David R. Lapen

Seasonal relationships among indicator bacteria, pathogenic bacteria, Cryptosporidium oocysts, Giardia cysts, and hydrological indices for surface waters within an agricultural landscape

The South Nation River basin in eastern Ontario, Canada is characterized by mixed agriculture. Over 1600 water samples were collected on a bi-weekly basis from up to 24 discrete sampling sites on river tributaries of varying stream order within the river basin between 2004 and 2006. Water samples were analyzed for: densities of indicator bacteria (Escherichia coli, Clostridium perfringens, enterococci, total and fecal coliforms), the presence of pathogenic bacteria (Listeria monocytogenes, E. coli O157:H7, Salmonella spp., Campylobacter spp.), and densities of parasite Giardia cysts and Cryptosporidium oocysts. Relationships between indicator bacteria, pathogens, and parasite oocysts/cysts were overall weak, seasonally dependent, site specific, but primarily positive. However, L. monocytogenes was inversely related with indicator bacteria densities. Campylobacter, Salmonella, Giardia cysts and Cryptosporidium oocysts were most frequently detected in the fall. E. coli O157:H7 was detected at a very low frequency. Exploratory decision tree analyses found overall that E. coli densities were the most utilitarian classifiers of parasite/pathogen presence and absence, followed closely by fecal coliforms, and to a lesser extent enterococci and total coliforms. Indicator bacteria densities that classified pathogen presence and absence groupings, were all below 100 CFU per 100 mL(-1). Microorganism relationships with rainfall indices and tributary discharge variables were globally weak to modest, and generally inconsistent among season, site and microorganism. But, overall rainfall and discharge were primarily positively associated with indicator bacteria densities and pathogen detection. Instances where a pathogen was detected in the absence of a detectable bacterial indicator were extremely infrequent; thus, the fecal indicators were conservative surrogates for a variety of pathogenic microorganisms in this agricultural setting. The results from this study indicate that no one indicator or simple hydrological index is entirely suitable for all environmental systems and pathogens/parasites, even within a common geographic setting. These results place more firmly into context that robust prediction and/or indicator utility will require a more firm understanding of microorganism distribution in the landscape, the nature of host sources, and transport/environmental fate affinities among pathogens and indicators.

Runoff of pharmaceuticals and personal care products following application of biosolids to an agricultural field

Municipal biosolids are a source of nutrients for crop production. Beneficial Management Practices (BMPs) can be used to minimize the risk of contamination of adjacent water resources with chemical or microbial agents that are of public or environmental health concern. In this field study, we applied biosolids slurry at a commercial rate using either subsurface injection or broadcast application followed by incorporation. Precipitation was simulated at 1, 3, 7, 22, 36 and 266 days post-application on 2 m(2) microplots to evaluate surface runoff of 9 model pharmaceuticals and personal care products (PPCPs), atenolol, carbamazepine, cotinine, gemfibrozil, naproxen, ibuprofen, acetaminophen, sulfamethoxazole and triclosan. In runoff from the injected plots, concentrations of the model PPCPs were generally below the limits of quantitation. In contrast, in the broadcast application treatment, the concentrations of atenolol, carbamazepine, cotinine, gemfibrozil, naproxen, sulfamethoxazole and triclosan on the day following application ranged from 70-1477 ng L(-1) in runoff and generally declined thereafter with first order kinetics. The total mass of PPCPs mobilized in surface runoff per m(2) of the field ranged from 0.63 microg for atenolol to 21.1 microg for ibuprofen. For ibuprofen and acetaminophen, concentrations in runoff first decreased and then increased, suggesting that these drugs were initially chemically or physically sequestered in the biosolids slurry, and subsequently released in the soil. Carbamazepine and triclosan were detected at low concentrations in a runoff event 266 days after broadcast application. Overall, this study showed that injection of biosolids slurry below the soil surface could effectively eliminate surface runoff of PPCPs.

Pharmaceutical and personal care products in groundwater, subsurface drainage, soil, and wheat grain, following a high single application of municipal biosolids to a field

Dewatered municipal biosolids (DMBs) were applied to a field at a rate of ~22 Mg dw ha(-1) in October 2008. Pharmaceuticals and personal care products (PPCPs) were monitored in groundwater, tile drainage, soil, DMB aggregates incorporated into the soil post-land application, and in the grain of wheat grown on the field for a period of ~1 year following application. Over 80 PPCPs were analyzed in the source DMB. PPCPs selected for in-depth monitoring included: antibiotics (tetracyclines, fluoroquinolones), bacteriocides (triclosan, triclocarban), beta-blockers (atenolol, propranolol, metaprolol), antidepressants (fluoxetine, citalopram, venlafaxine, sertraline), antifungals (miconazole), analgesics (acetaminophen, ibuprofen) and anticonvulsants (carbamazepine). PPCPs in tile were observed twice, ~3 weeks and 2 months post-application. Of all PPCPs measured in tile drainage, only carbamazepine, ibuprofen, acetaminophen, triclosan, triclocarban, venlafaxine, and citalopram were detected (5-74 ng L(-1)). PPCPs were not detected in groundwater >2 m depth below the soil surface, and concentrations above detection limits at 2 m depth were only observed once just after the first rain event post-application. In groundwater, all compounds found in tile, except carbamazepine, acetaminophen and citalopram, were detected (10-19 ng L(-1)). PPCPs were detected in DMB aggregates incorporated in soil up to 1 year post-application, with miconazole and fluoxetine having the lowest percent reductions over 1 year (~50%). For several compounds in these aggregates, concentration declines were of exponential decay form. No PPCPs were detected in the grain of wheat planted post-application on the field. No PPCPs were ever detected in water, soil or grain samples from the reference plot, where no DMB was applied.

Pharmaceutical and personal care products in tile drainage following land application of municipal biosolids.

Land application of municipal biosolids (sewage) is a common farming practice in many parts of the world. There is potential for transport of pharmaceuticals and personal care products (PPCPs) from agricultural fields to adjacent surface waters via tile drainage systems. In this study, liquid municipal biosolids (LMB) (total solids=11,933 mg L(-1)), supplemented with selected PPCPs and the fluorescent dye tracer rhodamine WT (RWT), were applied to tile drained fields using two land application approaches. Objectives included evaluating the relative benefits of land application practices with respect to reducing PPCP loadings to tile drains, evaluating PPCP persistence in tile water, and determining whether rhodamine WT can be used to estimate PPCP mass loads in tile. The PPCPs examined included an antibacterial agent used in personal care products (triclosan), a metabolite of nicotine (cotinine), and a variety of drugs including two sulfonamide antimicrobials (sulfapyridine, sulfamethoxazole), a beta-blocker (atenolol), an anti-epileptic (carbamazepine), an antidepressant (fluoxetine), analgesic/anti-inflammatories (acetaminophen, naproxen, ibuprofen), and a lipid-regulator (gemfibrozil). Maximum observed PPCP concentrations in the spiked LMB were about 10(3) ng g(-1) dry weight. PPCPs were shown to move rapidly via soil macropores to tile drains within minutes of the land application. Maximum observed PPCP concentrations in tile effluent associated with the LMB application-induced tile flow event were approximately 10(1) to 10(3) ng L(-1). PPCP mass loads, for the application-induced tile-hydrograph event, were significantly (p<0.1) higher for surface spreading over non-tilled soil (incorporation tillage occurring 20 h post-application), relative to aerating soil immediately prior to surface spreading using an AerWay slurry deposition system. PPCP concentrations that were detected above the limit of quantitation (LOQ) in tile water during several precipitation-induced tile flow events that occurred post-application, included: triclosan (max. approximately 1.5 x 10(2) ng L(-1)), carbamazepine (max. approximately 7 x 10(1) ng L(-1)), atenolol (max approximately 4 x 10(1) ng L(-1)), and cotinine (max approximately 2 x 10(1) ng L(-1)). In spite of their presence in biosolids, the other PPCPs were not observed above LOQ concentrations during these events. PPCP concentrations were predicted from RWT concentrations over a 40 day study period. Tile mass loads as a percent of PPCP mass applied to soil ranged from 4.2%+/-SD of 9.2% to 7.1%+/-10.9% for the AerWay system and surface spreading plus incorporation treatments, respectively.

Uptake of pharmaceuticals, hormones and parabens into vegetables grown in soil fertilized with municipal biosolids

Several recent greenhouse studies have established the potential for uptake of human pharmaceuticals from soil fertilized with municipal biosolids into a variety of crops. In the present study, a field experiment was undertaken to evaluate the uptake of organic micropollutants from soil fertilized with municipal biosolids at a regulated application rate into tomatoes, carrots, potatoes and sweet corn produced under normal farming conditions. The vegetables were grown according to farming practices mandated by the province of Ontario Canada, the key feature being a one-year offset between biosolid application and the harvest of crops for human consumption. Biosolids at application, and crop samples following harvest were analyzed for 118 pharmaceuticals and transformation products, 17 hormones or hormone transformation products, and 6 parabens. Analyte concentrations in the biosolids were consistent with those detected in other surveys. Eight of the 141 analytes were detected in one or two crop replicates at concentrations ranging from 0.33 to 6.25 ng/g dry weight, but no analytes were consistently detected above the detection limit in all triplicate treated plots. Overall, this study suggests that the potential for micropollutant uptake into crops under normal farming conditions is low.

Associations among pathogenic bacteria, parasites, and environmental and land use factors in multiple mixed-use watersheds

Over a five year period (2004-08), 1171 surface water samples were collected from up to 24 sampling locations representing a wide range of stream orders, in a river basin in eastern Ontario, Canada. Water was analyzed for Cryptosporidium oocysts and Giardia cyst densities, the presence of Salmonella enterica subspecies enterica, Campylobacter spp., Listeria monocytogenes, and Escherichia coli O157:H7. The study objective was to explore associations among pathogen densities/occurrence and objectively defined land use, weather, hydrologic, and water quality variables using CART (Classification and Regression Tree) and binary logistical regression techniques. E. coli O157:H7 detections were infrequent, but detections were related to upstream livestock pasture density; 20% of the detections were located where cattle have access to the watercourses. The ratio of detections:non-detections for Campylobacter spp. was relatively higher (>1) when mean air temperatures were 6% below mean study period temperature values (relatively cooler periods). Cooler water temperatures, which can promote bacteria survival and represent times when land applications of manure typically occur (spring and fall), may have promoted increased frequency of Campylobacter spp. Fifty-nine percent of all Salmonella spp. detections occurred when river discharge on a branch of the river system of Shreve stream order = 9550 was >83 percentile. Hydrological events that promote off farm/off field/in stream transport must manifest themselves in order for detection of Salmonella spp. to occur in surface water in this region. Fifty seven percent of L. monocytogenes detections occurred in spring, relative to other seasons. It was speculated that a combination of winter livestock housing, silage feeding during winter, and spring application of manure that accrued during winter, contributed to elevated occurrences of this pathogen in spring. Cryptosporidium and Giardia oocyst and cyst densities were, overall, positively associated with surface water discharge, and negatively associated with air/water temperature during spring-summer-fall. Yet, some of the highest Cryptosporidium oocyst densities were associated with low discharge conditions on smaller order streams, suggesting wildlife as a contributing fecal source. Fifty six percent of all detections of ≥ 2 bacteria pathogens (including Campylobacter spp., Salmonella spp., and E. coli O157:H7) in water was associated with lower water temperatures (<∼ 14 °C; primarily spring and fall) and when total rainfall the week prior to sampling was >∼ 27 mm (62 percentile). During higher water temperatures (>∼ 14 °C), a higher amount of weekly rainfall was necessary to promote detection of ≥ 2 pathogens (primarily summer; weekly rainfall ∼>42 mm (>77 percentile); 15% of all ≥ 2 detections). Less rainfall may have been necessary to mobilize pathogens from adjacent land, and/or in stream sediments, during cooler water conditions; as these are times when manures are applied to fields in the area, and soil water contents and water table depths are relatively higher. Season, stream order, turbidity, mean daily temperature, surface water discharge, cropland coverage, and nearest upstream distance to a barn and pasture were variables that were relatively strong and recurrent with regard to discriminating pathogen presence and absence, and parasite densities in surface water in the region.

Distribution and Characteristics of Listeria monocytogenes Isolates from Surface Waters of the South Nation River Watershed, Ontario, Canada

ABSTRACT Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA, and hlyA. Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes, but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 ± 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.

Tracking Host Sources of Cryptosporidium spp. in Raw Water for Improved Health Risk Assessment

ABSTRACT Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.

Pharmaceutical and personal care products in tile drainage following surface spreading and injection of dewatered municipal biosolids to an agricultural field

Land application of municipal biosolids can be a source of environmental contamination by pharmaceutical and personal care products (PPCPs). This study examined PPCP concentrations/temporally discrete mass loads in agricultural tile drainage systems where two applications of biosolids had previously taken place. The field plots received liquid municipal biosolids (LMB) in the fall of 2005 at an application rate of approximately 93,500 L ha (-1), and a second land application was conducted using dewatered municipal biosolids (DMB) applied at a rate of approximately 8Mg dw ha (-1) in the summer of 2006 [corrected].The DMB land application treatments consisted of direct injection (DI) of the DMB beneath the soil surface at a nominal depth of approximately 0.11 m, and surface spreading (SS) plus subsequent tillage incorporation of DMB in the topsoil (approximately 0.10 m depth). The PPCPs examined included eight pharmaceuticals (acetaminophen, fluoxetine, ibuprofen, gemfibrozil, naproxen, carbamazepine, atenolol, sulfamethoxazole), the nicotine metabolite cotinine, and two antibacterial personal care products triclosan and triclocarban. Residues of naproxen, cotinine, atenolol and triclosan originating from the fall 2005 LMB application were detected in tile water nearly nine months after application (triclocarban was not measured in 2005). There were no significant differences (p>0.05) in PPCP mass loads among the two DMB land application treatments (i.e., SS vs. DI); although, average PPCP mass loads late in the study season (>100 days after application) were consistently higher for the DI treatment relative to the SS treatment. While the concentration of triclosan (approximately 14,000 ng g(-1) dw) in DMB was about twice that of triclocarban (approximately 8000 ng g(-1) dw), the average tile water concentrations for triclosan were much higher (43+/-5 ng L(-1)) than they were for triclocarban (0.73+/-0.14 ng L(-1)). Triclosan concentrations (maximum observed in 2006 approximately 235 ng L(-1)) in tile water resulting from land applications may warrant attention from a toxicological perspective.

Runoff of pharmaceuticals and personal care products following application of dewatered municipal biosolids to an agricultural field.

Municipal biosolids are a useful source of nutrients for crop production, and commonly used in agriculture. In this field study, we applied dewatered municipal biosolids at a commercial rate using broadcast application followed by incorporation. Precipitation was simulated at 1, 3, 7, 21 and 34 days following the application on 2 m(2) microplots to evaluate surface runoff of various pharmaceuticals and personal care products (PPCPs), namely atenolol, carbamazepine, cotinine, caffeine, gemfibrozil, naproxen, ibuprofen, acetaminophen, sulfamethoxazole, triclosan and triclocarban. There was little temporal coherence in the detection of PPCPs in runoff, various compounds being detected maximally on days 1, 3, 7 or 36. Maximum concentrations in runoff ranged from below detection limit (gemfibrozil) to 109.7 ng L(-1) (triclosan). Expressing the total mass exported as a percentage of that applied, some analytes revealed little transport potential (<1% exported; triclocarban, triclosan, sulfamethoxazole, ibuprofen, naproxen and gemfibrozil) whereas others were readily exported (>1% exported; acetaminophen, carbamazepine, caffeine, cotinine, atenolol). Those compounds with little transport potential had log K(ow) values of 3.18 or greater, whereas those that were readily mobilized had K(ow) values of 2.45 or less. Maximal concentrations of all analytes were below toxic concentrations using a variety of endpoints available in the literature. In summary, this study has quantified the transport potential in surface runoff of PPCPs from land receiving biosolids, identified that log K(ow) may be a determinant of runoff transport potential of these analytes, and found maximal concentrations of all chemicals tested to be below toxic concentrations using a variety of endpoints.