Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments

This study compares five genera of free-living amoebae (FLA) hosts by Legionella spp. in the fixed and floating biofilm samples from spring environments. Detection rate of Legionella spp. was 26.9% for the floating biofilms and 3.1% for the fixed biofilms. Acanthamoeba spp., Hartmanella vermiformis, and Naegleria spp. were more frequently detected in floating biofilm than in fixed biofilm samples. The percentage of pathogenic Acanthamoeba spp. among all the genus Acanthamoeba detected positive samples was 19.6%. The potential pathogenic Naegleria spp. (for example, Naegleria australiensis, Naegleria philippinensis, and Naegleria italica) was 54.2% to all the Naegleria detected positive samples. In the study, 12 serotypes of possible pneumonia causing Legionella spp. were detected, and their percentage in all the Legionella containing samples was 42.4%. The FLA parasitized by Legionella included unnamed Acanthamoeba genotype, Acanthamoeba griffini, Acanthamoeba jacobsi, H. vermiformis, and N. australiensis. Significant differences were also observed between the presence/absence of H. vermiformis and Legionella parasitism in FLA. Comparisons between the culture-confirmed method and the PCR-based detection method for detecting FLA and Legionella in biofilms showed great variation. Therefore, using these analysis methods together to detect FLA and Legionella is recommended.     

Mortality rates of autochthonous and fecal bacteria in natural aquatic ecosystems

Bacterial mortality has been investigated in freshwater (River Seine) and in marine (North Sea) systems using a method based on following the disappearance of radioactivity from the DNA of assemblages of bacteria previously labeled with tritiated thymidine. Measurement of bacterial mortality of autochthonous and various types of fecal bacteria allowed direct comparisons between their respective first-order mortality rates. Mortality rates obtained for the different types of bacteria in the River Seine were, respectively, 7.9-33.9 x 10(-3)h(-1) for Escherichia coli, 12.2-29.2 x 10(-3)h(-1) for S. faecium and 7.0-18.3 x 10(-3)h(-1) for the autochthonous bacteria. In the Belgian coastal waters, these rates were 4.6-27.3 x 10(-3)h(-1) for E. coli, 6.0-22.0 x 10(-3)h(-1) for S. typhimurium, 10.0-18.9 x 10(-3)h(-1) for S. faecium and 1.0-13.9 x 10(-3)h(-1) for autochthonous bacteria. In both environments, the overall mortality rates of autochthonous and the different fecal bacteria were in the same order of magnitude and overall mortality rates of E. coli were on average about twice as high for autochthonous bacteria. Grazing by protozooplankton was the dominant process of mortality for fecal and autochthonous bacteria in both environments. Except in a few situations, grazing by protozooplankton was responsible for more than 90% of the overall mortality rate of fecal and autochthonous bacteria in the river and in the coastal area.     

Comparison of ammonia volatilisation rates in algae and duckweed-based waste stabilisation ponds treating domestic wastewater

Quantification of ammonia volatilisation from wastewater stabilisation ponds is important in order to understand its significance for overall nitrogen removal in these widely applied low-cost treatment systems. Ammonia volatilisation rates were measured in pilot plant facilities consisting of one line of four algae-based ponds in series and a parallel line of four ponds with a floating mat of duckweed (Lemna gibba). Ammonia volatilisation was assessed during a period of one and a half years. The method applied is accurate, convenient and is proposed for analysis of a wide range of gasses emitted from stabilisation ponds and possibly other aquatic systems. The ammonia volatilisation rates in algae-based ponds (ABPs) were higher than in duckweed-based ponds (DBPs). This can be explained by the lower values of NH(3) in DBPs due to shading and lower pH values, since the volatilisation rate highly correlated with free ammonia concentration (NH(3)) in pond water. The duckweed cover appeared not to provide a physical barrier for volatilisation of unionised ammonia, because whenever NH(3) concentrations were equal in ABP and DBP also the volatilisation rates were equal. Volatilisation was in the range of 7.2-37.4 mg-Nm(-2)d(-1) and 6.4 -31.5 mg-Nm(-2)d(-1) in the ABPs and DBPs, respectively. Average influent and effluent ammonium nitrogen measurements showed that the ammonia volatilisation during the study period in any system did not exceed 1.5% of total ammonium nitrogen removal. Therefore this study confirmed results from simultaneous experimental work in our laboratory indicating that nitrification/denitrification, rather than ammonia volatilisation, is the most important mechanism for N removal in ABPs and DBPs.     

Performance of CHROMagar™ Staph aureus and CHROMagar™ MRSA for detection of Staphylococcus aureus in seawater and beach sand - Comparison of culture, agglutination, and molecular analyses

Beach seawater and sand were analyzed for Staphylococcus aureus and methicillin resistant S. aureus (MRSA) for samples collected from Avalon, and Doheny Beach, CA. Membrane filtration followed by incubation on CHROMagar™ Staph aureus (SCA) and CHROMagar™ MRSA (C-MRSA) was used to enumerate S. aureus and MRSA, respectively. Media performance was evaluated by comparing identification via colony morphology and latex agglutination tests to PCR (clfA, 16S, and mecA genes). Due to background color and crowding, picking colonies from membrane filters and streaking for isolation were sometimes necessary. The specificity of SCA and C-MRSA was improved if colony isolates were identified by the presence of a matte halo in addition to mauve color; however routine agglutination testing of isolates did not appear warranted. Using the appearance of a colony on the membrane filter in conjunction with isolate appearance, the positive % agreement, the negative % agreement, and the % positive predictive accuracy for SCA was 84%, 95%, and 99% respectively, and for C-MRSA it was 85%, 98%, and 92%, respectively. Sensitivity and specificity of SCA and C-MRSA with membrane-filtered beach samples were optimized through identification experience, control of filter volume and incubation time, and isolation of colonies needing further identification. With optimization, SCA and C-MRSA could be used for enumeration of S. aureus and MRSA from samples of beach water and sand. For the sites studied here, the frequency of detection of S. aureus ranged from 60 to 76% and 53 to 79% for samples of beach seawater and sand, respectively. The frequency of detection of MRSA ranged from 2 to 9% and 0 to 12% for samples of seawater and sand, respectively.     

Occurrence and persistence of enteroviruses, noroviruses and F-specific RNA phages in natural wastewater biofilms

Enteroviruses and noroviruses are pathogenic viruses excreted by infected individuals. Discharged in wastewaters, some of these viruses can be captured by biofilms. In the present study, we assessed the occurrence and persistence of these viruses in wastewaters and in corresponding biofilms. Natural wastewaters and biofilms were analyzed monthly from January to July using real-time RT-PCR. Enterovirus RNA was detected in wastewater in June while norovirus RNA was detected from January to March. In contrast, biofilm analysis revealed the presence of both enterovirus and norovirus genomes throughout the study period. For instance, enterovirus and norovirus genogroups (GG) I and II were detected in 50, 46 and 37% of the biofilm samples, respectively (n = 24). In a laboratory experiment, persistence of norovirus GGI RNA (quantified using molecular techniques) and F-specific bacteriophages (quantified using both culture and molecular techniques) was assessed in wastewater and corresponding naturally-contaminated biofilms at both 4 and 20 °C. The concentrations of viral genomes (norovirus GGI and F-specific RNA phage) were very stable in biofilms. Indeed, no significant decrease was observed during the persistence experiment that lasted 49 days. Furthermore, regardless of our experimental conditions, viral genome and infectious F-specific bacteriophages persisted longer in biofilm than in wastewater. According to our results, wastewater biofilms may contribute to the persistence and dispersal of pathogenic viruses outside of epidemic periods.     

Influence of acid volatile sulfides and simultaneously extracted metals on the bioavailability and toxicity of a mixture of sediment-associated Cd, Ni, and Zn to polychaetes Neanthes arenaceodentata

Laboratory microcosm experiments were conducted to investigate the influence of acid volatile sulfides (AVS) and simultaneously extracted metals (SEM) in sediments on the bioavailability and toxicity of Cd, Ni, and Zn in sediments to polychaete worms Neanthes arenaceodentata. Cohorts of juvenile N. arenaceodentata were exposed to sediments spiked with metal mixtures containing Cd, Ni, and Zn (0.5-15 micromol x g(-1) of total SEM) with Low- (approximately 1 micromol x g(-1)), Medium- (approximately 5 micromol x g(-1)), and High-AVS concentrations (approximately 10 micromol x g(-1)) for 20 days to determine mortality, growth rate, and metal bioaccumulation. Tissue Cd and Zn concentrations at the end of the exposure were significantly higher in sediments with the low-AVS concentration at a given SEM concentration due to the increased dissolved metal concentrations in overlying water (OW). However, tissue Ni concentrations were not related to dissolved Ni in the OW. AVS concentrations also influenced the toxicity of metals to the worms. Significant mortality was observed only at the highest SEM treatments at Low-AVS series. Most individuals survived at the highest SEM treatments at Medium- and High-AVS series. Similarly, the growth rates of worms were reduced in treatments having higher molar differences between SEM and AVS ([SEM-AVS]). Overall, the bioavailability and toxicity of metals in sediments was not well predicted by sediment metal concentrations only, but considering the influence of geochemical factors (AVS) on the metal bioavailability improved the prediction of toxicity. Also, the relationship between tissue metal concentration and toxicity was used to determine which contaminant was most responsible for the observed toxicity of the metal mixture.     

Pollution of montane soil with Cu, Zn, As, Sb, Pb, and nitrate in Kanto, Japan

Soil cores and rainwater were sampled under canopies of Cryptomeria japonica in four montane areas along an atmospheric depositional gradient in Kanto, Japan. Soil cores (30 cm in depth) were divided into 2-cm or 4-cm segments for analysis. Vertical distributions of elemental enrichment ratios in soils were calculated as follows: (X/Al)_i/(X/Al)_BG (where the numerator and denominator are concentration ratios of element-X and Al in the i- and bottom segments of soil cores, respectively). The upper 14-cm soil layer showed higher levels of Cu, Zn, As, Sb, and Pb than the lower (14-30 cm) soil layer. In the four areas, the average enrichment ratios in the upper 6-cm soil layer were as follows: Pb (4.93) ≥ Sb (4.06) ≥ As (3.04) > Zn (1.71) ≥ Cu (1.56). Exogenous elements (kg/ha) accumulated in the upper 14-cm soil layer were as follows: Zn (26.0) > Pb (12.4) > Cu (4.48) ≥ As (3.43) ≥ Sb (0.49). These rank orders were consistent with those of elements in anthropogenic aerosols and polluted (roadside) air, respectively, indicating that air pollutants probably caused enrichment of these elements in the soil surface layer. Approximately half of the total concentrations of As, Sb, and Pb in the upper 14-cm soil layer were derived from exogenous (anthropogenic) sources. Sb showed the highest enrichment factor in anthropogenic aerosols, and shows similar deposition behavior to NO₃⁻, which is a typical acidic air pollutant. There was a strong correlation between Sb and NO₃⁻ concentrations in rainfall (e.g., in the throughfall under C. japonica: [NO₃⁻] = 21.1 [dissolved Sb], r = 0.938, p < 0.0001, n = 182). Using this correlation, total (cumulative) inputs of NO₃⁻ were estimated from the accumulated amounts of exogenous Sb in soils, i.e., 16.7 t/ha at Mt. Kinsyo (most polluted), 8.6 t/ha at Mt. Tsukuba (moderately polluted), and 5.8 t/ha at the Taga mountain system (least polluted). There are no visible ecological effects of these accumulated elements in the Kanto region at present. However, the concentrations of some elements are within a harmful range, according to the Ecological Soil Screening Levels determined by the U.S. Environmental Protection Agency.     

Sediment reworking rates in deep sediments of the Mediterranean Sea

Different pelagic areas of the Mediterranean Sea have been investigated in order to quantify physical and biological mixing processes in deep sea sediments. Herein, results of eleven sediment cores sampled at different deep areas (> 2000 m) of the Western and Eastern Mediterranean Sea are presented.²¹⁰Pb_xs and ¹³⁷Cs vertical profiles, together with ¹⁴C dating, are used to identify the main processes characterising the different areas and, finally, controlling mixing depths (SML) and bioturbation coefficients (D_b). Radionuclide vertical profiles and inventories indicate that bioturbation processes are the dominant processes responsible for sediment reworking in deep sea environments.Results show significant differences in sediment mixing depths and bioturbation coefficients among areas of the Mediterranean Sea characterised by different trophic regimes. In particular, in the Oran Rise area, where the Almeria-Oran Front induces frequent phytoplankton blooms, we calculate the highest values of sediment mixing layers (13 cm) and bioturbation coefficients (0.187 cm² yr⁻¹), and the highest values of ²¹⁰Pb_xs and ¹³⁷Cs inventories. Intermediate values of SML and D_b (~ 6 cm and ~ 0.040 cm² yr⁻¹, respectively) characterise the mesothrophic Algero-Balearic basin, while in the Southern Tyrrhenian Sea mixing parameters (SML of 3 cm and D_b of 0.011 cm² yr⁻¹) are similar to those calculated for the oligotrophic Eastern Mediterranean (SML of 2 cm and D_b of ~ 0.005 cm² yr⁻¹).     

Short-term harmful effects of unionised ammonia on natural populations of Moina micrura and Brachionus rubens in a deep waste treatment pond

Populations of Moina micrura and Brachionus rubens in a deep waste treatment pond were exposed to the natural short-term fluctuations of unionised ammonia (90-min intervals of monitoring) that occur in the course of a day during a summer algal bloom. Under natural conditions, three replicate experiments were conducted in which water temperature, pH, dissolved oxygen, total ammonia, unionised ammonia, phytoplankton biomass and zooplankton (number of living and dead organisms, mortality rate and instant mortality) were studied. The time-course of unionised ammonia concentration was consistent with those shown by temperature, pH, phytoplankton biomass, dissolved oxygen, Moina micrura mortality and Brachionus rubens mortality. On the other hand, temperature, pH and dissolved oxygen never exceeded the tolerance ranges described for Moina and Brachionus, which led us to attribute the cause of zooplankton mortality to unionised ammonia toxicity. Mortality rates of 63%, 27% and 34% were recorded for Moina in each replicate experiment. Brachionus was less affected, with mortalities of 7.3%, 6.2% and 6.0%.These results confirm previous field observations (Water Res. 34(14) (2000) 3666; Water Res. 37(5) (2003) 1048) that attributed a reduction in zooplankton biomass during certain periods of summer (algal blooms) to a harmful side-effect of an excessive increase in phytoplankton biomass: high photosynthetic activity during these periods of proliferation of algae gives rise to an increased pH (>/=8) and, subsequently, leads to production of unionised ammonia (toxic for aquatic organisms) from its ionised fraction.     

Simultaneous effects of environmental factors on motile Aeromonas dynamics in an urban effluent and in the natural seawater

Seasonal dynamics of motile Aeromonas in a treated urban effluent and in natural seawater along the Sfax coast (Mediterranean sea, Tunisia) were measured over a year concurrently with seven environmental factors, and compared with those of faecal coliforms. Counts for Aeromonas from a standard plate count method, ranged from 1.48 x 10(5)CFU.100 ml(-1) to 2.2 x 10(8)CFU.100 ml(-1) in the effluent and from 7.9 x 10(3)CFU.100 ml(-1) to undetectable level in the surface marine waters. Contrary to faecal coliforms, the Aeromonas dynamics exhibited a seasonal distribution in seawater which was inverse of the seasonal distribution in the sewage: From the end of November 1998 to April 1999 (cold period), Aeromonas counts increased in the treated effluent, while it decreased very rapidly in seawater. From May to October (warm period), Aeromonas abundance decreased in the effluent but showed an increasing fluctuating trend in the marine waters with a maximum in late summer/early autumn when the temperatures were around 22-23 degrees C. Multiple correlation and regression analyses suggest, by the coefficient of determination (R(2)), that 42% of variance in Aeromonas number changes in the treated effluent, may be explained by only turbidity, radiation and Aeromonas density in the previous sample, while 37% of variance in marine ecosystem were explained by radiance and conductivity. Furthermore, the t statistics and their p values and the coefficient of partial determination (r(2)) indicated that radiance contributed the most (r(2)=0.3184, t=-3.2, p=0.0041) to the dynamics of motile Aeromonas in seawater, when combined with conductivity.The models relevant for changes in faecal coliforms abundance incorporated turbidity, radiance in the effluent and conductivity, pH, radiance, turbidity in coastal marine environment. These models explain 66% and 73% of the observed cell number fluctuation, with turbidity (r(2)=0.529, t=5.08, p=0.0001) and conductivity (r(2)=0.5407, t=4.97, p=0.0001) as dominant factors in the multivariate model proposed, respectively, for the two sampling sites. The results presented here suggest that the combination of negative effects of sunlight and conductivity in natural seawater mainly affects the colony-forming capacity and make the motile Aeromonas nonrecoverable during cold months.     

Comparison of six different sewage treatment processes—Reduction of estrogenic substances and effects on gene expression in exposed male fish

Treated sewage effluents often contain a mixture of estrogenic compounds in low concentrations. The total combined activity of these, however, may be sufficiently high to affect the reproduction of aquatic vertebrates. The introduction of advanced treatment technologies has been suggested as a way to remove micro-contaminants, including estrogenic substances. In this study, one municipal influent was treated with six different processes in parallel on a semi-large scale in order to assess their potential to reduce substances that could contribute to estrogenic effects in male fish. The effluent from a conventional, activated sludge treatment line was compared to a similarly treated effluent with a final sand-filtering step. The addition of ozonation (15 g O₃/m³), a moving bed biofilm reactor (MBBR) or both in combination was also evaluated. There was also a separate treatment line that was based on a membrane bioreactor. A small battery of hepatic estrogen-responsive genes was measured in the exposed fish using quantitative PCR. Concentrations of steroid estrogens and estrogenic phenols in the effluents were measured by GC-ECNI-MS. The ozonated effluents were the only tested effluents for which all measured biological effects in exposed fish were removed. Chemical data suggested that the MBBR technology was equally effective in removing the analyzed estrogens; however, elevated expression of estrogen-responsive genes suggested that some estrogenic substances were still present in the effluent. The membrane bioreactor removed most of the measured estrogens and it reduced the induction of the estrogen-responsive genes. However, fish exposed to this effluent had significantly enlarged livers. Given that the same influent was treated in parallel with a broad set of technologies and that the chemical analyses were combined with an in vivo assessment of estrogenic responses, this study provides valuable input into the assessment of advanced treatment processes for removing estrogenic substances.     

A multi-beach study of Staphylococcus aureus, MRSA, and enterococci in seawater and beach sand

Incidences of Staphylococcus aureus and methicillin resistant S. aureus (MRSA) have risen worldwide prompting a need to better understand routes of human exposure and whether standard bacterial water quality monitoring practices adequately account for this potential threat. Beach water and sand samples were analyzed during summer months for S. aureus, enterococci, and MRSA at three southern California beaches (Avalon, Doheny, Malibu Surfrider). S. aureus frequently was detected in samples of seawater (59%, n = 328) and beach sand (53%, n = 358). MRSA sometimes was detected in seawater (1.6%, n = 366) and sand (2.7%, n = 366) at relatively low concentrations. Site specific differences were observed, with Avalon Beach presenting the highest concentrations of S. aureus and Malibu Surfrider the lowest in both seawater and sand. S. aureus concentrations in seawater and sand were correlated to each other and to a variety of other parameters. Multiple linear regression on the combined beach data indicated that significant explanatory variables for S. aureus in seawater were S. aureus in sand, water temperature, enterococci in seawater, and the number of swimmers. In sand, S. aureus concentrations were related to S. aureus in seawater, water temperature, enterococci in seawater, and inversely to surf height classification. Only the correlation to water temperature held for individually analyzed beaches and for S. aureus concentrations in both seawater and sand. To provide context for these results, the prevalence of S. aureus in sand was compared to published fomite studies, and results suggested that beach prevalence was similar to that in homes.     

Detection of Legionella spp. and some of their amoeba hosts in floating biofilms from anthropogenic and natural aquatic environments

Floating biofilms develop at the water-air interface and harbor numerous microorganisms, some of which are human pathogens like Legionella pneumophila. The presence of Legionella spp. and especially L. pneumophila in such biofilms was investigated. In parallel, the occurrence of Naegleria spp., Acanthamoeba spp., Willaertia spp., Vahlkampfia spp. and Hartmanella spp. was determined and it was examined whether Acanthamoeba spp. isolates were naturally infected with L. pneumophila bacteria. Eight anthropogenic and 37 natural aquatic environments were sampled between June and August 2005. Both Legionella spp. and L. pneumophila were present in 100% of the floating biofilms of the anthropogenic aquatic systems. Eighty-one percent of all natural floating biofilm samples were positive for Legionella spp. and 70% of these samples were positive for L. pneumophila. Legionella concentrations were in the range of 10(1)-10(2)cells/cm(2). Naegleria spp. and Acanthamoeba spp., two well-known L. pneumophila amoeba hosts, were present in 50-92% and 67-72% of floating biofilm samples, respectively. Acanthamoeba spp. isolates appeared to be naturally infected with L. pneumophila bacteria as proved by fluorescent in situ hybridization.     

Sublethal toxicity and biotransformation of pyrene in Lumbriculus variegatus (Oligochaeta)

The aim of this work was to study the toxicity and biotransformation of polyaromatic hydrocarbon (PAH) pyrene in the oligochaete aquatic worm, Lumbriculus variegatus. PAHs are ubiquitous environmental pollutants that pose a hazard to aquatic organisms, and metabolizing capability is poorly known in the case of many invertebrate species. To study the toxicity and biotransformation of pyrene, the worm was exposed for 15 days to various concentrations of water-borne pyrene. The dorsal blood vessel pulse rate was used as a sublethal endpoint. Pyrene biotransformation by L. variegatus was studied and the critical body residues (CBR) were estimated for pyrene toxicity. The toxicokinetics of pyrene uptake was evaluated. A combination of radiolabeled (¹⁴C) and nonlabeled pyrene was used in the exposures, and liquid scintillation counting (LSC) and high-pressure liquid chromatography were employed in both water and tissue residue analyses. The results showed that L. variegatus was moderately able to metabolize pyrene to 1-hydroxypyrene (1-HP), thus demonstrating that the phase-I-like oxidizing enzyme system metabolizes pyrene in L. variegatus. The amount of the 1-HP was 1-2% of the amount of pyrene in the worm tissues. The exposure to pyrene reduced the blood vessel pulse rate significantly (p < 0.05), showing that pyrene had a narcotic effect. The estimated CBRs remained constant during the exposure time, varying from 0.120 to 0.174 mmol pyrene/kg worm wet weight. The bioconcentration factors (BCF) decreased as exposure concentration increased. It was suggested that the increased toxicity of pyrene accounted for the decrease in BCFs by lowering the activity of the organism.     

Effect of periodic feeding in sequencing batch reactor on substrate uptake and storage rates by a pure culture of Amaricoccus kaplicensis

A pure culture of Amaricoccus kaplicensis was aerobically cultured at a long culture residence time (Theta(C)>12d), under periodic acetate feeding in a sequencing batch reactor (SBR). The cycle length and, correspondingly, the volumetric organic load rate (vOLR) were varied in the range 4-24h and 0.76-0.12gCODl(-1)d(-1), respectively. The transient response of the microorganism to the acetate spike was investigated throughout batch tests, as a function of SBR cycle length and vOLR. In all tested conditions, a rapid transient response was observed, mainly due to acetate storage in the form of polyhydroxybutyrate, since growth (production of active biomass) played a minor role. Apart from this general trend, the maximum rates under transient conditions increased as the cycle length increased from 4 to 24h. In the SBR, the longest cycle also caused a decrease in floc size and settleability as well as an increase in the observed yield. The observed effect of SBR operating conditions on the physiological state of cells and their related transient response may have great significance on the performance of full scale activated sludge processes.     

Toxicity of chlorpyrifos and TCP alone and in combination to Daphnia carinata: the influence of microbial degradation in natural water

The acute toxicity of chlorpyrifos and its principal metabolite 3,5,6-trichloropyridinol (TCP) alone and in combination to a cladoceran, Daphnia carinata, was studied in both cladoceran culture medium and natural water collected from a local suburban stream. TCP was found to be more toxic than its parent chemical chlorpyrifos to Daphnia survival in cladoceran culture medium. However, TCP in natural water was not toxic to D. carinata up to 2 microgL(-1). The LC(50) values for chlorpyrifos, TCP and chlorpyrifos+TCP were 0.24, 0.20 and 0.08 microgL(-1), respectively, in cladoceran culture medium. Although the parent chemicals and their degradation products co-exist in natural waters, the existing guidelines for water quality are based on individual chemicals. The results of this investigation suggest that chlorpyrifos and TCP can interact synergistically, additively or antagonistically, resulting in an increase or decrease in the overall toxicity of the mixture compared to individual compounds. The indigenous microorganisms in natural water could play a significant role in degradation of these compounds thereby influencing their toxicity in receiving waters. This study clearly suggests that the joint action of pesticides and their degradation products should be considered in the development of water quality guidelines. To our knowledge, this is the first study on the interactive effect of chlorpyrifos and TCP to a cladoceran and suggests that these two compounds are non-toxic when present together at concentrations up to 0.12 microgL(-1). However, these compounds together act additively at and above 0.5 microgL(-1) to fresh water invertebrates and therefore pollution with these compounds may adversely affect natural ecosystems.     

Persistence of microbial and chemical pig manure markers as compared to faecal indicator bacteria survival in freshwater and seawater microcosms

Natural seawater and freshwater microcosms inoculated with pig manure were set up to determine the persistence of pig faecal microbial and chemical markers in these two types of surface water. The concentrations of Lactobacillus amylovorus, the Bacteroidales Pig-2-Bac 16S rRNA genetic marker, five stanols and the evolution of two ratios of stanols, R₁ (coprostanol to the sum of coprostanol and 24-ethylcoprostanol) and R₂ (sitostanol to coprostanol) were analyzed during two months along with the concentration of Faecal Indicator Bacteria (FIB). Pig manure was inoculated to unfiltered water microcosms incubated aerobically at 18 °C in the dark. The faecal contamination load represented by the concentrations of culturable Escherichia coli and/or enterococci remained for two months in the freshwater and seawater microcosms water column. These concentrations followed a biphasic decay pattern with a 97% reduction of the initial amount during a first rapid phase (<6 days) and a remaining proportion undergoing a slower or null second decline. The L. amylovorus marker and five stanols persisted as long as the indicators in both treatments. The Pig-2-Bac marker persisted 20 and 27 days in seawater and freshwater, respectively. The ratios R₁ and R₂ were in the range specific to pig manure until day 6 in both types of water. These results indicate that Pig-2-Bac, L. amylovorus and stanol ratios might be used in combination to complement FIB testing to determine the pig source of fecal pollution. However, stanol ratios are to be used when the time point of the discharge is known.     

Genotype diversity of Escherichia coli isolates in natural waters determined by PFGE and ERIC-PCR

Most library-dependent bacterial source tracking studies using Escherichia coli (E. coli) have focused on strain diversity of isolates obtained from known human and animal faecal sources for library development. In contrast, this study evaluated the genotype variation of E. coli isolated from natural surface water using pulsed field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) to better understand these naturally occurring populations. A total of 650 water samples were collected over a nine month period from eleven sampling stations from Lake Waco and Belton Lake in Central Texas. Of the 650 water samples collected, 412 were positive for E. coli, yielding a total of 631 E. coli isolates (1-12 isolates collected per sample). PFGE and ERIC-PCR patterns were successfully generated for 555 isolates and were compared using the curve-based Pearson's product-moment correlation coefficient. The 555 E. coli isolates represented 461 PFGE genotypes, with 84% (386/461) of the genotypes being represented by individual isolates. The remaining 75 genotypes were represented by 2-5 isolates each. Using ERIC-PCR, the 555 E. coli isolates represented 175 genotypes, with 63% (109/175) of the genotypes being represented by individual isolates. In contrast to the PFGE results, two ERIC-PCR genotypes represented 37% of the E. coli isolates, (83 and 124 isolates, respectively), and were found throughout the watersheds both spatially and temporally. Based on the PFGE genotype diversity of water isolates, there is little evidence that a small number of environmentally-adapted E. coli represent dominant populations in the studied waterbodies. However, with the lower discriminatory power technique ERIC-PCR, an opposing conclusion might have been drawn. These results emphasize the importance of considering the resolving power of the source tracking technique being used when assessing strain diversity and geographical stability.     

Elucidation and short-term forecasting of microcystin concentrations in Lake Suwa (Japan) by means of artificial neural networks and evolutionary algorithms

Non-supervised artificial neural networks (ANN) and hybrid evolutionary algorithms (EA) were applied to analyse and model 12 years of limnological time-series data of the shallow hypertrophic Lake Suwa in Japan. The results have improved understanding of relationships between changing microcystin concentrations, Microcystis species abundances and annual rainfall intensity. The data analysis by non-supervised ANN revealed that total Microcystis abundance and extra-cellular microcystin concentrations in typical dry years are much higher than those in typical wet years. It also showed that high microcystin concentrations in dry years coincided with the dominance of the toxic Microcystis viridis whilst in typical wet years non-toxic Microcystis ichthyoblabe were dominant. Hybrid EA were used to discover rule sets to explain and forecast the occurrence of high microcystin concentrations in relation to water quality and climate conditions. The results facilitated early warning by 3-days-ahead forecasting of microcystin concentrations based on limnological and meteorological input data, achieving an r(2)=0.74 for testing.     

Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis

Cell densities of the fecal pollution indicator genus, Enterococcus, were determined by a rapid (3 h or less) quantitative polymerase chain reaction (QPCR) analysis method in 100 ml water samples collected from recreational beaches on Lake Michigan and Lake Erie during the summer of 2003. Measurements by this method were compared with counts of Enterococcus colony-forming units (CFU) determined by Method 1600 membrane filter (MF) analysis using mEI agar. The QPCR method had an estimated 95% confidence, minimum detection limit of 27 Enterococcus cells per sample in analyses of undiluted DNA extracts and quantitative analyses of multiple lake water samples, spiked with known numbers of these organisms, gave geometric mean results that were highly consistent with the spike levels. At both beaches, the geometric means of ambient Enterococcus concentrations in water samples, determined from multiple collection points during each sampling visit, showed approximately lognormal distributions over the study period using both QPCR and MF analyses. These geometric means ranged from 10 to 8548 cells by QPCR analysis and 1-2499 CFU by MF culture analysis in Lake Michigan (N=56) and from 8 to 8695 cells by QPCR and 3-1941 CFU by MF culture in Lake Erie (N=47). Regression analysis of these results showed a significant positive correlation between the two methods with an overall correlation coefficient (r) of 0.68.