High throughput in vitro and in vivo screening of inland waters of Southern California.

The impact of unmonitored contaminants, also known as contaminants of emerging concern (CECs), on freshwater streams remains largely uncharacterized. Water samples from 31 streams representing urban, agricultural and undeveloped (i.e., open space) land use in Southern California (USA) were analyzed for in vitro and in vivo bioactivity. The extent and magnitude of bioactivity screened using endocrine-responsive cell bioassays and a fish embryo screening assay were low. In contrast, a wider gradient of responses for the aryl hydrocarbon receptor (AhR) assay was observed, which was negatively correlated with a measure of benthic community structure. Both aromatic and non-aromatic CECs were tentatively identified in these samples, but polycyclic aromatic hydrocarbons (PAHs), known AhR agonists in urban environments, were not present at detectable levels. These results suggest that a combination of in vitro and in vivo show potential as screening techniques for biological condition in situ, but that more advanced, comprehensive analytical methods are needed to identify bioactive contaminants.

Designing monitoring programs for chemicals of emerging concern in potable reuse--what to include and what not to include?

This study discussed a proposed process to prioritize chemicals for reclaimed water monitoring programs, selection of analytical methods required for their quantification, toxicological relevance of chemicals of emerging concern regarding human health, and related issues. Given that thousands of chemicals are potentially present in reclaimed water and that information about those chemicals is rapidly evolving, a transparent, science-based framework was developed to guide prioritization of which compounds of emerging concern (CECs) should be included in reclaimed water monitoring programs. The recommended framework includes four steps: (1) compile environmental concentrations (e.g., measured environmental concentration or MEC) of CECs in the source water for reuse projects; (2) develop a monitoring trigger level (MTL) for each of these compounds (or groups thereof) based on toxicological relevance; (3) compare the environmental concentration (e.g., MEC) to the MTL; CECs with a MEC/MTL ratio greater than 1 should be prioritized for monitoring, compounds with a ratio less than '1' should only be considered if they represent viable treatment process performance indicators; and (4) screen the priority list to ensure that a commercially available robust analytical method is available for that compound.

Residues of toxaphene in insectivorous birds (Petrochelidon spp.) from the Rio Grande, Texas.

Although it has been documented that wildlife in the Rio Grande Valley (RGV) contain increased concentrations of organochlorine (OC) contaminants, particularly DDE, little has been published on residues of toxaphene throughout this major North American watershed. In this study, 28 liver composites from adult swallows (Petrochelidon spp.) collected along the Rio Grande from 1999 through 2000 were analyzed for toxaphene residues using congener-specific gas chromatography-electron-capture negative ionization-mass spectrometry. Estimated total toxaphene concentrations ranged from 12 to 260 ng/g wet wt and were highest in samples from the lower RGV near Llano Grande Lake in Hidalgo and Cameron counties (Texas). Toxaphene congener profiles were relatively invariant throughout the watershed and were dominated by 2,2,5-endo,6-exo,8,8,9,10-octachlorobornane (P-42a or B8-806) with lesser amounts of several other Cl(7)-Cl(9) compounds, many of which remain unidentified. Petrochelidon spp. liver profiles appear to be intermediate in complexity between those in invertebrates and fish (more complex) and mammals (less complex) and differs somewhat from those reported for other avian species. In addition to other legacy OC contaminants, toxaphene residues were most concentrated in the lower RGV and accumulated at up to hundreds of parts per billion in these insect-eating birds, underscoring their utility as avian bioindicators of persistent organic pollutants.

Dehalogenation of 2,6-dibromobiphenyl and 2,3,4,5,6-pentachlorobiphenyl in contaminated estuarine sediment.

Estuarine sediments from a USEPA Superfund site in coastal Georgia were extensively contaminated with Aroclor 1268, a mixture of highly chlorinated polychlorinated biphenyls used by a former chlor-alkali plant. Batch slurries of contaminated sediment were incubated for 1 yr with amendments of 2,6-dibromobiphenyl (26-BB) and 2,3,4,5,6-pentachlorobiphenyl (23456-CB) under anaerobic, sulfate-reducing conditions and different pH (5.5-7.5). Organic extracts of slurry sub-samples in a time series were analyzed by congener-specific GC-MS. Dechlorination of 23456-CB was pH dependent and occurred via two routes with the sequential loss of (1) meta and para chlorines and (2) para, ortho, and meta chlorines. Quantitative dehalogenation of 26-BB was observed at all pH. Supplementation of nonachlorobiphenyls (as primers) did not induce dechlorination of native Aroclor 1268 nor of the primers themselves. While contaminated estuarine sediments possess microbial consortia with diverse dehalogenating activities, lack of dechlorination of Aroclor 1268 and spiked nonachlorobiphenyl congeners suggests a bioavailability limitation or enzyme-substrate incompatibilities.

Characterization of the B12- and iron-sulfur-containing reductive dehalogenase from Desulfitobacterium chlororespirans.

The United Nations and the U.S. Environmental Protection Agency have identified a variety of chlorinated aromatics that constitute a significant health and environmental risk as "priority organic pollutants," the so-called "dirty dozen." Microbes have evolved the ability to utilize chlorinated aromatics as terminal electron acceptors in an energy-generating process called dehalorespiration. In this process, a reductive dehalogenase (CprA), couples the oxidation of an electron donor to the reductive elimination of chloride. We have characterized the B12 and iron-sulfur cluster-containing 3-chloro-4-hydroxybenzoate reductive dehalogenase from Desulfitobacterium chlororespirans. By defining the substrate and inhibitor specificity for the dehalogenase, the enzyme was found to require an hydroxyl group ortho to the halide. Inhibition studies indicate that the hydroxyl group is required for substrate binding. The carboxyl group can be replaced by other functionalities, e.g. acetyl or halide groups, ortho or meta to the chloride to be eliminated. The purified D. chlororespirans enzyme could dechlorinate an hydroxylated PCB (3,3',5,5'-tetrachloro-4,4'-biphenyldiol) at a rate about 1% of that with 3-chloro-4-hydroxybenzoate. Solvent deuterium isotope effect studies indicate that transfer of a single proton is partially rate-limiting in the dehalogenation reaction.

Structure of a persistent heptachlorobornane in toxaphene (b7-1000) agrees with molecular model predictions.

A Cl(7) component of technical toxaphene (CTT), previously detected in marine mammals and fish and referred to as "7-1", was isolated from contaminated estuarine sediment using preparative solid-liquid chromatography followed by reversed-phase HPLC. The structure of this compound, elucidated by GC/MS and (1)H NMR, was 2-endo,3-exo,5-endo,6-exo,8,8,10-heptachlorobornane (hereafter referred to as B7-1000). This newly identified CTT eluted in the nonpolar fraction from silica and shares the alternating endo-exo chlorine substitution pattern with other relatively nonpolar, persistent congeners (e.g., B8-1413 and B9-1679). Based on ECNI-MS response, levels of B7-1000 in tissue samples of various higher organisms including humans were as high as 16% of B8-1413. Enantioselective determination of B7-1000 using a modified cyclodextrin chiral stationary phase (beta-BSCD) resulted in enantiomer ratios that were depleted in adipose tissue of a marine bird (skua) and Weddell seal blubber (0.3 and 0.5, respectively), but not in elephant seal blubber (1.1). Elucidation of the structure of B7-1000 thus validates previous predictions of persistence based on structure-activity relationships, chromatographic properties, and molecular modeling.

Induction of the P450 reporter gene system bioassay by polycyclic aromatic hydrocarbons in Ulsan Bay (South Korea) sediments.

Polycyclic aromatic hydrocarbons (PAHs) and induction of the P450 reporter gene system (RGS) for 6- and 16-h exposure periods were determined in organic extracts of Ulsan Bay (South Korea) sediments to assess the utility of this bioassay as a screening tool for PAH contamination. The sum of the concentrations of 23 individual PAHs in 30 sediment samples (sigma PAH) based on GC-MS analysis ranged from 0.05 to 6.1 micrograms/g dry wt. P450 RGS fold induction ranged from 4.0 to 320 micrograms/g based on benzo[a]pyrene toxic equivalents (BaPEq). P450 RGS BaPEq and the 'chemical BaPEq', defined as the sum of the products of individual PAH concentrations and pre-determined toxic equivalency factors, exhibited very strong positive correlations with sigma PAH (r2 > 0.90; P < 0.001). Fold induction did not increase (and in some cases decreased) after the optimal incubation period (6 h) for PAHs, indicating that other compounds known to induce the P450 RGS (e.g. chlorinated organics) were not present at levels effecting significant induction. This was supported by GC-ECD analysis where non-ortho and mono-ortho polychlorinated biphenyls (PCBs) known to be strong P450 RGS inducers were found to be at very low or non-detectable levels in samples with the highest P450 RGS responses. The profound difference in PAH profiles for the two most contaminated sites suggested that this assay is especially sensitive for selected PAHs with greater than four rings. Combined with previous results, the P450 RGS shows promise as a useful screening tool for predicting deleterious biological effects resulting from CYP1A1-inducing, sediment-associated chemicals, particularly high molecular weight PAHs.

Interpreting nonracemic ratios of chiral organochlorines using naturally contaminated fish.

Although reports of nonracemic proportions of chiral organochlorine pollutants in the environment are widespread, the interpretation of such data is not well developed. Using GC/MS and a chiral stationary phase consisting of 25% tert-butyldimethylsilylated beta-cyclodextrin in PS086 (beta-BSCD), we followed the change in the enantiomeric signature of 2-exo,3-endo,6-exo,8,9,10-hexachlorobornane (B6-923) in naturally contaminated fish maintained under toxaphene-free conditions. Whereas the enantiomeric ratio (ER) of B6-923 was near racemic at the start of the elimination experiment, it had increased severalfold by the end of 60 d. On the basis of first-order kinetics, one enantiomer of B6-923 was eliminated twice as fast as its mirror image, resulting in half-lives of 7 and 13 d, respectively. Enantioselective elimination by our test fish (Fundulus sp.) strongly suggests active biotransformation of B6-923; however, bioprocessing throughput estimates suggest a very low in situ rate of natural attenuation. These results confirm that the relatively constant ERs observed for chiral organochlorines in a given species are the result of competing processes, e.g., uptake vs elimination. Our experiments also further illustrate the utility of enantioselective analysis in characterizing the biotransformation of persistent organochlorine pollutants.

Fungal biomass in saltmarsh grass blades at two contaminated sites.

Ascomycetous fungi are the principal drivers of the decomposition of shoots of smooth cordgrass (Spartina alterniflora). Shoots of smooth cordgrass move into the saltmarsh food web via the decomposition system. Therefore, influences on saltmarsh ascomycetes by pollutants of saltmarshes could have far-reaching impacts. Earlier examination of impacts of severe contamination of a Georgia saltmarsh by mercury and polychlorinated biphenyls (PCBs) revealed little or no influence of the toxicants on living standing crops or sexual productivities of cordgrass ascomycetes. Extension of the examination of saltmarsh-ascomycete response to sites containing other toxic pollutants (the chlorinated organocyclic insecticide toxaphene; chromium, copper, and lead; and polycyclic aromatic hydrocarbons [PAHs]) has shown that none of the additional toxicants engendered saltmarsh-fungal responses in the form of reduced living standing crops or sexual productivities. Thus the ascomycetes of the cordgrass-decay system appear to be as resistant to anthropogenic-pollutant poisoning as smooth cordgrass itself. Unless the fungal and plant resistance mechanisms involve degradation of the toxicants, this may imply that saltmarshes are especially dangerous as receiving sites for toxic waste because they may have the potential to readily move toxicants into the food web.

DNA strand breaks (comet assay) and embryo development effects in grass shrimp (Palaemonetes pugio) embryos after exposure to genotoxicants.

Grass shrimp embryos develop in egg sacs (stages 1-10) attached to the female for 14-20 days after which they 'hatch' from the egg sacs into a swimming zoea stage (stage 11). Until they emerge from the egg sacs, embryos depend on lipids and lipovitellin stored within the egg. The percent of embryos which hatch after exposure to toxicants relative to controls was the basis of an embryo development assay. Exposure of embryos to chromium(III) chloride, sodium chromate, mercuric chloride, and 2-methyl-1,2-naphthoquinone (MNQ) resulted in a reduced hatching rate. In addition to effects on embryo development, DNA strand damage tests were carried out on contaminant-exposed embryos, using the single-cell electrophoresis method often referred to as comet assay. Development of stage 4 embryos was more affected by MNQ exposure than stage 7 embryos. The hatching rates of stages 4 and 7 embryos exposed to MNQ (172 micrograms/l) were 0 and 90%, respectively. DNA strand damage, measured as DNA tail moments, were 3.4 and 4.4, respectively. Thus, exposure of an early embryo stage to MNQ prevented full embryo development while development of later embryo stages was not affected. It may be that the DNA repair systems are more efficient in later embryo stages than in early stages and thus DNA damaged in the early stages affects development.