In fissured aquifers, hydrodynamic phenomena combined with a network of permeable fissures imparts varying degrees of vulnerability depending on the type of contaminant. The study presented here examines the impact on a karst aquifer of a river which receives effluents from a sewage treatment plant just upstream from the point of recharge. This recharge constitutes a source of bacteriological and chemical pollution. The aquifer is accessible by boreholes at discrete points, facilitating investigation of the evolution of three of the contaminants characteristic of treatment station effluents, NH4+ and two biological contaminants with different survival capacities. The study was undertaken both under normal climatic conditions and during a flood occurring after a long dry period. The rate of infiltration, of subsurface transport, and of dissemination of contaminants into a zone usually protected from contamination was recorded. The results confirm the vulnerability of fissured aquifers to contamination.

Aims:The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Française de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters. Methods and Results:The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation. The recovery was 30-50% for both parasites at seeding levels from 30 to 230 (oo)cysts. The method is linear from 0 to around 400 seeded (oo)cysts and the yield does not significantly vary for turbidity levels from 10 to 40 Formazin Nephelometric Units (FNU). The results were obtained using manual microscopic enumeration of the (oo)cysts. The ChemScan-RDI yielded counts that were at least equivalent to those obtained using manual microscopy for both parasites in raw and potable water concentrates, for seeding levels of 10-300 or 10-100, respectively. The purification and labelling method proposed by the supplier of the ChemScan-RDI (Chemunex) reached very similar recoveries to the AFNOR protocol (70-86% in both cases). Conclusions: Laser scanning cytometry can be used as a more standardized alternative to manual enumeration as part of the new AFNOR standard method. Significance and Impact of the Study: By using laser scanning cytometry instead of manual microscopy, laboratories could circumvent the limitations of manual microscopy, namely: low sample throughput, operator subjectivity and operator fatigue. The study further supports the drive to incorporate laser scanning cytometry in the standard methods for Giardia and Cryptosporidium enumeration.

Experiments were carried out to investigate the potential for bacterial colonization of different substrates in karst aquifers and the nature of the colonizing bacteria. Laboratory batch experiments were performed using limestone and PVC as substrates, a natural bacterial isolate and a known laboratory strain (Escherichia coli [E. coli]) as inocula, and karst ground water and a synthetic formula as growth media. In parallel, fragments of limestone and granite were submerged in boreholes penetrating two karst aquifers for more than one year; the boreholes are periodically contaminated by enteric bacteria from waste water. Once a month, rock samples were removed and the colonizing bacteria quantified and identified. The batch experiments demonstrated that the natural isolate and E. coli both readily colonized limestone surfaces using karst ground water as the growth medium. In contrast, bacterial colonization of both the limestone and granite substrates, when submerged in the karst, was less intense. More than 300 bacterial strains were isolated over the period sampled, but no temporal pattern in colonization was seen as far as strain, and colonization by E. coli was notably absent, although strains of Salmonella and Citrobacter were each observed once. Samples suspended in boreholes penetrating highly fractured zones were less densely colonized than those in the borehole penetrating a less fractured zone. The results suggest that contamination of karst aquifers by enteric bacteria is unlikely to be persistent. We hypothesize that this may be a result of the high flow velocities found in karst conduits, and of predation of colonizing bacteria by autochthonous zooplankton.