James P. Sherry

Environmental immunoassays and other bioanalytical methods: Overview and update

Immunoassays and bioanalytical techniques can aid the cost effective detection and quantification of trace contaminants in the environment, food, and human and animal populations. This overview of recent progress shows that rapid advances have occurred in the development and validation, of assays for many contaminants of both industrial and agricultural origin. Promising antibody based techniques such as immunoaffinity chromatography, biosensors, and flow injection immunoanalysis continue to evolve. Such techniques can not only help lower costs and improve efficiency, but can also allow the range of hypotheses that can be tested in many environmental studies to be broadened by permitting the determination of trace residues in small volume samples that would be otherwise difficult to analyze.

An ELISA for brown trout (Salmo trutta) vitellogenin and its use in bioassays for environmental estrogens

An enzyme linked immunosorbent assay (ELISA) was developed for the detection of the egg yolk precursor vitellogenin (Vg) in plasma of brown trout (Salmo trutta). Purified Vg from a 17 beta-estradiol-induced trout was used as the competing antigen in the ELISA which is based on polyclonal antibodies. The ELISAs performance was optimized and characterized. The assays working range was (25-500 ng ml-1), its sensitivity was (10.5 ng ml-1), and it had an intra-assay coefficient of variation of less than 10% between 30 and 1000 ng ml-1. The ELISA was used in bioassays for the detection of environmental estrogens, including estrogen mimics, in whole and fractionated industrial waste waters. Those bioassays were based on intraperitoneal (i.p.) injection-, static renewal-, and flow through exposure systems. The response threshold of both bioassays is limited to 1-2 micrograms ml-1 Vg by a low level plasma interference that was regularly detected in plasma from non-induced male fish. The responsiveness of the bioassays was characterized using progressive doses of 17 beta-estradiol. The i.p.-based assay, which was responsive to at least 100 micrograms kg-1 of 17 beta-estradiol, was used to screen extracts of pulp mill effluent and black liquor for estrogenic effects. Neither extract induced Vg in our assay. The i.p. assay was also used to test 4-tert-octylphenol (OP) and the PAH derivative, retene, for estrogenic activity. OP induced Vg in the i.p.-exposed fish; no Vg induction was detected in the retene-exposed fish. The static renewal bioassay, which was responsive to at least 0.1 microgram ml-1 of 17 beta-estradiol over a 15-day exposure period, was used to screen whole pulp mill effluents for estrogenic effects. No Vg induction was detected in the effluent-treated fish.

Ability of fractionated petroleum refinery effluent to elicit cyto- and photocytotoxic responses and to induce 7-ethoxyresorufin-O-deethylase activity in fish cell lines

The ability of fractionated petroleum refinery effluent to cause cellular responses in fish cell lines was evaluated. The cellular responses, which included direct and indirect cytotoxicity, photocytotoxicity and induction of 7-ethoxyresorufin-O-deethylase (EROD) activity, may potentially be linked to sublethal effects observed in effluent-exposed fish and fish larvae. In order to be able to quantify cellular responses rapidly, microtitre plates were used along with fluorescent probes. For the quantification of cyto- and photocytotoxicity, the fluorescent probes were alamar Blue and carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which monitor metabolic activity and cell membrane integrity, respectively. EROD activity was measured as the rate of conversion by EROD of the substrate 7-ethoxyresorufin to its fluorescent product, resorufin. Effluent from an Ontario refinery was fractionated into aqueous and particulate phase. As well, a solid phase extract (SPE) was used to prepare concentrated effluent for testing in the cell lines. The effluent was able to elicit all of the responses of interest although significant cyto- and photocytotoxicity required effluent equivalent concentrations above 100% effluent and could only be revealed upon exposure of cells to the SPE concentrated effluent. Based on their retention on C18, the cytotoxicants are likely to be non-polar to moderately polar chemicals. The presence of polar compounds affecting cellular metabolism was indicated by the responses of exposed cells to a 90% aqueous phase effluent. In contrast to cyto- and photocytotoxicity, EROD induction occurred at effluent equivalent concentrations well below 100% effluent and was elicited by the SPE and the particulate fraction thereby suggesting that most EROD-inducers were particle-bound. Among other applications, the described techniques could help to determine the source of causative agents of sublethal effects in the refining process.

Impacts of pollution in feral Mya arenaria populations: the effects of clam bed distance from the shore.

This study examined the relationships between population characteristics and the expression of physiological biomarkers of stress in an intertidal clam population under pollution at sites differing in thermal history and coastline distance. The clam population metrics were age distribution, growth, condition factor, distance of the clam beds from the shore, and gonad development. Physiological biomarkers comprised biomarkers of defence such as superoxide dismutase, labile IIb metals in tissues, redox status of metallothioneins and glutathione S-transferase, of tissue damage such as lipid peroxidation and DNA strand breaks, of reproduction as determined by vitellogenin-like proteins and gonadosomatic index and immunocompetence such as phagocytosis and hemocyte viability. Age-related pigments were also examined to compare the physiological age of the clams with their chronological age. The results showed that all the above biomarkers were significantly affected at one of the two polluted sites at least. Distance from the shore was significantly correlated with most (81%) of the biomarkers examined. Clams collected at one polluted site were physiologically older than clams from the corresponding reference site. Canonical and adaptive regression (artificial neural networks) analyses found that the biomarkers measured in this study were able to predict the ecologically relevant endpoints. Biomarkers implicated in defense mechanisms, tissue damage and age-related pigments were most closely related to the clam population characteristics. Sensitivity analysis of the learning algorithm found that the following physiological and biochemical markers were the most predictive, in decreasing order, of clam population characteristics: glutathione S-transferase, phagocytosis, age pigments, lipid peroxidation in the gills, labile IIb metals and total MT levels. These biomarkers were affected by the distance of the clam beds from the shore, site quality (pollution) and reproduction activity.

Investigation of the sublethal effects of some petroleum refinery effluents

In Canada, environmental regulations for protection of the biota from the adverse effects of effluents from petroleum refineries have tended to focus on acute toxicity. There is concern those effluents may have other subtle, but still deleterious, long-term effects on aquatic ecosystems. We have used a battery of toxicity tests to assess the acute toxicity, genotoxicity, and chronic toxicity of effluent samples from two Ontario refineries. The test organisms included representatives of the bacterial, algal, plant, cladoceran, and fish communities. The results of our preliminary study indicate that the effluent samples had little acute toxicity to the test organisms. There were indications of some sublethal toxicity to Ceriodaphnia dubia, Panagrellus redivivus, and Pimephales promelas. One of the effluents inhibited the growth of Selanastrum capricornutum (IC50 of 59.9%) and Lemna gibba (IC25 of 73.3%) and also caused a 15 percent reduction in the germination of Lactuca sativa seeds. The SOS-Chromotest, a commercially available test that measures the activity of a bacterial DNA repair system, detected genotoxic effects in a single effluent that had been concentrated ten fold. There was no apparent relationship between several chemical parameters and the observed sublethal effects. Further research is needed to establish whether or not the observed toxic effects are typical of effluents from Ontario refineries.

The use of radioimmunoassay for the detection of polychlorinated dibenzo-p-dioxins in fish samples

Abstract Because of the increasing numbers of environmental samples requiring analysis for polychlorinated dibenzo-p-dioxin (PCDD) contamination, a need exists for screening techniques, such as radioimmunoassay (RIA), that will facilitate the elimination of PCDD free samples from time-consuming conventional analysis. The RIA for the detection of PCDDs was interfaced with an extraction and cleanup procedure, and its performance was assessed using extensively and minimally cleaned-up Lake Trout samples. Sample size appeared to influence assay performance, probably because of its relationship to the specific detection limit: the larger the sample size that can be analyzed without adversely affecting the amount of TCDD detectable, the lower will be the specific detection limit. However, larger than optimal samples narrowed the assays working range, adversely affected dose response, and raised the detection limit. The working range of the assay results from a compromise between the required degree of cleanup and sample size.

Temporal Distribution of Faecal Pollution Indicators and Opportunistic Pathogens at a Lake Ontario Bathing Beach

Abstract In order to examine the temporal relationship between bather load and water quality indicator levels, faecal pollution indicator (faecal coliforms and faecal streptococci) and opportunistic pathogen (Candida albicans and Pseudomonas aeruginosa) levels were monitored hourly in the nearshore waters of a shallow Lake Ontario bathing beach on a hot August day when the bather load was high, and on two overcast August days when the bather load was negligible. On the high bather load day, an increase occurred in the numbers of faecal coliforms and faecal streptococci in the beach water in conjunction with an increase in the bather load; P. aeruginosa levels increased later in the day. There was little apparent relationship between C. albicans levels and the other parameters on the high bather load day. No corresponding increases were observed for any of the monitored parameters on the low bather load days. The results indicate that bather load and sample collection time may influence estimations of recreational water quality based on faecal pollution indicator and P. aeruginosa levels.

Enzyme-immunoassay techniques for the detection of atrazine in water samples: Evaluation of a commercial tube based assay

Abstract Environmental immunoassays can help lower the operating costs and improve the effectiveness of residue laboratories. The present study assesses the ability of a commercially available enzyme immunoassay (EIA) to detect triazine herbicides in water. The tube based EIA could detect atrazine in lake and river water with detection limits of 62 pg/mL and 180 pg/mL respectively. The assays ability to quantify atrazine in a set of 124 water samples taken from many parts of Canada was compared with a reference method that used gas chromatographic separation combined with a nitrogen phosphorous detector (GC-NPD) (R=0.919). A 71 % reduction in analytical load was achieved at a threshold concentration of 1 ng/mL. There were 2.4 % false negative and 0.8 % false positive results associated with that load reduction. The variability of the assay control parameters was generally within two standard deviations of the mean response for 65 assays. The EIA for atrazine is recommended for use as a screening technique and as an inexpensive way to monitor triazine levels in waters that are known to be contaminated with those herbicides.

Use of dimethyl sulfoxide as solubilization agent in the detection of 2,3,7,8-TCDD by radioimmunoassay

Abstract Immunoassays are potentially valuable tools for use in screening environmental samples for a broad range of contaminants, such as polychlorinated dibenzo-p-dioxins (PCDDs). The performance of the radioimmunoassay (RIA) for PCDDs was characterized using 4 solubilization systems: Cutscum, Triton, horse serum, and dimethyl sulfoxide (DMSO). The DMSO based assay appeared to perform best at low 2,3,7,8-TCDD levels. The effects of assay incubation time and hapten storage conditions on the DMSO based assay were assessed. The separation of bound from unbound radioactivity was accelerated without adversely affecting assay performance. Further assay development through the use of an alternative labelled hapten is considered.

An MSD-based method for the detection of chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans in fish

Abstract A stream-lined method for the detection of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in fish samples is described. The samples were mechanically blended with anhydrous sodium sulphate and then packed in a glass column. The lipid fraction was eluted from the column with dichloromethane. The described extraction technique recovered 13 C-labelled surrogate spikes as effectively as a method based on acidic digestion and liquid phase extraction. Lipids were removed from the fish extracts by size exclusion chromatography followed by chromatography on a mini acid/base silica column. The extract was further enriched by high performance liquid chromatography (HPLC) on basic alumina and activated carbon. The analytes were identified and quantified by gas chromatography combined with mass selective detection. Two sets of fish samples (n = 16 & n = 6) were used to evaluate the method: surrogate recoveries ranged from 67.5% to 75.5%, and analytical precision ranged from 13.9 to 26.4%. Tritiated 2,3,7,8-T 4 CDD was used to conveniently trouble shoot the method.