Lynda H. McCarthy

Evaluation of low-copy genetic targets for waterborne bacterial pathogen detection via qPCR.

Recent developments in water quality research have highlighted difficulties in accurately predicting the incidence of pathogens within freshwater based on the viability, culturability and metabolic activity of indicator organisms. QPCR-driven assays are candidates to replace standard culture-based methods, however, protocols suitable for routine use have yet to be sufficiently validated. The objective of this study was to evaluate five oligonucleotide primers sets (ETIR, SINV, exoT, VS1 and ipaH2) for their potential applicability in qPCR assays to detect contamination from five waterborne bacterial pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, Pseudomonas aeruginosa, and Shigella flexneri). An enrichment-free qPCR protocol was also tested using S. Typhimurium-seeded source water, combining membrane filtration and mechanical, chemical and enzymatic lysis techniques to recover the bacterial cells. All five primer sets were found to have high specificity and sensitivity for the tested organisms. Four of the primers were able to detect pathogen loads as low as 10 cells/mL while 200 cells/mL of C. jejuni were detectable in pure culture. Although sensitivity decreased in an artificially contaminated environmental matrix, it was still possible to detect as few as 10 S. Typhimurium cells without enrichment. The primers and protocols evaluated in this study have demonstrated potential for further validation for possible application alongside traditional indicator techniques.

Utilization of multiple organisms in a proposed early-warning biomonitoring system for real-time detection of contaminants: preliminary results and modeling

During past decades, biomonitors were deployed in lakes and rivers to rapidly detect hazardous chemicals by measuring the endpoints of a single aquatic species at defined short intervals. Most biomonitors, however, are only capable of indicating a departure from baseline water conditions without identifying the cause. In order to provide a more comprehensive assessment, a biomonitoring system which features a library of stereotyped responses of multiple aquatic species in various water conditions is proposed. A preliminary library was constructed by characterizing the behavioural and physiological responses of Daphnia magna, Hyalella azteca, Lumbriculus variegatus, and Pseudokirchneriella subcapitata to various concentrations of atrazine and tributyltin. By employing multivariate statistical tools such as principal component analysis (PCA) and discriminant analysis, this library (which contained responses after 6h of exposure to contaminants) was used as a template to classify and to model other sets of earlier measurements at 2 and 4h, resulting in an accuracy of 73 and 97%, respectively. These findings demonstrated the potential capability of the proposed early-warning biomonitoring system to provide real-time water quality assessment and early-warning contaminant detection.

Vertical Phosphorus Migration in a Biosolids-Amended Sandy Loam Soil in Laboratory Settings: Concentrations in Soils and Leachates

The impacts of biosolids land application on soil phosphorus and subsequent vertical migration to tile drainage were assessed in a laboratory setup. Soil, representing typical “nonresponse” Ontario soil as specified by Ontario Ministry of Agriculture, Food, and Rural Affairs (OMAFRA), was amended with anaerobically digested biosolids at a rate of 8 Mg ha−1 (dry weight). Over five months, these amended soil samples from two different depths were sequentially fractionated to determine various inorganic and organic phosphorus pools in order to evaluate phosphorus vertical migration within a soil profile. Soil leachate was analyzed for soluble reactive phosphorus. The results indicated that biosolids application did not significantly affect phosphorus concentrations in soil and did not cause phosphorus vertical migration. The concentrations of soluble reactive phosphorus also were not significantly affected by biosolids.

Automated image analysis of Euglena gracilis Klebs (Euglenophyta) for measuring sublethal effects of three model contaminants.

The short-term impacts of atrazine (herbicide), tributyltin (organometal) and copper on the behaviour of Euglena gracilis Klebs (Euglenophyta) were assessed. First, the ECOTOX automated image analysis system was used, which measured swimming velocity, cell shape, percentage of cells swimming upwards, and randomness of swimming. Next, visual observation by microscopy was used to measure percentage of cell motility and cell shape. Behavioural changes can be used as an indicator of stress in less than 24 h, potentially making them suitable for inclusion in early-warning systems for water quality. Findings indicate that E. gracilis is a very sensitive organism to copper, showing inhibition of motility with visual observation at 0.8 μmol/L within 1 h. The image analysis system was in general less sensitive than visual observation for detecting behavioural changes after incubation in copper. In contrast, after exposure to organic contaminants atrazine and tributyltin, the ECOTOX system detected small changes in the number of cells swimming upwards (antigravitactic behaviour) at higher concentrations.