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Many nanotoxicological studies have assessed the acute toxicity of nanoparticles (NPs) at high exposure concentrations. There is a gap in understanding NP chronic environmental effects at lower exposure concentrations. This study reports life-cycle chronic toxicity of sublethal exposures of polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) relative to dissolved silver nitrate (AgNO3) for the estuarine meiobenthic copepod, Amphiascus tenuiremis, over a range of environmentally relevant concentrations, i.e., 20, 30, 45, and 75 µg-Ag L−1. A concentration-dependent increase in mortality of larval nauplii and juvenile copepodites was observed. In both treatment types, significantly higher mortality was observed at 45 and 75 µg-Ag L−1 than in controls. In AgNO3 exposures, fecundity declined sharply (1.8–7 fold) from 30 to 75 µg Ag L−1. In contrast, fecundity was not affected by PVP-AgNPs exposures. A Leslie matrix population-growth model predicted sharply 60–86% of decline in overall population sizes and individual life-stage numbers from 30–75 µg-Ag L−1 as dissolved AgNO3. In contrast, no population growth suppressions were predicted for any PVP-AgNPs exposures. Slower release of dissolved Ag from PVP-AgNPs and/or reduced Ag uptake in the nanoform may explain these sharp contrasts in copepod response.

Crude oil and its water-accommodated fractions (WAF) have the potential to cause catastrophic damage to aquatic ecosystems when released. A new nanomaterial (NM), composed of polyvinylpyrrolidone-coated iron oxide, has been successfully used to remediate oil-contaminated waters. In this study, the toxicity of these NMs, crude oil WAF, and their mixtures was evaluated using an acute toxicity assay based on the estuarine copepod Amphiascus tenuiremis. Results showed that the NMs had no significant effect on copepod survival up to concentrations of 25 mg L−1 over 4 days. For comparison, optimal oil removal from the aqueous phase was shown to be at 18 mg L−1 over 1 hour. WAF was highly-toxic to copepods (mortality 95 ± 5%). Mixing and magnetic removal of NMs within 1 hour of exposure to WAF resulted in a >90% reduction in toxicity compared to the control sample that contained WAF with no NMs. Addition of NMs to WAF at times up to 72 hours without magnetic separation also resulted in significantly reduced toxicity, suggesting that the physical form of the oil : NM mixture influenced toxicity. The addition of oil degrading-bacteria to WAF treated with NMs resulted in significant stimulation of bacterial growth relative to WAF without NMs, likely because of the input of additional available Fe and C sources. These initial data suggest that under realistic environmental conditions, these NMs could be added rapidly to oil spills in marine systems to reduce acute oil toxicity and increase oil degradation.

Robust sediment quality criteria require chemistry and toxicity data predictive of concentrations where population/community response should occur under known geochemical conditions. Understanding kinetic and geochemical effects on toxicant bioavailability is key, and these are influenced by infaunal sediment bioturbation. This study used fine-scale sediment and porewater measurement of contrasting infaunal effects on carbon-normalized SEM-AVS to evaluate safe or potentially toxic nickel concentrations in a high-binding Spartina saltmarsh sediment (4%TOC; 35-45 μmol-S2-·g(-1)). Two crustaceans producing sharply contrasting bioturbation--the copepod Amphiascus tenuiremis and amphipod Leptocheirus plumulosus--were cultured in oxic to anoxic sediments with SEM[Ni]-AVS, TOC, porewater [Ni], and porewater DOC measured weekly. From 180 to 750 μg-Ni·g(-1) sediment, amphipod bioturbation reduced [AVS] and enhanced porewater [Ni]. Significant amphipod uptake, mortality, and growth-depression occurred at the higher sediment [Ni] even when [SEM-AVS]/foc suggested acceptable risk. Less bioturbative copepods produced higher AVS and porewater DOC but exhibited net population growth despite porewater [Ni] 1.3-1.7× their aqueous [Ni] LOEC. Copepod aqueous tests with/without dissolved organic matter showed significant aqueous DOC protection, which suggests porewater DOC attenuates sediment Ni toxicity. The SEM[Ni]-AVS relationship was predictive of acceptable risk for copepods at the important population-growth level.

Acute toxicities of crude oil and crude oil water accommodated hydrocarbon fraction (WAF) are relatively well documented, but data on the biological effects of chronic exposures to WAF on species and populations are scarce. South Louisiana Sweet crude oil was used to assess the effects of crude oil WAF on the copepod Amphiascus tenuiremis’ survival, development and reproduction. Effects were evaluated using a 96-well microplate full life-cycle toxicity test, a test that allows tracking of individuals from the nauplius stage to sexual maturation and reproduction. Briefly, 24-h hatched nauplii were followed to adulthood (ni=z120 nauplii/treatment) in individual glass-coated microplate wells containing 200 AL of seawater solution. Treatments consisted of 10%, 30%, 50% and 100% Louisiana WAF, with seawater used as control. Nauplii were monitored through development to adulthood, and sexually mature virgin copepods were mated pairwise in wells containing original rearing treatments. Nauplius-tocopepodite survival was reduced by 57% in exposures to 100% WAF, relative to controls (88F3%), and copepodite-to-adult survival was reduced by 18% in the 50% WAF, relative to controls (98F3%). Analysis of development curves showed that nauplii in the 10% WAF developed significantly faster into copepodites, while nauplii in the 50% WAF developed significantly slower than controls. Although the naupliar developmental rate in the 100% WAF was not significantly different from the control, these nauplii showed an average 1.4 day delay in development into copepodites. Similarly, copepodite development into mature females and males was significantly enhanced by 1.2 to 1.8 days and delayed by 1.9 to 2.2 days (pb0.05) in the 10% and 50% WAFs, respectively, compared to controls. Although the copepodite developmental rate in the 100% WAF was not significantly different from the control, these copepodites still showed an average 1.5 and 2.1 day delay in development into females and males, respectively. Analysis of reproductive endpoints showed that fertility was the only endpoint negatively affected by WAFs; reproductive failure increased by 30% and 41% in exposures to 30% and 100% WAF, respectively, compared to controls (3.33F4.71%). Leslie matrix population projections based on empirical microplate data indicated lower production rates through three generations of exposure to WAFs. Furthermore, a comparison between NIST and Louisiana crude oil WAFs using the same life-cycle approach indicated a greater chronic toxicity for the Louisiana WAF and an overall developmental delay in exposures to high WAFs (50% and 100% WAFs) from both crude oil types. D 2005 Elsevier B.V. All rights reserved.

Atrazine is a widely used herbicide in the US found at levels ranging from10 ng/L to 62.5 microg/L in estuaries throughout the southeast. Effects of atrazine on estuarine meiobenthic assemblages chronically exposed to environmentally relevant concentrations are unknown. The purpose of our research was to assess effects of atrazine on meiobenthos at concentrations near the proposed USEPA SWQC (26 microg/L) using modular estuarine salt marsh mesocosms as a field surrogate. Indigenous copepod and nematode densities were assessed after 28 days of exposure in transplanted colonization chambers. Cluster analysis showed a group characterized by low copepod densities, mostly atrazine exposed chambers, and a group containing all but one control chamber. The later group included chambers with high densities of the copepods Paronychocamptus wilsoni and Enhydrosoma baruchi. Compared to controls, copepod densities was approximately 70% lower in atrazine chambers, with three of the most common copepod species (E. baruchi, Onychocamptus sp. and P. wilsoni) showing an average of 50-70% reduction in population densities (p0.05). Although nematode density did not differ between atrazine and control chambers, the nematode-to-copepod ratio was significantly higher in atrazine (9.95+/-7.61; p=0.011) than in control chambers (0.61+/-0.35). Our findings suggest that chronic exposures over multiple generations to atrazine at concentrations near the proposed USEPA SWQC could have significant effects on the abundance and composition of estuarine meiobenthic copepod assemblages.

Dissolved organic matter (DOM) represents an important carbon phase in coastal environments and influences the partitioning of organic contaminants. In this study, we evaluated the role of salt-marsh sediment-derived DOM vs. DOM-free seawater on the acute and chronic toxicity of three pesticides (chlorothalonil—CHTH, chlorpyrifos—CHPYand fipronil—FIP) to the meiobenthic copepod Amphiascus tenuiremis. Acute toxicity was evaluated via standard 96-h median lethal concentration (LC50), while chronic toxicity was evaluated for 16 days using a 96-well microplate life-cycle bioassay. DOM significantly reduced (pb0.05) acute toxicity of CHTH and CHPY to male and female copepods relative to copepods exposed in DOM-free seawater. In contrast, DOM elevated the acute toxicity of FIP to male copepods. In chronic exposures with/without DOM, CHTH and CHPY did not significantly affect copepodite-to-adult development. In these treatments, plus controls, the majority (95%) of copepodites developed into adults by 8 days. Individuals exposed to FIP in the presence of DOM showed a slower development rate than FIP individuals in DOM-free seawater. Overall, FIP exposed copepodites developed into adult copepods 4 days later than controls. CHTH, CHPY and FIP significantly reduced reproductive success by 33%, 31% and 89%, respectively. DOM, however, mitigated 30% (p=0.006) and 20% (p=0.05) of the reproductive failure attributed to CHTH and CHPY. FIP-induced reproductive failure was high (~80%) in all exposures regardless of DOM presence/absence. An exponential growth model predicted that exposure to CHTH, CHPY and FIP in the absence of DOM significantly reduced (z38%) projected naupliar production relative to DOM-free controls. In the presence of DOM, naupliar production under CHTH and CHPYexposures was not significantly different (pN0.05) from control projections. These results indicate that DOM generally reduced the acute and chronic toxicity of CHTH and CHPY to A. tenuiremis, while certain compound-specific pesticide (FIP): organic associations may enhance acute toxicity of FIP, particularly to male copepods. D 2005 Elsevier B.V. All rights reserved.

Coastal urbanization supplies surrounding estuarine environments with urban-related contaminants such as polycyclic aromatic hydrocarbons (PAHs), metals and pesticide mixtures. In our study, adult female and male copepods (Amphiascus tenuiremis) were chronically exposed to 18 sediment samples collected from sites influenced by different land-uses. Sediment samples were collected from three major suburban areas (Hilton Head, Kiawah Island and the Okatee River watershed) and a pristine site (North Inlet) in coastal South Carolina. Three-sediment bioassays (six sites per bioassay) were conducted by culturing copepods for 14 days in quadruplicate test sediments under flow-through conditions at 20 � C and 12:12 LD cycles. Adult survival and copepod reproductive output were quantified. Sediment samples were also analyzed by GC–MS for low and high molecular weight PAHs. Minimal adult mortality was observed in most sediment samples. However, sediments from Hilton Head Island and the Okatee River showed significant effects on copepod reproductive output (i.e., nauplii, copepodites and clutch size). Thus, we determined that reproductive endpoints rather than adult copepod survivorship were more sensitive to effects of contaminated sediments on A. tenuiremis. Furthermore, six (33%) of the 18 sites had a >25% reduction in copepod bioassay endpoints relative to controls, suggesting a high risk to long term A. tenuiremis population maintenance. � 2004 Elsevier Ltd. All rights reserved.

†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

The harpacticoid copepod Microarthridion littorale (Poppe) was tested for interaction effects between salinity change and acute pesticide exposure on the survival and genotypic composition of a South Carolina population. Previous data suggested a significant link between a combined exposure to chlorpyrifos (CHPY) and dichloro-diphenyl-trichloroethane (DDT) and mitochondrial haplotype in the cytochrome b apoenzyme for this euryhaline species when exposed at 12-ppt salinity seawater. Our tests demonstrate a significant non-linear survival response for M. littorale to short-term immersion (24 h) in 3-, 12- and 35-ppt seawater, with copepods transferred to 12-ppt seawater having the lowest survival. There was significant statistical interaction between salinity and pesticide exposure for the dependent variable “survival.” However, changes in genetic composition of survivors were not significant, and they were complicated by extremely low survival in the pesticide/3 ppt and pesticide/36 ppt treatments. As noted for many studies of harpacticoids, males faired worse than females in all treatments, with none surviving pesticide exposure at 45 μg/l CHPY and 6 μg/l DDT.

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.

We compared a 21 day full life-cycle bioassay with an existing 14 day partial life-cycle bioassay for two species of meiobenthic copepods, Microarthridion littorale and Amphiascus tenuiremis. We hypothesized that full life-cycle tests would better simulate chronic exposures to copepods and provide more sensitive and realistic information about contaminant effects on growth of copepod populations. Copepods were exposed to sediments from two sites, non-contaminated North Inlet Estuary (NIE), SC (USA), and highly contaminated Shipyard Creek (SYC), SC, and measured for lethal and sublethal test points. Fourteen and 21 day bioassays showed similar negative effects in SYC sediments for both species. Survival and reproductive success were higher in the 14 day than in the 21 day bioassay for both species, in both sediment types. The lower 21 day overall response was not indicative of enhanced bioassay sensitivity. The 14 day bioassay provides sufficient f 1 generation information to detect negative population level responses to contaminant mixtures.

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.

Two experiments were conducted using laboratory cultured meiofauna to measure bioturbation effects on cadmium partitioning. The benthic harpacticoid copepod, Amphiascus tenuiremis cf. Mielke, and the foraminiferan, Ammonia beccarii cf. Linne' served as bioturbators. The first experiment utilized single and combined species treatments which were compared to controls having no fauna. All treatments and controls used sediment spiked with Cd at the concentration at which 5% mortality occurs in the copepods (LC 5 ). Cadmium concentrations were measured in the overlying water, pore water, and sediment after 96 h to see if bioturbation altered the partitioning of cadmium. The second experiment employed the same design as the first except that the number of organisms used was tripled to simulate reported field densities. Results from the first experiment revealed significantly higher amounts of Cd in the pore waters of all treatments relative to the controls. There were also between treatment differences showing that copepod-only treatments resulted in the highest concentration of pore water Cd. With the second experiment, it was assumed that this bioturbation effect would be even greater due to the increased number of organisms used. However, this was not seen. Treatment pore water Cd concentrations of Experiment 2 were not significantly higher than like treatments from Experiment 1. Both experiments demonstrated that meiofaunal bioturbation has a significant effect on the partitioning of Cd in muddy sediments.

Estuarine sediments sequester heavy metals and non-ionic pollutants at concentrations manyfold greater than those in the water column. Sediment-associated pollutants probably have a greater impact on the wholly sediment-dependent meiobenthos than any other invertebrate group. In this study we compared acute effects of a highly lipophilic synthetic pyrethroid pesticide, fenvalerate (i.e. FV, log Kow = 6·20), on survival of a sediment-cultured meiobenthic copepod, Amphiascus tenuiremis, exposed to aqueous and sediment-associated FV phases. Empirical results were compared to predictions generated by the equilibrium partitioning approach to sediment quality criteria. Additionally, sediment-associated FV effects on a generic group of marine nematodes and one other species of field-collected benthic copepod, Paronychocamptus wilsoni, were tested. For sediment-associated FV, nematodes showed the highest acute sensitivity (96-h LC50 = 26·1 < 33·2 < 40·5 μg-FV g−1 carbon), followed by the field-collected copepod. Paronychocamptus wilsoni (96-h LC50: 61·9 < 73·9 < 91·7), and cultured copepod Amphiascus tenuiremis (96-h LC25: 56·2 < 84·2 < 147·4). In a major departure from equilibrium partitioning theory (EqPT) predictions, A. tenuiremis exposed to aqueous FV concentrations 7300 x higher than EqPT porewater values (i.e. 0·017 to 0·068 μg-FV liter−1) exhibited only 1–27% higher mean mortalities than in sediment-associated FV exposures. Based on EqPT, the high sediment-carbon (3·8%) particulate phase probably reduced porewater FV concentrations. However, the particulate phase in this study appeared a more important FV exposure route to the sediment-ingesting A. tenuiremis than porewater or even direct aqueous exposures.

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.

The benthic harpacticoid copepod, Amphiascus tenuiremis, was cultured through one generation at four concentrations (600, 300, 150, and 0 micrograms/kg) of sediment-associated fenvalerate, a synthetic pyrethroid insecticide, to assess effects on reproductive output and age structure. Two static renewal experiments with four replicates of each treatment were conducted. At the end of 21 days, surviving copepods were sorted and counted into adult males, adult females, copepodites, and nauplii. Fenvalerate was extracted from the sediment and measured by GC-MS. The number of individuals in each life-history stage in fenvalerate treatments was not significantly different from controls, except for increased adult females in fenvalerate in the second experiment. The overall lack of a fenvalerate toxic effect on A. tenuiremis was most likely because fenvalerate tightly binds to sediments and was probably not bioavailable to the copepods. Sediment-associated pesticides with large octanol:water partitioning coefficients (K(ow)'s) such as fenvalerate appear to be less toxic to infaunal copepods than those exhibiting smaller K(ow)'s.

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.

Attached bacteria inhabit the surfaces of many marine animals--a process that may play important roles in the survival and transport through aquatic systems. However, efficient detection of these bacteria has been problematic, especially small aquatic animals such as benthic harpacticoid copepod. Quantum dots (QD) have recently emerged as a significant tool in immunofluorescence detection because of their unique properties compared to other fluorescent probes. In the present study, a polyclonal antibody was raised against the Gram-negative marine bacterium, Alteromonas sp. A microplate-based immunofluorescence bioassay using QD strepavidin conjugates was developed for quantifying putative Alteromonas sp. cells located on the surfaces of a marine harpacticoid copepod, Microarthridion littorale. The number of attached Alteromonas sp. was estimated to be 10(2)+/-8 CFU using this method. The QD approach, coupled to a microplate assay can potentially provide an efficient and accurate method for rapidly detecting multiple bacteria species attached to small invertebrate animals because of their unique excitation and emission characteristics.

Pure, microcosm-cultured populations of benthic copepods were established from pristine or pesticide-impacted Spartina marsh creeks and used as efficient bioassay groups to assess lethal and sublethal effects of sediment-bound pesticide residues. Naturally-weathered sediments contaminated with the synthetic pyrethroid insecticide fenvalerate were collected by traps moored in a tidal creek receiving major pesticide-laced runoff from an agricultural watershed, and used as dosing material. Silty sediments with fenvalerate residues reaching 100 ppb were trapped and then diluted with uncontaminated sediments to achieve an exposure range of 0, 25, 50 and 100 ppb (i.e. no dilution). Despite a broad database showing extreme sensitivity to water-solubilized fenvalerate by many marine invertebrates and fishes, a 7-day exposure to sediment-bound residues as high as 100 ppb caused no significant mortality for any life stages (i.e. nauplii, copepodites or adults) of the benthic harpacticoid copepods Microarthridion littorale or Paronychocamptus wilsoni, and no mortality for adults of Enhydrosoma propinquum. However, sediment-bound residues as low as 25 ppb significantly depressed egg production (50–100% reduction) and mean clutch sizes (40–100% reduction) of fertile M. littorale and P. wilsoni. If sedimenting fenvalerate depresses copepod reproduction in the field, then lowered recruitment of new individuals will lead inevitably to a decline in population growth.

Single-walled carbon nanotubes (SWNT) are finding increasing use in consumer electronics and structural composites. These nanomaterials and their manufacturing byproducts may eventually reach estuarine systems through wastewater discharge. The acute and chronic toxicity of SWNTs were evaluated using full life-cycle bioassays with the estuarine copepod Amphiascus tenuiremis (ASTM method E-2317-04). A synchronous cohort of naupliar larvae was assayed by culturing individual larvae to adulthood in individual 96-well microplate wells amended with SWNTs in seawater. Copepods were exposed to "as prepared" (AP) SWNTs, electrophoretically purified SWNTs, or a fluorescent fraction of nanocarbon synthetic byproducts. Copepods ingesting purified SWNTs showed no significant effects on mortality, development, and reproduction across exposures (p0.05). In contrast, exposure to the more complex AP-SWNT mixture significantly increased life-cycle mortality, reduced fertilization rates, and reduced molting success in the highest exposure (10 mg x L(-1)) (p0.05). Exposure to small fluorescent nanocarbon byproducts caused significantly increased life-cycle mortality at 10 mg x L(-1) (p0.05). The fluorescent nanocarbon fraction also caused significant reduction in life-cycle molting success for all exposures (p0.05). These results suggest size-dependent toxicity of SWNT-based nanomaterials, with the smallest synthetic byproduct fractions causing increased mortality and delayed copepod development over the concentration ranges tested.

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

Confocal laser scanning microscopy (CLSM) represents a powerful, but largely unexplored ecotoxicologic tool for rapidly assessing in vivo effects of toxicants on marine invertebrate embryo quality and development. We describe here a new semiquantitative CLSM approach for assessing relative yolk quantity in marine invertebrate embryos (harpacticoid copepods) produced by parents reared from hatching to adult in the polycylic aromatic hydrocarbon chrysene. This method is based on fluorogenic labeling of embryo yolk and subsequent statistical analysis of areal pixel intensities over multiple Z-series using a general linear model (GLM)-nested analysis of variance. The fluorescent yolk-labeling method described here was able to detect statistically significant differences in yolk concentrations in marine copepod (Amphiascus tenuiremis) eggs or embryos from females exposed to ultraviolet light and chrysene-contaminated sediments. Yolk intensities in embryos from females cultured throughout their life cycles in clean sediments were statistically identical with or without UV exposure. In contrast, yolk intensities in embryos of females cultured throughout their life cycle in chrysene-contaminated sediments were significantly higher in the non-UV-exposed treatment with chrysene at 2500 ng/g sediment (65.7% higher) and the UV-exposed treatment with chrysene at 500 ng/g sediment (76.6% higher).

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.

Copepods are the most abundant arthropods on earth and are often the most important secondary producers in estuarine/marine food webs. The new GABA (gamma-aminobutyric acid)-disrupting insecticide fipronil (FP) induces unique sex-specific reproductive dysfunction in male meiobenthic copepods, leading to trans-generational population depression at environmentally realistic concentrations (0.63 microg/L). Using a newly developed 96-well microplate lifecycle bioassay, more than 700 individual Stage-I juveniles were reared to adulthood in as short as 12 days in only 200 microL of control (CTL) or 0.63 microg-FP/L seawater solution. Individual virgin male: female pairs were then cross-mated for all possible combinations within and across rearing treatments and allowed to mate for an additional 12 days in CTL or 0.63 microg-FP/L solution. FP at 0.63 microg/L caused no significant lethality to any mating combinations but evoked 73% or 89% inhibition of reproduction when FP-reared males were mated with either a control- or FP-reared female in FP solution, respectively. In contrast, when CTL-reared males were mated with FP-reared females in FP solution, there was no difference in reproductive success compared to FP-free controls. When FP-reared males were mated with either female group in FP-free solution, these mating pairs displayed a 3-day delay in time to brood sac extrusion but ultimately did reproduce. As fipronil (1) has a high K(ow), (2) is persistent in sediments where meiobenthic copepods live, and (3) has been detected in estuarine waters >0.7 microg/L, it may pose high risk to copepod production in estuarine systems.

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).

Endocrine function in arthropods has principally been characterized in insects and malacostracan crustaceans. However, meiofauna represent the most abundant metazoan marine taxa, with harpacticoid copepods comprising the second most abundant taxon. In addition, their diminutive biomass has made characterization of endocrine components difficult, so little is known about endocrine control of reproduction, molting, and growth in meiofauna. In this study, a sensitive fluorometric enzyme immunoassay (EIA) was utilized to quantify and compare the arthropod molting hormone, 20-hydroxyecdysone (20E), in various life-cycle and developmental stages of a laboratory reared meiobenthic copepod, Amphiascus tenuiremis, and in an amphipod, Leptocheirus plumulosus. In copepods, gravid females carrying late stage pre-hatch embryos contained significantly more 20E (390+/-252 fmol/female) than gravids carrying early (Stage-I) embryos (172+/-83 fmol/female). In contrast, ecdysteroid levels in Stage-I L. plumulosus gravid females (277+/-83 fmol/female) was greater than pre-hatch gravid females (146+/-42). Stage-I embryos of both copepods (19+/-10) and amphipods (11+/-5 fmol/embryo) possessed lower ecdysteroid content than copepod (35+/-15) and amphipod (43+/-33 fmol/embryo) pre-hatch embryos. Ecdysteroid levels were also assessed in naupliar, juvenile, adult male and non-gravid female copepod life-stages. In addition, ecdysteroids measured in field collected copepod species indicated gravid females possessed ecdysteroid levels similar to gravid A. tenuiremis. However, upon normalization of egg sac 20E content by brood size, embryos from larger broods contained lower levels of ecdysteroids when compared to embryos from smaller clutch sizes-indicating an inverse embryo/ecdysteroid relationship may exist across species.

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.

The cosmopolitan benthic foraminiferan, Ammonia beccarii, is a fervent microfloral predator which often forms densely-populated 2–4 cm2 aggregates in the field. Sediments within aggregate patches become extensively pelletized, mucus bound and depleted in microfloral food. On a West German Wattenmeer mudflat, copepodite and naupliar densities of a predominant harpacticoid copepod, Amphiascoides limicola, were significantly depressed in sediments containing>100 A. beccarii·3 cm-2 suggesting a possible foraminiferal: copepod amensalism. Therefore, I cultured A. beccarii and A. limicola separately in sediment microcosms and then tested if A. limicola's seemingly negative reaction to sediments containing A. beccarii occurs under controlled conditions, how various life stages of A. limicola are affected, and what the repulsive mechanisms of A. beccarii may be. In natural field sediments seeded with a latin-square dispersion of sterile sediment patches containing 0 or 100 A. beccarii, mean A. limicola naupliar and copepodite densities were 2 to 6 times lower in Ammonia-rich patches than Ammonia-poor patches (i.e. patches containing

Two mechanisms of muddy-bottom meiofaunal dispersal, waterborne suspended transport and holobenthic infaunal immigration, were compared as to their rate and effectiveness in mediating community reestablishment after small-scale defaunation. Colonizing meiofauna were quantitatively sampled in winter and summer from 16 replicates of two azoic sediment chamber designs on 2 and 29 days postplacement. The chambers were ≈ 3750 cm 3 ; one design allowed colonization via suspended movement through an open top, while the other design permitted entry only by infaunal crawling through subsurface open sides. After 48 h, mean harpacticoid copepod and naupliar densities in sediment chambers open to colonization exclusively by meiofauna in suspended transport were not significantly different from background sediment densities. Sediment chambers allowing colonization exclusively via infaunal immigration through the sediment, however, contained copepod and naupliar densities that were significantly less than densities in background sediments and suspension-colonized chambers. In contrast, nematode densities in both suspension- and infaunally colonized chambers were significantly less than in background sediments, but densities were not significantly different between the chamber treatments. Thus for a small-scale defaunation, copepods most rapidly and completely recolonize sediments via suspended transport. Nematode dispersal occurs equally well via suspended or infaunal movement; however nematodes never seemed to utilize the chambers fully because densities did not reach background levels even after 29 days.