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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.

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.

Silver nanomaterials are the major components of healthcare products largely because of their antimicrobial effects. However, their unintended toxicity to biological organisms and its mechanism are not well understood. Using medaka fish embryo model, the toxic effects and corresponding mechanisms of silver nanocolloids (SNC, particle size 3.8 ± 1.0-diameter nm) were investigated. SNC caused morphological changes in embryos including cardiovascular malformations, ischemia, underdeveloped central nervous system and eyes, and kyphosis at exposures of 0.5 mg/L. Interestingly, SNC were observed inside the eggs at a level of 786.1 ± 32.5 pg/mg egg weight, and TEM analysis showed that SNC adhered to the surface and inside of the chorion. Meanwhile, medaka oligo DNA microarray and qRT-PCR were used for gene expression analysis in the embryos exposed to 0.05 mg/L SNC for 48 h. As a result, expressions of six of the oxidative stress-, embryogenesis- and morphogenesis-related genes, ctsL, tpm1, rbp, mt, atp2a1, and hox6b6, were affected by the SNC exposure, and these genes' involvement in those malformations was implied. Thus, SNC could potentially cause malformations in the cardiovascular and central nervous systems in developing medaka embryo through SNC-induced differential expression of the genes related to oxidative stress, embryonic cellular proliferation, and morphological development.

The marine copepod, Amphiascus tenuiremis (A. tenuiremis), is a well characterized invertebrate model for the screening and evaluation of endocrine and reproductive toxins using life-cycle assays. These tests evaluate phenotypic endpoints related to development and reproduction, which are utilized to predict population outcomes. Some of these endpoints in arthropods, including sexual maturation and molting, are controlled by the hormone ecdysone which acts through its cognate receptor, the ecdysone receptor. The purpose of this research was to obtain and characterize sequence information for the A. tenuiremis ecdysone receptor and investigate modulation of expression levels by fipronil, an insecticide that causes infertility in males and reduced egg extrusion in female copepods, and ponasterone, a natural ecdysone receptor agonist. Results show successful cloning and phylogenetic analysis of the ecdysone receptor for A. tenuiremis, providing the first genetic information for a hormone receptor in this species. Exposure of copepodites to fipronil for 1, 2, 4, 18 and 30 h caused a significant increase in ecdysone receptor transcriptional expression at 30 h compared to control unexposed animals. This work illustrates a potential mechanism whereby exposure to fipronil, and potentially other endocrine disrupting compounds, results in impacted reproduction. Furthermore, this exemplifies the potential utility of ecdysone receptor transcriptional measurement as a sensitive and rapid biomarker of ecological relevance when linked to traditional A. tenuiremis bioassays.

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) is comprised of a myriad of macromolecules with specific physical and chemical properties that may influence the bioavailability of hydrophobic pesticides to animals. This study was conducted to assess the role of various forms of DOM on the uptake and bioconcentration of the organophosphate insecticide chlorpyrifos (CHPY) to the bivalve Mercenaria mercenaria. Bivalves were exposed to DOM-free seawater (30 per thousand) or to seawater containing a single form of DOM. DOM forms included two filtrate fractions of natural salt-marsh sediment DOM (DOM-(0.45 mum) and DOM-(3 kDa)); natural purified humic (HA) and fulvic (FA) acids; and water soluble cyclic oligosaccharides alpha and beta cyclodextrins (CD-alpha and CD-beta). In (14)C-CHPY uptake and elimination experiments, juvenile bivalves were exposed to uniformly-labeled (14)C-CHPY and collected at time intervals during 48 h. The remaining bivalves were transferred to (14)C-CHPY-free elimination chambers with bivalve collection at time intervals over 144 h. Total uptake of (14)C-CHPY by bivalves in DOM-free seawater was40% greater than in bivalves exposed to (14)C-CHPY in the presence of most DOM forms. These results are consistent with much faster (14)C-CHPY uptake rates estimated using a simple two parameter model. After the elimination period, bivalves exposed to DOM-free seawater had (14)C-CHPY body residue concentrations between 25% and 86% greater than bivalves in the presence of DOM forms. Experiments with larger bivalves showed that pulse-chase exposures with a 1.5 h exposure period to (14)C-CHPY was not long enough to detect differences in (14)C-CHPY tissue accumulation efficiencies across treatments. Our findings suggest that natural forms of DOM, at environmentally realistic organic carbon concentrations, reduced pesticide uptake and bioconcentration, consistent with much lower uptake rates relative to bivalves exposed to (14)C-CHPY in the absence of DOM. Interestingly, at the tested organic carbon concentrations CD-alpha and CD-beta did not reduce (14)C-CHPY bioconcentration in M. mercenaria.

The predominant data used in ecological risk assessment today are individual-based rather than population-based; yet environmental policies are usually designed to protect populations of threatened species or communities. Most current methods in ecotoxicology are limited by largely logistic/ technology-driven requirements that yield data for a relatively small number of test species and end points that focus on acute lethality or sublethal nonproduction-based parameters (e.g., biomarkers, mutagenesis, genetic change, physiological condition). A contrasting example is presented here showing the predictive ability of meiobenthos-based full life cycle toxicity testing to extrapolate multi-generational effects of chemicals on variables of import to population growth and maintenance. Less than 24-h-old larvae of a meiobenthic copepod were reared individually in 96-well microplate exposures to parent and degradates of the phenylpyrazole insecticide fipronil. Survival, development rates, sex ratio change, fertility, fecundity, and hatching success were tracked daily for 32 d through mating and production of three broods in spiked seawater. These data were then inserted in a Leslie (Lefkovitch) matrix stage-based population growth model to predict relative rates of population increase (lambda) and changes in net population growth with time and toxicant concentration. Field-reported test concentrations produced strong reproductive (52-88%) and net production (40-80%) depressions for parent (at 0.25 and 0.5 microg/L), desthionyl (0.25 and 0.5 microg/L), and sulfide (0.15 microg/L) moieties as compared to controls. Spiked sediment exposures of 65-300 ng of fipronil/g of dry sediment yielded significantly reduced production rates per female that were 67-50% of control production. The consistent reproductively linked impacts of fipronil and its degradation products at the population maintenance levels suggest risks to sediment-dwelling crustaceans at concentrations well below noneffects for most aquatic test species based on risk assessment data from primarily acute and sub-life cycle toxicity tests.

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.

Although much is known regarding photoperiodic effects on crustacean egg production, the effects of ultraviolet (UV) light on reproduction has not been investigated. Likewise, little is known concerning the interaction between UV and xenobiotic exposure on crustacean reproductive cycles. In this study, male and female grass shrimp, Palaemonetes pugio, were exposed to sublethal concentrations of endosulfan (200 ng/l and 400 ng/l ES) under both white fluorescent (WF) and UV-A (315-400 nm) light conditions for 50 days in laboratory bioassays. Female endocrine (vitellogenin, ecdysteroids, and cholesterol), reproductive (percent gravid, clutch size), and embryo (days to hatch, hatching success, and hatching survival) responses were assessed. UV-exposure alone caused a significant (>4-fold) increase in total Palaemonetes pugio female egg production over the course of 50 days. Exposure to ES and UV significantly lowered the percentage of gravid females relative to UV controls, whereas ES-exposed shrimp under WF lighting did not exhibit these trends. Although higher vitellogenin concentrations and lower ecdysteroid titers were correlated with increased female egg production, cholesterol titers only exhibited a dose-dependent change when exposed to ES. Embryos from females exposed to UV had significantly lower ecdysteroid titers and shorter hatching times but there were no differences in embryo vitellogenin concentrations, hatching success, or hatching survival. These results indicate that UV-A exposure has a pronounced effect on grass shrimp (Palaemonetes pugio) reproduction and is likely mediated through 5-hydroxytrptamine (5-HT)-related neuroendocrine pathways. The implications for decapod aquaculture and evaluating chronic contaminant effects are discussed.

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

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.

Single-walled carbon nanotubes (SWNT) have extremely high affinity for hydrophobic organic contaminants, considerably higher than natural or refractory (e.g., soot and detrital) carbon found in sediments. To evaluate the effect of sediment-associated SWNT on contaminant uptake from sediments by infaunal deposit feeders, we have conducted a comparative bioaccumulation study using two infaunal estuarine invertebrates. The deposit-feeding meiobenthic copepod Amphiascus tenuiremis and the deposit/suspension-feeding polychaete Streblospio benedicti were exposed to hydrophobic organic contaminants (HOCs) including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, and polybrominated diphenyl ethers for 14 days in the presence of sediment amended with (1) SWNTs, (2) NIST diesel soot, or (3) no carbon amendment. Coaddition of SWNT to sediments significantly reduced bioaccumulation of HOCs in S. benedicti; however, soot addition tended to increase the bioaccumulation of these same compounds in the polychaete worm. Bioaccumulation of HOCs from sediments by copepods (A. tenuiremis) was less dependent on black carbon addition to sediment; neither SWNT nor soot significantly impacted bioaccumulation of PAHs from sediment by this organism. Exposure of both copepods and polychaetes to radiolabeled (14C) SWNT in estuarine sediments revealed that these organisms did not assimilate these materials into their tissues, although S. benedicti did ingest 14C-SWNT, as fecal rods from this organism contained identical 14C activity as that of the sediment to which the worms were exposed.

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.

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.

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).

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 (

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.

Vitellin is a major yolk protein that plays a significant role in the embryonic development of crustacean embryos. This protein was rapidly purified from embryos of the estuarine amphipod, Leptocheirus plumulosus, by subjecting the crude protein homogenate to high affinity column chromatography. SDS-PAGE revealed a single band with an approximate molecular weight of 200,000 daltons. Vitellin was characterized by SDS-PAGE techniques and amino acid composition analysis. L. plumulosus vitellin is a lipoglycophosphoprotein with serine, glutamic acid/glutamine, alanine, and aspartic acid/asparagine accounting for almost 66% of all amino acid residues. Polyclonal antibodies were raised against L. plumulosus vitellin and antibody reactivity was verified by dot-blotting and immuno-fluorescence confocal microscopy. These antibodies are specific for purified vitellin and show little cross-reactivity with other embryonic proteins.

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.