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Little is known about environmental biodegradability or biotransformations of single-walled carbon nanotubes (SWNT). Because of their strong association with aquatic organic matter, detailed knowledge of the ultimate fate and persistence of SWNT requires investigation of possible biotransformations (i.e., biodegradation) in environmental media. In the present study, [14C]SWNT were utilized to track biodegradation over 6 mo by pure liquid culture of the fungus Trametes versicolor and mixed bacterial isolates from field-collected sediment or aerated wastewater treatment plant sludge. The mixed cultures were chosen as more environmentally relevant media where SWNT will likely be deposited under both aerobic and anaerobic conditions. Activity of [14C] was assessed in solid, aqueous, and 14CO2 gaseous phases to determine amounts of intact SWNT, partially soluble SWNT degradation products, and mineralized SWNT, respectively, during the 6 mo of the experiment. Mass balances based on radiocarbon activity were approximately 100% over 6 mo, and no significant degradation of SWNT was observed. Approximately 99% of the [14C] activity remained in the solid phase, 0.8% in the aqueous phase, and less than 0.1% was mineralized to 14CO2, regardless of culture type. These results suggest that SWNT are not readily biodegraded by pure fungal cultures or environmental microbial communities, and are likely persistent in environmental media. Environ Toxicol Chem 2014;9999:1–5. © 2014 SETAC

As the use of single-walled carbon nanotubes (SWNTs) increases over time, so does the potential for environmental release. This research aimed to determine the toxicity, bioavailability, and bioaccumulation of SWNTs in marine benthic organisms at the base of the food chain. The toxicity of SWNTs was tested in a whole sediment exposure with the amphipod Ampelisca abdita and the mysid Americamysis bahia. In addition, SWNTs were amended to sediment and/or food matrices to determine their bioavailability and bioaccumulation through these routes in A. abdita, A. bahia, and the estuarine amphipod Leptocheirus plumulosus. No significant mortality to any species via sediment or food matrices was observed at concentrations up to 100 ppm. A novel near-infrared fluorescence spectroscopic method was utilized to measure and characterize the body burdens of pristine SWNTs in nondepurated and depurated organisms. We did not detect SWNTs in depurated organisms but quantified them in nondepurated A. abdita fed SWNT-amended algae. After a 28-d exposure to [14C]SWNT-amended sediment (100 µg/g) and algae (100 µg/g), [14C]SWNT was detected in depurated and nondepurated L. plumulosus amphipods at 0.50 µg/g and 5.38 µg/g, respectively. The results indicate that SWNTs are bioaccessible to marine benthic organisms but do not appear to accumulate or cause toxicity. Environ Toxicol Chem 2013;32:1270–1277. © 2013 SETAC

†University of South Carolina, Norman J. Arnold School of Public Health, Department of Environmental Health Sciences, Columbia,South Carolina, USA 29208‡State University of New York–Stony Brook, Marine Sciences Research Center, Stony Brook, New York, USA 11794(Received 10 September 2001; Accepted 11 April 2002)Abstract—Microtechniques for polycyclic aromatic hydrocarbon (PAH) body burden and total lipid analysis were developed andapplied to determine the first lipid-normalized bioaccumulation factors for a hydrophobic organic toxicant in a meiobenthicorganism(0.063–0.500 mm) living in field-contaminated sediments. The total lipid microtechnique combines the standard Bligh–Dyer ex-traction method with a colorimetric quantification method for analysis of samples containing 1 to 50mg lipid. The microtechniquefor body burden analysis quantifies PAHs from tissue samples containing as little as 10 pg PAH. Fluoranthene, benz[a]anthracene,and benzo[a]pyrene biota-sediment accumulation factors (BSAFs) were determined for the meiobenthic copepodMicroarthridionlittorale living in an estuarine U.S. Environmental Protection Agency Superfund site. Gravid female, nongravid female, and maleBSAFs were 0.82, 0.54, and 0.36, respectively, for fluoranthene; 0.50, 0.44, and 0.40, respectively, for benz[a]anthracene; and0.09, 0.12, and 0.15, respectively, for benzo[a]pyrene. Comparison of nonlipid-normalized bioaccumulation factors (BAFs) toBSAFs indicates that M. littorale bioaccumulated PAHs on a gram lipid basis. The BSAFs declined consistently with increasingPAH log K

Little is known about environmental biodegradability or biotransformations of single-walled carbon nanotubes (SWNT). Because of their strong association with aquatic organic matter, detailed knowledge of the ultimate fate and persistence of SWNT requires investigation of possible biotransformations (i.e., biodegradation) in environmental media. In the present study, [(14)C]SWNT were utilized to track biodegradation over 6 mo by pure liquid culture of the fungus Trametes versicolor and mixed bacterial isolates from field-collected sediment or aerated wastewater treatment plant sludge. The mixed cultures were chosen as more environmentally relevant media where SWNT will likely be deposited under both aerobic and anaerobic conditions. Activity of [(14)C] was assessed in solid, aqueous, and (14)CO2 gaseous phases to determine amounts of intact SWNT, partially soluble SWNT degradation products, and mineralized SWNT, respectively, during the 6 mo of the experiment. Mass balances based on radiocarbon activity were approximately 100% over 6 mo, and no significant degradation of SWNT was observed. Approximately 99% of the [(14)C] activity remained in the solid phase, 0.8% in the aqueous phase, and less than 0.1% was mineralized to (14)CO2, regardless of culture type. These results suggest that SWNT are not readily biodegraded by pure fungal cultures or environmental microbial communities, and are likely persistent in environmental media.

In chronic bioassays of sediment organic compounds, toxicant exposures often decline through time, such that the beginning of a test yields disproportionately higher exposures than the end. Thus, those life stages initiating a test often are exposed to the highest concentrations, and for rapidly maturing test fauna, this may lead to varying conclusions regarding compound toxicities depending on the initial life stage chosen. This problem can be addressed by comparative full life-cycle tests initiated with different test-organism life stages. Thus, a full life stage-to-life stage toxicity test was developed for the rapidly maturing meiobenthic copepod Amphiascus tenuiremis to assess the importance of developmental stage at the onset of sediment toxicant exposure relative to reproduction, net population growth, and sex and age structure. Tests were conducted with a model spiked-sediment insecticide, chlorpyrifos, for each of the major life stages (P1) of A. tenuiremis (nauplius, copepodite, and adult). Each P1 stage was allowed to mature and reproduce in low chlorpyrifos concentrations (6–33% of stage-specific 96-h LC50s; 4–22 ng chlorpyrifos/g dry sediment) for 26 d. Test endpoints were numbers of surviving adult females, males, eggs per female (clutch), first generation (F1) nauplii, F1 copepodites, F1 total production, and realized F1 production per surviving female. Only the copepodite P1 test showed a significant decline in survival of an adult age class: females declined by 28% at 22 ng/g. Reductions in total production ranged from 33–96% of controls from nauplius to adult. The P1 naupliar stage was most sensitive, with F1 production being 33–47% of that in controls. However, on a realized production per female basis, both the copepodite and naupliar P1 yielded significantly reduced F1S of 23 and 40% of controls at 11 and 22 ng/g.

Meiobenthic copepod populations ( Microarthridion littorale) were collected from three South Carolina, USA, estuaries having different pollution stress histories (i.e., pristine sediments, high polycyclic aromatic hydrocarbon (PAH) sediments, high metals/moderate PAH sediments) and then assayed for survival and reproductive output in 14-d exposures to pristine and heavily PAH/metals-contaminated sediments. Whole-sediment reproduction bioassays were used to determine whether copepods exposed to a highly contaminated sediment mixture exhibited differential survival and reproductive outputs as a function of previous environmental histories and whether genetic relatedness among populations measured as DNA sequences of the mitochondrial gene, cytochrome apoenzyme b, were linked to copepod contaminant tolerance. Overall, adult survival and reproductive success in contaminated sediments were significantly reduced relative to controls for all three populations irrespective of environmental histories. Differential resistance to sediment-contaminant mixtures by the two copepod populations inhabiting the contaminated sites was not found, despite their previous exposures to mixed contaminants at SPAH and SMetal concentrations of 7,287 to 2,467 ng/g dry wt and 461 to 3,497 mg/g, respectively. Significant genetic differentiation, however, was found between copepod populations from the control and the two contaminated sites. Generally, cross-population survival and reproductive outputs were not significantly different and could not be linked to genetic differentiation at the population level.

Microbial extracellular polymeric substances (EPS) are ubiquitous features in aquatic environments. Produced by surface-adherent bacteria and microalgae. EPS are often present as coatings on surfaces of sediment particles and exhibit high affinities for divalent cationic metals. Thus, EPS sediment coatings may participate in the fate of potentially toxic metals. We coated particulate silica (8-10 μm) with EPS produced by NISCI, a bacterium isolated from estuarine sediments, in order to measure the metal binding characteristics of these coatings. We used the radioisotope 109 Cd to measure effects of salinity (salinity = 2.5, 7.5, and 25‰). Cd concentration ([Cd] = 0.038, 5, and 50 mg/L). and pH (pH = 5, 6, 7, 8, and 9) on Cd sorption to EPS-coated (EPS-silica) silica and to noncoated silica (NC-silica). Also, Cd sorption by NISCI EPS coatings was compared to coatings of polymers formed by the bacterium Alteromonas atlantica and the alga Macrocystis porifera. Under all circumstances. EPS coatings increased the affinity of silica for Cd. Extracellular polymeric substance-particulate aggregates rapidly sorbed up to 90% of Cd from aqueous solution. Extracellular polymeric substance sediment coatings exhibited a maximum log distribution coefficient (K d ) of 6.5 at 2.5‰. Sorption of Cd to NC-silica was affected by salinity and metal concentration, whereas sorption of Cd to EPS-silica was only affected by salinity under high metal concentrations. Changes in pH had a dramatic effect on Cd sorption, with the proportion of free Cd to sorbed Cd changing from approximately 90% at pH 5 to 5% at pH 9. Desorption of Cd from EPS-silica was enhanced with increasing salinity. These experiments suggest that EPS coatings actively participate in binding dissolved overlying and pore-water metals in estuarine sediments.

Miobenthos occur at densities of >106/m2 in almost every uncontaminated estuarine sediment of the world, yet few studies have explored their utility for a multiple-species chronic bioassay of sediment contaminant effects. A new approach using laboratory-reared, whole-sediment meiobenthic microcosms was developed to holistically test the sublethal effects of chlorpyrifos on benthic copepod survival, age structure, and reproduction. Entire sediment-dwelling meiofaunal communities from a pristine estuarine preserve were cultured (i.e., net population gains were achieved in controls) for at least a full generation to the known 96-h copepod adult 25% lethal concentration and larval 50% lethal concentration of sediment-associated chlorpyrifos (21–33 μg/kg sediment). As an internal bioassay control, we added and cultured 100 gravid benthic copepods (Amphiascus tenuiremis) in each microcosm to assess the exact rather than relative effects of chlorpyrifos on population growth and dynamics. All but one copepod species reproduced and was cultured successfully for at least 21 d using these techniques. We found no chlorpyrifos effects on total meiobenthic copepod densities, but the predominant naturally occurring copepod Microarthridion littorale, known to be a major prey item of many juvenile fish, was significantly reduced in some chlorpyrifos-spiked microcosms. Other naturally occurring copepods were either unaffected or their growth was enhanced in the presence of chlorpyrifos. Amphiascus tenuiremis, known to be adversely affected at this concentration in 96-h static bioassays, increased dramatically in every microcosm. Species-specific responses of endemic copepods were masked when combined into the more commonly used coarse taxonomic category of total copepods.

The life-stage-specific acute toxicity of sediment-associated chlorpyrifos to the marine, infaunal copepod, Amphiascus tenuiremis, was assessed. Chlorpyrifos is a common-use, high K ow , organophosphate pesticide. Duplicate 96-h tests were conducted with each major life stage of the copepod (adult, copepodite, and nauplius) exposed to sediments spiked with chlorpyrifos. Calculated median lethal concentrations (LC50s) were 66, 74, and 40 μg chlorpyrifos per kilogram sediment for the adult, copepodite, and nauplius stages, respectively. Analysis of covariance and logistical regression analysis on the mortality data revealed significant (α = 0.05) sensitivity differences for the life stages. The nauplius stage was significantly more sensitive than either the adult or copepodite stages. However, the copepodite stage showed no significant sensitivity difference versus the adult stage. The lowest-observed-effect concentrations (LOECs) also showed these relationships, with the naupliar LOEC being almost three times lower than that of either the adult or copepodite stages. A predicted sediment quality criterion (SQC) based on the equilibrium partitioning theory (EqPT) for chlorpyrifos was calculated to be 38 μg chlorpyrifos per kilogram sediment. This calculated criterion, supposedly protective of most sediment-dwelling invertebrates, was only 2 μg/g lower than the LC50 for the nauplius life stage and far above the LOEC. An EqPT-derived SQC for chlorpyrifos clearly would not be protective of this meiobenthic copepod.

As the use of single-walled carbon nanotubes (SWNTs) increases over time, so does the potential for environmental release. This research aimed to determine the toxicity, bioavailability, and bioaccumulation of SWNTs in marine benthic organisms at the base of the food chain. The toxicity of SWNTs was tested in a whole sediment exposure with the amphipod Ampelisca abdita and the mysid Americamysis bahia. In addition, SWNTs were amended to sediment and/or food matrices to determine their bioavailability and bioaccumulation through these routes in A. abdita, A. bahia, and the estuarine amphipod Leptocheirus plumulosus. No significant mortality to any species via sediment or food matrices was observed at concentrations up to 100 ppm. A novel near-infrared fluorescence spectroscopic method was utilized to measure and characterize the body burdens of pristine SWNTs in nondepurated and depurated organisms. We did not detect SWNTs in depurated organisms but quantified them in nondepurated A. abdita fed SWNT-amended algae. After a 28-d exposure to [(14) C]SWNT-amended sediment (100 µg/g) and algae (100 µg/g), [(14) C]SWNT was detected in depurated and nondepurated L. plumulosus amphipods at 0.50 µg/g and 5.38 µg/g, respectively. The results indicate that SWNTs are bioaccessible to marine benthic organisms but do not appear to accumulate or cause toxicity.

Critical body residues (CBRs) are the measured tissue toxicant concentrations yielding a median dose–response on a dry-weight or lipid-normalized basis. They facilitate management decisions for species protection using tissue analysis. Population CBR is the mean dose yielding 50% population suppression and was predicted here in Amphiascus tenuiremis for fipronil sulfide (FS) using lifetables and the Leslie matrix. Microplate bioassays (ASTM E-2317-14) produced biomass sufficient for dry mass and lipid-normalized CBR estimates of reproduction (fertility) and population growth suppression. Significant FS toxic effects were delayed naupliar development (at ≥0.10 µg L−1), delayed copepodite development (at 0.85 µg L−1), decreased reproductive success (at ≥ 0.39 µg L−1), and decreased offspring production (at 0.85 µg L−1). A reproductive median effective concentration (EC50) of 0.16 µg L−1 (95% CI: 0.12–0.21 µg L−1) corresponded to an adult all-sex CBR and lipid-normalized CBR of 0.38 pg FS · µg−1 dry weight (95% CI: 0.27–0.52 pg FS · µg−1) or 2.8 pg FS · µg−1 lipid (95% CI: 2.2–3.6 pg FS · µg−1), respectively. Copepod log bioconcentration factor (BCF) = 4.11 ± 0.2. Leslie matrix projections regressed against internal dose predicted fewer than five gravid females in a population by the third generation at 0.39 and 0.85 µg FS · L−1 (i.e., 9.6–10.2 µg FS · µg−1 lipid), and 50% population suppression at a CBR of 1.6 pg FS · µg−1 lipid. This more integrative population CBR as a management tool would fall 1.75 times below the CBR for the single most sensitive endpoint—fertility rate. Environ. Toxicol. Chem. 2012; 31: 1076–1082. © 2012 SETAC

Acute toxicity tests were performed on field-collected copepods (Microarthridion littorale) using the sediment-associated polychlorinated biphenyl (PCB) Aroclor 1254 (i.e., PCB concentrations in bulk sediments in the bound and/or unbound states). Three replicates of 50 adult copepods were exposed to five levels of PCB-contaminated sediments for 96 h and compared to untreated controls and solvent controls. LC50 concentrations were nearly twice as high for females as for males. To determine the effects of the PCB on reproductive output of the copepods, copulating pairs of Microarthridion littorale were allowed to reproduce in concentrations of Aroclor 1254-contaminated sediments below LC50 values. Two experimental trials with 10 and 15 replicates, each with one pair of Microarthridion littorale in copulus, were conducted for 12 d, the normal time needed for females to produce one set of nauplii and carry a second clutch of eggs. In both experiments, a significant decrease in number of nauplii was found with Aroclor contamination. Although NOECs were not determined, high concentrations of the sediment-associated Aroclor NOECs were required to affect mortality significantly, whereas lower levels impaired reproduction.

Comparative effects of aqueous-, pore-water-, and sediment-phase cadmium on mortality of an infaunal laboratory-cultured copepod, Amphiascus tenuiremis, were determined using acute 96-h bioassays. Experimental design included five cadmium concentrations, three replicates per concentration, and 50 adult copepods per replicate for each of the exposures. Exposures included cadmium solubilized in seawater only (aqueous), whole sediment (i.e., pore water and sediment), and pore water only. In addition, two whole-sediment bioassays were compared in which pore-water cadmium concentrations were altered experimentally but sediment concentrations remained the same. Results of these experiments showed that for Amphiascus tenuiremis, cadmium is most toxic in the aqueous phase, less toxic in the pore-water phase, and least toxic in the sediment-bound phase. The lowered toxicity of cadmium in the pore water was most likely due to complexation of cadmium with DOC, because concentrations of DOC were six times higher in the pore-water phase than in the aqueous phase. In whole sediments, pore-water-phase cadmium was the primary source of acute toxicity, as sediment-associated cadmium contributed negligible effects.

Endosulfan-contaminated muddy sediments were collected from a tidal creek bottom after rain runoff from an agricultural watershed and tested for their effects on larval settlement/growth, survival and reproduction of cultured meiobenthic polychaetes and copepods. Endosulfan (ES) strongly inhibited larval colonization and early juvenile growth of the cosmopolitan, euryhaline polychaete Streblospio benedicti. In laboratory microcosms, ES concentrations as low as 50 μg·kg−1 sediment significantly reduced the number of larval colonists by >50% relative to ES-free control sediments. Higher concentrations closer to actual field levels suppressed Streblospio benedicti colonization completely. Early growth of newly metamorphosed juveniles was depressed 36 and 40% relative to controls at ES concentrations of 50 and 100 μg·kg−1, respectively. In contrast, when the common benthic harpacticoid copepod Pseudobradya pulchella was chronically exposed to sediment ES as high as 200 μg·kg−1, survival and egg production were unaffected despite many examples of high ES toxicity to other crustaceans. Of more than 1,600 Pseudobradya pulchella tested, more than 95% survived ES concentrations of at least 200 μg·kg−1, and over 98% of 1,200 females produced normal clutches of eggs. Similarly, survival of another common benthic copepod, Nannopus palustris, was not significantly affected below a threshold concentration of 200 μg·kg−1 ES. At 200 μg·kg−1, however, Nannopus palustris' survival was significantly reduced relative to control and 50-μg·kg−1 ES treatments.

Fish embryo toxicology is important because embryos are considered more susceptible than adult fish to the effects of toxic chemicals. Recently, fish embryo bioassay was proposed to replace the conventional fish acute toxicity chemical test of the Organization for Economic Cooperation and Development guidelines because it offers the advantages of fewer reagents, easy handling, and efficient data production. To accelerate the establishment of a chemical toxicity database for the protection of environmental and human health, we need to determine whether the conventional toxicity test can safely be replaced by such fish embryo toxicity tests. For instance, it is unclear how the presence of the chorion moderates the toxic effects of some chemicals. If such chemical toxicities do differ between embryos and, for example, the larval stage, then different toxic effects should appear in later life. We tested the later-life effects of the neurotoxic insecticide carbaryl at sublethal concentrations (0 [control] and 5 and 10 mg/L) in embryos and posthatch larvae of the freshwater fish medaka, Oryzias latipes. Although embryos exposed until hatching showed multiple developmental malformations and reductions in subsequent survival rates over three months, no significant reduction was observed in tolerance to starvation for 7 d and in intrinsic population growth rate (r). Exposure of larvae for 96 h resulted in dose-responsive vertebral fracture, significant reduction in tolerance to starvation for 7 d, and reduced three-month survival rate; r was reduced significantly and consistently. These results suggested that posthatch larvae were more susceptible than embryos to carbaryl exposure and that the toxic cascades may differ between larvae and embryos. The influences of carbaryl exposure on population growth rate differed significantly with developmental stage.

Serial analysis of gene expression (SAGE) is a transcriptome-profiling technique that allows accurate gene expression profiles to be generated without any need for prior sequence knowledge, making it ideal for assaying toxicant-specific genetic responses in nonmodel species. We used SAGE to perform a large-scale survey of the Palaemonetes pugio transcriptome under different chemical xenobiotic stresses. Four libraries were constructed from fipronil-exposed, endosulfan-exposed, and cadmium-exposed shrimp, as well as from carrier-control-exposed shrimp. A total of 37,152 tags were sequenced from the four libraries, including 13,754 unique tags. Statistical analysis revealed that 117 tags were differentially expressed significantly in at least one of the three exposures. Cluster analyses of the altered tags showed that fipronil and endosulfan elicit transcriptome profiles that are more similar to each other than either one was to cadmium. Quantitative polymerase chain reaction (QPCR) was performed on a subset of 46 differentially expressed tags, and largely confirmed the SAGE results. The tags were filtered to identify tags for which both SAGE and QPCR agreed in regard to the direction and magnitude of affect, and which were informative about the exposure experienced. A subset of 16 tags was identified that represents a putative biomarker suite that is indicative of exposures to the three toxicants.

The U.S. National Institute of Standards and Technology (NIST) petroleum crude oil was used to generate NIST water-accommodated hydrocarbon fractions (WAFs) for standardized assessment of crude oil effects on the copepod Amphiascus tenuiremis. Effects were assessed using a 96-well microplate, full life-cycle test. Briefly, nauplii (age, 24 h) were reared individually to adults (nor =120 nauplii/treatment) in microplate wells containing 200 microl of treatment solution (seawater control [0%] or 10, 30, 50, or 100% NIST-WAF). Nauplii were monitored through development to adulthood, and mature virgin male:female pairs mated in wells containing original treatments (30 d). A second bioassay using 0, 10, 30, and 50% WAFs (nor =60 nauplii/treatment) was conducted to assess the effects of ultraviolet (UV) light on naupliar endpoints (16 d). In the first experiment, nauplius-to-copepodite survival in exposures to 100% WAF was 27% +/- 6% lower than in controls (92% +/- 1%), but copepodite-to-adult survival was greater than 90% across all treatments. Analysis of development curves showed that nauplii in the 10% WAF developed into copepodites 25% faster, whereas nauplii in the 50 and 100% WAFs developed 17% slower, than controls. Copepodite development into male and female copepods was significantly delayed (2 and 4 d, respectively) in the 100% WAF compared to controls. Although none of the WAF exposures had significant effects on fertilization success or total viable production (p0.05), embryo hatching in the 100% WAF was significantly less (70.0% +/- 21.2%) than that in controls (87.0% +/- 19.4%). Results from the UV bioassay showed that relatively short exposures (14 d) to 30 and 50% WAFs in the presence of UV light caused negative effects on copepod survival and development. Naupliar-stage survival and developmental endpoints were the most sensitive indicators of exposure to the NIST crude oil WAF

Vitellogenin (VTG) has been widely used as a biomarker of estrogenic exposure in fish, leading to the development of standardized assays for VTG quantification. However, standardized quantitative assays for invertebrate, particularly crustacean, lipovitellin (also known as vitellin [VTN]) are lacking. In this study, a fluorescence-based VTN enzyme-linked immunosorbent assay (ELISA) was developed to quantify microquantities of VTN in the estuarine, sediment-dwelling copepod Amphiascus tenuiremis. This ELISA utilizes a VTN-specific polyclonal antibody developed against amphipod (Leptocheirus plumulosus) embryo VTN and exhibits specificity toward female copepod proteins. In routine assays, the working range of the ELISA was 31.25 to 1,000 ng/ml (75-25% specific binding/maximum antibody binding [B/B0]) with a 50% B/B0 intra- and interassay variation of 3.9% (n = 9) and 12.5% (n = 26), respectively. This ELISA is capable of detecting VTN as low as 2 ng/ml, and can accurately detect VTN in as few as four copepods. The ELISA significantly discriminated positive (gravid female) and negative (male) samples, and was suitable for screening endocrine toxicity in copepods. Stage-I juvenile copepods were individually reared to adults in aqueous microvolumes of the phenylpyrazole insecticide, fipronil, and whole-body homogenate extracts were assayed for VTN levels. Fipronil-exposed virgin adult females, but not males, exhibited significantly higher levels of VTN relative to control males and females. This crustacean VTN ELISA is likely useful for evaluating endocrine activity of environmental toxicants in copepods and other crustacean species.

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 was90%. 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.

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).

Under estuarine conditions, hydrophobic pesticides undergo physical/chemical partitioning into dissolved and particulate fractions of sediments. Our experimental study using 14C-chlorpyrifos (14C-CHPY) showed that sorptive associations of this common insecticide with particulate (>0.2 microm) and dissolved fractions (

Sublethal test methods are being used with increasing frequency to measure sediment toxicity, but little is known about the relative sensitivity of these tests compared to the more commonly used acute tests. The present study was conducted to compare the sensitivity of several acute and sublethal methods and to investigate their correlations with sediment chemistry and benthic community condition. Six sublethal methods (amphipod: Leptocheirus plumulosus survival, growth, and reproduction; polychaete: Neanthes arenaceodentata survival and growth; benthic copepod: Amphiascus tenuiremis life cycle; seed clam: Mercenaria mercenaria growth; oyster: Crassostrea virginica lysosome destabilization; and sediment-water interface testing with mussel embryos, Mytilus galloprovincialis) and two acute methods (amphipod survival with Eohaustorius estuarius and L. plumulosus) were used to test split sediment samples from stations in California. The test with Amphiascus proved to be the most sensitive sublethal test and the most sensitive overall, identifying 90% of the stations as toxic. The Leptocheirus 10-d test was the most sensitive of the acute tests, identifying 60% of the stations as toxic. In general, the sublethal tests were not more sensitive to sediments than the acute tests, with the sublethal tests finding an average of 35% of the stations to be toxic while the acute found 44%. Of the sublethal tests, only the Amphiascus endpoints and Neanthes growth significantly (por= 0.05) correlated with sediment chemical concentrations. Poor correspondence occurred between the toxicity endpoints and the indicators of benthic community condition. Differences in test characteristics such as mode of exposure, species-specific contaminant sensitivity, changes in contaminant bioavailability, and influence of noncontaminant stressors on the benthos may have been responsible for variation in response among the tests and low correspondence with benthic community condition. The influence of these factors cannot be easily predicted, underscoring the need to use multiple toxicity methods, in combination with other lines of evidence, to provide an accurate and confident assessment of sediment toxicity.