Inhibition of swim bladder inflation in Japanese medaka (Oryzias latipes) embryos following exposure to select pharmaceuticals alone and in combination.

This study leveraged the Japanese medaka fish embryo model for the assessment of effects of select contaminants on early development in fish. Fish embryos were exposed to various pharmaceutical contaminants including synthetic hormones and non-steroidal anti-inflammatory drugs and their effects on development were observed. Initial screening determined that swim bladder inflation failure was the most common endpoint detected. Swim bladder inflation failure was first explored in a study demonstrating that medaka require access to the air-water interphase to inflate their swim bladders in a time-dependent manner, and swim bladder inflation failure was correlated with mortality. Fish embryos were exposed 24-hours post fertilization until hatch to concentration ranges of various pharmaceutical contaminants including: 17ß-estradiol, 17α-ethinylestradiol, and levonorgestrel (1 to 1000 µg/L), or diclofenac (0.32 to 100 mg/L). The main effect observed across all four compounds was a significant increase in failure of swim bladder inflation with increasing exposure concentration (24 to 72-hours post-hatch). Following single compound experiments combinatorial exposures using no-observed-effect concentrations were conducted. The main effect observed was a significant decrease in inflation success 24-hours post-hatch following a binary mixture of levonorgestrel and 17α-ethinylestradiol, as well as a significant decrease in swim bladder inflation success at all times following exposure to a quaternary mixture of all four compounds. This study demonstrated that embryonic exposure to pharmaceutical compounds, both alone and in combination, resulted in failure of swim bladder inflation in larval Japanese medaka.

Evaluating cumulative effects of anthropogenic inputs in Prince Edward Island estuaries using the mummichog (Fundulus heteroclitus).

Estuarine eutrophication as a result of agricultural land use, including the use of chemical fertilizers, is increasing worldwide. Prince Edward Island (PEI), Canada has very high agricultural intensity by international standards with approximately 44% of the land area under production, and some watersheds in excess of 75% agricultural land-use. The type of agriculture is also intensive with primarily row crops that have high chemical fertilizer and pesticide usage. In light of these stressors, the hypothesis of this study was that mummichog (Fundulus heteroclitus) population parameters would change with point and nonpoint source pollution, and that multivariate statistics could be used to draw associations with specific stressors. Fish were sampled on a monthly basis from May through August at 7 estuaries spanning a range of land use, nutrient, and contaminant loadings. A suite of environmental variables were simplified into 3 principal components: PC1 representing agricultural land use, N loading, and plant habitat, PC2 being dominated by sediment sand and silt distribution, and PC3 largely reflecting P loading and sediment organic matter. There were significant differences in abundance of both adult and young-of-the-year mummichog, and these changes associated most strongly with PC1, the largely N-driven agricultural influences. In contrast, somatic variables such as liver and gonad size did not show strong association with the environmental quality principal component scores. The sand and silt PC2 appeared to have the opposite association with the biological data, with siltier environments correlating to older, larger, less dense populations of mummichog. Although pesticide residues were detected in estuarine sediment, there was no clear relationship between these and watershed agricultural intensity or biochemical indicators. There was, however, a strong relationship between agricultural environmental variables (PC1) and in vitro steroid production that is suggestive of a potential chemical effect. Eutrophication appeared to be a primary stressor affecting mummichog populations, as nutrient enrichment was associated with changes in habitat variables and these in turn were associated with high mummichog density. Thus, mummichog population demographics appear to have use as an indicator of adverse or worsening conditions in estuaries. We concluded that, based on the subset of environmental factors evaluated, the nonpoint-source inputs of sediments and nutrients exerted the greatest influence on mummichog populations in PEI estuaries.

Trace metal burdens and expression of heat stable low molecular mass proteins in the female asteroid Coscinasterias muricata--Fluctuations throughout the reproductive cycle.

Concentrations of the essential trace metals copper and zinc were measured in the pyloric caeca of female Coscinasterias muricata sampled from Port Phillip Bay, Victoria, Australia at monthly intervals for 15 months. Results indicate that the concentrations of these metals appear to be regulated by the reproductive cycle. Peaks in zinc concentration occurred simultaneously with peaks in progesterone concentration in the pyloric caeca. These findings suggest that zinc plays a role in gametogenesis and ovarian development. Fluctuations in pyloric caeca copper concentration appear inversely related to the pyloric caecal index and associated with oocyte diameter, indicative of a role for this metal in oogenesis. The levels of cytosolic heat stable low molecular mass (LMM) proteins were examined throughout the sampling period. Heat stable LMM proteins (metallothionein-like) of 12 kDa and 7.5 kDa (apparent molecular mass) and the concentrations of copper appear to be related; although the exact nature of these proteins remains unknown. No such relationship was observed between the levels of heat stable LMM proteins and zinc.

Effect of crude oil contaminated sediment exposure on cytochrome P450 enzymes in the Australian asteroid Coscinasterias muricata.

Levels of cytochrome P450 enzymes were measured in pyloric caeca microsomes of the asteroid Coscinasterias muricata following exposure to sediment with nominal concentrations of 0, 0.1 or 2 ml crude oil kg(-1) (dry weight) and subsequent depuration. No significant differences were observed in total cytochrome P450 levels or cytochrome P418 levels following the exposure period. However after five days of depuration, levels of total P450 in the pyloric caeca of C. muricata exposed to the highest oiled sediment concentration were significantly lower than in specimens exposed to the other treatments. Cytochrome P418 levels were inversely related to total P450 levels following exposure and subsequent depuration. Preliminary results show that levels of CYP1A-like immunopositive protein (CYP1A-like IPP) in exposed asteroids exhibited a concentration response relationship following the exposure period. Variations in CYP1A-like IPP levels observed during the depuration period may be influenced by the sublethal toxicity of hydrocarbons within the crude oil.

Avoidance of crude-oil contaminated sediment by the Australian seastar, Patiriella exigua (Echinodermata: Asteroidea).

This study attempted to determine whether Patiriella exigua, an Australian seastar, could detect and/or avoid oiled sediment when given an equal choice of unoiled sediment. The sediment was spiked once to produce one of three concentrations of oiled sediment used in the test chambers versus unoiled sediment. Behavioral observations were repeated over a 32 day period to test the effects of aging the oiled sediment. Results show that Patiriella exigua was capable of detecting oiled sediment and/or an oiled environment. Seastars avoided oiled sediment, with significantly higher numbers choosing either to reside on the clean sediment (p<0.05) or to travel up the glass sides of the tanks (p<0.001). Avoidance of oiled sediment increased with increasing sediment oil concentrations. Aging the oiled sediment decreased the oil content of the sediment and increased the number of seastars able to inhabit it (p<0.001). A potential narcotic effect of exposure to oiled sediment was observed.

The impact of oil-derived products on the behaviour and biochemistry of the eleven-armed asteroid Coscinasterias muricata (Echinodermata).

The present study examines the impact of exposure to oil-derived products on the behaviour and physiology of the Australian 11-armed asteroid Coscinasterias muricata. Asteroids were exposed to dilutions of water-accommodated fraction (WAF) of Bass Strait stabilised crude oil, dispersed oil or burnt oil (n = 8) for 4 days whereby, prey-localisation behaviour was examined immediately after exposure, and following 2, 7, and 14 days depuration in clean seawater. The prey-localisation behaviour of asteroids exposed to WAF and dispersed oil was significantly affected though recovery was apparent following 7 and 14 days depuration, respectively. In contrast, there was no significant change in the prey-localisation behaviour of asteroids exposed to burnt oil. Behavioural impacts were correlated with the total petroleum hydrocarbon concentrations (C6-C36) in each exposure solution, WAF (1.8 mg l(-1)), dispersed oil (3.5 mg l(-1)) and burnt oil (1.14 mg l(-1), respectively. The total microsomal cytochrome P450 content was significantly lower (P(Dunnett test) < 0.01) in asteroids exposed to dispersed oil than in any other asteroids, whilst asteroid alkaline phosphatase activity was not significantly affected (P(ANOVA) = 0.11). This study further documents the deleterious impact of dispersed oil to marine organisms and supports further research in the area of in situ burning as a less damaging oil spill response measure towards benthic macro-invertebrates.

A rapid response toxicity test based on the feeding rate of the tropical cladoceran Moinodaphnia macleayi.

Recent research has reported that the feeding rate of the cladoceran Daphnia magna (measured over 24 h) is a sensitive indicator of toxicity. Using a methodology similar to that used for D. magna, a feeding test for the Australian tropical cladoceran Moinodaphnia macleayi was developed and the sensitivity of the test was compared with the currently used reproduction test. The results of the study revealed that the feeding rate of M. macleayi was a more sensitive indicator of toxicity than reproduction for cadmium; a mean concentration of 1.4 microg/L caused significant inhibition of feeding (P < or = 0.05), while reproduction was significantly reduced at 3.1 microg/L (P < or = 0.05). No feeding inhibition was detected when M. macleayi were exposed to copper, despite significant mortality. The difference in feeding and reproductive responses of M. macleayi to cadmium and copper suggest differing modes of action for the two metals. The feeding test was validated on two samples of mine release water containing various metals. Results demonstrated that a cladoceran feeding test, performed over a 20-h period, was comparable in sensitivity to a reproduction test performed over a 5- to 6-day period.

EROD activity, serum SDH and PAH biliary metabolites in sand flathead (Platycephalus bassensis) collected in Port Phillip Bay, Australia.

In order to evaluate the health status of fish inhabiting Port Phillip Bay, Australia, southern sand flathead (Platycephalus hassensis, N = 133) were collected at six stations throughout the Bay. Fish had a similar serum sorbitol dehydrogenase (SDH) activity level (p = 0.12), indicating that they were not experiencing hepatocellular injuries. Ethoxyresorufin-O-deethylase (EROD) activity was generally lower in the non-urbanized and non-industrialized southern part of the Bay. The highest EROD activity was observed in Hobson Bay, the closest station from Melbourne city. Naphthalene-type biliary metabolites were also highest in Hobson Bay with intermediate levels found in Corio Bay where refineries are present. An opposite trend was observed with the pyrene-type bile metabolites, the highest levels being observed in Corio Bay while intermediate levels were found in Hobson Bay. The ratio of naphthalene-type to benzo(a)pyrene (B(a)P)-type metabolites indicate that relatively to other sites sampled in Port Phillip Bay, Corio Bay is subjected to enriched petroleum hydrocarbons of pyrolytic origin. Temporal trends indicate that the availability of xenobiotics to fish remained unchanged over the 1990s.

Comparative sensitivity of three populations of the cladoceran Moinodaphnia macleayi to acute and chronic uranium exposure.

Assessment of differences in the response of three different populations of the tropical cladoceran Moinodaphnia macleayi to uranium exposure was evaluated. The populations tested included a laboratory stock (maintained for 10 years), a wild population collected from Bowerbird Billabong (an uncontaminated environment), and a population collected from Djalkmara Billabong (a relatively contaminated environment with elevated levels of uranium), located on the Ranger uranium mine site, Jabiru East, NT, Australia. Chronic and acute toxicity of uranium was determined for all three populations. The no-observed-effect-concentration (NOEC; reproduction) and lowest observed-effect-concentration (LOEC; reproduction) for uranium ranged between 8-31 micrograms L-1 and 20-49 micrograms L-1, respectively, for all three populations. The 48 h EC50 (immobilization-lethality) for uranium ranged between 160-390 micrograms L-1 for all three populations. There was little difference in the response of the three populations of M. macleayi to acute and chronic uranium exposure, although the response of the laboratory population to chronic uranium exposure appeared more variable than the "wild" populations. There was no apparent tolerance in the population of M. macleayi obtained from Djalkmara Billabong when exposed to elevated levels of uranium. M. macleayi was significantly more sensitive to uranium exposure than other species previously tested. It was concluded that the sensitivity of the laboratory population (to uranium) is still representative of natural M. macleayi populations.

The acute and chronic toxicity of cadmium and zinc to two hydra species.

The potential of two hydra species, Hydra vulgaris (pink) and Hydra viridissima (green), for use as invertebrate models for toxicity testing of waterborne metals was investigated. The acute and subchronic toxicities of cadmium (a nonessential metal) and zinc (an essential metal) were determined. Results showed that both the hydra species were more sensitive to cadmium than to zinc, and that green hydra were more sensitive than pink hydra. The mean (SE) 96 h LC50 values of cadmium and zinc for pink hydra were 83 (8.5) and 2300 (150) micrograms/L, respectively. For green hydra, the respective 96 h LC50 values for cadmium and zinc were 3.0 (0.0) and 935 (46.5) micrograms/L. The respective 7-day no-observed-effect-concentrations (NOEC) and lowest-observed-effect-concentrations (LOEC) for pink hydra were < 13 and 13 micrograms/L for cadmium, and < 250 and 250 micrograms/L for zinc. The respective 7-day NOEC and LOEC values for green hydra were 0.4 and 0.8, microgram/L for cadmium, and 38 and 75 micrograms/L for zinc. Neither 1, 2, or 3 x 90-min pulse-exposures to 0.4, 0.8, or 1.5 micrograms/L of cadmium had any significant deleterious effect on total green hydra numbers after seven days in clean water. Green hydra appeared to be excellent freshwater invertebrate models for testing dissolved metals based on their sensitivity and the ability to rapidly assess population reproduction in the laboratory.

EROD induction and biliary metabolite excretion following exposure to the water accommodated fraction of crude oil and to chemically dispersed crude oil.

Immature Atlantic salmon (Salmo salar) were exposed to water accommodated fraction (WAF) of Bass Strait crude oil or to Corexit 9527-dispersed crude oil for 6 days, followed by a depuration period of 29 days. Serum sorbitol dehydrogenase (SDH) levels, indicator of liver damages, remained low during the experiment. Hepatic EROD activity was induced within 2 days following the onset of the exposure in both treatments, and persisted for 2-4 and 4-6 days after transfer to clean sea water in the WAF and dispersed oil treatment, respectively. Naphthalene-type metabolites, determined by fixed-wavelength fluorescence detection, appeared in the bile of the fish with 2 days' delay compared to EROD induction. In both treatments, EROD activity induction and levels of naphthalene-type metabolites in the bile were significantly related. The biliary levels of naphthalene-type metabolites were over 15 times higher in fish exposed to dispersed crude oil relative to fish exposed to the WAF of Bass Strait crude oil. BaP-type metabolites appeared only in the bile of the fish exposed to the WAF, possibly due to BaP-type compounds remaining associated with the dispersant in the water column or to an inhibition of Phase II detoxification enzymes by the dispersant. Bile metabolites as determined by fixed-wavelength fluorescence and EROD induction appear to be sensitive and complementary biomarkers of exposure to PAH.

Metabolic enzyme activities in fish gills as biomarkers of exposure to petroleum hydrocarbons.

Metabolic effects of low-level exposure of Atlantic salmon (Salmo salar) to the water accommodated fraction (WAF) of crude oil and to dispersed crude oil were studied. Aerobic enzymes citrate synthase and cytochrome C oxidase, and anaerobic enzyme lactate dehydrogenase were measured in gills during a 4-day exposure to low concentrations of dispersed Bass Strait crude oil and WAF, and during the following 8 days of depuration in clean seawater. Relative to pre-exposure levels, citrate synthase and lactate dehydrogenase exhibited a significant inhibition of activity during exposure to the WAF of crude oil, and to dispersed crude oil, while activity of cytochrome C oxidase remained unchanged. Citrate synthase activities returned to preexposure levels after 4 days following termination of exposure for the WAF-exposed fish, and after 2 days for the dispersed-oil-exposed fish. After the termination of exposure to both treatments, lactate dehydrogenase activity remained low relative to levels measured prior to exposure, which indicated that the activity of this enzyme may be a sensitive medium to long-term biomarker of exposure to petroleum-contaminated water bodies.

Potential of two hydra species as standard toxicity test animals.

The potential for using pink hydra (Hydra vulgaris) and green hydra (Hydra viridissima) as a model invertebrate for the toxicity testing of xenobiotics was investigated. Test compounds were 4-chlorophenol, endosulfan, and copper. The reference toxicant 4-chlorophenol was used as a standard during toxicity testing to ensure the sensitivity of hydra did not change over time. Hydra had a low sensitivity to 4-chlorophenol and endosulfan compared to other freshwater species. The 96-h LC50 (SE) values for 4-chlorophenol and endosulfan were 32 mg/L (1.3) and 0.81 mg/L (0.1), respectively, for pink hydra, and 45 mg/L (6.1) and 0.67 mg/L (0.02), respectively, for green hydra. Based on population growth rates, the 6-day NOEC and LOEC results for pink hydra exposed to 4-chlorophenol and endosulfan were <1.1 and 1.1 mg/L, and 0.044 microg/L and 0.080 mg/L, respectively; results for green hydra were 10.3 and 22.3 mg/L, and 0.060 and 0.080 mg/L, respectively. Following exposure to copper, the 96-h LC50 (SE) values were 26 microg/L (3.4) for pink hydra and 8.5 microg/L (0.3) for green hydra, respectively. Based on population growth rates, the 7-day population growth NOEC and LOEC values for both pink and green hydra exposed to copper were 4 and 8 microg/L, respectively. Results indicate that hydra have the potential for use in acute and subchronic toxicity testing of inorganic toxicants, but have a low sensitivity to organic toxicants.

Chronic toxicity of diethylenetriamine pentaacetic acid to crimson-spotted rainbowfish (Melanotaenia fluviatilis): effects on reproduction, condition, and ethoxyresorufin O-deethylase activity.

The chronic effects of the chelating agent diethylenetriamine pentaacetic acid (DTPA) on reproduction, condition factor, liver somatic index (LSI), gonad somatic index (GSI), and ethoxyresorufin O-deethylase (EROD) activity of adult Australian crimson-spotted rainbowfish (Melanotaenia fluviatilis) were assessed. Breeding groups of three females and two males were exposed to 0, 1, 10, or 100 mg/liter DTPA (nominal) in a 28-day "static-renewal" experiment. Overall, the toxicity of DTPA to adult crimson-spotted rainbowfish was relatively low. Reproduction was not affected at concentrations up to 100 mg/liter DTPA, although an early effect on hatchability was potentially attributed to direct toxicity to rainbowfish eggs. DTPA also had little effect on the condition of adult rainbowfish, with condition factor and GSI being unaffected at concentrations up to 100 mg/liter, the latter finding supporting the reproduction results. However, LSI in male rainbowfish exposed to 100 mg/liter was significantly lower than in those exposed to 1 mg/liter DTPA (P

The effects of exposure duration and feeding status on fish bile metabolites: implications for biomonitoring.

Biliary metabolites of 2-chlorosyringaldehyde (2-CSA), the major chlorinated phenol found in chlorine dioxide bleached eucalypt pulp effluent, have been found to be sensitive biomarkers of effluent exposure in the sand flathead (Platycephalus bassensis). Before this method of biomonitoring can be applied in the field, the influences of exposure duration, depuration time, and fish feeding status on the level of this metabolite should be determined. In this study, sand flathead were exposed to a measured concentration of 0.3 microgram/1 of 2-CSA for 1, 2, 4, 8, 12, or 16 days. Fish previously exposed to 2-CSA were then held in sea-water alone for 1, 2, 3, 4, or 6 days. Fish were fed ad libitum throughout the experiment, and the fullness of the fish's stomach at the time of sampling was noted. There were no effects of exposure on biotransformation enzyme activities, either between exposure times or between the exposure and depuration periods. The major metabolite of 2-CSA, 2-chloro-4-hydroxy-3,5-dimethoxybenzylalcohol (2-CB-OH), was first detected in the bile of some fish sampled after 24 h of exposure, and the mean concentration of 2-CB-OH in the bile increased over the exposure period. The mean concentration (+/- SE) of 2-CB-OH in the bile was strongly influenced by fish feeding status, being 94 +/- 18 ng/ml bile in fish with empty stomachs and undetectable in fish with full stomachs. Bile volume was also influenced by fish feeding status, being greatest in fish with empty stomachs at the time of sampling. Results indicate that the feeding status of fish should be taken into consideration when using biliary metabolites as biomarkers of effluent exposure in the field, and methods to establish this are discussed.

Comparative acute and chronic toxicity of diethylenetriamine pentaacetic acid (DTPA) and ferric-complexed DTPA to Daphnia carinata.

The acute and chronic toxicity of diethylenetriamine pentaacetic acid (DTPA) and ferric complexed DTPA (Fe[III]-DTPA) to Daphnia carinata were compared, while the effects of DTPA exposure prior to and/or during 1st brood embryogenesis were also assessed. For chronic exposures, daphnids were exposed to DTPA at high or low food levels, or to Fe(III)-DTPA at high food level until the 6th reproductive instar. The 48 h LC50S of DTPA and Fe(III)-DTPA to D. carinata were 245 mg/L and > 1,000 mg/L, respectively. Chronic exposure to 10 mg/L DTPA resulted in a significant reduction in all individual brood sizes, while it increased the age at each reproductive instar. Ten mg/L DTPA also significantly decreased the cumulative number of offspring per adult at high and low food level from 161.3 +/- 14.6 to 11.3 +/- 4.9 offspring, and 56.4 +/- 1.8 to 0 +/- 0 offspring, respectively, while a similar effect was observed for the number of offspring per adult per day. Both the 3rd and 5th brood sizes were also significantly reduced at 1 mg/L DTPA, but only at high food level, from 39.0 +/- 2.9 to 27.6 +/- 3.8 offspring, and 49.3 +/- 5.0 to 39.9 +/- 4.2 offspring, respectively. Chronic exposure to Fe(III)-DTPA had little effect on D. carinata, but there was a significant negative relationship between Fe(III)-DTPA and the number of offspring per adult per day (y = -0.024x + 14.048, r2 = 0.20, n = 26, P < 0.02). This was due to a 25% reduction in reproduction at 134 mg/L Fe(III)-DTPA, the highest test concentration, compared to controls. The no-observed-effect concentrations (NOEC) and lowest-observed-effect concentrations (LOEC) for DTPA and Fe(III)-DTPA following chronic exposure to D. carinata were 1.0 and 10 mg/L, and 67 and 134 mg/L, respectively, although the possibility of effects occurring below 10 mg/L DTPA could not be discounted. Exposure to DTPA prior to 1st brood embryogenesis significantly decreased the 1st brood size but did not affect the 2nd brood size, while exposure during 1st brood embryogenesis significantly decreased the 2nd brood size, but did not affect the 1st brood size, indicating the reproductive impairment was due to maternally-mediated factors and not direct toxicity to the eggs. The decrease in DTPA toxicity when complexed with Fe(III) was attributed to preferential binding with that metal, thereby limiting any further chelating ability. Concentrations of DTPA in receiving waters are unlikely to be toxic to D. carinata.

Metabolites of chlorinated syringaldehydes in fish bile as biomarkers of exposure to bleached eucalypt pulp effluents.

Metabolites of chlorinated phenolic compounds in fish bile have been found to be sensitive biomarkers of bleached pulp mill effluent exposure. Chlorinated syringaldehydes are largely unstudied chlorophenolics found in bleached hardwood effluent. Sand flathead (Platycephalus bassensis), Australian marine fish, were exposed to 100% chlorine dioxide-bleached eucalypt pulp effluent at concentrations of 0.5, 2, and 8% (v/v) for 4 days. Metabolites of 2-chlorosyringaldehyde (2-CSA), the predominant chlorophenolic in this effluent, were measured in the bile. The major metabolite was the conjugate of 2-chloro-4-hydroxy-3,5-dimethoxy-benzylalcohol (2-CB-OH), the reduced product of 2-CSA. 2-CB-OH was found in all fish exposed to diluted effluent and was concentrated in the bile over 1000 times above 2-CSA levels in the effluent. A separate experiment examined the metabolic fate of 2,6-dichlorosyringaldehyde (2,6-DCSA), which is one of the major chlorophenolics in chlorine-bleached eucalypt pulp effluent. Sand flathead were exposed to 2,6-DCSA by intraperitoneal injection at 15 mg/kg or through the water to 0.5, 2, or 8 micrograms/liter for 4 days. Analysis of the bile revealed the major metabolite of 2,6-DCSA to be the conjugate of 2,6-dichloro-4-hydroxy-3,5-dimethoxybenzylalcohol, which was found in all exposed fish and was concentrated in the bile over 20,000 times above 2,6-DCSA exposure levels. Results reveal that the analysis of metabolites of chlorinated syringaldehydes in fish bile can provide a biomarker of bleached hardwood effluent exposure that is sensitive to low levels of exposure, specific to certain bleaching sequences, and correlates well with exposure concentrations.

Effect of algal food concentration on toxicity of two agricultural pesticides to Daphnia carinata.

The effects of algal concentration (Selanastrum capricornatum) on the toxicity of the organochlorine pesticide endosulfan and the synthetic pyrethroid esfenvalerate to Daphnia carinata was investigated. The study progressed through four stages: (1) A dose-response experiment on the effects of endosulfan and esfenvalerate on the survival, growth, and reproduction of D. carinata at a single nonlimiting food level. (2) An experiment to investigate the effects of five different food concentrations on survival, growth, and reproduction of D. carinata at sublethal concentrations of endosulfan and esfenvalerate compared with nonexposed controls. (3) An experiment to investigate the effects of route of exposure (water, food-borne, or water+food-borne exposure) on the toxicity of endosulfan to D. carinata. (4) An experiment to investigate the effects of algal concentration on persistence of endosulfan in the water column and on the relative toxicity of the alpha and beta isomers and of endosulfan sulfate to D. carinata. In the first experiment all daphnids exposed to 500 ng/liter esfenvalerate died within 3 days. There was a significant effect of esfenvalerate on reproduction at 50 ng/liter by the second brood. Endosulfan did not cause significant mortality to daphnids but brood size was reduced at 320 micrograms/liter. In the second experiment the toxicity of esfenvalerate increased significantly with decreasing food concentration. In contrast, the toxicity of endosulfan to D. carinata was greatest at the higher food concentrations. Direct water-borne exposure to endosulfan was the most toxic route of exposure and the presence of algae decreased toxicity of this pesticide. The total amount of endosulfan (alpha + beta + endosulfan sulfate) persisting in the water column after 24 hr was greater at high food levels, suggesting that this may be one mechanism for increased toxicity at high food concentrations. The 48-hr LC50s of technical endosulfan, endosulfan sulfate, alpha-endosulfan, beta-endosulfan, and a 50:50 mixture of alpha, and beta endosulfan were 478, 756, 249, 205, and 234 micrograms/liter, respectively.

Metabolites of 2-chlorosyringaldehyde in fish bile: indicator of exposure to bleached hardwood effluent.

1. 2-Chlorosyringaldehyde (2-CSA) is the major chlorinated phenol produced by the 100% chlorine dioxide bleaching of eucalypt pulp and is found in other bleached hardwood effluents. Almost nothing is known of the environmental or metabolic fates of this chemical. 2. Sand flathead (Platycephalus bassensis) was given 2-CSA by intraperitoneal injection at 0.15, 1.5, 15 and 75 mg/kg doses and, 4 days later, bile was collected and solvent extracted before and after enzymatic cleavage of conjugates. The acetate derivatives of bile extracts were analysed by gas chromatography/mass spectrometry. 3. The major metabolite 4 days after administration was the glucuronide or sulphate conjugate of 2-chloro-4-hydroxy-3,5-dimethoxy-benzylalcohol (2-CB-alcohol). The identity of 2-CB-alcohol was confirmed by chemical synthesis. 4. The quantity of 2-CB-alcohol in the bile was linearly related to dose of 2-CSA and was detected at all dose levels. Minor metabolites identified were conjugated 2-CSA, unchanged 2-CSA and 2-chloro-4-hydroxy-3,5-dimethoxy-benzoic acid. 5. The amount of 2-CB-alcohol in bile has the potential to be a sensitive and specific indicator of fish exposure or bleached hardwood effluent.