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Archives of Environmental Contamination and Toxicology (v.35, #3)

Editorial by Richard J. Wenning (pp. 369-369).

Human Cell Line Toxicity of Binary and Ternary Chemical Mixtures in Comparison to Individual Toxic Effects of Their Components by G. Malich; B. Markovic; C. Winder (pp. 370-376).

Contaminated site projects involve health risk assessment procedures that must consider potential interactive effects of present contaminants. In order to establish a quick and reliable method that accounts for smaller than additive, additive, or greater than additive toxic effects, this study compared the cytotoxicity of 34 binary and ternary chemical mixtures of four structurally different chemicals to HeLa cells. Further, five blind samples of these mixtures or their components were tested to determine the ability to identify unknown mixtures. The colorimetric MTS in vitro cytotoxicity assay was used to detect cytotoxic effects after the cells were exposed for 1 h to serial dilutions of the mixtures. Experimental cytotoxicity data were compared and set against data predicted by a mathematical algorithm. They mainly showed additive effects of the components in mixture but also identified smaller than additive and greater than additive effects. A subjective classification scheme allowed evaluation of the toxicity of the blind samples in comparison to results from the study on binary and ternary mixtures tested before. This scheme focused on quantitative cytotoxicity data as well as on the slope of the concentration-effect curves and demonstrated the use of the MTS assay for human health risk assessments in the context of contaminated sites.

Spatial and Temporal Trends of Paraquat, Diquat, and Difenzoquat Contamination in Water from Marsh Areas of the Valencian Community (Spain) by M. Fernández; M. Ibáñez; Y. Picó; J. Mañes (pp. 377-384).

The levels and distribution of diquat, paraquat, and difenzoquat were determined by solid phase extraction (SPE) and high-performance liquid chromatography (HPLC) in water samples from irrigation channels, rivers, and lagoons taken during 1 year from three different marsh areas of the Valencian community. These areas are representative of the typical Mediterranean coastal ecosystems. All three compounds were detected. Diquat was found most frequently at all the sampling sites. Although the spatial distribution of diquat and paraquat showed a maximum concentration near the fields where they were originally applied, their location fluctuated due to irregular large spill and/or loading. The herbicide concentration tended to be highest during the summer (June, July, and August) because these are the months with the least rainfall and highest evaporation rates, when weeds grow best and pesticides are needed more often. The average concentration found for diquat was 0.09 μg/L, with a maximum of 3.10 μg/L. The average concentration for paraquat was 0.01 μg/L, with a maximum of 3.95 μg/L. Samples that were below the method detection limit are included in the mean calculation as zero. Difenzoquat was only detected in one sample at a concentration of 1.75 μg/L.

The Environmental Occurrence of Herbicides: The Importance of Degradates in Ground Water by D. W. Kolpin; E. M. Thurman; S. M. Linhart (pp. 385-390).

Numerous studies are being conducted to investigate the occurrence, fate, and effects on human health and the environment from the extensive worldwide use of herbicides to control weeds. Few studies, however, are considering the degradates of these herbicides in their investigations. Our study of herbicides in aquifers across Iowa found herbicide degradates to be prevalent in ground water, being detected in about 75% of the wells sampled. With the exception of atrazine, the frequencies of detection in ground water for a given herbicide increased multifold when its degradates were considered. Furthermore, a majority of the measured concentration for a given herbicide was in the form of its degradates—even for a relatively persistent compound such as atrazine. For this study, degradates comprised from 60 to over 99% of a herbicide's measured concentration. Because herbicide degradates can have similar acute and chronic toxicity as their parent compounds, these compounds have environmental significance as well as providing a more complete understanding of the fate and transport of a given herbicide. Thus, it is essential that degradates are included in any type of herbicide investigation.

Organophosphate and Carbamate Insecticides in Agricultural Waters and Cholinesterase (ChE) Inhibition in Common Carp (Cyprinus carpio) by S. J. Gruber; M. D. Munn (pp. 391-396).

Cholinesterase (ChE) activity was used as a biomarker for assessing exposure of common carp (Cyprinus carpio) to organophosphate and carbamate insecticides from irrigated agricultural waters. Carp were collected from a lake (Royal Lake) that receives most of its water from irrigation return flows and from a reference lake (Billy Clapp Lake) outside of the irrigation system. Results indicated that the mean whole-brain ChE activity of carp from Royal Lake (3.47 μmol/min/g tissue) was 34.2% less than that of carp from Billy Clapp Lake (5.27 μmol/min/g tissue) (p = 0.003). The depressed ChE activity in brain tissue of Royal Lake carp was in response to ChE-inhibiting insecticides detected in water samples in the weeks prior to tissue sampling; the most frequently detected insecticides included chlorpyrifos, azinphos-methyl, carbaryl, and ethoprop. Neither sex nor size appears to be a covariable in the analysis; ChE activity was not correlated with fish length or weight in either lake and there was no significant difference in ChE activity between the two sexes within each lake. Although organophosphate and carbamate insecticides can break down rapidly in the environment, this study suggests that in agricultural regions where insecticides are applied for extended periods of the year, nontarget aquatic biota may be exposed to high levels of ChE-inhibiting insecticides for a period of several months.

Photolysis of Phloxine B in Water and Aqueous Solutions by L. Wang; W.-F. Cai; Q. X. Li (pp. 397-403).

Phloxine B (2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachlorofluorescein disodium salt) as a potential photoactive insecticide was rapidly photodegraded in water under various light sources. Two major photolytic products characterized were 2′,4′,5′-tribromo-4,5,6,7-tetrachlorofluorescein and 4′,5′-dibromo-4,5,6,7-tetrachlorofluorescein. The photolysis rates of phloxine B were influenced by various factors including salts in medium, sample pH, and light sources. Half-lives (t_½) of phloxine B spiked in different water samples and 2% NaCl solution at 29 ± 1°C ranged from 0.70 to 1.28, 26.3 to 115, and 14.1 to 46.2 hours under 254 nm, 365 nm, and cool white fluorescent lights, respectively. Half-lives of phloxine B in tap, stream, or seawater in a beaker were from 10 to 13 min under sunlight at ambient air temperature. In a range of buffer pH 6–8 at 29 ± 1°C, phloxine B photodegraded slightly faster in acidic solution than in basic solution. The photolysis t_½ of phloxine B at 29 ± 1°C was 25, 32, 128, and 755 min in the buffered NaF, NaCl, NaBr, and NaI solutions, respectively. The t_½ of phloxine B was 31 min when phloxine B was dissolved in the sodium phosphate buffer as control. Sodium iodide and ammonium iodide photostabilized phloxine B 24 and 27 folds, respectively, when it was compared with the buffer control.

Influence of Trophic Status on the Toxic Effects of a Herbicide: A Microcosm Study by J. R. Pratt; R. Barreiro (pp. 404-411).

Naturally derived microbial communities were developed in the laboratory under three nutrient regimes by manipulating phosphate and nitrate concentrations. Resulting communities differed in both functional and structural attributes. Low nutrient microcosms (0.05 mg N-NO₃ ⁻/L + 0.01 mg P-PO₄ ⁻³/L) showed the sharpest differences. Medium (0.5 mg N-NO₃ ⁻/L + 0.1 mg P-PO₄ ⁻³/L) and high (5.0 mg N-NO₃ ⁻/L + 1.0 mg P-PO₄ ⁻³/L) nutrient treatments differed in total algal biomass and algal community composition. After a 25-day acclimation period, a single dose of the herbicide diquat (3.5 mg/L) was added to test the response of the microbial communities to herbicide stress. Regardless of nutrient regime, diquat-dosed microcosms had decreased electron transport system activity (ETSA), an almost complete absence of cyanobacteria, and reduced gross photosynthesis (GP), respiration, and pH relative to undosed microcosms. Inorganic nutrients (PO₄ ⁻³, NO₃ ⁻) were released from the stressed algal communities, probably as a result of their altered metabolism. Alkaline phosphatase activity (APA), total microbial biomass (estimated as protein), algal biomass (estimated as chlorophyll), and relative abundance of green algal taxa proved highly insensitive to herbicide action. Nutrient treatments had a small influence on toxicant effects; the magnitude of the herbicide effects was comparable across nutrient levels. Only the capacity of recovery from the toxic stress was affected by trophic status. At the end of the study period, ETSA had recovered to control values in high nutrient microcosms but not in medium and low ones. Microcosm pH, and to a lesser extent GP, showed recovery under both high and medium nutrient treatments. Trophic status affected the diquat disappearance rate; the herbicide persisted longer in low nutrient microcosms than in high and medium nutrient ones. Differences in recovery capacity may stem from higher nutrient level microcosms reaching less toxic herbicide levels in a shorter period of time.

Characterization of Organotin-Resistant Bacteria from Boston Harbor Sediments by A. Pain; J. J. Cooney (pp. 412-416).

Organotins are widely used in agriculture and industry. They are toxic to a variety of organisms including bacteria, although little is known of their physiology and ecology. Bacteria resistant to six organotins—tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPT), diphenyltin (DPT), and monophenyltin (MPT)—were isolated from Boston Harbor sediments, Massachusetts, USA. Bacteria resistant to each of the organotins, except DPT, were isolated directly from estuarine sediments. Viability of the organotin-resistant bacteria on serial transfer in the laboratory ranged from 80 to 91%. Each isolate was screened for resistance to the other organotins. All of 250 isolates were resistant to at least two organotins. No DPT-resistant isolates were found on initial isolation on DPT, although there was DPT resistance among the other organotin-resistant bacteria. Eighty percent of TBT-resistant bacteria were TPT-resistant, suggesting that antifouling paints containing TPT will not be a suitable substitute for TBT in paints designed to inhibit microbial biofilms. Debutylation reduced toxicity in some cases while dephenylation did not. Thus, even though trisubstituted organotins are generally believed to be more toxic than di- or monosubstituted organotins, this may not always be the case, and more than one mechanism of resistance may be involved. All the bacteria were resistant to at least six of eight heavy metals tested, suggesting that resistance to heavy metals may be associated with resistance to organotins.

Sensitivity to Copper in a Ciliate as a Possible Component of Biological Monitoring in the Lagoon of Venice by O. Coppellotti (pp. 417-425).

The impact of copper was studied in cultures of the microbenthic organism Euplotes vannus (Ciliophora, Hypotrichida). This ciliate was isolated from sediment collected from a particular area in the Lagoon of Venice, which may be considered almost unpolluted by heavy metals. The effects of copper exposure in the laboratory on growth, metal accumulation, total acid-soluble thiol levels, and glutathione levels were examined, together with morphological alterations. E. vannus exhibited tolerance toward copper up to a concentration exposure of 0.2 μg Cu/ml, at which cell growth rate overlapped that of controls. Copper accumulated up to 239 μg/g dry wt after exposure to 0.4 μg Cu/ml, and morphological alterations were evident in cells exposed to concentrations from this value upward. Processes of vegetative reorganization involving nuclear apparatus, membranelles, cirri, and cortex could be observed after 4 days of exposure to 0.4 μg Cu/ml. Laboratory experiments with cultures of ciliates of cosmopolitan distribution such as Euplotes species in controlled conditions indicated the value of these organisms, which constitute a simple model for a monitoring system suitable for prediction in multicellular organisms.

The Toxicity of Margosan-O, a Product of Neem Seeds, to Selected Target and Nontarget Aquatic Invertebrates by I. M. Scott; N. K. Kaushik (pp. 426-431).

Margosan-O, an insecticide formulated from extracts of neem tree (Azadirachta indica) seed kernels, besides being toxic, also has feeding, oviposition-deterring, and growth-inhibitory effects on insects. This product, registered in the United States for ornamental plants, has been proposed for food crop use. However, little information exists on its effects on aquatic organisms. This study investigated toxicity of Margosan-O to the mosquito Culex spp., a possible target species, and to nontarget species—two crustaceans, Daphnia magna, Hyalella azteca, and a dipteran, Chironomus riparius. The 48-h EC50 value of 105 mg L⁻¹ for Culex spp. was significantly more toxic than for C. riparius (281 mg L⁻¹), not significantly different from D. magna (125 mg L⁻¹) but was significantly less toxic than for H. azteca (71 mg L⁻¹). A concentration of 20–30 mg L⁻¹ caused growth inhibitory effects in Culex spp. and C. riparius larvae and 40 and 84 mg L⁻¹ affected growth and reproduction in H. azteca and D. magna, respectively. Margosan-O may not be suitable for mosquito control since the concentrations required to control emergence may have some nontarget effects. Alternatively, the agricultural application of Margosan-O is also not expected to reduce the survival or produce growth and reproductive effects in nontarget aquatic organisms. However, based on estimated concentrations of less than 10 mg L⁻¹ in adjacent shallow bodies of water and recommendations for repeated applications, there should be concern that the threshold for chronic toxicity is too narrow.

The Acute Toxicity of Lindane to Hyalella azteca and the Development of a Sublethal Bioassay Based on Precopulatory Guarding Behavior by S. J. Blockwell; S. J. Maund; D. Pascoe (pp. 432-440).

Acute and sublethal toxicity of the organochlorine insecticide lindane to the amphipod crustacean Hyalella azteca was investigated. Acute experiments were conducted for a maximum test exposure period of 240 h with adult and neonate H. azteca. Median lethal concentrations (LC50s) determined for adult Hyalella included a 48-h LC50 of 47.6 μg/L and 240-h LC50 of 26.9 μg/L. For neonate H. azteca 24-, 48-, and 240-h LC50s were 29.5, 14.8, and 9.8 μg lindane/L, respectively. Neonate H. azteca were approximately three times more sensitive than adults. Two sublethal toxicity bioassays were developed based on the direct and indirect disruption of the precopulatory or mate guarding behavior of Hyalella. This reproductive behavior is readily quantifiable and of ecological significance as it is a vital component of the mating success of the species. The direct disruption bioassay examined the separation of precopulatory pairs maintained in control water and a range of lindane concentrations during a 24-h exposure period. Median separation times (ST50s) were determined and the LOEC was 24.4 μg lindane/L. The indirect disruption bioassay consisted of a test exposure period of just 4 h after which an invertebrate anesthetic solution was administered to induce separation of precopulatory pairs. The LOEC was 17.3 μg lindane/L, suggesting that the indirect precopulatory separation bioassay was comparable to the 24-h direct separation study. Both bioassays are rapid, relatively simple to perform, and have yielded effect concentrations that correspond with LC50 values determined using adult and neonate H. azteca life stages over more prolonged lindane exposures. Following some modification, these behavioral bioassays may be suitable for use in the hazard evaluation of sediments and for deployment as in situ toxicity tests.

Chronic Effects of the Herbicide Diuron on Freshwater Cladocerans, Amphipods, Midges, Minnows, Worms, and Snails by A. V. Nebeker; G. S. Schuytema (pp. 441-446).

The chronic effects of the herbicide diuron on survival and reproduction of Daphnia pulex, and survival and growth of the amphipod Hyalella azteca, the midge Chironomus tentans, juvenile and embryo/larval fathead minnows, Pimephales promelas, annelid worms, Lumbriculus variegatus, and snails, Physa gyrina, were determined in laboratory static and static-renewal tests. D. pulex 96-h and 7-day LC50 values were 17.9 and 7.1 mg/L; 7-day LOAEL and NOAEL values based on mortality and reproduction were 7.7 and 4.0 mg/L. H. azteca 96-h and 10-day LC50 values were 19.4 and 18.4 mg/L; 10-day LOAEL and NOAEL values based on survival and reduced weight were 15.7 and 7.9 mg/L. C. tentans 10-day LC50 value was 3.3 mg/L; 10-day LOAEL and NOAEL values based on growth were 7.1 and 3.4 mg/L, and 3.4 and 1.9 mg/L based on mortality. Juvenile fathead minnows had a 10-day LC50 of 27.1 mg/L and 10-day LOAEL and NOAEL values based on growth of 3.4 and <3.4 mg/L. The fathead minnow embryo-larval test had a 7-day LC50 value of 11.7 mg/L and 7-day LOAEL and NOAEL values based on reduced growth of 8.3 and 4.2 mg/L. L. variegatus had 10-day LOAEL and NOAEL values based on reduced weight of 3.5 and 1.8 mg/L. P. gyrina had 10-day LOAEL and NOAEL values based on reduced weight of 22.8 and 13.4 mg/L. Laboratory effects concentrations were higher that those found in normal field application situations, except in areas of localized pooling after recent herbicide applications, indicating that there would probably be little harm to these fish and invertebrates from diuron exposure in the field.

Heavy Metals Alter the Survival, Growth, Metamorphosis, and Antipredatory Behavior of Columbia Spotted Frog (Rana luteiventris) Tadpoles by H. Lefcort; R. A. Meguire; L. H. Wilson; W. F. Ettinger (pp. 447-456).

Amphibian populations appear to be declining around the world. Although there is no single cause, one factor may be pollution from heavy metals. As a result of mining in the Silver Valley of Idaho, heavy metals have been released into habitats containing many species of sensitive organisms, including spotted frogs (Rana luteiventris). While the gross extent of pollution has been well documented, the more subtle behavioral effects of heavy metals such as lead, zinc, and cadmium are less well studied. We tested the effects of heavy metals on the short-term survival (LC₅₀) of spotted frog tadpoles. Compared to single metals, metals presented together were toxic at lower doses. We also raised the tadpoles in outdoor mini-ecosystems containing either a single heavy metal or soil from an EPA Superfund site in the Silver Valley known to be composed of numerous heavy metals. Exposure to Silver Valley soil resulted in delayed metamorphosis. We tested the ability of metal-exposed tadpoles to detect and respond to chemical cues emanating from predacious rainbow trout. We found that high levels of Silver Valley soil, medium levels of zinc, and medium and high levels of lead resulted in a decreased fright response. Low levels of cadmium, zinc, and lead did not cause a significant effect, but low levels of soil did result in a decreased fright response. Heavy metals may alter interactions between tadpoles and their predators.

Heavy Metals Inhibit Limb Regeneration in Horseshoe Crab Larvae by T. Itow; T. Igarashi; M. L. Botton; R. E. Loveland (pp. 457-463).

We studied the effects of heavy metals on the regeneration of walking legs in horseshoe crabs (Limulus polyphemus). The second walking leg was amputated in embryos (stage 20 and 21) and first instar (trilobite) larvae, and the length and morphology of the regenerated appendage was observed after molting to the second instar stage. Regeneration following continuous exposure to TBT (0.001–100 mg/L), mercury (0.001–100 mg/L), cadmium (0.01–100 mg/L), chromium (0.1–100 mg/L), lead (0.1–100 mg/L), and copper (1–100 mg/L) was measured relative to regeneration in seawater. Although regeneration was incomplete in controls, treatment with heavy metals led to smaller and/or malformed legs. The impacts of heavy metals on survival, molting, and regeneration of horseshoe crab larvae were ranked as follows: organotin > Hg > Cd > Cr > Zn > Pb ≧ Cu. Cu and Pb did not inhibit regeneration, even at 100 mg/L. TBT, Hg, Cd, Cr, and Zn inhibited the regeneration of appendages, although first instar larvae successfully molted into second instars even after treatment. Regeneration was comparable to seawater controls in less than 2.5 mg/L Zn. In 5.0 and 10.0 mg/L Zn, regeneration was inhibited and the length of regenerated appendages remained shorter in all second instars. Larvae treated with 10 mg/L Zn for 1-week intervals during the molt cycle showed similar patterns of regeneration. The regeneration of claws was not all or none, and formation of the claw was proportional to the length of regenerated appendages. Limb regeneration in horseshoe crab larvae may be a useful model system for the study of pollutant impacts.

Altered Growth and Metabolism of an Estuarine Shrimp (Palaemonetes pugio) During and After Metamorphosis onto Fenvalerate-Laden Sediment by C. L. McKenney, Jr.; D. E. Weber; D. M. Celestial; M. A. MacGregor (pp. 464-471).

Dry weight (W), carbon (C), nitrogen (N), and energy (E) (calculated) accumulation were measured in the estuarine grass shrimp, Palaemonetes pugio, throughout larval development and during the first 2 weeks as postlarvae in seawater over sediment containing the pyrethroid insecticide fenvalerate (SCF; nominal concentrations of 1, 10, and 100 μg fenvalerate kg⁻¹ sediment). The influence of fenvalerate-laden sediment on shrimp growth and utilization patterns of C, N, and E was dependent on fenvalerate concentration, age of shrimp, and whether shrimp were premetamorphic or postmetamorphic in development. The fenvalerate concentration in the sediment, which ultimately inhibited larval metamorphosis (100 μg fenvalerate kg⁻¹ sediment), significantly reduced W accumulation in developing larvae and in postlarvae growing on the sediment for an equivalent time. Accumulation of C, N, and E varied not only with concentration of SCF, but differed between pelagic larvae developing in water above SCF and newly settled postlarvae growing in direct contact with SCF. Larvae developing above ≥10 μg kg⁻¹ SCF contained significantly less N, while postlarval shrimp settling onto ≥10 μg kg⁻¹ SCF accumulated significantly less C and E. Measurable variations in growth and energy reserves of toxicant-sensitive life stages in response to environmentally realistic insecticide exposures have a direct link to ecological consequences of toxic stress and may be useful as biomarkers to diagnose early damage in estuarine populations.

A Study of the Lethal and Sublethal Toxicity of Polyphase P-100, an Antisapstain Fungicide Containing 3-Iodo-2-Propynyl Butyl Carbamate (IPBC), on Fish and Aquatic Invertebrates by A. P. Farrell; E. Stockner; C. J. Kennedy (pp. 472-478).

The acute toxicity of Polyphase P-100, an antisapstain wood preservative that contains 97% 3-iodo-2-propynyl butyl carbamate (IPBC), was determined for three species of fish (coho salmon, rainbow trout, and starry flounder) and three species of aquatic invertebrates (Daphnia magna, Hyalella azteca, and Neomysis mercedis). The 96-h LC₅₀ values for the various fish species exposed to Polyphase P-100 ranged from 95 ppb for coho smolts (Oncorhynchus kisutch) to 370 ppm for juvenile starry flounder (Platichthys stellatus). The sensitivity of coho to Polyphase P-100 was altered by their developmental stage. Coho embryos were six to nine times more tolerant of Polyphase P-100 than coho alevins, which were twice as tolerant as coho smolts. The 48-h LC₅₀ values for the invertebrates D. magna, H. azteca, and N. mercedis were 40 ppb, 500 ppb, and 2,920 ppb, respectively. In addition to a wider range of sensitivity to Polyphase P-100 compared with the fish species, the invertebrate species were characterized by a shallower concentration-response. In acute, 24-h sublethal tests with juvenile starry flounder and rainbow trout, there was no primary or secondary stress response (changes in hematocrit, leucocrit, hemoglobin concentration, plasma lactate concentration, and plasma cortisol concentration) at concentrations up to 50% of the 96-h LC₅₀ value. The acute toxicity of a 1:8 mixture of Polyphase P-100 and Bardac 2280 (another antisapstain compound that contains didecyldimethylammonium chloride [DDAC] as the active ingredient) was close to additive for fish, but not for invertebrate species. The acute toxicity of the mixture was seven to eight times more than additive for H. azteca, but two to three times less than additive for D. magna. Some sublethal stress responses were revealed with the mixture that were not observed with the test chemicals alone.

Acute 2,4-D Poisoning in Tench (Tinca tinca L.): Lesions in the Hematopoietic Portion of the Kidney by L. Gómez; J. Masot; S. Martínez; E. Durán; F. Soler; V. Roncero (pp. 479-483).

An experimental model was designed to study the acute lesions caused by a continuous exposure to 2,4-dichlorophenoxyacetic acid (2,4-D) disolved in water (400 mg/L) in hematopoietic kidney tissue in tench (Tinca tinca L). Fifty fish were used in this study, 15 for calculating LC₅₀ and 35 were euthanized 1, 2, 5, 8, and 12 days postpoisoning (five treated and two controls each time). Tissue samples, fixed in 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for histopathological examination, revealed marked alteration of hematopoietic tissue, characterized by progressive swelling and cell necrosis, activation of the phagocyte system, and subsequent formation of myelin figures. Variations recorded in hematocrit and hemoglobin levels in blood samples indicated changes in membrane permeability, complementing the findings on hematopoietic tissue. The lethal dose (LC₅₀) at 96 h demonstrated the importance of the species and chemical form used as factors in calculating a product's toxicity.

Behavioral Responses to Atrazine and Diuron in Goldfish by P. Saglio; S. Trijasse (pp. 484-491).

Experiments were performed in goldfish to determine the effects of a short-term exposure (24 h) to atrazine or diuron (0.5, 5, 50 μg/L) on some behavior endpoints related to swimming and social activities. Observations were also made to assess the influence of such exposure on the behavioral responses of fish to the flow of a crude skin extract solution from conspecifics, active in social chemocommunication and producing alarm behaviors. Additive tests were run to check the behavioral responses of previously unexposed goldfish to the flow of a solution of atrazine- or diuron-contaminated water, at three concentrations (0.1, 1, 10 mg/L). Significant burst swimming reactions appeared in response to a 24-h exposure to atrazine, at the lowest concentration tested (0.5 μg/L). A 24-h exposure to 5 μg/L atrazine or diuron was found to induce various significant behavioral alterations in fish. At this concentration, both herbicides decreased grouping behavior and atrazine also increased surfacing activity. Herbicide-exposed fish showed a decreased grouping behavior during the flow of the skin extract solution. Sheltering was also decreased during the flow of the biological solution in fish exposed to atrazine. Moreover, fish exposed to diuron clearly displayed attraction responses to the flow of the skin solution. Previously unexposed fish showed a significant increase in burst swimming reactions in response to the flow of a solution of atrazine- or diuron-contaminated water, at all concentrations tested (0.1, 1, 10 mg/L). Furthermore, the diuron-contaminated flow was found to be significantly attractive at the highest concentration. These results indicate that a short-term exposure to a relatively low concentration (5 μg/L) of atrazine or diuron can affect various behaviors of fish not only directly but also indirectly by altering the chemical perception of natural substances of eco-ethological importance. In consideration of the basic role of olfaction in fish behavior, these results also emphasize the need for further developments on the possible effects of aquatic toxicants on olfactory-mediated behaviors.

Contaminants in Eggs of Colonial Waterbirds and Hepatic Cytochrome P450 Enzyme Levels in Pipped Tern Embryos, Washington State by L. J. Blus; M. J. Melancon; D. J. Hoffman; C. J. Henny (pp. 492-497).

Eggs of Forster's terns (Sterna forsteri) collected in 1991 from nesting colonies on Crescent Island (Columbia River) and the Potholes Reservoir in south central Washington generally contained low residues of organochlorine pesticides and metabolites, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and polychlorinated biphenyls (PCBs). Hepatic cytochrome P450 enzyme activity in pipped embryos of Forster's terns from the two colonies seemed unaffected by contaminants. At Crescent Island, examination of 23 Forster's tern eggs with large embryos (19 viable [10 pipped] and four dead [two pipped]) revealed developmental abnormalities in two viable pipped embryos (missing maxilla and deformed pelvic girdle) and a viable prepipping embryo (shortened beak). Our limited sample sizes and number of compounds analyzed preclude us from determining whether or not the abnormalities are related to contaminants. No abnormalities were noted in 10 pipped eggs (nine viable and one dead at collection) of Forster's terns collected from the Potholes Reservoir colony. Eggs of Caspian terns (Sterna caspia) collected from Crescent Island in 1991 also contained generally low residues of contaminants, only one developmental abnormality was noted, and limited data indicated that cytochrome P450 enzyme activity apparently was unaffected by contaminants. Organochlorine contaminants were generally low in addled eggs of American white pelicans (Pelecanus erythrorhynchos) collected from Crescent Island in 1994.

Subchronic Dietary Toxicity of Strychnine: Bobwhite Quail Are Less Sensitive than Mallard Ducks by R. T. Sterner; C. A. Pedersen; B. R. Helsten; M. J. Goodall (pp. 498-505).

Separate, 28-day, subchronic studies of strychnine dietary toxicity were conducted using northern bobwhite quail (Colinus virginianus) and mallard ducks (Anas platyrhynchos). Five groups (five males five females/group) of 29-week-old quail were fed Purina® Game Bird Breeder Layena® diets containing mean (±SD) 484.2 (±17.0), 972.6 (±54.0), 1,870.8 (±176.1), 3,516.7 (±68.0), and 6,083.3 (±269.6) μg/g strychnine; whereas five groups of 27-week-old mallards (five males five females/group) were fed similar diets containing mean (±SD) 18.8 (±1.3), 91.1 (±27.3), 235.0 (±33.8), 484.2 (±17.0), and 972.6 (±54.0) μg/g strychnine. Separate “vehicle control” (0.0 μg/g strychnine) groups (five males, five females/group) were included in each study. Strychnine toxicity was much less pronounced in quail; no observed effect concentrations (NOECs) were 972.6 (±54.0) and 91.1 (±27.3) μg/g strychnine for quail and ducks, respectively. Several possible explanations for the species effects are offered, and some practical issues affecting the conduct of long-term, dietary toxicity studies are discussed.

Retrospective Study of the Diagnostic Criteria in a Lead-Poisoning Survey of Waterfowl by W. N. Beyer; J. C. Franson; L. N. Locke; R. K. Stroud; L. Sileo (pp. 506-512).

Between 1983 and 1986 the National Wildlife Health Center (NWHC) conducted a nationwide study of lead poisoning of waterfowl from federal and state refuges. This survey was done to assist in identifying zones with lead-poisoning problems. One thousand forty one moribund or dead waterfowl were collected and examined. The presence or absence of 13 gross lesions selected as indicators of lead poisoning and three lesions indicating body condition was recorded. Lead-poisoning diagnoses were based on the finding of at least 6–8 ppm (wet weight) lead in the liver and either lead shot in the gizzard content or at least one convincing gross lesion indicative of lead poisoning. Four hundred twenty-one of these waterfowl were diagnosed as lead poisoned. The NWHC survey provided a comprehensive basis for estimating the sensitivities, specificities, and likelihood ratios of the gross lesions of lead poisoning and the associated hepatic lead concentrations for several species of waterfowl. Some of the 13 defined gross lesions were more common than others; frequencies ranged from 3% to 80% in the 421 lead-poisoned waterfowl. The most reliable indicators of lead poisoning were impactions of the upper alimentary tract, submandibular edema, myocardial necrosis, and biliary discoloration of the liver. Each of the 13 lesions occurred more frequently in the lead-poisoned birds, but each of the lesions also occurred in waterfowl that died of other causes. The number of lead shot present in a bird's gizzard was only weakly correlated with its hepatic lead concentration; however, this weak correlation may have been adequate to account for differences in hepatic lead concentrations among species, once the weights of the species were taken into account. Although lead-poisoned ducks tended to have higher hepatic mean lead concentrations than did lead-poisoned geese or swans, the differences were probably a result of a greater dose of shot per body weight than to kinetic differences between species. Hepatic lead concentrations were independent of age and sex. Ninety-five percent of waterfowl diagnosed as lead poisoned had hepatic lead concentrations of at least 38 ppm dry weight (10 ppm wet weight). Fewer than 1% of the waterfowl that died of other causes had a concentration that high. This fifth percentile, of 38 ppm dry weight (10 ppm wet weight), is a defensible criterion for identifying lead-poisoned waterfowl when interpreting hepatic lead concentrations in the absence of pathological observations.

Subacute and Reproductive Effects in Mink from Exposure to Fusarium fujikuroi Culture Material (M-1214) Containing Known Concentrations of Moniliformin by M. K. Morgan; S. J. Bursian; G. E. Rottinghaus; G. A. Bennett; J. A. Render; R. J. Aulerich (pp. 513-517).

This study was conducted to ascertain the subacute and reproductive effects in mink (Mustela vison) resulting from exposure to moniliformin, a toxic mycotoxin produced by Fusarium fungi. In a preliminary trial, adult mink were presented diets that contained targeted concentrations of 10, 20, 40, 80, 160, or 240 ppm moniliformin provided by F. fujikuroi culture material (M-1214). The mink fed diets that contained more than 40 ppm moniliformin refused to eat significant quantities of feed. Feeding adult mink diets that contained 8.1 or 17.0 ppm (wet weight) moniliformin, provided by F. fujikuroi culture material, in a 30-day subacute trial produced no significant adverse effects on feed consumption, body weights, hematologic parameters, or serum chemical values, and notable histologic changes in tissues that were examined. In the reproduction trial, female mink were exposed to the same dietary concentrations of moniliformin provided by F. fujikuroi culture material as in the subacute test from 2 weeks prior to the breeding season until their offspring (kits) were 8 weeks old. Consumption of the high-dose (17 ppm) diet resulted in significant neonatal mortality and reduced kit body weights at birth and at 8 weeks of age. Necropsy of 8-week-old kits from the control and high-dose groups revealed no gross or histologic lesions or alterations in liver, lung, or heart tissues that could account for the mortality observed in the kits exposed to the culture material. These results indicate that long-term (105–135 days) dietary exposure to F. fujikuroi culture material containing 17 ppm moniliformin is not lethal to adult female mink, but can have adverse effects on neonatal mink.

Age-Dependent Accumulation of Heavy Metals in Baikal Seal (Phoca sibirica) from the Lake Baikal by I. Watanabe; S. Tanabe; M. Amano; N. Miyazaki; E. A. Petrov; R. Tatsukawa (pp. 518-526).

Concentrations of Fe, Mn, Zn, Cu, Cd, and Hg were determined in the liver, kidney, and muscle of 60 Baikal seals collected from Lake Baikal in 1992 to investigate age-dependent accumulation. Among essential elements, Fe concentrations in the muscle, liver, and kidney increased with age, suggesting development of diving ability. The concentrations of Mn, Zn, and Cu decreased with age, especially at immature stages. Toxic elements such as Hg and Cd decreased in adult males and thus the male-female difference was clearly observed in their concentrations, which differed from patterns usually found in marine mammals. Such accumulation patterns were due to difference in the feeding rates between males and females under low exposure to Hg and Cd. In addition, a greater excretion of Hg than that of Cd through molting and parturition was estimated.

Lifetime Health Risk Assessment from Exposure of Recreational Users to Polycyclic Aromatic Hydrocarbons by M. Hussain; J. Rae; A. Gilman; P. Kauss (pp. 527-531).

In order to assess the lifetime risk of skin cancer for recreational users from dermal exposure to polycyclic aromatic hydrocarbons (PAHs), sediment samples were collected from beach sites along the St. Marys River near Sault Ste. Marie, Ontario, and in Hamilton Harbor and Toronto Harbor, Ontario, and analyzed for PAHs. Dermal exposure and lifetime skin cancer risk were estimated as follows: Concentrations of 11 PAHs with sufficient or limited evidence of carcinogenicity or mutagenicity were converted to benzo(a)pyrene (BaP) equivalents using toxic equivalency factors (TEFs). Lifetime dermal exposure values were derived based on the BaP equivalents in the silt + clay fraction taken as representative of suspended sediment particulates to which recreational users would be exposed. The lifetime health risk of skin cancer associated with such exposures was above the negligible risk level of 1.0 × 10⁻⁶ at offshore Rytac, Lake George Channel, and Bell Point beaches in the St. Marys River; at Pier 4 Park in Hamilton Harbor; and at Humber Bay, Sunnyside Beach, Cherry Beach, and Water Rats Sailing Club in Toronto Harbor. Risk was negligible inshore at the Rytac and Bell Point beaches and at Squirrel Island and Ojibway Trailer Park along St. Marys River, at Lax Beach in Hamilton Harbor; and at Centre Island in Toronto Harbor. Strategies to reduce risk were developed with these communities; a key recommendation was to take a bath or shower within 24 h after a swim because virtually all the PAHs on the skin would be removed.

Assessment of Asbestos Burden in the Placenta and Tissue Digests of Stillborn Infants in South Texas by A. K. Haque; D. M. Vrazel; T. Uchida (pp. 532-538).

The primary aim of this prospective study was to examine the tissues and placentas of autopsied stillborn infants for presence of asbestos fibers. Asbestos burden of lung, liver, skeletal muscle, and placenta digests of 82 stillborn infants was determined using standard bleach digestion technique. The digests were examined by electron microscopy, and the types of fibers determined using energy dispersive x-ray analysis and selected area diffraction analysis. Digests of 45 placentas collected from deliveries of liveborn healthy infants were processed and examined similarly as controls. Asbestos fibers were detected in 50% of the fetal digests and 23% of the placental digests of stillborn infants. Of the fibers present, 88% were chrysotile, 10% were tremolite, and 2% were actinolite and anthophyllite. Fibers measured 0.5–16.73 μm in length (mean 1.55 μm), and 0.03–0.8 μm in width (mean 0.098 μm). Lungs were most frequently positive for fibers (50%), followed by muscle (37%), placenta (23%), and liver (23%). Mean fiber counts were highest in the liver (58,736 f/g), followed by placenta (52,894 f/g), lungs (39,341 f/g), and skeletal muscle (31,733 f/g). Digests of 15% of the control placentas also showed asbestos fibers, although in very small numbers. The mean fiber count of the stillborn placentas (52,894 f/g) was significantly higher than the mean fiber count of the control placentas (mean 19 f/g) (p = 0.001). A highly significant association was found between fiber presence in stillborns and a maternal history of previous abortions (p = 0.007). A significant association was also found between fiber presence and placental diseases (p = 0.041). An association was suggested between working mothers and fiber presence (p = 0.19), although it did not reach statistical significance. The study documents the presence of small and thin asbestos fibers in stillborn fetal tissues and placenta. Significantly higher number of fibers were found in stillborn tissues compared to controls (liveborn placenta). The absence of a maternal history of asbestos-related occupations suggests that the fibers may have been acquired through environmental exposure.

Exposure to Methyl Bromide During Greenhouse Fumigation on Crete, Greece by J. A. F. de Vreede; J. den Boeft; J. J. van Hemmen (pp. 539-547).

In agricultural areas where greenhouses and dwellings are intermixed, the general population as well as the professional applicators may be exposed to pesticides. In a field study on Crete, exposure to methyl bromide during soil fumigation was assessed. Exposure of applicators (both contractors and farmers) were measured with personal air sampling equipment. Environmental monitoring inside and outside greenhouses combined with meteo data formed the basis for calculating the exposure of the general population with a computer aided dispersion model. Exposure of contractors exceeded the TLV value. The safe limit for the general population living close to a fumigated greenhouse is also exceeded.

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Archives of Environmental Contamination and Toxicology (v.35, #3)