Lawrence R. Curtis

Endosulfan I and endosulfan sulfate disrupts zebrafish embryonic development

Fish in agricultural and remote areas may be exposed to endosulfan and its degradation products as a result of direct runoff, atmospheric transport and deposition. The following study used the zebrafish developmental model to investigate the responses to endosulfan I and endosulfan sulfate, the major degradation product of endosulfan I and II. Embryos were dechorionated and waterborne exposed to the endosulfan I or endosulfan sulfate from 6 to 120h post-fertilization (hpf). Endosulfan I exposure concentrations ranged from 0.01 to 10microg/L and endosulfan sulfate from 1 to 100microg/L. Water solutions were renewed every 24h and fish were scored for overt developmental and behavioral abnormalities. Chemical analysis was performed on water, whole embryo, and larvae samples to determine waterborne exposure concentrations and tissue concentrations throughout the 5-day period. The most sensitive toxicity endpoint for both endosulfan I and endosulfan sulfate was an abnormal response of the embryo/larvae to touch, suggesting that endosulfan I and sulfate are developmentally neurotoxic. The waterborne exposure EC(50)s for inhibition of touch response for endosulfan I and endosulfan sulfate were 2.2microg/L and 23microg/L, respectively. The endosulfans were highly concentrated by the organisms, and the inhibition of touch response tissue EC(50), determined from the measured tissue concentrations, was 367ng/g for endosulfan I and 4552ng/g for endosulfan sulfate.

Biomarker responses and disease susceptibility in juvenile rainbow trout Oncorhynchus mykiss fed a high molecular weight PAH mixture

Juvenile rainbow trout were fed a diet containing an environmentally relevant mixture of 10 high molecular weight polycyclic aromatic hydrocarbons (PAHs) at a dose of 0.66 or 7.82 µg PAH · g fish(-1) · d(-1). At 3, 7, 14, and 28 d, biomarkers of aryl hydrocarbon receptor activation (AHR), hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity, and cytochrome P4501A (CYP1A)-associated staining increased 14- to 26-fold and 6- to 14-fold, respectively, in fish fed 7.82 µg PAH · g fish (-1) · d(-1). Cytochrome P4501A-associated staining increased 2- to 9-fold on days 3, 7, and 28 in fish fed 0.66 µg PAH · g fish(-1) · d(-1). Bile fluorescent aromatic compounds served as a biomarker of exposure and confirmed that PAH exposure was consistent over 50 d. DNA damage in blood cells, protein oxidation, and lipid peroxidation in the kidney were biomarkers of oxidative stress and all increased in fish fed 7.82 µg PAH · g fish(-1) · d(-1). Fish fed 0.66 µg PAH · g fish(-1) · d(-1) had elevated DNA damage in blood cells but increased protein oxidation or lipid peroxidation in the kidney were not observed. Challenge with Aeromonas salmonicida, at lethal concentration (LC) 20, decreased survival in fish previously fed either 0.66 µg PAH · g fish(-1) · d(-1) or 7.82 µg PAH · g fish(-1) · d(-1) relative to fish fed the control diet. In general, biomarkers of both AHR activation and oxidative stress peaked at 3 to 14 d then declined at 28 to 50 d of PAH exposure and an increase in susceptibility to disease was observed at 50 d. These results link PAH exposure to biomarker responses that may be useful as early indicators of population level responses, such as mortality resulting from an increase in disease susceptibility.

Toxicity of fenvalerate to developing steelhead trout following continuous or intermittent exposure

Environmental toxicant exposure commonly vary in terms of duration and concentration. However, laboratory toxicity tests usually entail continuous exposures to constant concentrations. We compared survival, growth, and toxicant accumulation in early life-stage steelhead trout intermittently or continuously exposed to fenvalerate (FV) for 70 d after fertilization. Acute lethality was assessed in ancillary 96-h LC50 determinations with juvenile fish. Intermittent exposures were daily 4.5-h introductions of toxicant, and continuous exposures were to constant concentrations. All tests were conducted in a flow-through dilution apparatus, and mean concentrations for the entire exposure period were calculated for comparisons between regimens. The respective 96-h LC50 values for intermittently and continuously exposed were 88 and 172 ng/l. In the subchronic study, marked lethality (32%) and reduced terminal weight (50%) were found following exposures to cyclic FV concentrations that yielded an average of 80 ng/l (peak of 461). Continuous exposure to 80 ng FV/l did not affect these parameters. At mean FV concentrations above 20 ng/l, bioaccumulation was greater following intermittent than continuous exposure. Interaction of partitioning and elimination processes may partially explain differences in FV accumulation and subsequent toxicity.

Disposition and elimination of 17α-methyltestosterone in Nile tilapia (Oreochromis niloticus)

Abstract 17α-Methyltestosterone (MT) is of potential use in commercial production of all male cohorts of food fish. Sexually undifferentiated Oteochromis niloticus received control ration or 30 mg MT/kg ration for 30 days. On day 31 control and MT pretreated fish received a single dietary dose of 3 H-MT and were killed 1, 3, 7, and 10 days later. At 1 day after dosing only 2.5-3% of whole body 3 H-MT residues were identified as parent compound. The concentration of residues remaining 1–10 days after 3 H-MT dosing were similar in control and MT pretreated fish. 3 H-MT whole body residues decreased logarithmically during this period and had a 1-day half-life. At day 3 after dosing, 95% of 3 H-MT had been convened to polar metabolites, which decreased to 70% by 7 days, and to only trace concentrations by day 10. Five months after MT pretreatment control and pretreated fish received a single oral dose of 3 H-MT and were killed 1, 3, 7, and 10 days thereafter. In both groups, concentrations of 3 H-MT residues were biliary ≫ liver > kidney > muscle at all times points. Bile contained 97–99% polar metabolites of 3 H-MT in all cases and appeared a major route of excretion. These data indicated MT was readily eliminated by Oreochromis niloticus and that the pretreatment regimen proposed for commercial use did not substantially alter disposition of subsequent doses.

A Digenean Metacercaria (Apophallus sp.) and a Myxozoan (Myxobolus sp.) Associated with Vertebral Deformities in Cyprinid Fishes from the Willamette River, Oregon

Abstract A high prevalence of vertebral deformities has been observed in various fishes, especially cyprinids, from certain regions of the Willamette River for many years. One proposed source of these deformities is exposure to toxicants. Histological evaluation of affected chiselmouth Acrocheilus alutaceus revealed that all lesions associated with vertebrae were associated with metacercariae of digenean trematodes. Approximately half of the northern pikeminnow Ptychocheilus oregonensis had infections in which metacercariae were associated with these lesions. Metacercariae were also associated with vertebral lesions in three of four affected peamouth Mylocheilus caurinus. Many metacercariae that were present within the vertebral bodies were associated with bony dysplasia and bony proliferation in all three species. We also evaluated the association of the metacercariae with the vertebral deformities, using intact fish that had been cleared with trypsin. Fish from the affected regions had a much higher pre...

Effects of diet and temperature on food consumption, growth rate and tissue fatty-acid composition of triploid grass carp.

Abstract Food consumption, growth rate, and tissue fatty-acid composition of juvenile triploid grass carp varied markedly when fed different aquatic plants or a commercial ration at 20°C. Highest consumption, growth rate and ω 3 fatty-acid (especially 18:3n−3) concentrations in muscle and liver occurred in grass carp fed the aquatic plant, Elodea . Fish fed Elodea for 32 days and then fed commercial ration for 33 more days at 14.4°C, 18.8°C or 24.4°C were also studied. Growth rate and food consumption, but not assimilation efficiency, increased with environmental temperature. When Elodea -fed fish were subsequently fed commercial ration, growth rate and feed conversion efficiency were maintained for the first 23 days and dropped suddenly in the last 10 days at all temperatures. Decreased growth rate and feed conversion efficiency were coincident with reduced muscle and liver ω 3 fatty acids. This coincidence suggested the importance of 18:3n−3 in their growth. When the diet of fish at 14.4°C and 18.8°C was changed from Elodea to commercial ration the ratio of total unsaturated to total saturated fatty acids increased while the total number of fatty-acid double bonds in muscle and liver did not increase. This was primarily due to decreased 16:0 and increased 16:1. Feeding grass carp Elodea at warm temperatures enhanced the value of the fish as a source of ω 3 fatty acids for human consumption.

Reduced cytochrome P4501A activity and recovery from oxidative stress during subchronic benzo[a]pyrene and benzo[e]pyrene treatment of rainbow trout

This study assessed the role of aryl hydrocarbon receptor (AHR) affinity, and cytochrome P4501A (CYP1A) protein and activity in polyaromatic hydrocarbon (PAH)-induced oxidative stress. In the 1-100nM concentration range benzo[a]pyrene (BaP) but not benzo[e]pyrene (BeP) competitively displaced 2nM [(3)H]2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin from rainbow trout AHR2α. Based on appearance of fluorescent aromatic compounds in bile over 3, 7, 14, 28 or 50days of feeding 3μg of BaP or BeP/g fish/day, rainbow trout liver readily excreted these polyaromatic hydrocarbons (PAHs) and their metabolites at near steady state rates. CYP1A proteins catalyzed more than 98% of ethoxyresorufin-O-deethylase (EROD) activity in rainbow trout hepatic microsomes. EROD activity of hepatic microsomes initially increased and then decreased to control activities after 50days of feeding both PAHs. Immunohistochemistry of liver confirmed CYP1A protein increased in fish fed both PAHs after 3days and remained elevated for up to 28days. Neither BaP nor BeP increased hepatic DNA adduct concentrations at any time up to 50days of feeding these PAHs. Comet assays of blood cells demonstrated marked DNA damage after 14days of feeding both PAHs that was not significant after 50days. There was a strong positive correlation between hepatic EROD activity and DNA damage in blood cells over time for both PAHs. Neither CYP1A protein nor 3-nitrotyrosine (a biomarker for oxidative stress) immunostaining in trunk kidney were significantly altered by BaP or BeP after 3, 7, 14, or 28days. There was no clear association between AHR2α affinity and BaP and BeP-induced oxidative stress.

Temperature influences on water permeability and chlorpyrifos uptake in aquatic insects with differing respiratory strategies

Aquatic insects have evolved diverse respiratory strategies that range from breathing atmospheric air to breathing dissolved oxygen. These strategies result in vast morphological differences among taxa in terms of exchange epithelial surface areas that are in direct contact with the surrounding water that, in turn, affect physiological processes. This paper examines the effects of acute temperature shifts on water permeability and chlorpyrifos uptake in aquatic insects with different respiratory strategies. While considerable differences existed in water permeability among the species tested, acute temperature shifts raised water influx rates similarly in air-breathing and gill-bearing taxa. This contrasts significantly with temperature-shift effects on chlorpyrifos uptake. Temperature shifts of 4.5 degrees C increased 14C-chlorpyrifos accumulation rates in the gill-bearing mayfly Cinygma sp. and in the air-breathing hemipteran Sigara washingtonensis. However, the temperature-induced increase in 14C-chlorpyrifos uptake after 8 h of exposure was 2.75-fold higher in Cinygma than in Sigara. Uptake of 14C-chlorpyrifos was uniformly higher in Cinygma than in Sigara in all experiments. These findings suggest that organisms with relatively large exchange epithelial surface areas are potentially more vulnerable to both osmoregulatory distress as well as contaminant accumulation. Temperature increases appear more likely to impact organisms that have relatively large exchange epithelial surface areas, both as an individual stressor and in combination with additional stressors such as contaminants.

Subacute toxicity of the water-soluble fractions of Kuwait crude oil and partially combusted crude oil on Menidia beryllina and Palaemonetes pugio

As a consequence of the 1991 Gulf War, a substantial amount of crude oil (CO) and partially combusted crude oil (PCO) were emitted into the environment. Therefore, the study objective was to evaluate the toxicity of the water soluble fraction (WSF) of CO and PCO on a fish, Menidia beryllina and an invertebrate, Palaemonetes pugio in 16-d flow-through tests. Specific growth rate (SGR) was studied as a function of total petroleum hydrocarbon (TPHC) concentration in water. Reductions in SGR were observed in fish exposed to PCO and CO WSFs, with TPHC water concentration being 10-fold higher in CO exposures (67–145 μg/L) than in PCO exposures (4–12 μg/L). Significant negative correlations were observed between TPHC concentration and fish SGR in both CO (r2=0.730) and PCO (r2=0.867) exposures, with the slope being significantly lower for PCO exposures (−0.169) than CO exposures (−0.009). Differences between CO and PCO toxicity were not as clear in shrimp exposures due to slow growth rates and variability in TPHC concentrations. Qualitative PAH analysis indicated that naphthalene was present in the CO WSF whereas chrysene and benzo(a)pyrene were present in the PCO WSF. Heavy metal analysis of concentrated stock solutions indicated that the PCO WSF had substantially higher concentrations of some metals (Sr=2,521 μg/L, B=556 μg/L, and Ba=130 μg/L) than the CO WSF in which concentrations were less than 55 μg/L. Fish and shrimp tissue analysis did not reveal any uptake of parent PAH compounds from the water, which may be attributed to the formation of PAH metabolites.

Evaluation of the methoxytriazine herbicide prometon using a short‐term fathead minnow reproduction test and a suite of in vitro bioassays

Prometon is one of the most consistently detected herbicides in the U.S. environment. However, no previous assessment of the potential for prometon or related methoxytriazine herbicides to act as endocrine-disrupting chemicals has been conducted. This study used an array of in vitro bioassays to assess whether prometon, atraton, terbumeton, or secbumeton might act as potent (ant)agonists of the aryl hydrocarbon, estrogen, androgen, or glucocorticoid receptors or as aromatase inhibitors or inducers in vitro. Potential effects of prometon were also evaluated using a 21-d fathead minnow reproduction assay. Concentrations of methoxytriazines, as great as 1 mg/L (4.4 microM), did not induce significant dioxin-like responses in H4IIE-luc cells, estrogenic responses in MVLN cells, or androgen or glucocorticoid receptor-mediated responses in MDA-kb2 cells, nor did the methoxytriazines significantly affect aromatase activity in vitro. In the fathead minnow assay, exposure to 20, 200, or 1,000 microg prometon/L significantly reduced the weight of the male fat pad (an androgen-responsive tissue) relative to body weight. Exposure to 20 microg prometon/L significantly increased female plasma testosterone concentrations, but the effect was not observed at greater concentrations. Overall, prometon did not significantly reduce fecundity over the 21-d exposure, nor were other endpoints, including plasma vitellogenin and estradiol concentrations, brain and ovary aromatase activity, and male tubercle index, significantly affected. Evidence from our work suggests that prometon may cause subtle endocrine and/or reproductive effects in fathead minnows, but no clear mechanism of action was observed. The relevance of these effects to hazard assessment for the pesticide is uncertain.