Follicular fluid concentrations of free insulin-like growth factor (IGF)-I during follicular development in mares.

The objective of the present study was to evaluate changes in concentrations of free insulin-like growth factor (IGF)-I in follicular fluid (FFL) during follicle development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6-15 mm; n = 54), medium (16-25 mm; n = 23) or large (>25 mm; n = 15) and FFL was collected. Free IGF-I levels in FFL in large follicles of follicular phase mares were greater (P < 0.05) than in large follicles of luteal phase mares and small or medium follicles of luteal and follicular phase mares. Free IGF-I concentrations were greater (P < 0.05) in large follicles of luteal phase mares than small but not medium follicles of luteal phase mares. FFL ratio of estradiol:progesterone paralleled changes in free IGF-I. Free IGF-I concentrations were negatively correlated (P < 0.05) with insulin-like growth factor binding protein (IGFBP)-2, -4 and -5 but not IGFBP-3 levels. In addition, free IGF-I concentrations in FFL were positively correlated (P < 0.01) with FFL estradiol, progesterone, androstenedione, estradiol:progesterone ratio, total IGF-I and total IGF-II. We conclude that increases in intrafollicular levels of bioavailable (free) IGF-I are associated with increased steroidogenesis in developing mare follicles.

Changes in follicular fluid steroids, insulin-like growth factors (IGF) and IGF-binding protein concentration, and proteolytic activity during equine follicular development.

The objective of the present study was to evaluate changes in equine follicular fluid insulin-like growth factor binding protein (IGFBP) proteolytic activity as well as steroid, IGF, and IGFBP concentrations during follicular development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6 to 15 mm; n = 54), medium (16 to 25 mm; n = 23), or large (> 25 mm; n = 15), and follicular fluid was collected. Estradiol and androstenedione levels in follicular fluid were greater (P < 0.05), and IGFBP-3 concentrations tended to be greater (P < 0.10) in large than in small or medium follicles, whereas IGFBP-2, -4, and -5 levels were less (P < 0.05) in large than in small or medium follicles. Estradiol and androstenedione concentrations were negatively correlated (P < 0.01) with IGFBP-2, -4, and -5 but not IGFBP-3 concentrations. To evaluate proteolysis of IGFBP, follicular fluid was incubated with human 125I-labeled IGFBP-2, -3, and -5 and protein separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of 125I-lableled IGFBP-2 or -3, and the small amount of proteolysis of IGFBP-2 and -3 did not differ (P > 0.10) among follicle classes. However, more 125I-labeled IGFBP-5 was cleaved (P < 0.05) by follicular fluid from large follicles collected during the follicular phase than large follicles during the luteal phase, and small or medium follicles from follicular and luteal phase mares indicating that a protease to IGFBP-5 exists in estrogen-dominant equine follicles. This IGFBP-5 protease was inhibited by kallikrein/serine protease and metalloprotease inhibitors. We conclude that the tendency of estrogen-dominant follicles of mares to have greater levels of IGFBP-3 and lesser levels of IGFBP-2 does not appear to be due to differences in proteolysis, whereas changes in IGFBP-5 levels are likely due to changes in activity of a serine protease or metalloprotease. Changes in IGFBP may alter levels of bioavailable IGF that stimulate steroidogenesis and mitogenesis in developing mare follicles.

Toxicity of sediment-associated nitroaromatic and cyclonitramine compounds to benthic invertebrates.

The toxicity of nitroaromatic (2,4-diaminonitrotoluene [2,4-DANT] and 1,3,5-trinitrobenzene [TNB]) and 14C-labeled cyclonitramine compounds (hexahydro-1,3,5-trinitro-1,3,5-triazine [RDX] and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine [HMX]) to the marine polychaete Neanthes arenaceodentata and the estuarine amphipod Leptocheirus plumulosus following 10- or 28-d exposures to spiked sediments was investigated. Organismal-level effects on survival, growth, and reproduction and cellular-level effects on apoptosis (programmed cell death) were evaluated. Because cyclonitramines have low affinity for sediment, overlying water was not exchanged in the RDX and HMX exposures. Nitroaromatics sorbed strongly to sediment, resulting in near complete resistance to solvent extraction. Cyclonitramines sorbed weakly to sediment, as more 14C-activity was found in the overlying water than in the sediment at exposure termination. No significant decrease in survival or growth was observed with cyclonitramines at initial sediment concentrations as high as 1,000 microg/g. Survival was significantly affected by nitroaromatics at nominal sediment concentrations as low as 200 microg/g, with L. plumulosus being more sensitive than N. arenaceodentata. Growth was significantly decreased at sublethal concentrations of 2,4-DANT for N. arenaceodentata. Reproduction, measured only with L. plumulosus, was significantly decreased only in the highest RDX treatment and also in the lower TNB treatment. However, no decrease was observed in higher concentrations of TNB. Body burden at exposure termination was below detection limit (1 microg/kg) for all compounds. Significant inhibition of apoptosis was not accompanied by significant decreases in growth or reproduction. Because of its critical function in many biological processes. alterations in this endpoint may result in adverse effects on the organism and could be used as an early indicator of toxicity.

Towards environmental toxicogenomics -- development of a flow-through, high-density DNA hybridization array and its application to ecotoxicity assessment.

Assessment of the environmental hazard posed by soils/sediments containing low to moderate levels of contaminants using standard analytical chemical methods is uncertain due (in part) to a lack of information on contaminant bioavailability, the unknown interactive effects of contaminant mixtures, our inability to determine the species of a metal in an environmental matrix, and the relative sensitivity of bioassay species. Regulatory agencies compensate for this uncertainty by lowering cleanup goals, but in this process they effectively exclude otherwise attractive cleanup options (i.e. bioremediation). Direct evaluations of soil and sediment toxicity preclude uncertainty from most of these sources. However, the time and cost of chronic toxicity tests limits their general application to higher levels of tiered toxicity assessments. Transcriptional level (mRNA) toxicity assessments offer great advantages in terms of speed, cost and sample throughput. These advantages are currently offset by questions about the environmental relevance of molecular level responses. To this end a flow-through, high-density DNA hybridization array (genosensor) system specifically designed for environmental risk assessment was developed. The genosensor is based on highly regular microchannel glass wafers to which gene probes are covalently bound at discrete (200-microm diameter spot) and addressable (250-microm spot pitch) locations. The flow-through design enables hybridization and washing times to be reduced from approximately 18 h to 20 min. The genosensor was configured so that DNA from 28 environmental samples can be simultaneously hybridized with up to 64 different gene probes. The standard microscopic slide format facilitates data capture with most automated array readers and, thus high sample throughput (> 350 sample/h). In conclusion, hardware development for molecular analysis is enabling very tractable means for analyzing RNA and DNA. These developments have underscored the need for further developmental work in probe design software, and the need to relate transcriptional level data to whole-organism toxicity indicators.

Uncertainty and variability in risk from trophic transfer of contaminants in dredged sediments.

The risks associated with bioaccumulative contaminants must be considered when evaluating dredged material disposal alternatives. The bioaccumulation of organochlorines and other contaminants by higher trophic level organisms represents one of the most significant sources of uncertainty in risk assessment. Both population variability (e.g. true population heterogeneity in body weight, lipid content, etc.) and uncertainty (e.g. measurement error) in trophic transfer can lead to large errors in predicted risk values for ecological receptors. This paper describes and quantitatively evaluates sources of uncertainty and variability in estimating the risk to an ecological receptor (osprey) from the trophic transfer of polychlorinated biphenyls (PCBs) in sediments from the New York-New Jersey (NY-NJ) Harbor. The distribution of toxicity quotients is obtained using a food chain model for the osprey and specifying distributions for input parameters, which are disaggregated to represent either uncertainty or variability. PCB concentrations in sediment and water are treated as predominantly uncertain, whereas lipid content in fish, feeding preferences, and fish weight are assumed to contribute primarily to population variability in PCB accumulation. The analysis shows that point estimates of reasonable maximum exposure (RME) exceed the uncertainty bounds on the 95th percentile of variability. The analysis also shows that uncertainties in the sediment and water contaminant concentrations contribute more to the range of risk estimates than does the variability in the population exposure parameters. The separation of uncertainty and variability in food chain models can help to support management decisions regarding dredged material disposal by providing a quantitative expression of the confidence in ecological risk estimates. A rationale is provided for the distinction between uncertain and variable parameters based on management goals and data availability.

DDT toxicity and critical body residue in the amphipod Leptocheirus plumulosus in exposures to spiked sediment.

The lethal and sublethal toxicity of dichlorodiphenyltrichloroethane (DDT) to the estuarine amphipod Leptocheirus plumulosus was determined using sediment spiked with (14)C-labeled compound. Juvenile amphipods were exposed to concentrations up to 9.9 nmol/g dry weight (3.5 microg/g). Acute effects on survival were determined in a 10-day experiment. Chronic effects on survival, growth, and reproduction were assessed in a 28-day experiment. The DDT in the sediments transformed to dichlorodiphenyldichloroethane (DDD), dichlorodiphenyldichloroethylene (DDE), and polar metabolites during the 14-day sediment storage prior to exposing the amphipods. The mixture of DDT and its breakdown products (tDDT) was comprised mostly of DDT at the beginning of the exposures. DDD was the prevalent compound at termination of the 28-day exposure. Complete mortality occurred at sediment concentrations of tDDT as low as 7 nmol/g (2.3 microg/g) in both acute and chronic experiments. Most of the mortality appeared to have occurred within the first 4 days of exposure. No sublethal reductions in growth or reproduction were observed in the 28-day experiment. In the 10-day experiment, where amphipods did not receive supplemental food, growth was significantly increased in DDT treatments where survival was not affected. The concentration of tDDT in amphipod tissues was determined at exposure termination. In the 10-day experiment, a mean body residue of 14 nmol/g wet weight was associated with significant mortality (30%). Lower critical body residues were observed in the 28-day experiment, where the median lethal tissue residue (LR(50)) was 7.6 (6.8--8.4, 95% confidence interval) nmol/g wet weight. Based on previous studies, the lethal critical body residue for L. plumulosus is similar to those determined for freshwater amphipods and substantially lower than those determined for cladocerans and polychaetes.

Chronic toxicity of Great Lakes sediments to Daphnia magna: elutriate effects on survival, reproduction and population growth.

: Seventeen Great Lakes sediments ranging in degree of expected toxicity were evaluated using a 21 day sediment elutriate bioassay with Daphnia magna. Sediments differed in their effects on survival, age at first reproduction, the number of broods produced and the total number of young produced per adult. Sediments producing low survivorship (50-60%) also had negative effects on reproduction. However, both positive and negative effects on reproduction were found among sediments producing high survivorship. To integrate all test end-points, a stochastic matrix population model was constructed and parameterized with survival and reproduction data from each sediment. By including estimates of variability in vital rates, the model output provided quantitative estimates of uncertainty in projected population size. Sediment effects on survival and reproduction translated into large differences in projected population growth; mean estimates of projected population size at day 28 of the simulations ranged over two orders of magnitude among the 17 sediments. Reproductive timing (e.g. age at first reproduction), followed by fecundity and survivorship, had the largest effect on population growth. Results of this study also indicate that the presence of suspended sediment in elutriates may confound toxicity evaluations using cladocerans. The concentration of total suspended solids was negatively correlated with age at first reproduction and positively correlated with measures of fecundity and population growth. In order to realize the potential benefits of chronic testing we must develop ecologically relevant ways of interpreting sediment bioassay results and expressing the uncertainty associated with our estimates of ecological risk.

Reproductive Investment in Four Developmental Morphs of Streblospio (Polychaeta: Spionidae).

Per brood and per offspring C and N investment were examined in four developmental morphs of the spionid polychaete Streblospio: S. shrubsolii (direct development, D), S. benedicti (lecithotrophic, L), S. benedicti (planktotrophic, P), and Streblospio n. sp. (planktotrophic, P). Large differences were apparent among these morphs in fecundity and embryo size. S. shrubsolii (D) and S. benedicti (L) invested about 10 x more C and N in each brood in each offspring and 30% more C and N in each brood than did the morphs with planktotrophic development. C and N concentration (µg per unit volume) was significantly greater in S. benedicti (L) than in the other morphs, though no general relationship with embryo size was evident. The C:N ratio of offspring did not differ among the four morphs. Comparisons of estimated lifetime reproductive investment made by the two developmental types of S. benedicti indicated that lecithotrophic development involved twice the C and N investment in reproduction. Positive, significant regressions were evident between embryo C and N content and embryo volume at the intermorph level. Significant intra-morph regressions were evident in all morphs but Streblospio n. sp. (P). However, the large amount of variation unaccounted for by embryo volume calls into question the use of embryo size as a predictor of parental investment in offspring.