Using accelerated life testing procedures to compare the relative sensitivity of rainbow trout and the federally listed threatened bull trout to three commonly used rangeland herbicides (picloram, 2,4-D, and clopyralid)

We conducted 96-h static acute toxicity studies to evaluate the relative sensitivity of juveniles of the threatened bull trout (Salvelinus confluentus) and the standard cold-water surrogate rainbow trout (Onchorhyncus mykiss) to three rangeland herbicides commonly used for controlling invasive weeds in the northwestern United States. Relative species sensitivity was compared using three procedures: standard acute toxicity testing, fractional estimates of lethal concentrations, and accelerated life testing chronic estimation procedures. The acutely lethal concentrations (ALC) resulting in 50% mortality at 96 h (96-h ALC50s) were determined using linear regression and indicated that the three herbicides were toxic in the order of picloram acid > 2,4-D acid > clopyralid acid. The 96-h ALC50 values for rainbow trout were as follows: picloram, 41 mg/L; 2.4-D, 707 mg/L; and clopyralid, 700 mg/L. The 96-h ALC50 values for bull trout were as follows: picloram, 24 mg/L; 2.4-D, 398 mg/L; and clopyralid, 802 mg/L. Fractional estimates of safe concentrations, based on 5% of the 96-h ALC50, were conservative (overestimated toxicity) of regression-derived 96-h ALC5 values by an order of magnitude. Accelerated life testing procedures were used to estimate chronic lethal concentrations (CLC) resulting in 1% mortality at 30 d (30-d CLC1) for the three herbicides: picloram (1 mg/L rainbow trout, 5 mg/L bull trout), 2,4-D (56 mg/L rainbow trout, 84 mg/L bull trout), and clopyralid (477 mg/L rainbow trout; 552 mg/L bull trout). Collectively, the results indicated that the standard surrogate rainbow trout is similar in sensitivity to bull trout. Accelerated life testing procedures provided cost-effective, statistically defensible methods for estimating safe chronic concentrations (30-d CLC1s) of herbicides from acute toxicity data because they use statistical models based on the entire mortality:concentration:time data matrix.     

Effects of atrazine and cyanazine on chlorpyrifos toxicity in Chironomus tentans (Diptera: Chironomidae)

Toxicities of two triazine herbicides (atrazine and cyanazine) and an organophosphate insecticide (chlorpyrifos) were evaluated individually and with each herbicide in binary combination with chlorpyrifos using fourth-instar larvae of the aquatic midge, Chironomus tentans. Chlorpyrifos at 0.25 microg/L resulted in an effect in less than 10% of midges in 48-h acute toxicity bioassays. Neither atrazine nor cyanazine alone at relatively high concentrations (up to 1,000 microg/L) caused significant acute toxicity to C. tentans. However, atrazine and cyanazine caused significant synergistic effects on the toxicity of chlorpyrifos when midges were exposed to mixtures of atrazine or cyanazine (10, 100, 1,000 microg/L) with chlorpyrifos (0.25 microg/L). At fixed concentrations (200 microg/L) of the herbicides, toxicity of chlorpyrifos was enhanced by 1.8- and 2.2-fold by atrazine and cyanazine, respectively, at the 50% effective concentration levels. Although atrazine and cyanazine are not effective inhibitors of acetylcholinesterase (AChE) in vitro, the synergism of the two triazine herbicides with chlorpyrifos was associated with increased in vivo inhibition of AChE in midges. We observed a positive correlation between the degree of inhibition of AChE and the concentration of atrazine or cyanazine in the presence of a fixed concentration of chlorpyrifos. It is possible that these herbicides may affect cytochrome P450 enzymes to confer synergistic effects on the toxicity of chlorpyrifos.     

Acute and chronic toxicity of zinc to the mottled sculpin Cottus bairdi

The acute and chronic toxicity of zinc to wild mottled sculpin (Cottus bairdi) was measured with 13-d and 30-d flow-through toxicity tests, respectively. Exposure water hardness was 48.6 mg/L as CaCO3 and 46.3 mg/L as CaCO3 in the acute and chronic tests, respectively; pH was slightly above neutral; and temperature near 12 degrees C. The median lethal concentration (LC50) after 96 h was 156 microg Zn/L, but decreased with exposure duration to a median incipient lethal level (ILL50) of 38 microg Zn/L after 9 d, the lowest zinc LC50 reported for any fish species. The 30-d chronic no-effect and lowest-effect concentrations were 16 microg Zn/L (no mortality) and 27 microg Zn/L (32% mortality), respectively. The ILL50 was 32 microg Zn/L. No sublethal growth differences were observed during the chronic test. Analysis of the results from these tests suggested that mottled sculpin may experience acute and chronic toxicity at zinc concentrations lower than any other fish species tested to date. Protection of aquatic communities in stream reaches contaminated by metals seem to require determination of zinc toxicity to lotic species other than trout and other species amenable to aquaculture.     

Toxicity of perfluorooctane sulfonic acid and perfluorooctanoic acid to Chironomus tentans

Two perfluorinated surfactants, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), were evaluated for their toxicity to the aquatic midge, Chironomus tentans. Impetus for this laboratory study originated from a 10-d, in situ field assessment in which C. tentans was exposed to PFOS at concentrations ranging from 300 to 30,000 microg/L. No midges survived these exposures. Midge survival in a preliminary, acute 10-d laboratory test with nominal PFOS concentrations ranging from 0.1 to 100,000 microg/L showed similar toxicity with respect to survival (median lethal concentration [LC50], 45.2 microg/L) and growth (median effective concentration [EC50], 27.4 microg/L). A parallel test using PFOA indicated no significant impacts on survival or growth. A definitive 10-d assay with PFOS concentrations ranging from 1 to 150 microg/L produced an EC50 for growth (87.2+/-11.6 microg/L) of the same order of magnitude as that in the preliminary findings. The same was not true for survival, however, with the LC50 falling outside the range of test concentrations. To further investigate the sensitivity of C. tentans to PFOS, we conducted a chronic life-cycle test using a nominal concentration range of 1 to 100 microg/L. Three of the four endpoints measured-survival, growth, and emergence-were significantly affected, with EC50 values of 92.2+/-3.1, 93.8+/-2.6, and 94.5+/-3.2 microg/L, respectively. Reproduction was not affected by those PFOS concentrations at which females emerged. The results of the present study indicate that PFOS toxicity thresholds for C. tentans are as much as three orders of magnitude lower than those reported for other aquatic organisms but, at present, are approximately two orders of magnitude higher than those concentrations typically observed in aquatic environments.     

Chronic effects of silver exposure on ion levels, survival, and silver distribution within developing rainbow trout (Oncorhynchus mykiss) embryos

Rainbow trout embryos were chronically exposed to silver (as AgNO3) in moderately hard water (120 mg CaCO3/L, 0.70 mM Cl-, 1.3 mg/L dissolved organic matter. 12.3+/-0.1 degrees C) at nominal concentrations of 0.1, 1, and 10 microg/L (measured = 0.117+/-0.008, 1.22+/-0.16, and 13.51+/-1.58 microg/L, respectively) to investigate the effects on mortality, ionoregulation, and silver uptake and distribution of the embryo. Mortalities in the low concentrations (0.1 and 1.2 microg/L) were not significantly different from controls throughout embryonic development (days 1-32 postfertilization). Mortalities of embryos in the 13.5-microg/L treatment reached 56% by day 32 postfertilization (33% when accounting for control mortality), by which time more than 50% of surviving embryos had hatched. Accumulation of silver in whole embryos of 1.2- and 13.5-microg/L treatments reached the highest concentrations of 0.13 and 0.24 microg/g total silver, respectively, by day 32, but whole embryo silver burden was not correlated with mortality. Silver concentrations in different compartments of the whole embryo (chorion, dissected embryo, and yolk) were greatest just before hatch and were higher in the chorion for all experimental treatments. Up to 85% of total whole embryo silver content was bound to the chorion, which acts as a protective barrier during silver exposure. Whole embryo Na+ concentration in the 13.5-microg/L treatment was significantly reduced relative to controls from days 23 to 32 postfertilization, and levels in the embryo were reduced by 40% at day 32 postfertilization, indicating that silver toxicity in the whole embryo is associated with an ion regulatory disturbance that is similar to the acute effect of AgNO3 in juvenile and adult trout.     

Toxicity of fluoroquinolone antibiotics to aquatic organisms

Toxicity tests were performed with seven fluoroquinolone antibiotics, ciprofloxacin, lomefloxacin, ofloxacin, levofloxacin, clinafloxacin, enrofloxacin, and flumequine, on five aquatic organisms. Overall toxicity values ranged from 7.9 to 23,000 microg/L. The cyanobacterium Microcystis aeruginosa was the most sensitive organism (5-d growth and reproduction, effective concentrations [EC50s] ranging from 7.9 to 1,960 microg/L and a median of 49 microg/L), followed by duckweed (Lemna minor, 7-d reproduction, EC50 values ranged from 53 to 2,470 microg/L with a median of 106 microg/L) and the green alga Pseudokirchneriella subcapitata (3-d growth and reproduction, EC50 values ranged from 1,100 to 22,700 microg/L with a median 7,400 microg/L). Results from tests with the crustacean Daphnia magna (48-h survival) and fathead minnow (Pimephales promelas, 7-d early life stage survival and growth) showed limited toxicity with no-observed-effect concentrations at or near 10 mg/L. Fish dry weights obtained in the ciprofloxacin, levofloxacin, and ofloxacin treatments (10 mg/L) were significantly higher than in control fish. The hazard of adverse effects occurring to the tested organisms in the environment was quantified by using hazard quotients. An estimated environmental concentration of 1 microg/L was chosen based on measured environmental concentrations previously reported in surface water; at this level, only M. aeruginosa may be at risk in surface water. However, the selective toxicity of these compounds may have implications for aquatic community structure.     

Long-term sediment bioassay of lead toxicity in two generations of the marine amphipod Elasmopus laevis, S.I. Smith, 1873

Sediments are evaluated for toxicity by measuring mortality in a single cohort of amphipods in either acute (10-d) or chronic (28-d) bioassays. This investigation differed from conventional bioassays in four ways: Sublethal effects (fecundity) were estimated; the testing period was 60+ d; two successive generations were examined; and Elasmopus laevis Smith, 1873, amphipods were employed. Four test sediments were created between 58 and 424 microg/g of lead using the 30-microg/g whole-sediment as the control. Bioaccumulated lead at 60 d varied as a linear function of lead concentration in the sediments. Fecundity, as estimated by offspring-per-chamber and/or percent reproductive success, was reduced as sediment lead concentrations increased and reproduction was delayed compared with the control. The reduction in offspring production per test chamber varied significantly as an inverse function of lead sediment concentration, best described by a curvilinear exponential equation. It was concluded that E. laevis exposed to 118 microg/g and higher could not maintain a population as large as that in the control. Although the current sediment quality guideline for lead stipulates that adverse biological effects likely will occur above 218 microg/g, this study revealed a statistically significant negative reproductive response at 118 microg/g lead, and suggests that the current regulatory guideline for lead, based on lethality, should be reconsidered.     

Effects of ammonia on juvenile unionid mussels (Lampsilis cardium) in laboratory sediment toxicity tests

Ammonia is a relatively toxic compound generated in water and sediments by heterotrophic bacteria and accumulates in sediments and pore water. Recent data suggest that unionid mussels are sensitive to un-ionized ammonia (NH3) relative to other organisms. Existing sediment exposure systems are not suitable for ammonia toxicity studies with juvenile unionids; thus, we modified a system to expose juveniles to ammonia that was continuously infused into sediments. This system maintained consistent concentrations of ammonia in pore water up to 10 d. Juvenile Lampsilis cardium mussels were exposed to NH3 in pore water in replicate 96-h and 10-d sediment toxicity tests. The 96-h median lethal concentrations (LC50s) were 127 and 165 microg NH3-N/L, and the 10-d LC50s were 93 and 140 microg NH3-N/L. The median effective concentrations (EC50s) (based on the proportion affected, including dead and inactive mussels) were 73 and 119 microg NH3-N/L in the 96-h tests and 71 and 99 microg NH3-N/L in the 10-d tests. Growth rate was substantially reduced at concentrations between 31 and 76 microg NH3-N/L. The lethality results (when expressed as total ammonia) are about one-half the acute national water quality criteria for total ammonia, suggesting that existing criteria may not protect juvenile unionids.     

Acute and chronic toxicity of lead in water and diet to the amphipod Hyalella azteca

We evaluated the influence of waterborne and dietary lead (Pb) exposure on the acute and chronic toxicity of Pb to the amphipod Hyalella azteca. Test solutions were generated by a modified diluter with an extended (24-h) equilibration period. Acute (96-h) toxicity of Pb varied with water hardness in the range of 71 to 275 mg/L as CaCO3, despite similar dissolved Pb concentrations. Acute toxicity was greatest in soft test water, with less than 50% survival at the lowest dissolved Pb concentration (151 microg/L). Survival also was significantly reduced in medium-hardness water but not in hard test water. In chronic (42-d) studies, amphipods were exposed to waterborne Pb and fed either a control diet or a diet equilibrated with waterborne Pb levels. For animals fed the control diet, the median lethal concentration (LC50) for Pb was 24 degrees g/L (as dissolved Pb), and significant reductions in survival occurred at 16 microg/L. Exposure to Pb-treated diets significantly increased toxicity across a wide range of dissolved Pb concentrations, with a LC50 of 16 microg/L and significant reductions in growth and reproduction at 3.5 microg/L. Significant effects on growth and reproduction occurred at dissolved Pb concentrations close to the current U.S. chronic water-quality criterion. Our results suggest that both aqueous- and dietary-exposure pathways contribute significantly to chronic Pb exposure and toxic effects in aquatic biota.     

Influence of salinity and organic carbon on the chronic toxicity of silver to mysids (Americamysis bahia) and silversides (Menidia beryllina)

Tests were conducted with mysids (Americamysis bahia) and silversides (Menidia beryllina) to evaluate the influence of salinity and organic carbon on the chronic toxicity of silver. During 7- and 28-d tests conducted at 10, 20, and 30% per hundred salinity, higher concentrations of dissolved silver generally were required to cause a chronic effect as the salinity of the seawater was increased. The 28-d mysid and silverside 20%-effective concentration values (expressed as dissolved silver) ranged from 3.9 to 60 and from 38 to 170 microg/L, respectively, over the salinity range. This pattern was not observed when the same test results were evaluated against the concentrations of free ionic silver (measured directly during toxicity tests), as predicted by the free-ion activity model. Increasing the concentration of dissolved organic carbon from 1 mg/L to the apparent maximum achievable concentration of 6 mg/L in seawater caused a slight decrease in chronic toxicity to silversides but had no effect on the chronic toxicity to mysids. The possible additive toxicity of silver in both food and water also was investigated. Even at the maximum achievable foodborne concentration, the chronic toxicity of silver added to the water was not affected when silver was also added to the food, based on the most sensitive endpoint (growth). However, although fecundity was unaffected at all five tested concentrations during the test with silver in water only, it was significantly reduced at the two highest waterborne silver concentrations (12 and 24 microg/L) during the test with silver dosed into food and water.     

Toxicity and Bioavailability of Copper Herbicides (Clearigate, Cutrine-Plus, and Copper Sulfate) to Freshwater Animals

In designing aquatic herbicides containing copper, an important goal is to maximize efficacy for target species while minimizing risks for nontarget species. To have a margin of safety for nontarget species, the concentration, duration of exposure (i.e., uptake), and form (i.e., species) of copper used for herbicidal properties should not elicit adverse effects on populations of nontarget species. To determine the potential for risk or adverse effects (conversely the margin of safety), data regarding the comparative toxicity of copper-containing herbicides are crucial. A series of comparative toxicity experiments was conducted, including baseline estimates of toxicity (LC50s, LOECs), sensitive species relationships (thresholds and exposure-response slopes), and bioavailability of toxic concentrations and forms of copper 7 days after initial herbicide application. Aqueous 48-h toxicity experiments were performed to contrast responses of Daphnia magna Strauss, Hyalella azteca Saussure, Chironomus tentans Fabricius, and Pimephales promelas Rafinesque to copper herbicides: Clearigate(R), Cutrine(R)-Plus, and copper sulfate. D. magna was the most sensitive aquatic animal tested for all three herbicides; 48-h LC50s for organisms exposed to Clearigate, Cutrine-Plus, and copper sulfate were 29.4, 11.3, and 18. 9 microg Cu/L, respectively. In terms of potency (calculated from the linearized portion of the exposure-response curves, which included 50% mortality), D. magna was the most sensitive animal tested. Organisms exposed to Clearigate, Cutrine-Plus, and copper sulfate had exposure-response slopes of 2.55, 8.61, and 5.07% mortality/microg Cu/L, respectively. Bioavailability of Clearigate and Cutrine-Plus was determined by comparing survival data (LC50s) of test organisms exposed to herbicide concentrations during the first and last 48-h of a 7-day exposure period. Even in these relatively simplified water-only exposures, a transformation of copper to less bioavailable species over time was observed with a 100-200% decrease in toxicity (i.e., an increase in 48-h LC50s) for all four test animals. This series of laboratory experiments provides a worst-case scenario for determining the risk associated with the manufacturer's recommended application rates of Clearigate (100-1,000 microg Cu/L), Cutrine-Plus (200-1,000 microg Cu/L), and copper sulfate (100-500 microg Cu/L) in natural waters for four nontarget freshwater animals.     

Fipronil effects on estuarine copepod (Amphiascus tenuiremis) development, fertility, and reproduction: a rapid life-cycle assay in 96-well microplate format

Fipronil is a novel gamma-aminobutyric acid receptor-specific phenylpyrazole insecticide commonly used near estuarine environments for rice production, turf-grass management, and residential insect control. In this study, we evaluated the acute, developmental, and reproductive toxicity of fipronil to the estuarine harpacticoid copepod Amphiascus tenuiremis. Fipronil was highly toxic to A. tenuiremis (adult 96-h median lethal concentration [LC50] = 6.8 microg/L) and was more toxic to male copepods (96-h LC50 = 3.5 microg/L) than to nongravid female copepods (96-h LC50 = 13.0 microg/L). By using a newly developed 96-well microplate-based life-cycle toxicity test, we successfully reared single individuals of A. tenuiremis to adulthood in 200-microl microwells and concurrently assessed developmental and reproductive effects (after paired virginal matings) of environmentally relevant aqueous fipronil concentrations (0.16, 0.22, and 0.42 microg/L measured). Throughout the entire life cycle, copepod survival in all treatments was >90%. However, fipronil at 0.22 microg/L and higher significantly delayed male and female development from stage 1 copepodite to adult by approximately 2 d. More importantly, fipronil significantly halted female egg extrusion by 71% in the 0.22-microg/L fipronil treatment, and nearly eliminated reproduction (94% failure) in the 0.42-microg/L fipronil treatment. A three-generation Leslie matrix-based population growth model of fipronil reproductive and life-cycle impacts predicted a 62% decline in population size of A. tenuiremis relative to controls at only 0.16 microg/L.     

Toxicity of silver in water and sediment to the freshwater amphipod Hyalella azteca

Hyalella azteca was exposed to Ag as AgNO3 over a 10-d period in water and two lake sediments that were selected on the basis of their differences in metal-binding properties. The median lethal concentrations (LC50s) for waterborne exposures were 5.4 and 4.9 microg/L for total and dissolved Ag, respectively. In the sediment containing a lesser quantity of total Ag-binding ligands (i.e., Bond Lake, Douglas County, WI, USA, sediment), an Ag-amended sediment toxicity test resulted in a 10-d LC50 of 0.084 g (i.e., 84,000 microg) Ag/kg dry sediment or 8.6 microg Ag/L of pore water (PW). The no-observed-effect concentration (NOEC) to lowest-observed-effect concentration (LOEC) range was 0.012 to 0.031 g Ag/kg dry sediment, or less than 5.0 to 6.0 microg Ag/L of PW. In the sediment with a greater quantity of total Ag-binding ligands (i.e., West Bearskin Lake, Cook County, MN, USA, sediment), the 10-d LC50 was 2.98 g Ag/kg dry sediment, and the NOEC to LOEC range was 2.15 to 4.31 g Ag/kg dry sediment. Because "dissolved" concentrations of Ag in PW were less than 5.0 microg/L at the critical exposures in the latter test, the bioavailable and toxic form of Ag may have been a weakly associated coprecipitate or colloidal complex with hydrous iron oxides that competitively partitioned to the surface of the gills.     

Isoxaben Soil Biodegradation in Pear Tree Orchard After Repeated High Dose Application

During the past nine years, each of the plots of a pear tree orchard were treated annually with the same herbicide treatment. The following herbicide treatments were compared, each being made by application of a mixture of two or three herbicides: 1a, no herbicide at all, weeds being hoed (control 1a); 2, diuron + paraquat 3 + 1 kg/ha; 3, simazine + paraquat 2 + 1 kg/ha; 4, isoxaben + diuron + paraquat 0.5 + 1.6 + 1 kg/ha; and 5, isoxaben + simazine + paraquat 0.5 + 1.25 + 1 kg/ha. In March 1996, one year after the final orchard herbicide treatment, isoxaben could not be detected in the soils of any field plots; isoxaben was incorporated at 0.74 mg/kg in the loamy soils sampled separately in each of the field plots, and the soils were incubated in the laboratory. Isoxaben soil half-lives were 92 days in the soils treated previously with herbicide treatments 1a, 2, or 3 and 42 days in the soils treated with herbicide treatments 4 and 5. The repeated isoxaben treatments applied in the past thus enhanced the isoxaben soil biodegradation; diuron, simazine, and paraquat had no influence on this rate enhancement. On the other hand, herbicide treatments 4 and 5 were applied in the orchard in April 1996 on the corresponding plots treated in this manner for the last nine years. Isoxaben + paraquat 0.5 + 1 kg/ha was applied simultaneously on other plots (control 1b) not treated in the past with isoxaben. During the growth season in the orchard, the isoxaben soil half-lives in the control plots 1b was 101 days, and 41 days in the plots where herbicide treatments 4 or 5 were applied. Received: 25 March 1997/Accepted: 15 July 1997     

Laboratory and field responses to cadmium: an experimental study in effluent-dominated stream mesocosms

Although select stream flows in the southwestern United States are dominated by effluent discharges, metal hazards have not been experimentally evaluated in effluent-dominated streams. Lotic mesocosms were designed to assess cadmium effects on multiple levels of biological organization, to determine relevance to regulatory criteria of standard laboratory toxicity tests, and to link laboratory tests to stream responses. Replicate streams were treated with 15 or 143 microg/L Cd during a 10-d study. Streams were sampled on days 0 and 10 for benthic macroinvertebrates, periphyton, and ecosystem metabolism. Concurrent Ceriodaphnia dubia and Pimephales promelas laboratory tests were performed with water from streams. Individual organism, population, and community response variables were affected by 143 microg/L Cd but not by the I5-microg/L treatment level. A biotic ligand model for cadmium predicted a 48-h median effective concentration (EC50) value of 280 microg/L Cd for C. dubia in these effluent-dominated streams: an EC50 value of 38.3 microg/L Cd was estimated for C. dubia in tests performed with reconstituted hard water. Our findings generally support use of the biotic ligand model for establishing site-specific, acute water quality criteria for cadmium. However, future effluent-dominated stream research is required to evaluate relationships between chronic cadmium exposure and organismal and community responses.     

Pigments as biomarkers of exposure to the vineyard herbicide flazasulfuron in freshwater algae

Weed control in Champagne vineyards has long relied on the use of diuron and substituted triazines; these compounds are now being replaced by flazasulfuron, a sulfonylurea that is used at a much lower dosage. The vineyards of Champagne are planted on steep slopes and runoff is important, and even though low doses of these herbicides are used, they may present some potential risk for freshwater ecosystems. Therefore, the effects of the sulfonylurea herbicide, flazasulfuron (formulated as Katana) was investigated on the unicellular green alga Scenedesmus obliquus. The pigment content of the algal suspensions was followed as a biomarker of exposure to the herbicide. The results demonstrate that flazasulfuron induced a reduction in chlorophyll content at concentrations of 10 microg/L, while the increase of pigment content in the culture was reduced with the lowest concentration tested (0.1 microg/L). Among the three pigments tested, chlorophyll a appeared to be the most sensitive biomarker. In the algal medium, flazasulfuron was slowly degraded (DT(50) approximately 8 days) in a compound that was tentatively identified. The toxicity of this herbicide for the algae was comparable to that of older herbicides which are used at a much higher rate. Therefore, we may speculate that even if flazasulfuron comes into contact with freshwater ecosystems, its effects on algae will be less deleterious than that of traditional herbicides.     

Reproductive and developmental effects of atrazine on the estuarine meiobenthic copepod Amphiascus tenuiremis

Atrazine is one of the most widely used herbicides in the United States. Atrazine concentrations in coastal environments chronically range from 90 ng/L to 46 microg/L, with rare but measured concentrations near 60 microg/L at edge-of-field conditions. Chronic atrazine effects on estuarine benthos exposed to environmentally relevant concentrations are unknown. The purpose of this research was to assess atrazine reproductive and developmental effects over multiple-generation exposures of the copepod Amphiascus tenuiremis. Copepods were chronically exposed to two environmentally relevant nominal atrazine concentrations (2.5 and 25 microg/L, and to an environmentally unrealistic concentration (250 microg/L). Chronic exposures were performed using a 96-well microplate life cycle bioassay. Individual stage I copepodites (C1, n = 60/treatment) were reared through two generations (F0 and F1) to sexual maturity and individually mated in microwells containing 200 microl of atrazine solution. Copepod survival across all treatments and generations was >95%. Atrazine did not affect development to reproductive maturity, time to egg extrusion, or time to egg hatch (p > 0.05). However, reproductive failures increased across generations with increasing atrazine concentrations. Reproductive failures in the 0-, 2.5-, 25-, and 250-microg/L atrazine treatments were 11, 11, 20, and 24% for the F0 and 4, 9, 26, and 38% for the F1, respectively. Compared to controls, total nauplii production per female was reduced by approximately 22% in F0 females exposed to 250 microg/L atrazine (p < 0.05), and by approximately 23%, approximately 27%, and approximately 32% in F1 females exposed to 2.5-, 25-, and 250-microg/L atrazine treatments, respectively (p < 0.05). The combined effect of reproductive failure and reduced offspring production significantly reduced total population growth in the F1 generation (p < 0.05) even at atrazine concentrations lower than that considered safe for seawater chronic exposure (26 microg/L).     

Chlorine toxicity to early life stages of freshwater mussels (Bivalvia: Unionidae)

Chlorine (Cl) is a highly toxic, widely used halogen disinfectant that is present in point-source pollution discharges from wastewater treatment plants and industrial facilities. The U.S. Environmental Protection Agency freshwater criteria for Cl are 19 microg total residual Cl (TRC)/L as a maximum 1-h average concentration and 11 microg TRC/L as a maximum 4-d average; however, toxicological data for unionids were not used in these calculations. To address this void in the data, we conducted acute tests with glochidia from several species and 21-d bioassays with three-month-old Epioblasma capsaeformis and three-, six-, and 12-month-old Villosa iris juveniles. The 24-h lethal concentration 50 values for glochidia were between 70 and 220 [Lg TRC/L, which are 2.5 to 37 times higher than those reported in other studies for cladocerans. Significant declines in growth and survivorship were observed in the 21-d test with E. capsaeformis at 20 microg TRC/L. Lowest-observed-adverse-effects concentrations in bioassays with juvenile V. iris were higher (30-60 microg TRC/L) but showed a significant trend of declining toxicity with increased age. Although endpoints were above water quality criteria, the long life spans of unionids and potential implications of chronic exposure to endangered juvenile mussels still warrant concern.     

Effects of dissolved organic carbon concentration and source, pH, and water hardness on chronic toxicity of copper to Daphnia magna

The effects of pH (5.3-8.7), water hardness (CaCO3 at 25-500 mg/L), dissolved organic carbon (DOC) concentration (1.6-18.4 mg/L), and DOC source on the chronic toxicity of copper to Daphnia magna were investigated by using a multifactorial, central composite test design. Natural dissolved organic matter (DOM) was collected at three sites in Belgium and The Netherlands by using reverse osmosis. For a total number of 35 toxicity tests performed, 21-d no-observed-effect concentrations (NOECs) of copper based on reproduction ranged from 29.4 to 228 microg/L and 21-d concentrations of copper causing 50% reduction of reproduction (EC50s) ranged from 41.5 to 316 microg/L. Statistical analysis revealed that DOC concentration and pH had a significant effect on copper toxicity but hardness (at the levels tested) did not. In general, an increase in pH or DOC resulted in a linear increase of 21-d NOEC and EC50 values. All DOMs (originating from three different sources) reduced copper toxicity to the same extent. Multiple linear regression analysis on the results of all 35 toxicity tests revealed that DOC concentration is the most important factor for chronic toxicity of copper to D. magna, explaining about 60% of the observed variability, whereas pH only explained about 15% of the observed variability. Regression models were developed (with DOC and pH as parameters) that were capable of predicting NOECs and EC50s within a factor of 1.9 from observed NOEC and EC50 values obtained with eight natural surface waters spiked with copper. Until future research further elucidates the mechanisms underpinning the observed bioavailability relations, these empirical regression models can become a first simple tool for regulatory applications.     

Effect of maternal exposure to tributyltin on reproduction of the pearl oyster (Pinctada fucata martensii)

We examined the effect of tributyltin (TBT) on reproduction of the pearl oyster (Pinctada fucata martensii). In a maternal exposure test, five female pearl oysters were exposed to TBT at measured concentrations of 0 (control), 0.092, or 0.191 microg/L at 25 degreesC for one week, and the embryo developmental success (the ratio of normal D-larvae to all larvae) was measured. The embryo developmental success was significantly decreased in the 0.191-microg/L treatment group (65.5%) compared to that in the control group (82.5%; p = 0.031). Concentrations of TBT in the ovary reached 0.088 microg/g in the 0.191-microg/L treatment group. In a waterborne exposure test, inseminated eggs were exposed to TBT at measured concentrations of 0 (control), 0.020, 0.045, 0.091, 0.192, or 0.374 microg/L for 24 h. The embryo developmental success also was significantly decreased in the 0.192-microg/L treatment group (78.3%; p = 0.020) and no development at all was observed in the 0.374-microg/L treatment group compared with that in the control group (95.4%). These results clearly demonstrate that TBT accumulating in the bodies of bivalves has the potential to inhibit reproduction.