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Adsorption of hydrophobic organic contaminants (HOCs) to black carbon is a well-studied phenomenon. One emerging class of engineered black carbon materials are single-walled carbon nanotubes (SWNTs). Little research has investigated the potential of SWNT to adsorb and sequester HOCs in complex environmental systems. This study addressed the capacity of SWNT, amended to polychlorinated biphenyl (PCB)-contaminated New Bedford Harbor (NBH) sediment, to reduce the toxicity and bioaccumulation of these HOCs to benthic organisms. Overall, SWNT amendments increased the survival of two benthic estuarine invertebrates, Americamysis bahia and Ampelisca abdita, and reduced the accumulation of PCBs to the benthic polychaete, Nereis virens. Reduction in PCB bioaccumulation by SWNT was independent of Kow. Further, passive sampling-based estimates of interstitial water concentrations indicated that SWNT reduced PCB bioavailability. Results from this study suggest that SWNT are a good adsorbent for PCBs and might be useful for remediation in the future once SWNT manufacturing technology improves and costs decrease.

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.

Ontogenetic (developmental stage) measurements of Mg/Ca and Sr/Ca were made on the benthic foraminifer Bulimina aculeata , which were cultured under controlled physicochemical conditions of temperature, pH, alkalinity, salinity, and trace- and minor-element concentrations. We utilized two methods of ontogenetic sampling—whole specimens progressively increasing in length and laser microdissection of a single specimen with subsequent analysis of dissected portions. A novel high-resolution laser-microdissection (HRLM) method allowed for precise (10 μm) cuts of the foraminiferal tests (shells) along the geometrically complex sutures distinguishing individual chambers. This new microdissection method limited sample loss and cross-contamination between foraminiferal chambers. Little or no variation in D Sr was observed at different foraminiferal developmental stages. Conversely, D Mg was enriched during a mid-developmental stage of whole-specimen samples (150–225 μm D Mg = 1.6 × 10 −3 ) compared to earlier and later stages ( 225 μm D Mg = 8.3 × 10 −4 ). Further analysis of HRLM ontogenetic samples showed a larger, age-dependent D Mg signature variation. This increase in shell Mg/Ca may contribute substantially to the measured inter-individual variability in Mg/Ca temperature prediction for cultured B. aculeata . Due to relatively large Mg/Ca inter- and intra-individual variability, measuring similar-size foraminiferal samples may improve the precision of paleotemperature prediction. Additionally, partial dissolution of the highest ontogenetically Mg-enriched calcite ( D Mg = 1.3 × 10 −2 –1.6 × 10 −2 ) may occur in undersaturated bottom-water environments or during reductive cleaning procedures. Thus, the calcite phases remaining after partial dissolution by either natural or laboratory cleaning processes may not accurately represent the calcification environment.

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.

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.

We describe a novel application of the fluorescent compound calcein (Bis[N,N-bis(carboxymethyl)aminomethyl]-fluorescein), which was used to fluorescently label foraminiferal calcite. Foraminifers that were incubated in a 10 mg L−1 solution of calcein and seawater precipitated normal-looking chambers during and after calcein incubation, which lasted up to three weeks. The survival rate of specimens incubated in calcein was similar to that of control specimens; some specimens reproduced during or after calcein exposure. Thus, this calcein-tagging method is non-lethal. Chambers precipitated during calcein incubation fluoresced a yellow-green when viewed with epifluorescence or laser scanning confocal microscopy (470 nm excitation, 500 nm emission). When viewed alternatively with reflected light, chambers formed after calcein incubation were easily distinguished from calcein-marked chambers, because calcite precipitated after calcein exposure does not fluoresce. Fluorescence is retained through fixation and air drying, thus the signal can be viewed in archived specimens. The method was executed on specimens from 15 species collected from three habitats with diferent environmental conditions. Results indicate that calcein is incorporated by all 15 species. The method has a number of potential applications, including experiments aimed at identifying benthic foraminifers that are faithful recorders of paleoceanographic proxies, as well as field studies to assess locations and chronology of foraminiferal calcification.

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.

†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.

Polycyclic aromatic hydrocarbon (PAH) bioaccumulation by the polychaete worm Streblospio benedicti (Webster) was measured under exposure to PAH-contaminated sediments in the field and for 28 d in the laboratory. Streblospio benedicti collected from field sediments contaminated at 2.94, 1.07, and l.52 μg g −1 fluoranthene (FLU), benz[a]anthracene (BAA), and benzo[a]pyrene (BAP), bioaccumulated those PAHs at 1.53, 0.215, and 0.332 μg g −1 , while worms isolated from less contaminated field sediments (0.399, 0.228, 0.288 μg g −1 FLU, BAA, and BAP) had FLU, BAA, and BAP body burdens of 0.543, 0.236, and 0.083 μg g −1 . Worms incubated for 28 d in PAH-spiked sediments (1.52, 0.991, 0.504 μg g −1 FLU, BAA, and BAP) bioaccumulated those PAHs at 0.382, 0.966, and 0.602 μg g −1 , respectively. Data normalization to organism lipid and sediment organic carbon (biota-sediment accumulation factors [BSAFs] strongly suggest that Streblospio PAH bioaccumulation was directly related to percent sediment organic carbon, but BSAFs were substantially lower than predicted by equilibrium partitioning theory. BSAFs decreased with increasing PAH log K ow , in worms collected from field sediments, but in spiked sediments BSAFs increased with increasing PAH hydrophobicity. This disparity may have been caused by insufficient spiked-sediment equilibration time (1.5h) in the case of the laboratory test sediments.

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

Macrobenthos of the pristine North Inlet, SC, USA and urbanized Murrell's Inlet, SC, estuaries are predominated by the cosmopolitan spionid polychaete Streblospio benedicti. (Webster) Laboratory experiments were conducted to measure larval settlement and metamorphosis, and sub-adult bioaccumulation/growth rates of S. benedicti when exposed to controlled concentrations of sediment-associated polynuclear aromatic hydrocarbons. Larval settlement/metamorphosis was reduced, but not significantly affected (relative to controls) by IX, 5X and 10X background PAH mixture concentrations of the six most abundant sediment-associated PAHs found in urbanized Murrell's Inlet (1X = 0.9 μg PAH per g dry sediment). Bioaccumulation of the most abundant sediment-associated PAH, fluoranthene (FL), was very high in this PAH tolerant species—9.5–13.7X FL sediment concentrations after 28-day exposures. 28-day exposures to 0.26 and 2.4 μg FL · g−1 caused no significant mortality, and significantly effected positive average weight gains in S. benedicti up to 18 days exposure. However, dramatic and significant weight declines occurred from days 18–28 in both FL treatments. Such a strong tolerance of PAH may explain why these polychaetes are not limited to pristine geographic areas, but can, as numerous field studies have shown, recruit well into hydrocarbon-contaminated and organically enriched sediments.

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.

Environmentally controlled sedimentary microcosms were used to experimentally determine temperature, salinity, and depth-horizon effects on 613C DIC profiles of dissolved inorganic carbon (DIC) in sediment pore water (PW) and near-bottom water (BW). Simultaneously, benthic foraminifera1 populations of Ammonia beccarii were cultured and assayed for calcite 613C to establish how accurately foraminifera1 shells recorded BW-PW 813CDIC. One treatment population was restricted to the O-l -cm sediment depth; in another treatment, the population could freely "roam" up and down in the sediment microcosm. Pore-water DIC between the uppermost O-0.5-cm sediment layer and the overlying BW (i.e. 3 cm above the sediment-water interface) showed steep gradients of core-top AS13Cpw averaging -3.6-11.6Ym at 25°C and -2.6+0.5% at 20°C. Cor- respondingly, shell calcite 613C for A. beccarii collected from sediment microhabitats recorded a strong PW influence; V3C shell values averaged 1.1+0.8% lighter than BW V3C,, but 1.6 -L 0.6% enriched relative to average O-5-mm PW 613CDIC. Foraminifera restricted to the uppermost l-cm sediment depth throughout their lives exhibited shell calcite AV3C values not significantly different from foraminifera allowed to free- roam through the sediment column.

A study was undertaken to measure Aroclor 1254 body burdens in two benthic marine copepods, Amphiascus tenuiremis and Microarthridion littorale. A microextraction technique was developed which utilizes 100 μL of total extraction volume and as few as 20 copepods (approximately 25 μg of dry tissue mass). Copepods were homogenized and extracted in a 100-μL closed vial with alkaline media (4 M NaOH, 50 μL) and isooctane (50 μL) spiked with an internal standard (hexachlorobenzene), creating a colloidal suspension. After centrifugation, Aroclor 1254 quantification by GC-ECD was directly determined from the organic solvent phase

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.

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

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

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.

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.

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.

Objective: To evaluate the safety and efficacy of orally administered ruboxistaurin (RBX) as a mesylate salt in patients with diabetic macular edema (DME). 1Design: Multicenter, double-masked, randomized, placebo-controlled study of 686 patients receiving placebo or RBX orally ( 4, 16, or 32 mg/d) for 30 months. At baseline, patients had DME farther than 300 mu m from the center of the macula, an Early Treatment Diabetic Retinopathy Study retinopathy severity level from 20 to 47A without prior photocoagulation, and an Early Treatment Diabetic Retinopathy Study visual acuity of 75 or more letters in the study eye. The primary study outcome was progression to sight-threatening DME or application of focal/grid photocoagulation for DME.Main Outcome Measure: Masked grading of stereoscopic fundus photographs.Results: The delay in progression to the primary outcome was not statistically significant ( 32 mg of RBX vs placebo, P=.14 [unadjusted]; Cox proportional hazards model adjusted for covariates, hazards ratio=0.73; 95% confidence interval, 0.53-1.0; P=.06). However, application of focal/ grid photocoagulation prior to progression to sight-threatening DME varied by site, and a secondary analysis of progression to sight-threatening DME alone showed that 32 mg of RBX per day reduced progression, compared with placebo (P=.054 [ unadjusted]; Cox proportional hazards model, hazards ratio= 0.66; 95% confidence interval, 0.47-0.93; P=.02).Conclusions: Although progression to the primary outcome was not delayed, daily oral administration of RBX may delay progression of DME to a sight-threatening stage. Ruboxistaurin was well tolerated in this study.

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.