitness of the colony.     

Method for detection and quantitation of fathead minnow vitellogenin (Vtg) by liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry

Vitellogenin (Vtg) is a well-recognized biomarker of estrogen exposure in many species, particularly fish. This large protein shares a high degree of sequence homology across a large number of species. Quantitative measurement is currently done using antibody-based assays. These assays frequently require purification of Vtg and antibody production from each species because there is poor cross reactivity between antibodies for different fish. Therefore, complementary methods of measuring Vtg are desirable. Mass spectrometric (MS) analysis coupled to database searching offers the promise of a general method for protein identification. In this study, we used the well-characterized Vtg from rainbow trout (Oncorhynchus mykiss) to evaluate the analytical parameters for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the intact protein. The low sensitivity observed for the intact protein demonstrated that a proteolytic digestion would be necessary for MALDI-MS identification of Vtg. An analytical scale high performance liquid chromatography (HPLC) separation combined with MALDI-MS was used to measure and confirm the identity of Vtg from the plasma of an important species for regulatory agencies, fathead minnow (Pimephales promelas). The small volume requirement of this method (<10 microL) was found to be compatible with the plasma volume obtained from a few minnows. Importantly, the HPLC-MALDI-MS measurement of fathead minnow Vtg abundance after exposure to estradiol was similar to previously obtained enzyme linked immunosorbant assays (ELISA) data.     

The relationships among CYP1A induction, toxicity, and eye pathology in early life stages of fish exposed to oil sands

Exposure of the early life stages of fish to oil sands constituents is associated with mortality and larval malformations such as edemas, hemorrhages, and skeletal, craniofacial, and eye defects. In fathead minnow (Pimephales promelas) and white sucker (Catostomus commersoni) larvae, indices of total eye pathology increased significantly following oil sands exposure. Structural, cytoplasmic, inflammatory, and degenerative eye alterations included poor retinal differentiation, microphthalmia, optic fissures, dysphasic retinas and lenses, inflammatory infiltrates, retinal epithelial lifting, and necrotic foci. Cytochrome P-4501A (CYP1A) was expressed in ocular (retina, lens) and kidney endothelial tissues, as indicated by immunohistochemistry. Although the kinetics of exposure-response curves for mortality and CYP1A expression were similar in both species, species differences in the magnitude and sensitivity of the responses were observed. Oil sands were twofold more toxic to fathead minnows (TPAH LC50 = 47-330 microg/g) than to white sucker (TPAH LC50 = 95-860 microg/g) larvae. For both species, larval mortality was significantly related to CYP1A protein concentrations in kidneys, and severity of these effects rose with oil sands exposure. The relationships among eye damage, mortality, and CYP1A indices warrants further investigation, and may lead to the use of CYP1A induction as an indicator of adverse effects rather than just contaminant exposure.     

Acute toxicity of chlorinated organic chemicals to the embryos and larvae of the marine worm Platynereis dumerilii (Polychaeta: Nereidae)

The acute toxicity of a range of organic reference chemicals has been evaluated using the embryo-larval stages of the marine polychaete Platynereis dumerilii (Audouin and Milne-Edwards). After exposure under static conditions at 20±1°C, EC₅₀ values were calculated based upon reductions in normal embryo development. After exposure of 7 day old larvae under the same conditions, LC₅₀ values were calculated based on larval survival. The embryo development 48 h EC₅₀ and larval 48 h LC₅₀ values were, respectively, 6.39 and 8.94 mg L⁻¹ for 3,4-dichloroaniline, 122 and 104 mg L⁻¹ for phenol, 23.6 and 14.4 mg L⁻¹ for 4-chlorophenol, 4.24 and 3.20 mg L⁻¹ for 3,5-dichlorophenol, 2.55 and 4.24 mg L⁻¹ for 2,4,5-trichlorophenol, and 0.52 and 1.33 mg L⁻¹ for sodium pentachlorophenate (all based on nominal values). Comparison of these data with other published values suggest that, for these reference toxicants, the embryo–larval stages of P. dumerilii are of similar sensitivity to the early life stages of several other marine invertebrate species. These observations add to the refinement of a suite of bioassay techniques using P. dumerilii which, linked to previous studies, can be used to evaluate the developmental toxicity, cytotoxicity, and genotoxicity of marine contaminants. © 1998 John Wiley & Sons, Inc. Environ Toxicol Water Qual 13: 149–155, 1998     

Physiological stress in fish during toxicological procedures: A potentially confounding factor

An increasing body of evidence suggests that stress may modulate the response of an animal to toxicological challenge. The degree of stress inherent in the procedures comprising a fish LC₅₀ study (OECD guidelines for the testing of chemicals, “Fish, Acute Toxicity Test” No. 203, adopted May 12, 1981) was assessed by measuring plasma cortisol levels (rainbow trout) and whole-body levels of immunoreactive corticosteroids (zebra fish). Aspects of the 96 h LC₅₀ protocol which were found to evoke corticosteroidogenic activity in the test fish included transfer from holding tanks to test tanks, tank transfer during semistatic procedures, and disturbance associated with water quality measurement. There was evidence for acclimation of the fish to repetitive stimuli such as tank transfer, but, overall, the data suggest that the 96 h test protocols evoke a state of chronic stress in the test population. The removal of fish from test vessels during longer term studies was also found to evoke a stress response in fish remaining within the vessel, suggesting that these conditions may expose test fish to a regime of intermittent periods of acute stress. The possibility that physiological stress may interfere with the accurate estimation of toxicological end points was examined. In three independent LC₅₀ tests the toxicity of the arylmethane dye, malachite green, to rainbow trout was found to be markedly and consistently reduced when the degree of disturbance and handling during the test period was minimized. © by John Wiley & Sons, Inc.