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Microbial water quality and influences of fecal accumulation from a dog exercise area.


Waterborne disease outbreaks have been associated with dispersed sources of animal wastes, such as contamination from animal agriculture (Ferguson, Husman, Altavilla, Deere, & Ashbolt, 2003). Pathogens found in animal wastes may infect people following fecal contamination of drinking or recreational waters (Centers for Disease Control and Prevention [CDC], 1998). Dogs and other companion animals are a potential source of waterborne pathogens from feces (Maepherson, 2005). Canine-borne zoonoses include fecal salmonellosis (S. typhimurium), mycobacteria (M. bovis, M. tuburculosis), and protozoa including Giardia spp., Cryptosporidium parvum. Toxo-plasma gondii, and Leptospira (L. hardjo, L. ictero-haemorrhagiae) (Macpherson, 2005; Owen, 2005).

Zoonotic pathogens may affect human health, especially if they have environmentally resistant infective stages (Nithiuthai, Anantaphruti, Waikagul, & Gajadhar, 2004). These pathogens may be abundant in areas that have significant accumulations of fecal matter from infected animals. For example, approximately 36% of dogs in the United States are infected with helminths capable of causing human disease through contact with or ingestion of contaminated soils (CDC, 1995). Human enteric infections acquired from pets living in developed, urban communities are common (Croese, Loukas, Opdebeeck, Fairley, & Prociv, 1994). City parks and sidewalks used for dog exercise have been shown to have high accumulations of dog feces when owners do not collect and dispose of feces (Bonner & Agnew, 1983).

Frequently used dog parks with significant fecal loading could contaminate surface waters. Alta, Utah, cited this as a primary reason to limit the total number of dogs within town boundaries by restricting the number of licenses available (Foy, 2006). In 2004, the number of dog licenses in the California counties of Placer and Nevada alone led to an estimate of over 15,000 licenses in the Lake Tahoe basin watershed (Cobourn & Segale, 2004). Although some water quality monitoring efforts in the Lake Tahoe Basin have shown fecal coliform results ranging from 0 to 25,000 colony-forming units (CFU) per 100 ml (Tahoe Regional Planning Agency, 2007), little research has examined the sources or transport of these bacteria. This may be an important concern, especially in heavily used areas where residents and visitors exercise pets. The correlation between this type of fecal loading and microbial water quality, however, has not been explored.

This investigation examined fecal loading in a popular dog exercise area adjacent to a tributary (Burke Creek) to Lake Tahoe, Nevada (Figure 1). The intake for a public drinking water supply was approximately 200 meters offshore from the creek outlet. E. coli is a common fecal contamination indicator in water studies and monitoring efforts (Edberg, Rice, Karlin, & Allen, 2000) and was chosen as the microbial indicator for this study. Based on a statistically sufficient number of samples (not less than five samples equally spaced over a 30-day period), standards established by the U.S. Environmental Protection Agency (U.S. EPA) state that the geometric mean of E. coli should not exceed 126 CFU per 100 ml and no single sample should exceed 75% of a one-sided confidence limit if water is used for contact recreation (Emerson, 2003).



The study consisted of site mapping with repeated fecal matter collection and water sampling. The goal of the study was to characterize spatial and seasonal trends in, as well as any possible correlation between, fecal accumulation and E. coli in surface waters.

Site Description

The study area was within the U.S. Forest Service--managed Burke Creek Recreational Area (BCRA), located on the southeast side of Lake Tahoe (Figure 2). The BCRA lacked dog waste collection and disposal facilities and was heavily used by dog owners. A network of trails connected a parking lot and residential area to the lake through the BCRA. Burke Creek flows through the BCRA after descending through private and public lands from its origin near the Heavenly Mountain ski resort. At the study site, 0.75 miles (1.3 km) east of Lake Tahoe's south shore, the flow of Burke Creek was approximately 0.1 f[t.sup.3]/min (4x[10.sup.-3] [m.sup.3]/min). The creek flows through a 1 acre (0.4 hectare) constructed sedimentation pond and along a course with nearly a mile of meadow and riparian wetlands before reaching the lake. The predominant soils are alluvial tills, including loamy coarse sands with some gravel (Hanes, 1974). Plant cover included species common to arid alpine environments, such as cheatgrass, sagebrush, and rabbitbrush.


The boundaries for the study site were based upon the trail systems and topography. The bounded area was a portion of the lower watershed for Burke Creek, extending from Route 50 to approximately 1310 feet (400 m) downstream (Figure 2). Route 50 served as the upstream limit of the study area and the downstream-most water sampling site was below a wetland at a pedestrian bridge and stream crossing.

Fecal Accumulation and E. coli Burden Characterization

Fifteen circular plots of 7 ft (2.1 m) radius were established to estimate distribution of dog feces over the 8.8 acre (3.6 hectare) study site (Figure 2). All feces within the plots were collected semimonthly for 14 consecutive months. The plots were sited adjacent to heavily used trails and in unused portions of the study area. Plot locations were recorded with a Trimble Explorer 3 and corrected using a local geopositioning reference station.

Prior to establishing sampling plots we noted that fecal accumulation was localized, with more accumulation of fecal matter in several small areas than across the site as a whole, with some accumulation between such points. To capture this variability, we sited plots in close proximity, with a maximum distance of approximately 500 feet (152 m) between plots and the majority within 80 feet (24 m) of adjacent plots. The sampling network included plots in which no feces accumulated consistently through the study period, to serve as a boundary of zero accumulation for extrapolation from those plots where accumulation was consistently observed.

Feces were collected in tared paper bags and desiccated at 106[degrees]C for 24 hours prior to weighing. The dry mass divided by the total plot area and the time between collection events gave estimates of accumulation rates (mass dry matter. are[a.sup.-1]. tim[e.sup.-1]) for each plot. We used accumulation rates per plot to estimate total fecal accumulation rates in the BCRA using ESRI's 3D Analyst's Inverse Distance Weighting (IDW) function, with three nearest neighbors. The inverse distance weighting technique diminishes the influence of observations that are farther from a point of interest relative to those that are closer. This is accomplished by weighting each observation used to estimate a distribution between plots by the inverse of the distance between the plots' centroids, such that data from observations farther from a point of interest contribute less to estimates of accumulation than those that are in the immediate vicinity. The inverse distance weighting process is appropriate for use with quantities that are distributed unevenly in space, such as elevation at a landscape scale or vegetation types, especially if these occur in clumps or disconnected patches, as with the distribution of canine feces within this study area.

We characterized the E. coli burden in feces using fresh samples ([less than or equal to] 2 hours old) from dogs in Reno, Nevada. We also used these samples to determine average water content. We relied on these samples because those collected at the study site were of uncertain age and could have been exposed to environmental stresses for up to two weeks between collections. Characterization of the microbial burden in samples collected from the site would likely be biased low by losses from exposure to environmental stresses. We determined water content gravimetrically using fecal mass before and after desiccation at 106[degrees]C for 24 hours. We determined E. coli burden by adding small masses (approximately 0.1 mg) to 1 liter of sterile dilution water (Franson, 1998), of which varying sized aliquots (0.1-1 ml) were filtered through sterile 45 [micro]m filters and incubated using the same materials and procedures described below for water sampling.

Water Sampling Sites

We established water sampling sites to assess water quality upstream of, within, and downstream of the study area. The following sites on Burke Creek were tested for E. coli semimonthly for 14 months to provide water quality comparisons (Figure 2):

* "Below Highway" was at the upper border of the study area and provided information about influent water quality;

* "Inlet" reflected influences on creek water quality between Route 50 and the sedimentation basin;

* "Outlet" characterized water quality leaving the sedimentation basin;

* "Outlet & Creek 2" characterized dilution that may have occurred at the confluence of the outlet of the sedimentation pond and drainage from an unused area north of the study area; and

* "Below Bridge" was located below the study area, reflecting water quality leaving the study area, including effects of a crossing immediately upstream.

At each sampling, water was collected using standard method 9060A (Franson, 1998) in sterile plastic bottles. Samples were processed within eight hours of collection. Determinations of E. coli were made by membrane

filtration and cultured on CHROMagar ECC (Chromagar Ref. #EF322), according to manufacturer's instructions. Sample aliquots were diluted to 40 ml, as described in method 9050c (Franson, 1998), and passed through sterile 45 [micro]m filters (Fisher cat. #06-414-1A). Filters were incubated at 36[degrees]C for 24 hours, and colonies were reported as E. coli CFU/100 ml water. Duplicates comprised 10% of analyses. A 30% variance was considered acceptable for duplicate analytical results to be averaged and reported. Blank samples and positive controls were included with every sample set.

Data Analysis

We applied Minitab v. 14 for data analysis. For comparisons of water sampling results from "Below Highway" to "Below Bridge," representing the overall influence of the study area on microbial water quality, we applied linear regression. To evaluate the relationship between fecal accumulation and water sampling results at the lower most point on the study site we used linear regression, testing the hypothesis of no relationship between fecal accumulation, seasonal influences (using indicator variables and the definitions of seasons as blocks of months: summer [June-August], fall [September-October], winter [November-March], and spring [April-May]), and water sampling results. We also sought correlations between water sampling sites based on a full matrix comparison with Pearson's correlation coefficient, using [alpha] = .05 as the upper threshold for significant correlation. We evaluated temporal trends in fecal accumulation on the study site using one-way ANOVA, with seasons as factors, followed by an analysis of differences using Tukey's honest significant difference test (HSD) at [alpha] = .05.


Water Sampling Results

Results from fecal accumulation measurements and water sampling are shown in Figure 3. Regression analysis of E. coli levels in Burke Creek at several stations indicated correlation between the upstream-most and downstream-most sampling sites (BH and BB [Figure 4]), and no correlation with either fecal accumulation or season. The correlation (in units of CFU/100 ml) between upstream and downstream sampling sites was BB = 2.12 +0.273 BH (p = .034, [R.sup.2] = 20%).



Regression results with seasons (as defined above, represented by indicator variables) suggest that summer sampling results at BB were significantly higher than those observed during other seasons (p = .03), though not related to fecal accumulation rates (p = .125 for the regression relationship BB = f[[season.sub.i] fecal accumulation {kg}]).

The correlation matrix of sampling sites indicated significant results between the inlet to the study site (BH) and the outlet (BB, p = .011), though the correlation coefficient (0.48) indicated an overall reduction in CFU/100 ml at BB relative to BH, which corresponds with results of the regression analysis reported above. Similarly, the correlation coefficient between the inlet and outlet of the sedimentation pond (0.49) indicated reduction and was also significant (p = .008). Finally, samples taken from the confluence of Creek 2 and the outlet from the pond were significantly correlated, indicating reduced numbers of CFU/100 ml at BB relative to the confluence (p = .00+; correlation coefficient = 0.71).

Fecal Accumulation

Over the 14-month period, an estimated total of 100.1 lbs (45.5 kg) dry fecal mass accumulated in the study area. Fecal accumulation followed highly localized distribution patterns (see Figure 2 for an example, representing the accumulation within the BCRA in the two-week period prior to June 21, 2005). Total fecal accumulation in plots per two week period averaged 0.32 lb (147 g) dry mass, with a minimum of zero and maximum of 0.83 lb (377 g). A significant difference existed in fecal accumulations by season (p = .035), with accumulations during winter months (November-March, average 0.44 kg/mo.) significantly less than those estimated for any of the other seasons (spring = 1.66, summer = 2.25, fall = 2.8 kg/mo., respectively). Monthly accumulations in other seasons were not significantly different from each other, based on Tukey's honest significant difference test (HSD) at [alpha] = .05.

Fecal E. coli Burden

Based on results of 16 fecal analyses, canine feces contained an average of 47% water weight (standard deviation = 11%) and contained an average of 5x[10.sup.7] CFU/g (n = 20; range = 2x[10.sup.6] - 2x[10.sup.8] CFU/g) in fresh feces. The several orders of magnitude of variation can be attributed to the highly variable nature of dog food, digestive health, and variable diets.


We expected that downstream sites in the study area would have higher amounts of bacteria resulting from fecal accumulation in the study area. Correlations between "Below Highway" and "Below Bridge," however, showed an overall decrease in CFU/100 ml along the flow path from the point of inflow to the site to the point of outflow. This result may be due to the presence of a sedimentation pond and riparian wetlands. In-stream reservoirs and constructed wetlands have been shown to reduce bacterial contamination (Gannon et al., 2005), due to a complex interaction of physical, biological, and chemical factors (Vymazal, 2005). In fact, correlation coefficients between the inlet and outlet to the sedimentation pond and between the confluence of the outlet of the pond and Creek 2 and the below bridge sampling site reflect reductions in CFU/ml at each step (Inlet, Outlet: correlation coefficient 0.49, p = .008; Outlet:Creek 2, below bridge: correlation coefficient 0.71, p = .00+).

Over the 14-month period, an estimated total of 100.1 lbs (45.5 kg) dry fecal mass accumulated in the study area. Highly localized fecal loading patterns indicated that dogs concentrated wasted near access points and three main trail junctions. The results indicated that the majority of mass accumulated near a parking area and at the junctions of access trails from nearby residential areas. Fecal accumulation appeared to be controlled by the location of footpaths and their intersections, likely because of canine marking behavior.

Based on estimates of average water weight and the range of E. coli burden/g observed in fresh feces, (47% and 2 x [10.sup.6] -2 x [10.sup.8] CFU/g, respectively) we would expect to find ~2.x.10 [10.sup.11] - 2x[l0.sup.13] CFU E. coli in feces at the study site based on accumulations estimated over the course of 14 months, with an average loading rate of ~4 x [l0.sup.8] - 4x[10.sup.10] CFU/day. Given a flow rate of approximately 4 L/ min, and assuming 100% survival and 100% transport into the creek, we could expect a range of ~ 700--70,000 CFU E. coli/100 ml water. Samples from Burke Creek, however, remained low relative to U.S. EPA standard (126 CFU/100 ml, based on the geometricmean), with single samples at the BB site never exceeding the standard as opposed to samples from the BH site, which had single value samples that exceeded 126 CFU/100 ml in 4/29 samples.

The lack of correlation between fecal loading and bacterial CFU in water samples may be due to several factors that influence bacterial survival. Survival in feces is controlled by environmental factors including sunlight (Meays, Broersma, Nordin, & Mazumder, 2005), temperature (McFeters & Stuart, 2003), and moisture levels in the environment (Franson, 1998; Kudva, Blanch, & Hovde, 1998). Microbes may have been desiccated on soil surfaces before they could be transported into water by rainfall or snowmelt, which could explain the low CFU in stream samples despite fecal accumulation.

It is possible that E. coli were not present in accumulated feces, either when freshly deposited or after having been exposed to environmental stresses. The former seems unlikely, though we observed that E. coli CFU/g feces varied significantly between individual dogs and, in fact, within an individual dog through time (data not shown).

It is also possible that environmental stresses quickly and effectively reduced bacterial numbers in feces in this environment. The alpine climate of Lake Tahoe is arid, yet variable, with winter storms common from October through March and 250 to 300 sunny days per year. With very low relative humidity and ample sunshine, it is possible that feces desiccated quickly, leading to rapid losses of indicator organisms in the fecal matrix.


While fecal accumulation is an important aesthetic and potential public health problem, at this site no correlation appeared between accumulation and bacterial water quality. All samples taken from the critical sampling location of "Below Bridge," which reflected water quality closest to an intake for public drinking water, were below U.S. EPA standard of 126 CFU/100 ml.

Highly localized fecal loading patterns in the study area indicated that feces accumulated close to access gates and trail intersections. Canine marking behavior along trails and leash-walking habits of dog owners may contribute to this pattern, suggesting that the locations of access points and trails may determine where waste accumulates. This is consistent with findings that dog feces accumulations are partly controlled by owners' walking patterns (Bonner & Agnew, 1983).

In order to prevent pollution and minimize runoff, it would be beneficial to evaluate recreation sites for the location of the most appropriate toilet and collection areas. The placement of waste collection areas and facilities outside of the immediate creek watershed or upstream of features such as sedimentation ponds or wetlands that would sequester microorganisms and expose them to environmental stresses. Access points and trails could be manipulated to establish toilet areas where the influence on water quality is likely to be minimal and where exposure to environmental stresses is maximized. This "distribution tool" approach has been suggested as a way of managing livestock to minimize fecal contamination of water supplies (Tate, Atwill, McDougald, & George, 2003) and would be reasonable to apply to dog exercise areas.

The sediment basin and wetlands at the study site appear to have a beneficial effect on water quality. This is consistent with previous research indicating that wetlands remove bacteria from surface waters (Vymazal, 2005). Continued effectiveness of the basin, however, will depend upon maintenance. In this case, with a steep, mountainous water shed, basin maintenance, including periodic sediment removal, is critical to maintain adequate residence timeto allow contaminants to settle (Gannon et al., 2005) without increasing potential risk of contamination through flooding.

Zoonoses spread easily between infected animals, soils, uninfected animals, and human companions (Nithiuthai, Anantaphruti, Waikagul, & Gajadhar, 2004; Macpherson, 2005). It would therefore be beneficial to educate dog owners to promote voluntary waste pick up. Local veterinarians with an understanding of local canine zoonoses would make ideal partners in such an educational effort and could contribute to efforts to link domestic animal and public health, to limit the risk of zoonotic infections as a result of recreation in water.

It appears that during much of the year, especially periods of maximum accumulation when evaporative conditions are also highest (late spring, summer, and early fall), E. coli may not survive long enough to enter surface waters. This may explain the lack of correspondence between water sampling results and precipitation. Because the study site was in an alpine environment characterized by prolonged periods without precipitation, it is possible that E. coli were quickly lost from feces due to rapid changes in fecal water potential. To address this possibility, it would be relevant to evaluate bacterial degradation rates at different conditions and to relate evaporative conditions to changes in water potential within canine feces. This could be used to assess the risk of contamination based on environmental factors, which could be applied by land managers to evaluate seasonal needs for public outreach and education. With limited available resources to enforce litter laws and educate dog owners, this consideration may be useful for the protection of public health.

Acknowledgements: Many thanks to the USDA CSREES for financial support, the Tahoe Water Supplier's Association for site support, and Heidi Frantz and Emily Donaldson for technical support.

Corresponding Author: Mark Walker, Director, Department of Environmental Science, University of Nevada Reno, Department of Environmental Sciences, College of Agriculture, Biotechnology and Natural Resources, M/S 370, FA 132, Reno, NV 89557. E-mail:


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Lynell Garfield

Mark Walker, Ph.D.
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Title Annotation:FEATURES
Author:Garfield, Lynell; Walker, Mark
Publication:Journal of Environmental Health
Article Type:Report
Geographic Code:1USA
Date:Nov 1, 2008
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