Fish and amphibian embryos — A model system for evaluating teratogenicity

Developmental stages of fish and amphibians were used to evaluate reproductive impairments produced by environmental contaminants. Using staticrenewal and flow-through procedures, exposure was initiated at, or soon after, fertilization and maintained through 4 days posthatching. Trace concentrations of many environmental toxicants produced high frequencies of mortality and teratogenesis. When developmental stages of the trout were exposed to inorganic mercury, 10% control-adjusted mortality was observed at 1 μg/L and frequencies of terata in hatched populations ranged from 8% to 43% for exposure concentrations of 2 to 25 μg/L. In similar tests with the narrow-mouthed toad, 45% mortality occurred when mercury was administered at 1 μg/L; frequencies of teratogenesis ranged from 8% to 17% at exposure concentrations of 1 to 5 μg/L; and complete mortality occurred at 7 μg/L. Boron-induced teratogenesis in trout ranged from 5% at 1 μg/L to 51% at higher exposure levels. When catfish embryos were exposed to atrazine at 0.06, 4.8, and 46.7 mg/L, terata appeared in surviving populations at frequencies of 4%, 69%, and 100%, respectively. Fish and amphibian developmental stages constitute simple and effective models for 1) investigating mechanisms of teratogenesis, 2) evaluating the impact of environmental toxicants on aquatic biota, and 3) identifying environmental teratogens which may be of concern to human health.     

Developmental Toxicity of 2,3,4,7,8-Pentachlorodibenzofuran in the Fischer 344 Flat

Developmental Toxicity of 2,3,4,7,8-Pentachlordibenzofuran inthe Fischer 344 Rat. COUTURE, L. A,, HARRIS, M. W., AND BIRNBAUM,L. S. (1989). Fundam Appl Toxicol 12, 358–366. Fischer344 rats were exposed acutely to 2,3,4,7,8-pentachlordibenzofuran(4-PeCDF) during the organogenic period to evaluate its potentialas an inducer of teratogenic and embryolethal effects. All damswere treated by gavage with a single dose of 0, 30, 100, or300 µg 4- PeCDF/kg body wt on gestation Day (gd) 8, 10,or 12. An additional treatment group was included on gd 12 andadministered 10µg 4-PeCDF/kg body wt po. All animals werekilled on gd 20 and maternal and fetal toxicities were assessed.Determination of embryotoxicity involved both soft tissue andskeletal examinations. 4-PeCDF induced a dose-related decreasein corrected maternal weight gain following treatment on gd8 and 10, as well as resulted in a concomitant Increase in theliver/body weight ratios, first evident at 30 µg/kg forall 3 days of exposure. The maternal thymus weight decreasedrelative to body weight compared with those of controls. Embryo-fetaltoxicity was evident from the high mortality (>80%) observedat 300 µg/kg for all 3 days of exposure. Mean fetal weight,a sensitive indicator of fetal toxicity, decreased comparedto that of controls at 30, 100, and 300 µg/kg followingtreatment on either gd 8, 10, or 12. 4- PeCDF induced cleftpalate in survivors at a dose of 300 µg/kg for all 3 daysof exposure. In conclusion, 4-PeCDF is maternally and fetallytoxic regardless of the gestation day of exposure, but inducedterata only at doses where overt maternal and fetal toxicitywere observed, in contrast to previously reported studies inthe mice where teratogenic effects were observed at nonfetotoxicdose levels. Thus, the mouse may be a more sensitive model forevaluating specific toxic responses induced prenatally followingexposure to the structurally related polyhalogenated aromatichydrocarbons which include the dioxins, furans, biphenyls, andnaphthalenes.     

Characterization of the Peak Period of Sensitivity for the Induction of Hydronephrosis in C57BL/6N Mice following Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Characterization of the Peak Period of Sensitivity for the Inductionof Hydronephrosis in C57BL/6N Mice following Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.COUTURE, L. A., HARRIS, M. W., AND BIRNBAUM, L. S. (1990). Fundam.Appl. Toxicol. 15, 142–150. 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) is an extremely potent teratogen in mice. Hydronephrosisand cleft palate are the most sensitive measures of teratogenicityin mice following exposure to TCDD and other structurally relatedpolyhalogenated aromatic hydrocarbons. Despite a relativelylong half-life, investigators have identified a critical windowfor the induction of cleft palate in C57BL/6N mice. To characterizethe critical period for renal teratogenesis, pregnant C57BL/6Nmice were treated once by gavage with 0–24 µg TCDD/kgbody wt on Gestation Day (GD) 6, 8, 10, 12, or 14. All damswere killed on GD 18, and the fetuses were examined for thepresence of hydronephrosis and cleft palate. Maternal liver-to-bodyweight ratios were significantly elevated above controls onall days, while maternal weight gain was unaffected. Fetal mortalitywas increased relative to controls only at 24 µg TCDD/kgon GD 6. There was no significant difference in fetal body weightsbetween control and TCDD-treated fetuses. The incidence of cleftpalate increased in a dose-related fashion from GD 6 to GD 12,and identification of GD 12 as the critical window for inductionof clefting of the hard palate was confirmed. Hydronephrosiswas observed at all dose levels, regardless of exposure day,and the incidence was close to 100% at 3 µg TCDD/kg andhigher doses on GD 12 and earlier. At all doses on GD 14, boththe incidence and severity of hydronephrosis were decreasedrelative to all other days. There was a dose-related increasein the severity of the renal lesion on each day, but betweenGD 6 and 12 severity was constant Thus, while palatal sensitivityto TCDD increased with gestational age between GD 6 and 12,there was no difference among these days in development of hydronephrosis.The data suggest, however, that on GD 14 the urinary tract maybe less sensitive to TCDD.     

Hepatic CYP1A1 Induction in Rainbow Trout by Continuous Flowthrough Exposure to {beta}-Naphthoflavone

In order to assess the usefulness of CYP1A1 mRNA measurementas an environmental biomarker it was necessary to determineif hepatic P450 CYP1A1 mRNA induction is sustained during constantexposure to hepatic monooxygenase inducers. To accomplish this,rainbow trout (Oncorhynchus mykiss) were exposed, under flowthroughconditions, to ß-naphthoflavone (ß-NF),a known CYP1A1 inducer in fish. Trout were exposed to a ß-NFconcentration of 1.0 mg ß-NF/liter, using dimethylformamideas carrier, for 1, 2, 4, and 8 days, followed by depurationin clean water for 1, 8, 14, and 35 days. In a second experiment,trout were exposed to ß-NF concentrations of 0.05,0.10, and 0.50 mg ß-NF/liter, using dimethylformamideas carrier, for 1, 3, 7, and 14 days, followed by depurationfor 7 and 28 days. At the 1.0-mg ß-NF/liter concentration,ethoxyresorufin-O-deethylase (EROD) activity was significantlydecreased by 4 days of exposure when compared to controls. Atß-NF concentrations of 0.05 to 0.50 mg ß-NF/literEROD activity was increased compared to controls but was inverselyrelated to the ß-NF concentration. Hybridizable CYP1A1mRNA was increased approximately 40-fold over control levelsat concentrations of 0.05 to 0.50 mg ß-NF/liter for1, 3, and 7 days of exposure. In a third experiment, trout exposedto 0.05 mg ß-NF/liter for 2, 6, 12, 24, 32, 40, and48 hr had increased (45-to 167-fold) EROD activity by 18 and48 hr, respectively. Immunoreactive CYP1A1 protein was increased46-fold at 48 hr and CYP1A1 mRNA was increased 29-fold at 48hr of continuous ß-NF exposure. This is in contrastto previous experiments using intraperitoneal injection of ß-NFin which the induced CYP1A1 mRNA decreased to near control levelsby 48 hr after injection. These data indicate that both CYP1A1catalytic activity and immunoreactive protein are decreasedat high inducer concentrations while mRNA levels remain elevatedand continue to increase over time during continuous exposure.In a fourth experiment trout were continuously exposed to concentrationsof 0.625, 1.25, 2.5, 5.0, and 10.0µgß-NF/liter,using dimethylformamide as carrier, for 1, 3, 7, 14, and 21days, followed by clean water depuration for 1, 3, 7, 14, and21 days EROD activity was significantly increased in a concentration-dependentmanner over control by Day 1 of exposure with concentrationsof 2.5, 5.0, and 10.0 µg ß-NF/liter. EROD activitywas increased approximately 3-, 5-, 16-, 21-, and 58-fold introut exposed for 3 days to 0.625, 1.25, 2.5, 5.0, and 10.0µgß-NF/liter, respectively. Immunoreactive CYP1A1protein was also increased in a concentration-dependent mannerapproximately 1.2-, 1.9-, 3.4-, 5.2-, and 8.2-fold after 3 daysof exposure, respectively, and approximately 1.2-, 1.2-, 3.4-,5.1-, and 10.8-fold after 7 days of exposure, respectively.At Day 3, hybridizable CYP1A1 mRNA was increased over controls1.7-, 7.0-, 33-, 44-, and 62-fold, at concentrations of 0.625,1.25, 2.5, 5.0, and 10.0 µg ß-NF/liter, respectively.CYP1A1 mRNA levels were significantly increased over extendedtime periods during prolonged exposure. With the 10.0 µgß-NF/liter exposure, CYP1A1 mRNA levels were significantlyincreased approximately 7-, 17-, 20-, 16-, and 8-fold over controllevels at 1, 3, 7, 14, and 21 days, respectively. During a depurationperiod following exposure to 10.0 µg ß-NF/liter,EROD activity remained significantly increased compared to controlsfor 3 days and returned to control levels by 7 days. The resultsindicate that CYP1A1 mRNA remains elevated for at least 21 daysduring constant exposure to ß-NF, and the findingsprovide support for the feasibility of using of CYP1A1 mRNAlevels in biomonitoring of water for specific chemical contamination.     

Species Differences in 2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicity and Biotransformation in Fish

Species Differences in 2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicityand Biotransformation in Fish. KLEEMAN, J. M., OLSON, J. R.,AND PETERSON, R. E. (1988). Fundam. Appl. Toxicol 10, 206-213.Rainbow trout, yellow perch, carp, bluegill, largemouth bass,and bullhead were treated with graded doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD; 1, 5, 25, or 125 µg/ kg) or vehicle, ip. The lethalpotency of TCDD tended to be greater in yellow perch, carp,and bullhead than in the other three species (LD50 80 days post-treatment,3-5 versus 10-16 µg/kg, respectively). All species treatedwith the highest dose of TCDD (125 µg/kg) displayed alatency period of 1-4 weeks prior to death; longer latency periodswere produced by lower lethal doses. Effects of TCDD treatmenton body weight were both species-dependent and dose-dependent.Fin necrosis was observed in all fish species; however, cutaneoushemorrhage was observed only in TCDD-treated perch, carp, andbluegill, and cutaneous hyperpigmentation only in TCDD-treatedcarp and largemouth bass. Gallbladder bile was analyzed forTCDD and its metabolites 7 days after fish were injected with[¹⁴C]TCDD (60 µg/kg, ip). At least three TCDD metabolitesin addition to the parent compound were found in the gallbladderbile of all six species. In addition, the retention time ofthe major biliary TCDD metabolite (determined by HPLC) was similarin all species except yellow perch. ß-Glucuronidasetreatment of the bile from largemouth bass and bluegill suggestedthat at least two of the TCDD metabolites were glucuronide conjugates.Thus, species differences exist in the lethal potency, signsof overt toxicity, and biotransformation of TCDD among freshwaterfish.     

Clearance of Diesel Soot Particles from Rat Lung after a Subchronic Diesel Exhaust Exposure

Clearance of Diesel Soot Particles from Rat Lung after a ChronicDiesel Exhaust Exposure. Griffis, L.C., Wolff, R.K., Henderson,R.F., Griffith, W.C., Mokler, B.V. and McClellan, R.O. (1983).Fundam. Appl. Toxicol. 3:99-103. The participate exhaust ofdiesel engines consists of 0.1–0.2 µm mass mediandiameter particles composed of a carbonaceous core and adsorbedorganic compounds. A technique was needed to determine accumulatedlung burdens of particles in animals exposed to diesel exhaustas a determinant of dose. A method was developed for determininglung burdens of diesel soot particles in rats at 1 day, andat 1, 5, 15, 33 and 52 weeks after cessation of a subchronicexposure to diluted diesel exhaust. Lung tissue was dissolvedin tetramethylammonium hydroxide and the diesel soot separatedby centrifugation. The soot was suspended in water by sonicationand the light absorption of samples was compared to standardsuspensions of diesel soot. Recovery from lungs spiked with50–1000 µg of soot was 89±5%. Rats exposedover a period of 18 weeks to diluted diesel exhaust at averagenet diesel particle concentrations of 150, 940 and 4100 µg/m³had lung burdens of 35,220 and 1890 µg/g lung, respectively,one day after the last exposure. The long-term clearance ratesof soot had estimated half-times of 87±28, 99±4days, for the low and medium exposure groups, respectively.The clearance half-time for the high level exposure group of165 ± 8 days was significantly longer (P < 0.0001)than those of the other two groups.     

Potency of a Complex Mixture of Polychlorinated Dibenzo-p-dioxin, Dibenzofuran, and Biphenyl Congeners Compared to 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Causing Fish Early Life Stage Mortality

Use of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity equivalentsconcentration (TEC) assumes that polychlorinated di benzo-p-dioxins(PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) act additivelyand via a common mechanism to cause toxicity. To test theseassumptions, 11 TCDD-like congeners and three non-TCDD-likecongeners were combined at ratios typically found in Lake Michiganlake trout. The potency of the mixture, expressed as TEC basedon fish-specific toxic equivalency factors, was compared toTCDD for producing lake trout and rainbow trout early life stagemortality. Signs of toxicity following exposure of newly fertilizedeggs to the mixture or to TCDD were indistinguishable; sac frymortality associated with blue-sac disease, and slopes of thedose-response curves for percentage sac fry mortality versusegg TEC or versus egg TCDD were parallel. However, the mixturedose-response curves were significantly shifted to the rightof the TCDD dose-response curves by 1.3- to 1.8-fold as illustratedby LD₅₀ values. Following exposure to the mixture or TCDD, LD₅₀sfor lake trout early life stage mortality were 97 (89–110)pg TE/g egg and 74 (70–80) pg TCDD/g (LD₅₀, 95% fiduciallimits) and for rainbow trout were 362 (312–406) pg TE/gegg and 200 (148–237) pg TCDD/g egg. These data suggestthat TCDD-like congeners act via a common mechanism to causetoxicity during trout early development, but may not act strictlyadditively when combined in a mixture of TCDD- and non-TCDD-likecongeners at ratios found in Great Lakes fish. The deviationfrom additivity, however, is less than current safety factorsof 10-fold commonly applied in ecological risk assessments,providing support for the continued use of a TE additivity modelfor assessing risk posed by complex mixtures of PCDDs, PCDFs,and PCBs to fish.     

INFLUENCE OF TEMPERATURE AND DISSOLVED OXYGEN ON THE ACUTE TOXICITY OF PROFENOFOS TO FATHEAD MINNOWS (PIMEPHALES PROMELAS)

ABSTRACT

The purpose of this study was to investigate the influence of temperature and dissolved oxygen levels on the acute toxicity of profenofos to fathead minnows (Pimephales promelas). Exposure conditions were as follows: normal temperature and normal dissolved oxygen (NTNO; 20 ± 2°C and 6.0–9.0 mg/L, respectively); normal temperature and low dissolved oxygen (NTLO; 20 ± 2°C and 1.7–2.6 mg/L, respectively); high temperature and normal dissolved oxygen (HTNO; 30 ± 2°C and 6.6–6.9 mg/L, respectively); high temperature and low dissolved oxygen (HTLO; 30 ± 2°C and 1.5–3.0 mg/L, respectively). Initial 96-h acute toxicity studies with profenofos were conducted at NTNO and HTLO exposure conditions. The 96-h LC₅₀ at NTNO was 333 µg/L with 95% confidence limits ranging from 244 to 558 µg/L. However, the 96-h LC₅₀ at HTLO was significantly lower at 21.5 µg/L with 95% confidence limits ranging from 17.4 to 28.8 µg/L. Acetylcholinesterase (AChE) activity was measured in the head and torso of surviving fish at 96-h. A weak dose-related decrease in AChE was observed at NTNO but no dose–response relationship was observed at HTLO exposure condition. Additional experiments were conducted using 50 µg/L profenofos at NTNO, NTLO, HTNO, and HTLO exposure conditions. Mortality, sublethal effects (erratic and hyperactive swimming), and AChE activity in the head and torso were measured at 2, 4, and 12-h following exposure to profenofos. No mortality or significant sublethal effects were observed in controls or profenofos-treated groups in NTNO and NTLO exposure conditions. However, significant mortality and sublethal effects were observed in profenofos-treated fish in HTNO at 12 h and at all time points in HTLO. Both high temperature and low dissolved oxygen, as well as combinations of high temperature and low dissolved oxygen significantly decreased AChE activity in control fish. Exposure to 50 µg/L profenofos in all exposure conditions further decreased AChE activity, but no apparent correlations between mortality and AChE activity were observed. These results suggest that the acute toxicity of profenofos to fathead minnows may be exacerbated during summer conditions in southern U.S. aquatic ecosystems.     

Comparative study on sensitivity of higher plants and fish to heavy fuel oil

Laboratory tests were conducted on higher plants [garden cress (Lepidium sativum), great duckweed (Spirodela polyrrhiza), and Tradescantia clone BNL 02] and fish [rainbow trout (Oncorhynchus mykiss) at all stages of development: eggs, larvae and adults] to estimate their sensitivity to heavy fuel oil (HFO). A number of biological indices (survival, growth, and physiological and morphological parameters) as well as the genotoxic impact (Tradescantia) of HFO was evaluated by acute and chronic toxicity tests. Fish were found to be more sensitive to the toxic effect of HFO than were higher plants. EC(50) values obtained for higher plants ranged from 8.7 g/L (L. sativum) to 19.8 g/L (Tradescantia), and maximum-acceptable-toxicant concentration (MATC) values ranged from 0.1 to 1.0 g/L of total HFO for L. sativum and Tradescantia, respectively. The 96-h LC(50) values ranged from 0.33 g/L, for larvae, to 2.97 g/L, for adult fish, and the MATC value for fish was found to be equal to 0.0042 g/L of total HFO. To evaluate and predict the ecological risk of the overall effects of oil spills, studies should be performed using a set of acute and chronic bioassays that include test species of different phylogenetic levels with the most sensitive morphological, physiological, and genotoxic indices.     

Effects of depleted uranium on survival, growth, and metamorphosis in the African clawed frog (Xenopus laevis)

Embryos (stage 8-47, Nieuwkoop and Faber) of the African clawed frog (Xenopus laevis) were subjected to water-borne depleted uranium (DU) concentrations that ranged from 4.8 to 77.7 mg/L using an acute 96-h frog embryo teratogenesis assay-Xenopus (FETAX). In a chronic 64-d assay, X. laevis (from embryo through metamorphosis; stages 8-66) were subjected to concentrations of DU that ranged from 6.2 to 54.3 mg/L. Our results indicate DU is a non teratogenic metal. No effects on mortality, malformations, or growth were observed in the 96-h FETAX with concentrations of DU that ranged from 4.8 to 77.7 mg/L. From stage 8 to stage 47, X. laevis tadpoles do not actively feed and the gills are not well developed. Thus, uptake of DU was reduced despite exposure to elevated concentrations. The 64-d assay resulted in no concentration response for either mortality or malformations; however, a delay in metamorphosis was observed in tadpoles subjected to elevated DU concentrations (from 13.1 to 54.3 mg/L) compared to tadpoles in both the well-water control and reference. The delay in metamorphosis was likely due to increasing body burden of DU that ranged from 0.98 to 2.82 mg/kg.     

Subchronic Oral Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Guinea Pig: Comparisons with a PCB-Containing Transformer Fluid Pyrolysate

Subchronic Oral Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxinin the Guinea Pig: Comparisons with a PCB-Containing TransformerFluid Pyrolysate. DECAPRIO, A. P., MCMARTIN, D. N., O'KEEFE,P. W., REJ, R., SILKWORTH, J. B., AND KAMINSKY, L. S. (1986).Fundam. Appl. Taxicol. 6,454–463. In contrast to the well-characterizedacute toxicity of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin(2,3,7,8-TCDD) in the guinea pig, the effects of prolonged poexposure in this species are unknown. The present report describesthe results of administration to guinea pigs of 2,3,7,8-TCDDin the feed at levels of 0,2, 10, 76, or 430 ppt for up to 90days. Additional aims were to examine recovery following prolonged2,3,7,8-TCDD exposure in the guinea pig and to generate datato facilitate comparison of the previously reported toxicityof a transformer fluid pyrolysate with that of pure 2,3,7,8-TCDD.Animals receiving 430 pot 2,3,7,8-TCDD exhibited body weightloss, thymic atrophy, liver enlargement, and 60% mortality byDay 46 (males) and by Day 60 (females), when surviving animalsin this group were sacrificed. Total 2,3,7,8-TCDD consumptionwas approximately 1.3 and 1.9 µg/kg, respectively. Animalsreceiving 76 ppt 2,3,7,8-TCDD for 90 days (total 0.44 µg/kg)exhibited a decreased rate of body weight gain and increasedrelative (to body) liver weights. Male animals also displayeda reduction in relative thymus weights and elevated serum triglycerides,while females exhibited hepatocellular cytoplasmic inclusionbodies and lowered serum alanine arninotransferase activities.Toxic effects were generally similar to those observed afteracute 2,3,7,8-TCDD administration. No dose-related alterationswere seen in animals receiving either 10 ppt (total 0.06 µg/kg)or 2 ppt (total 0.01 µg/kg) for 90 days, establishinga no-observed-effect level of approximately 0.65 ng 2,3,7,8-TCDD/kg/day.In the recovery study, groups of guinea pigs were administered430 ppt 2,3,7,8-TCDD for 11,21, or 35 days and then allowedto recover for an additional 79,69, or 55 days, respectively.Treatment-related mortality in each group was 0, 10, and 70%,respectively, by Day 90. An effective LD5O of 0.8 µg 2,3,7,8-TCDD/kgfor prolonged exposure was calculated on the basis of theseresults, a value lower than those previously reported from thislaboratory for acute exposure. The results also suggested apossible lowering of the body weight "set point" following 2,3,7,8-TCDD exposure. Comparison of the present findings with thosepreviously reported for a trans former fluid pyrolysate containinga mixture of polychlorinated aromatic species indicated botha greater variety of toxic effects and flatter dose-responserelationships for the pyrolysate in the guinea pig.     

Aquatic risk assessment of copper in freshwater and saltwater ecosystems of South Florida

A screening-level aquatic risk assessment was conducted for copper in south Florida’s freshwater and saltwater environments. Risk was quantified by comparing the overlap between the probability distributions of copper exposure from surface water and sediment with the probability distributions of effects data obtained from laboratory studies. Copper concentrations in surface water and sediment in south Florida were summarized by county. For surface water, the highest concentrations of copper were found in Martin and St. Lucie counties for freshwater and saltwater, respectively. From the exposure probability distributions, the 90th centile values were estimated at 14.0 μg/L and 15.4 μg/L in freshwater and saltwater, respectively. Copper concentrations in sediment were evaluated from a probability distribution of predicted pore water concentrations. The 90th centile values of pore water concentrations from freshwater sediments ranged from 5.0 μg/L in Palm Beach County to 71.7 μg/L in Broward County. In saltwater sediments, the 90th centile values for pore water ranged from 26.1 μg/L in St. Lucie County to 27.3 μg/L in Miami-Dade County. Ecological effects data were obtained for acute and chronic copper effects in freshwater and saltwater. The 10th centile values for acute effects data were 21.2 μg/L and 9.8 μg/L for freshwater and saltwater species, respectively. For chronic effects, the 10th centile values were 3.8 μg/L and 3.9 μg/L for freshwater and saltwater species, respectively. The risk of acute copper exposure in surface water was generally low; however, the potential for ecological risk from chronic copper exposure was low to high in several counties including Lee, Martin, and St. Lucie counties. The risk of acute copper exposure in porewater from freshwater sediments also was low with the exception of St. Lucie and Broward counties. However, porewater from saltwater sediments posed a significant acute risk in Miami-Dade and St. Lucie counties. In porewater from freshwater and saltwater sediments chronic risk was high in counties with sufficient data available to calculate risk estimates.     

Comparative aquatic toxicology of aromatic hydrocarbons

Structure-toxicity relationships were investigated for six organic contaminants, representative of three chemical classes, likely to be found in coal conversion process waters and effluents. Using embryo-larval stages of the rainbow trout (Salmo gairdneri) and largemouth bass (Micropterus salmoides), continuous-flow toxicity tests were performed on hydroxylated aromatic hydrocarbons (phenol, β-naphthol), azaarenes (quinoline, acridine), and polycyclic aromatic hydrocarbons (naphthalene, phenanthrene). Exposure was initiated at fertilization and maintained through 4 days posthatching. Median lethal concentrations (LC₅₀), based on combined frequencies of embryo-larval mortality and teratogenesis, were used to rank the toxicity of the compounds to each fish species. With the trout, the order of decreasing toxicity was phenanthrene (0.04 mg/L), β-naphthol (0.07 mg/L), naphthalene (0.11 mg/L), phenol (0.15 mg/L), acridine (0.32 mg/L) and quinoline (11.0 mg/L). The toxicological ranking with the bass was phenanthrene (0.18 mg/L), naphthalene (0.51 mg/L), acridine (1.02 mg/L), β-naphthol (1.77 mg/L), phenol (2.80 mg/L) and quinoline (7.50 mg/L). For each class of compounds, the chemical with the greater number of aromatic rings always exerted the greater toxicity. In tests with both fish species, β-naphthol (two rings) was about twice as toxic as phenol (one ring), and phenanthrene (three rings) was nearly three times more toxic than naphthalene (two rings). Acridine (three rings) was seven times more toxic to bass and 34 times more toxic to trout than was quinoline (two rings). This relationship between ring number and toxicity was in excellent agreement with results from acute tests on the same compounds. Furthermore, a close correlation existed between toxicity and n-octanol:water partition coefficients within each class of compounds.     

Dose-Dependent Disposition of Sodium Arsenate in Mice Following Acute Oral Exposure

The effect of dose on arsenate disposition was studied in adultfemale B6C3FI mice, dosed po with 0.5 to 5000 µ/kg [⁷³As]-arsenatein water. Urine was collected at 1, 2, 4, 8, 12, 24, and 48hr and feces at 24 and 48 hr postexposure. The mice were euthanizedat 48 hr and tissues were removed. Recovery of ar senate-derivedradioactivity ranged from 83 to 89%; 66–79% of the dosewas excreted in urine, 10–18% in feces, and <1% remainedin the tissues. Although dose had no effect on the 48-hr excretionof radioactivity, the level of radioactivity in several tissuesincreased significantly with dose. The urine was analyzed forarsenic metabolites by using ion chromatography to analyze forarsenate, methylarsonic acid (MMA), and dimethylarsinic acid(DMA); ion-pairing high-performance liquid chromatogra phy wasused for arsenite analysis. Arsenate elimination ranged from3 to 15%. DMA was the predominant metabolite excreted (51-64%of dose), but no effect of dose on its elimination was detected.As the dose of arsenate increased, the amount of MMA excreted(0.1-1.0% of dose) significantly increased. At 5000 µg/kgarsenate, a significant increase in arsenite excretion was observed.At doses of arsenate <500 µg/kg, peak elimina tionof DMA occurred within 4 hr postexposure. At the 5000 µg/kgdose, DMA peak elimination shifted to 8 hr and a lower amountwas excreted. In addition, at the 5000 sg/kg dose, there wasan increase of arsenate and arsenite in the 1- and 2-hr urines.These results suggest that an acute dose of arsenate can affectthe metabolism of arsenicals. High doses lead to the accumulationof intermediates that are more reactive than DMA, and this responsemay lead to increased toxicity.     

30-Day Oral Toxicity Study of L-Selenomethionine in Female Long-Tailed Macaques (Macaca fascicularis)

Twenty female long-tailed macaques received nasogastric intubationof 0–600 µg/kg-day L-sele-nomethionine for up to30 consecutive days. Selenium ingestion was well tolerated atall dose levels until the second to third week of the studyat which time two animals given 600 µg/kg-day died. Oneanimal from the 300 µg/kg-day group was removed from studyon Treatment Day 19 due to selenium-induced hypothermia. Insome cases, administered doses were reduced at the 300 and 600µg/kg-day levels such that the final time-weighted averagedoses were 0, 25, 62–117, 150, 188–203, and 300µg/kg-day. Six animals at the 188 µg/kg-day levelor greater required nonscheduled fruit and dietary supplementationto prevent their impending demise. As the dose and durationof exposure increased, the incidence of anorexia, gastrointestinaldistress, mucocutaneous toxicity, and frequency of reduced bodytemperature also increased. A dose-dependent reduction in bodyweight was also observed. At the greater doses, disturbancesin menstrual function were evident, and were accompanied bythe absence of serum progesterone concentrations above 1.0 ng/ml,reduced luteal phase lengths, increased intermenstrual intervals,and lowered estrogen excretion. A maximum tolerated dose of150 µg/kg-day L-seleno-methionine for 30 days was identifiedbased on mean body weight reduction, hypothermia, dermatitis,xerosis, cheilitis, disturbances in menstruation, and the necessityof dietary intervention to prevent death at doses of 188 µg/kg-dayor greater.     

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on Pulmonary Influenza Virus Titer and Natural Killer (NK) Activity in Rats

Suppression of immune function and enhanced susceptibility toinfection in mice is one of the more sensitive indicators ofTCDD toxicity. Recent efforts to demonstrate similar effectsin the rat have shown that there are considerable differencesbetween the two species. The purpose of this study was to determinethe effect of TCDD exposure on (a) an influenza virus host resistancemodel in rats and (b) natural killer cell activity in the lungand spleen. Fischer 344 rats were treated with 10 µg TCDD/kgbody weight via gavage and infected intranasally with rat-adaptedinfluenza virus (RAIV) 7 days later. Virus-augmented NK activityassessed at 48 hr postinfection in the lung was significantlysuppressed in rats treated with 3, 10, or 30 µg TCDD/kgbody weight. Spontaneous NK activity in either lung or spleenwas not affected by TCDD exposure. Significantly higher virustiters were observed on Days 2, 3, and 4 postinfection in thelungs of rats treated with TCDD (10 µg/kg) TCDD had noeffect on the amount of virus recovered from nasal lavage. Acuteexposure to TCDD did not significantly affect lung and bodyweights in rats infected with RAIV except in the highest dose(30 µg/kg) treated rats. Rats exposed to repeated dosesof TCDD showed a significant increase of lung weights and L/Bratios when rats were infected with RAIV after TCDD exposure.Virus-augmented pulmonary NK activity in these rats was significantlysuppressed; however, the suppression was not more profound thanthat in rats exposed to a single dose of TCDD. However, an increaseof lung weights and lung/body weight ratios was observed inRAIV infected rats which were exposed to repeated doses of TCDD(cumulative dose of 10 µg/kg). Virus-augmented NK activitywas significantly sup pressed in rats exposed to repeated dosesof TCDD. Our results showed that TCDD suppressed virus-augmentedpulmonary NK activity and this effect may at least in part berelated to enhanced susceptibility of rats to influenza virus.     

Exposure to Inorganic Arsenic Metabolites during Early Human Development

Because of the lack of data on the exposure to and toxic effectsof inorganic arsenic during early human development, the transferof arsenic to the fetus and suckling infant was studied in anative Andean population, living in the village San Antoniode los Cobres in the North west of Argentina, where the drinkingwater contains about 200 µg/liter. The concentration ofarsenic in cord blood (median, 9 µg/liter) was almostas high as in maternal blood (median, 11 µg/liter), andthere was a significant correlation between the two. Thus, atleast in late gestation, arsenic is easily transferred to thefetus. The median concentration of arsenic in the placenta was34 µg/kg, compared with 7 µg/kg previously reportedfor nonexposed women. Interestingly, essentially all arsenicin the blood plasma of both the newboms and their mothers wasin the form of dimethylarsinic acid (DMA), the end product ofinorganic arsenic metabolism. Similarly, about 90% of the arsenicin the urine of both the newboms and mothers in late gestationwas present as DMA, compared with about 70% in nonpregnant women(p < 0.001). This may indicate that methylation of arsenicis increased during pregnancy and that DMA is the major formof arsenic transferred to the fetus. The increased methylationin late gestation was associated with lower arsenic concentrationsin blood and higher concentrations in urine, compared with afew months postpartum. The arsenic concentrations in the urineof the infants decreased from about 80 µg/liter duringthe first 2 days of life to less than 30 µg/liter at 4.4months (p = 0.025). This could be explained by the low concentrationsof arsenic in the breast milk, about 3 µg/kg.     

Differential effects of fluoxetine on the phototactic behavior of 3 amphipod species (Crustacea; Amphipoda)

We document phototactic responses in different amphipod populations of Gammarus minus, Stygobromus tenuis, and Crangonyx shoemakeri, each collected at 2–3 sites within the Washington DC area. We then assessed how baseline phototaxis was altered following either short-term (3-week) or long-term (6-week) exposure to 0.05 µg/L or 0.5 µg/L fluoxetine. Our results classify all species as significantly photonegative, a response that depended solely on the presence, not quality, of light. Short-term fluoxetine exposure caused some animals to become photoneutral, regardless of concentration, while others remained photonegative. Long-term exposure to 0.5 µg/L fluoxetine caused photoneutral behaviors in all surviving populations; exposure to 0.05 µg/L had variable effects. These differential effects were due to a significant effect of population/sampling location on photobehavior. Overall, these results identify species-specific effects of chronic fluoxetine exposure and underscore how the response to light in 7 geographically distinct populations is uniquely tuned to requirements for survival.     

Natural tissue concentrations in adult Ambystoma maculatum and larval DNA damage from exposure to arsenic and chromium

Arsenic (As) and chromium (Cr) are two contaminants that are detected in aquatic and terrestrial habitats. Using the spotted salamander, Ambystoma maculatum, to assess impacts from these contaminants may be advantageous as adults live and breed in such environments. Adult amphibians typically exhibit elevated tissue concentrations of contaminants present in their environment, while larval stages were found to exhibit increased sensitivity to pollutants. From January through March of 2015, during the spring breeding season, 5 adults and approximately 32 egg masses were collected from a local breeding site. Field levels of As and Cr ranged from 5.99 to 8.88 µg/L and 1.45 to 2 µg/L, respectively, while mean adult As tissue concentrations were 56.74 µg/g dry weight for heart, 0.92 µg/g for liver, and 1.21 µg/g for tail tissue. Mean tissue concentrations for Cr were 87.64 µg/g for heart, 1.47 µg/g for liver, and 6.92 µg/g for tail. Developing larvae that were collected from the field and exposed in a lab setting for 12 d to 0.2 or 20 mg/L of either As or Cr displayed little DNA damage attributed to As, but marked damage due to exposure to 20 mg/L Cr when assessed using the comet assay. Exposure to a mixture of either 0.25:0.1 or 25:10 mg/L As and Cr resulted in significant DNA damage at the lower concentration of 0.25:0.1 mg/L. As adult spotted salamanders were found to possess high concentrations of these contaminants in cardiac tissue, and larvae were shown to be susceptible to DNA damage from increased exposures, assessing impacts and potential declines of amphibian populations exposed to As and Cr is needed.     

Effects of Acute Lead Acetate Exposure on Adult Guinea Pigs: Electrophysiological Study of the Inner Ear

The effects on humans of lead acetate exposure may involve thecranial nerves, since vertigo and sensory neuronal deafnesshave been reported in lead workers; however, there exist onlya few reports concerning the dose effects of lead acetate bothon the cochlea and the eighth cranial nerve. The effects oflead acetate on the cochlea and the eighth nerve were investigatedsystematically using cochlear microphonics (CM), wholenerveaction potential (AP), and endocochlear potential (EP) in guineapigs (male albino Hartley). Guinea pigs were injected with 2ml of a 1% solution of lead acetate (20 mg) once a week for1–5 weeks. The threshold of whole-nerve AP (N₁) was elevatedby injection of lead acetate, even 40 mg, and whole-nerve AP(N₁) output voltage decreased after injection of 100mg of leadacetate. On the other hand, no change was observed in CM afterlead acetate injection (100 mg) or in EP after lead acetateexposure (40 mg). The blood concentrations of lead acetate wereas follows (mean): control, 4.5 µg/dl; Expt 1, 80 µg/dl;Expt 2, 126 µg/dl; Expt 3, 142 µg/dl;. We concludethat dysfunction of the eighth nerve is induced by high-doselead exposure, but that lead exposure does not induce electrophysiologicaldysfunction of the organ of Corti and the stria vascularis.