17-ß estradiol and testosterone mineralization and incorporation into organic matter in broiler litter-amended soils.

The presence of the hormones estradiol and testosterone in the environment is of concern because they adversely affect vertebrate sexual characteristics. Land spreading broiler litter introduces these hormones into the environment. We conducted two studies. The first study determined the mineralization of C-labeled estradiol and testosterone at three water potentials and three temperatures in four broiler litter-amended soils. With a few exceptions, the mineralization of each hormone either stayed the same or increased with increasing water content (both hormones) and increasing (estradiol) or decreasing (testosterone) temperature. Mineralization was dependent on soil type. The second study determined the incorporation of C-labeled estradiol and testosterone into (i) three soil organic matter (SOM) fractions (fulvic acid, humic acid, and humin) at two water potentials, two temperatures, and one sampling time, and (ii) at one water potential, one temperature, and seven sampling times. As time increased, higher temperature and water potential decreased percentages of C estradiol and testosterone in water- and acetone-soluble fractions and increased percentages in SOM fractions. However, the distribution of the two hormones in SOM fractions differed. For estradiol, higher temperature and water potential increased the percentage in all three SOM fractions. For testosterone, higher temperature and water potential increased the percentage of hormone in fulvic acid and humin. Although the mineralization studies suggest the potential for these hormones to still have environmental effects, the incorporation of the two hormones into SOM suggest that land spreading these hormones may actually be less of an environmental concern.

17beta-Estradiol and testosterone in drainage and runoff from poultry litter applications to tilled and no-till crop land under irrigation.

Thirteen million [corrected] metric tons of poultry litter are produced annually by poultry producers in the U.S. Poultry litter contains the sex hormones estradiol and testosterone, endocrine disruptors that have been detected in surface waters. The objective of this study was to evaluate the potential impact of poultry litter applications on estradiol and testosterone concentrations in subsurface drainage and surface runoff in irrigated crop land under no-till and conventional-till management. We conducted an irrigation study in fall of 2001 and spring of 2002. Four treatments, no-till plus poultry litter, conventional-till plus poultry litter, no-till plus conventional fertilizer, and conventional-till plus conventional fertilizer, were evaluated. Flow-weighted concentration and load ha(-1) of the two hormones were measured in drainage and runoff. Soil concentrations of estradiol and testosterone were measured. Based on comparisons to the conventional fertilizer (and control) treatments, poultry litter did not add to the flow-weighted concentration or load ha(-1) of either estradiol or testosterone in subsurface drainage or surface runoff. Significant differences were, however, observed between tillage treatments: flow-weighted concentrations of estradiol were greater for no-till than conventional-till plots of the June irrigation; and runoff loads of both estradiol and testosterone were less from no-till than conventional-till plots for the November irrigation. Although the differences between no-till and conventional-tillage appeared to affect the hydrologic transport of both hormones, the differences appeared to have inconsequential environmental impact.

Combining targeted sampling and fluorometry to identify human fecal contamination in a freshwater creek.

Many bacterial source tracking (BST) methods are too expensive for most communities to afford. In an effort to develop an inexpensive method of detecting human sources of fecal contamination in a freshwater creek during baseflow and stormflow conditions, we combined targeted sampling with fluorometry. Targeted sampling is a prelude to BST and finds sources of fecal contamination by continued sampling and resampling over ever-decreasing distances. Fluorometry identifies human fecal contamination in water by detecting fluorescing compounds, optical brighteners, from laundry detergents. Potato Creek, a freshwater creek in Georgia (U.S.A.), had three reaches identified as containing high numbers of fecal bacteria, and these reaches were sampled by targeted sampling and fluorometry. Targeted sampling quickly and inexpensively identified humans, cattle, and dogs as the major sources of fecal contamination in the first, second, and third reaches, respectively. Fluorometric values were consistent with these identifications, but high fluorometric values were sometimes observed in areas with no fecal contamination. One likely cause of these false-positive signals was fluorescence from organic matter. For targeted sampling, the cost of each sample was $6, with a one-time equipment cost of $3,650; for fluorometry, the cost of each sample was negligible, with a one-time equipment cost of $14,250. This was the first study of this relatively inexpensive combination in freshwater during both baseflow and stormflow conditions.

Geographic sharing of ribotype patterns in Enterococcus faecalis for bacterial source tracking.

The limited host range of Enterococcus faecalis may reduce its clonal diversity and thereby increase its geographic sharing of ribotype patterns. Such sharing would be advantageous for bacterial source tracking (BST). We determined the geographic sharing of ribotype patterns in 752 Ent. faecalis isolates obtained primarily from wastewater treatment plants in Delaware (15 locations; 490 isolates), Georgia (2 locations; 48 isolates), Idaho (1 location; 118 isolates), New York (2 locations; 48 isolates), and Puerto Rico (2 locations; 48 isolates). Isolates were ribotyped with a RiboPrinter. When pooled across all locations and analyzed at a similarity index of 100% and a tolerance level of 1.00%, the 752 Ent. faecalis isolates yielded 652 different ribotypes, of which 429 (66%) were unshared. Even when the matching criterion was relaxed by decreasing the tolerance level from 1% to 10% or lowering the similarity cutoff from 100% to 90%, half or almost half of the ribotypes were unshared. A Mantel test of zero correlation showed no statistically significant correlation between ribotype patterns and geographic distance among the 32 samples (one location at one time) at either the 1.00% (P = 0.91) or 10.00% (P = 0.83) tolerance levels. Therefore, the percentage of ribotype patterns shared between two locations did not increase as the distance between locations decreased. In the case of BST, a permanent host origin database sufficiently large to encompass these ribotype patterns would be time-consuming and expensive to construct.

Exposing water samples to ultraviolet light improves fluorometry for detecting human fecal contamination.

Fluorometry identifies human fecal contamination by detecting optical brighteners in environmental waters. Because optical brighteners are sensitive to sunlight, we determined if we could improve fluorometry by exposing water samples to ultraviolet (UV) light to differentiate between optical brighteners and other fluorescing organic compounds. Optical brighteners were likely present when the relative percentage difference in fluorometric value of the water before and after UV light exposure was >30% (glass cuvettes, 30 min exposure) or >15% (polymethacrylate cuvettes, 5 min exposure). In a blind study, we correctly identified the presence or absence of optical brighteners in 178 of 180 (99%) of the samples tested with a more expensive field fluorometer and in 175 of 180 (97%) of the samples tested with a less expensive handheld fluorometer. In the field, the method correctly identified two negative and three positive locations for human fecal contamination. When combined with counts of fecal bacteria, the new fluorometric method may be a simple, quick, and easy way to identify human fecal contamination in environmental waters.

Identifying sources of fecal contamination inexpensively with targeted sampling and bacterial source tracking.

Most bacterial source tracking (BST) methods are too expensive for most communities to afford. We developed targeted sampling as a prelude to BST to reduce these costs. We combined targeted sampling with three inexpensive BST methods, Enterococcus speciation, detection of the esp gene, and fluorometry, to confirm the sources of fecal contamination to beaches on Georgia's Jekyll and Sea Islands during calm and stormy weather conditions. For Jekyll Island, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. For the Sea Island beach during calm conditions, the most likely sources of fecal contamination were leaking sewer lines and wildlife feces. The leaking sewer lines were confirmed with fluorometry and detection of the esp gene. For the Sea Island beach during stormflow conditions, the most likely sources of fecal contamination were wildlife feces and runoff discharging from two county-maintained pipes. For the pipes, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. Sediments were also a reservoir of fecal enterococci for both Jekyll and Sea Islands. Combining targeted sampling with two or more BST methods identified sources of fecal contamination quickly, easily, and inexpensively. This combination was the first time targeted sampling was conducted during stormy conditions, and the first time targeted sampling was combined with enterococcal speciation, detection of the esp gene, and fluorometry.

Mineralization of hormones in breeder and broiler litters at different water potentials and temperatures.

When poultry litter is landspread, steroidal hormones present in the litter may reach surface waters, where they may have undesirable biological effects. In a laboratory study, we determined the mineralization of [4-14C]-labeled 17beta-estradiol, estrone, and testosterone in breeder litter at three different water potentials (-56, -24, and -12 MPa) and temperatures (25, 35, and 45 degrees C), and in broiler litter at two different water potentials (-24 and -12 MPa) and temperatures (25 and 35 degrees C). Mineralization was similar in both litters and generally increased with increasing water content and decreasing temperature. After 23 wk at -24 MPa, an average of 27, 11, and <2% of the radiolabeled testosterone applied to breeder litter was mineralized to 14CO2 at 25, 35, and 45 degrees C, respectively. In contrast, mineralization of the radiolabeled estradiol and estrone was <2% after 25 wk at all water potentials, except after 17 wk at 25 degrees C and -12 MPa, where up to 5.9% of the estradiol and 7.8% of the estrone was mineralized. The minimal mineralization suggests that the litters may still be potential sources of hormones to surface and subsurface waters.

Presence of Enterococcus faecalis in broiler litter and wild bird feces for bacterial source tracking.

When Enterococcus faecalis is isolated from fresh feces, its host range appears to be limited to humans and birds. Although E. faecalis is found in human sewage, the extent to which the bacterium is found in broiler litter and in the feces of wild birds is unclear. These results have implications for bacterial source tracking. We determined if media designed for the isolation of fecal enterococci affected this host range, and if E. faecalis was routinely found in broiler litter and in the feces of wild birds. Of five different isolation media, none affected the isolation of E. faecalis. Enterococcus faecalis was routinely found in fresh broiler feces (522 of 1092 isolates; 48%), but rarely in broiler litter (12 of 1452 isolates; <2%). Therefore, broiler litter selects against this bacterium, and broiler litter is an unlikely environmental source of this bacterium. The presence of E. faecalis in eight wild bird species was highly variable. Unless the fecal loading rate from migratory or resident wild birds is high, water samples collected during baseflow conditions with high numbers of E. faecalis may indicate human fecal contamination.

Targeted sampling protocol as prelude to bacterial source tracking with Enterococcus faecalis.

Recent studies suggest that host origin databases for bacterial source tracking (BST) must contain a large number of isolates because bacterial subspecies change with geography and time. A new targeted sampling protocol was developed as a prelude to BST to minimize these changes. The research was conducted on the Sapelo River, a tidal river on the Georgia coast. A general sampling of the river showed fecal enterococcal numbers ranging from <10 (below the limit of detection) to 990 colony-forming units (CFU) per 100 mL. Locations with high enterococcal numbers were combined with local knowledge to determine targeted sampling sites. Fecal enterococcal numbers around one site ranged from <10 to 24,000 CFU per 100 mL. Bacterial source tracking was conducted to determine if a wastewater treatment facility at the site was responsible for this contamination. The fecal indicator bacterium was Enterococcus faecalis. Ribotyping, automated with a RiboPrinter (DuPont Qualicon, Wilmington, DE), was the BST method. Thirty-seven ribotypes were observed among 83 Ent. faecalis isolates obtained from the Sapelo River and the wastewater lagoon. Sixteen ribotypes were associated with either the river or the lagoon, and only five ribotypes (14%) were shared. Nevertheless, these five ribotypes represented 39 of the 83 Ent. faecalis isolates, almost a majority (47%). These results suggest that the fecal contamination in the river came from the wastewater treatment facility. As a prelude to BST, targeted sampling minimized subspecies changes with geography and time, and eliminated the need for a permanent host origin database by restricting BST to a small geographic area and requiring sampling to be completed in one day.

Deer diet affects ribotype diversity of Escherichia coli for bacterial source tracking.

Ribotyping is one of a number of genotypic methods for bacterial source tracking. This method requires a host origin database of one bacterial species be established in order to identify environmental isolates. Researchers establishing these databases have observed considerable ribotype diversity within a specific bacterial species. One source of this diversity may be diet. We determined the effect of diet on ribotype diversity for Escherichia coli in penned and wild deer (Odocoileus virginianus) in a 13-ha forested watershed. A total of 298 E. coli isolates was obtained, 100 from penned deer, 100 from wild deer, and 98 from the stream in the watershed to which all deer had access. The wild deer had significantly more ribotypes (35) than the penned deer (11 ribotypes, p = 0.05). This result suggests that diet affected ribotype diversity, and that a host origin database for bacterial source tracking should contain bacterial isolates from wild rather than from captive animals. Also, 42 of 98 (42.9%) environmental isolates matched penned and wild deer ribotypes. If bacterial source tracking determines that fecal contamination is predominantly from wildlife, then it may be unnecessary to monitor these watersheds because control over wildlife is difficult.

Comparison of genotypic-based microbial source tracking methods requiring a host origin database.

Microbial source tracking (MST) results, obtained using identical sample sets and pulsed field gel electrophoresis (PFGE), repetitive element PCR (rep-PCR) and ribotyping techniques were compared. These methods were performed by six investigators in analysis of duplicate, blind sets of water samples spiked with feces from five possible sources (sewage, human, dog, cow and seagull). Investigators were provided with samples of the fecal material used to inoculate the water samples for host origin database construction. All methods correctly identified the dominant source in the majority of the samples. Modifications of some of these methods correctly identified the dominant sources in over 90% of the samples; however, false positive rates were as high as 57%. The high false positive rates appeared to be indirectly proportional to the levels of stringency applied in pattern analysis. All the methods produced useful data but the results highlighted the need to modify and optimize these methods in order to minimize sources of error.

Potential of Enterococcus faecalis as a human fecal indicator for microbial source tracking.

Regulatory agencies are interested in a fecal indicator bacterium with a host range limited to humans because human fecal contamination represents the greatest hazard to humans, yet is a relatively easy nonpoint source to remedy. Watersheds with human fecal contamination could be given first priority for cleanup. A fecal indicator bacterium with a host range limited to humans and a few other warm-blooded animal species would also simplify microbial source tracking because only a few animal species would be required for any host origin database. The literature suggests that the fecal indicator bacterium Enterococcus faecalis has a limited host range. On this basis, we selected this bacterium for study. Of 583 fecal streptococcal isolates obtained on Enterococcosel agar from Canada goose, cattle, deer, dog, human, chicken, and swine, 392 were considered presumptive enterococci and were subsequently speciated with the API 20 Strep system. Of these isolates, 22 were Ent. durans (5.6%), 61 were Ent. faecalis (15.6%), 98 were Ent. faecium (25.0%), 86 were Ent. gallinarum (21.9%), and 125 were unidentified (31.9%). The host range of the Ent. faecalis isolates was limited to dogs, humans, and chickens. Media were developed to isolate and identify Ent. faecalis quickly from fecal samples and this scheme eliminated Ent. faecalis isolates from dogs. When the remaining Ent. faecalis isolates were ribotyped, it was possible to differentiate clearly among the isolates from human and chicken. It may be that combining the potentially limited host range of Ent. faecalis with ribotyping is useful for prioritizing watersheds with fecal contamination.

Geographic variability of Escherichia coli ribotypes from animals in Idaho and Georgia.

Several genotypic methods have been developed for determining the host origin of fecal bacteria in contaminated waters. Some of these methods rely on a host origin database to identify environmental isolates. It is not well understood to what degree these host origin isolates are geographically variable (i.e., cosmopolitan or endemic). This is important because a geographically limited host origin database may or may not be universally applicable. The objective of our study was to use one genotypic method, ribotyping, to determine the geographic variability of the fecal bacterium, Escherichia coli, from one location in Idaho and three locations in Georgia for cattle (Bos taurus), horse (Equus caballus), swine (Sus scrofa), and chicken (Gallus gallus domesticus). A total of 568 fecal E. coli isolates from Kimberly, ID (125 isolates), Athens, GA (210 isolates), Brunswick, GA (102 isolates), and Tifton, GA (131 isolates), yielded 213 ribotypes. The percentage of ribotype sharing within an animal species increased with decreased distance between geographic locations for cattle and horses, but not for swine and chicken. When the E. coli ribotypes among the four host species were compared at one location, the percent of unshared ribotypes was 86, 89, 81, and 79% for Kimberly, Athens, Brunswick, and Tifton, respectively. These data suggest that there is good ribotype separation among host animal species at each location. The ability to match environmental isolates to a host origin database may depend on a large number of environmental and host origin isolates that ideally are not geographically separated.