STUDY 1 OF FECflL U. S. ENVIRONMENTAL PROTECTION AGENCY ARCTIC ENVIRONMENTAL RESEARCH LABORATORY COLLEGE, ALASKA 99701 ------- PRELIMINARY STUDY COMPARATIVE WINTER SURVIVAL OF FECAL BACTERIA IN A SUBARCTIC RIVER by Dale J. Van Donsel Ronald C. Gordon Charlotte V. Davenport WORKING PAPER NO. 28 U. S. Environmental Protection Agency Arctic Environmental Research Laboratory College, Alaska Associate Laboratory of National Environmental Research Center Corvallis, Oregon Office of Research and Development May 1974 ------- A Working Paper presents results of investigations which are, to some extent, limited or incomplete. Therefore, conclusions or recommendations, expressed or implied, are tentative. Mention of commercial products or services does not constitute endorsement. ------- m ABSTRACT A preliminary investigation of Salmonella survival in a subarctic Alaskan river was conducted during March 1973, when there was total ice cover and 0°C water temperature. Since most of the domestic pollution entered the river from one source and there were no additional sources downstream, it was possible to examine survival without interruption for seven days flow time. Six Salmonella serotypes were isolated, and salmonellae were still recoverable both quantitatively and qualitatively after seven days flow time in the presence of 330 total coliforms/100 ml and 60 fecal coliforms/100 ml. ------- TABLE OF CONTENTS PAGE INTRODUCTION MATERIALS AND METHODS 4 RESULTS AND DISCUSSION 12 CONCLUSIONS 19 REFERENCES 20 ------- LIST OF FIGURES NUMBER PAGE Map of the lower Tanana River showing the location of sample stations 5 Gasoline powered ice auger being used to cut through ice at each sample station 6 Samples for quantitative analysis of salmonellae and fecal indicator bacteria being dipped from ice hole 7 Setting up tripod to support line with attached qualitative Salmonella sampler and station marker flag 9 Method of retrieving line with attached qualitative Salmonella sampler 10 ------- VI LIST OF TABLES NUMBER PAGE Salmonellae/Liter and Fecal Indicator Bacteria/ 100 ml of Sample Obtained at Each Sample Station During the March 21, 1973. Sample Run 13 Salmonellae/Liter and Fecal Indicator Bacteria/ 100 ml of Sample Obtained at Each Sample Station During the March 27, 1973 Sample Run 14 Salmonella Serotypes Isolated at Each Sample Station from Samples Used for Quantitative and Qualitative Analysis 16 Most Frequently Isolated Salmonella Serotypes in the United States During 1972 (4) , 17 ------- INTRODUCTION Decreasing the suspending medium temperature has shown to increase survival time of enteric bacteria and viruses (1, 9, 11, 16, 17, 19, 20). Survival time appears to increase continuously as the temperature decreases, so the longest survival would be expected when the temperature is at or near 0°C. However, most of the survival studies described in these reports were conducted in the laboratory using pure cultures, rather than directly in rivers receiving the enteric microorganisms discharged in effluent from waste treatment systems. Thus, information to fill the practical need for survival time parameters, as stated by Berg et al. (3) in 1966* still re- mains to be obtained. It has also been stated that coliforms must be quan- titatively related to the presence of enteric pathogenic bacteria if coliform enumeration is to continue as a useful criterion of health risk from polluted waters (17). Increased enteric microogranism survival at low suspending medium temperatures becomes more significant when viewed in relation to current treated wastewater disinfection practice. Recent studies (12, 13) have demonstrated that chlorine disinfection at low effluent temperatures may not effectively remove the fecal indicator bacteria before the effluent is released into the receiving water. Therefore, disinfection under these circumstances may not provide an adequate barrier to the spread of disease caused by enteric pathogenic bacteria. There is also very little chance that enteric viruses are being removed in the disinfection process. In an effort to obtain low temperature survival time data, a survival study was conducted in a subarctic Alaskan river (11). This study demon- strated that a significant number of total coliforms, fecal coliforms and enterococci remained viable after seven days flow time when the water ------- temperature was essentially 0°C under total ice cover. The fecal coliform results were compared with temperate climate winter data (2), and the percent survival was found to be approximately five times greater at 0°C than at the -warmer temperatures. That study did not include salmonellae, so quantita- tive survival relationships are not available. In 1911, Ruediger (20) demonstrated that salmonellae survived in much greater numbers for a much longer period in an ice covered river than during warm weather. He used pure culture suspensions of the bacteria in celloidin sacs which he placed in the river and sampled at various time intervals. More recently, Gallagher et al. (9) found salmonellae to be extremely per- sistent in the Red River under ice cover. They isolated two Salmonella serotypes 73 miles and four days travel time downstream from the outfall source. In addition, salmonellae were consistently isolated from river water when there were 1000 or more fecal col iforms/100 ml (9), and were occasionally isolated when the fecal coliforms ranged from 4 to 20/100 ml (5, 6, 8, 9, 21). Approximately 36 percent of all surface waters examined throughout the world have been found to contain enteric viruses (1). Even though enteric viruses have been implicated as the etiological agent in numerous diseases, infectious hepatitis virus is the only one for which definite epidemiological evidence has been obtained showing water as the mode of transmission (17). However, the extended survival of viruses in water makes this a potential route for viral disease transmission (1). The need for survival time data and quantitative relationships in arctic and subarctic rivers, where the water temperature is 0°C for about six months each year, cannot be overemphasized since low temperatures enhance enteric microorganism survival causing an increased potential ------- health hazard downstream. As a result, this preliminary investigation was conducted to determine whether salmonellae could be isolated at the same stations where fecal indicator bacteria had previously been isolated and to establish, if possible, a quantitative relationship under low tempera- ture conditions. ------- MATERIALS AND METHODS Selection of River to be Studied The reach of the Tanana River studied is shown in Figure 1. The descrip- tion of the river and reasons for selecting this particular river for study have been described previously (11). Sample Station Selection and Sampling Schedule Nine sample stations were selected (Figure 1). T-100 through T-800 are essentially in the same locations as previously described (11). Station T-900 was selected to obtain background data upstream from all known sources of domestic pollution. Two sampling trips were conducted during this preliminary study. All stations were visited on March 21, 1973, for the purpose of collecting samples for quantitative fecal indicator bacteria and Salmonella enumera- tion, and placing long term qualitative Salmonella samplers (Moore swabs). A second trip was made on March 27, 1973, to obtain anot&er set of samples for quantitative analysis, and to retrieve the swabs. Sampling Techniques A gasoline powered ice auger was used to cut through the 1-1/2 to 4 feet of ice encountered at each sample station (Figure 2). Ice chips were skimmed from the water surface, and the samples for quantitative analysis collected by dipping from the water surface (Figure 3). Samples for fecal indicator bacteria enumeration were collected in sterile, wide mouthed, screw capped, 1-liter, polypropylene containers. These containers were transported in ice chests to minimize freezing problems. ------- '!"* • 8*. 64*10 TANANA RIVER LOWIR IECTIOH tn Figure 1. Map of the lower Tanana River showing the location of sample stations ------- .-. Figure 2. Gasoline powered ice auger being used to cut through ice at each sample station ------- Figure 3. Samples for quantitative analysis of salmonellae and fecal indicator bacteria being dipped from ice hole ------- 8 For Salmonella quantitation, samples were collected in 5-gallon Cubi- tal ners. There appeared to be little or no ice formation in these large volume samples. Qualitative Salmonella samples were obtained with the gauze swabs as described by Spion (22). After the samples for quantitative analysis were obtained, the line with the gauze swabs attached was lowered through the hole in the ice, and attached to a tripod for support while the hole was freezing closed (Figure 4). The tripod also supported a flag which marked the sample station. In order to retrieve the gauze swabs after a week in the water, it was necessary to cut another hole in the ice just downstream from the first hole. A steel rod with a hook on the end was used to catch the line and pull it up through the new hole (Figure 5). The guaze swabs were placed in 18-ounce Whirl-Pak bags, and transported to the laboratory in ice chests to minimize freezing. Fecal Indicator Bacteria Enumeration Total coliforms, fecal coliforms and fecal streptococci were enumera- ted by the membrane filter method (13). All sample volumes were filtered in triplicate, and volumes up to 200 ml were used in an effort to obtain numbers of colonies on each filter in the statistically valid range. Quantitative and Qualitative Salmonella Enumeration The presence of salmonellae was quantitatively determined by the most probable number (MPN) procedure. The diatomaceous earth method of Hammar- strom and Ljutov (14) was used to concentrate the water samples. Sterile dia- tomaceous earth (0.5 gm/1) was added to the water. For convenience the membrane filters were removed from sterile, disposable, membrane filter holders (Fal- con Plastics, number 7103) and replaced with filter pads of the type accom- panying membrane filters. A slurry of 0.5 gm diatomaceous earth was ------- , Figure 4. Setting up tripod to support line with attached qualitative Salmonella sampler and station marker flag ------- Figure 5. Method of retrieving line with attached qualitative Salmonella sampler ------- 11 spread on the filter pad, and 12 liters of sample was vacuum filtered. To remove substances that caused erratic salmonellae isolation, the diato- maceous earth plug on the pad was washed five times with sterile buffered distilled water and three times with Dulcitol Selenite Enrichment Medium (DSE) (18). The concentrate plug was then removed from the filter pad and homogenized in a small amount of DSE. Additional DSE was added to bring the total concentrate volume to 120 ml (100:1 concentration of the original sample). The concentrate was then thoroughly mixed, and one 50-ml, five 10-ml, five 1-rnl and five 0.1-ml portions were immediately planted into the MPN series in DSE and incubated at 41.5°C. These portions were equi- valent to 5,000, 1,000, 100 and 10 ml volumes of the original sample. After 24 and 48 hours of incubation, material from the MPN tubes was streaked on Hektoen Enteric Agar (HE) and Xylose Lysine Desoxycholate Agar (XLD), and incubated for 24 hours at 35°C. For the qualitative determination of salmonellae, each gauze swab was divided into three equal portions. One portion was placed in a flask of DSE and two in Tetrathionate Broth. Incubation was at 41.5°C and streaks were made on HE and XLD at 24 and 48 hours with incubation at 35°C. Suspect Salmonella colonies on HE and XLD were picked to Triple Sugar Iron Agar (TSIA) slants. Colonies producing reactions indicative of sal- monellae in TSIA tubes were further screened for urease, indoTe and lysine decarboxylase production before being identified by the serological methods of Edwards and Ewing (7). Additional biochemical tests were performed only if warranted by questionable screening or serological results. ------- 12 RESULTS AND DISCUSSION The objectives of this limited preliminary investigation were: [1] to determine whether salmonellae could be isolated as many days flow time downstream from any source of domestic pollution as previously isolated fecal indicator bacteria (11), and; [2] to establish whether or not a quan- titative relationship exists between salmonellae and fecal indicator bacteria. Because this quantitative relationship was to be based on the number of bacteria/unit volume of river water, discharge measurement were not necessary at any station. The results presented in Tables 1 and 2 show that the numbers of fecal indicator bacteria decreased progressively downstream from the T-700 to the T-100 station in a manner comparable with earlier results (11). The T-700 station (Figure 1) is located downstream from the last source of domestic pollution while T-900 is upstream from any source. The fecal coliform numbers were lower than expected from the previous study. However, the m-Fecal Coliform Broth Base (BBL lot #204625) may have been a contributing factor. This medium gave a final reaction pH of 7.2, which is within the accepted 7.2-7.6 pH range. On further checking, it was found that the broth base had a pH of about 6.4 before rosalic acid addition and heating. This is 0.5 to 0.7 pH units lower than obtained with other broth base .lots from the same manufacturer. Fecal coliform recovery from unchlorinated primary sewage effluent was examined using the above mentioned broth base lot, and recovery was compared directly with two other lots from the same manufacturer. The results indicated that the broth base used during this study recovered only 48-60 percent as many fecal coliforms as the other two lots. This suggested that the fecal coliform numbers reported in Tables 1 and 2 may be considerably lower than the actual number/unit volume present in the river. ------- 13 TABLE 1 Salmonellae/Liter and Fecal Indicator Bacteria/100 ml of Sample Obtained at Each Sample Station During the March 21, 1973 Sample Run Salmonellae Station No. T-900 T-800 T-700 T-600 T-500 T-400 T-300 T-200 T-100 Total Col i forms (No./lOO ml) 2300 9600 1700 920 920 370 410 320 Fecal Col i forms (No./lOO ml)* 130t 1700 89t 160 110 64 36 34 Fecal Streptococci (No./lOO ml) <0.5§ 43 130 9t 15t 15t 9t lit 13t No. /liter MPN 0.1# 0.1 <0.1# 0.1 0.1 0.1 0.8 1.7 <0.1# 95% Confidence Limits No. /liter <0.05 0.4 <0.05 0.4 <0.05 0.4 <0.05 0.4 0.2 1.9 0.5 4.7 * These numbers are probably 48-60 percent of the actual number; see text for explanation. § No colonies on any filter when triplicate filters were examined, # No salmonellae isolated from any water volume examined. t Average of less than 20 colonies per filter when triplicate filters were examined. ------- 14 TABLE 2 Salmonellae/Liter and Fecal Indicator Bacteria/100 ml of Sample Obtained at Each Sample Station During the March 27, 1973 Sample Run SaTmonellae Station No. T-900 T-800 T-700 T-600 T-500 T-400 T-300 T-200 T-100 Total Col i forms (No./lOO ml) ** 3600t 6600 1600 2300 960 470 630 330 Fecal Col i forms (No./lOO ml)* __** 480 170t 64t 260 120 43 63t 60t Fecal Streptococci (No./lOO ml) _** 73 50 26 26 12 10 21 6t No. /liter MPN ** <0.1# 0.3 0.1 0.1 0.3 0.1 0.1 0.3 95% Confidence Limits No. /liter <0.05 0.8 <0.05 0.4 <0.05 0.4 <0.05 0.8 <0.05 0.4 <0.05 0.4 <0.05 0.8 * These numbers are probably 48-60 percent of the actual number; see text for explanation. ** No samples obtained from this station because of open water. # No salmonellae isolated from any water volume examined. t Average of less than 20 colonies per filter when triplicate filters were examined. ------- 15 Salmonellae were quantitatively measurable in samples from the T-800 through T-100 stations during one or both sampling trips (Tables 1 and 2). Since these were the same stations at which fecal indicator bacteria were also found, it is apparent that low indicator bacteria numbers do not pre- clude quantitative measurement of salmonellae. The most probable number (MPN) of salmonellae present at the various sample stations suggests that the numbers of these bacteria do not decrease with flow time. However, the 95 percent confidence limits of the MPN indicate that the salmonellae may be decreasing in a manner similar to the indicator bacteria. A much more detailed study must be conducted before relative survival rates and quan- titative relationships between salmonellae and fecal indicator bacteria can be established. When Salmonella isolates from both quantitative and qualitative samples were examined, six serotypes were found (Table 3). Four serotypes were found in the quantitative samples and five were obtained from the swabs. S. thompson was the only serotype isolated at every station from T-700 to T-100 and was the most frequent isolate from quantitative samples. Thus. SN, thompson was probably present in much larger numbers than the other salmonellae. Insufficient data are available to ascertain the numerical relationship of the other isolates, or to determine if survival character- istics vary among the serotypes. The 10 most frequently isolated Salmonella serotypes from human and nonhuman sources during 1972 are shown in Table 4. Three of the serotypes found in the Tanana River were among those most frequently isolated from human sources and three from nonhuman sources. S. typhimurium and S. derby appear under both headings, while S. thompson was ranked among those most frequently isolated from human sources and S. senftenberg from nonhuman sources. ------- 16 TABLE 3 Salmonella Serotypes Isolated at Each Sample Station From Station Number T-Qnn T-800 1 / UU T-600 1 OUU T-200 T-100 Samples Used for Quantitative and Qualitative Quantitative Samples March 21, 1973 March 27, -1973 derby None thompson group .B non-motile LI lUIHJJo Ul 1 Li lUli i|p oU * 1 •fhnmnc nn "t" hnmncnn thompson thompson None thompson senftenberg Analysis Qualitative Samples ** derby * thompson senftenberg typh.imurium derby * * * thompson senftenberg thompson paratyphi B typhimurium ** No samples obtained from this station because of open water. * Swab not recovered. ------- 17 TABLE 4 Rank 1 2 3 4 5 6 7 8 9 10 in the United States During Human typhimurium* newport enteritidis infantis hei del berg saint-paul thompson* derby* oranienburg javiana 1972 C4) Source Nonhuman typhimurium* oranienburg senftenberg* saint-paul newport anatum montevideo eimsbuettel derby* hei del berg *Isolated from Tanana River. ------- 18 The results presented in Table 3 indicate that the method used to re- cover the gauze swabs was not entirely satisfactory since the swabs were not recovered from four stations. The problem was apparently caused by the insulating snow layer having been removed from the ice around the hole through which the line and attached swabs were lowered. During the ensuing week, a cone of ice developed below the lower edge of the ice cover and encased the line on which the swabs were attached. The cone extended deeper than could be reached with the hook so it was impossible to retrieve the swabs. Efforts are currently in progress to develop a means of overcoming this serious problem. Circumstances did not permit the examination of enteric viruses during this preliminary investigation. In view of the available information about extended virus survival at low water temperatures (1), isolation and identi- fication of enteric viruses should constitute a major effort during any detailed enteric microorganism survival study. Field methods for concen- tration of viruses from large volumes of water are available (15), and one or more of these should be applicable under low temperature conditions, making a virus survival study feasible. ------- 19 CONCLUSIONS 1. Salmonella serotypes survive for at least seven days flow time down- stream from the last source of domestic pollution when the water temperature is 0°C and there is total ice cover. 2. After seven days flow time, a variety of Salmonella serotypes are still recoverable both quantitatively and qualitatively when total and fecal coli- forms are present in low numbers. 3. The results from this preliminary study were too few to determine the extent to which fecal indicator bacteria numbers would indicate Salmonella density. The results do suggest that a survival relationship may exist. However, a more detailed study is necessary to assess the quantitative relationship. 4. 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Sewage and Industrial Wastes. 22_: 1261-1281. 20. Ruediger, G. F. 1911. Studies on the Self-Purification of Streams. J. American Public Health Association. 1_:411-417. 21. Smith, R. J., R. M. Twedt, and L. K. Flanigan. 1973. Relationships of Indicator and Pathogenic Bacteria in Stream Waters. J. Water Pollution Control Federation. 45_: 1736-1745. 22. Spino, D. F. 1966. Elevated-Temperature Technique for the Isolation of Salmonella From Streams. J. Applied Microbiology. 14:591-596. 23. Van Donsel, D- J. and E. E. Geldreich. 1971. Relationships of Salmonellae to Fecal Coliforms in Bottom Sediments. J. Water Research. 5:1079-1087. * U. S. GOVERNMENT PRINTING OFFICE: 1974-697-595 /54 REGION 10 ------- |