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QUALITY IN THE PUBLIC DISTRIBUTION SYSTEMS OF THE VIRGIN ISLANDS Robert H. Ruskin, Jr. Patrick S. Callender Henry He Smith January 1987 Project No. 03. “Agreement No. 14-08-0001-G1050 “Technical Report No. 28 Caribbean Research Institute University of the Virgin Islands St. Thomas, ULS.Ver. 00802 _The research on which this report is based was financed in part by the United States Department of the Interior, Geological Survey, through the. Virgin Islands Water Resources Research Center. Contents of this publication do not necessarily reflect the views and policies of the U.S. Department of the Interior, nor does mention . of trade names: or commercial products constitute their endorsement. by the United States. Government. . ABSTRACT - The public distribution systems of the Virgin Islands were first installed in 1949 through the’ mid-1950's. The cast iron pipes which comprise the system are subject to _ severe corrosion. Though the systems have undergone seyeral major repairs, questions have arisen about the quality of the water being distributed as a direct result of a typhoid epidemic at a public housing complex on St.. Croix in which _potable water was the suspected. carrier... These fears are. understandable in as. much as the systems can become contaminated by several different pathways. Avstudy of the ‘distribution systems of all three islands was conducted. The principal findings were that with the exception of. iron, probably due to residual rust in the lines, all sys ems” nad easily met the U.S. ELPA. physical and chemical parameters for drinking water. The réal threat to public health however. is due to inadequate or improper chlorination. Without. _ proper chlorination, | then the outbreak of some other water borne enteric disease is highly likely. ~iti-. TABLE OF CONTENTS Page ABSTRACT Lit LIST. OF FIGURES Se ae ene ee cas eee me ae em ere ee ams ae ome eine ene ine EY oe eons ce eee ow LIST OF TABLES Vi INTRODUCTION MATERIALS AND METHODS: ot ee me em oe io oem care em ee cme eee ee ees ere com eee ane aoe oven Site Selection Collection Procedures Chemistry and Physical Analyses: 11 Microbiological Analyses 13 RESULTS AND CONCLUSIONS- 27 ‘REFERENCES 40 om iv- “LIST OF FIGURES FIGURE Page 1. St. Thomas Water Distribution System St. Croix Water Distribution System 3. 10 St. John Water Distribution system -Vo LIST OF TABLES TABLE Chronological” Results. of Chemical and Physical Analyses for St. Thomas and St. Tokncacee ciehminded TOT TS iarnann agers Results of Chemical and Physical Analyses for St. Thomas and St. John Grouped “According. to. Sites -s-corecre teaeeteatetetecteteetad HHH. Chronological Results of Microbiological oa Analyses for St. Thomas. and St. Joharwec-ns Results of Microbiological Analyses. for. - $t.-Thomas and St. John Grouped According to. Bitters cron smencon nna rtcecsnan cece e oe Chronological Results of Microbiological cand. Physical ‘Analyses for St. Stodar Tare Results. of. Microbiological and ‘Physical. - Analyses for St. Croix Grouped According . an Ke) Site cor Sos par Sac ioccr aren tenon seta U.S. ELPLA. Physical-Chemical Standards “for Drinking Wat efonne case gamer e seco omy 295 30 31 34. INTRODUCTION. The _ public distribution systems of the Virgin Islands were first installed in 1949 through the | mid= -1950's. The system’ on St.. Croix serves. an area of 14 square miles with 3,000 connections; — in St. Thomas it serves 2.square miles with 2,150 connections} and, on St. John 0.3 square miles are served through 10 connections. The necessary pressure in the systems on St. Thomas and St. John is maintained by pumping while the st.Croix system is mainly a gravity operation. Maintenance of the distribution lines is difficult. Through the years these systems have undergone several major repairs. due to corrosive soils and the presence of most of the lines “in areas in close proximity to sea water. , Water in the public distribution systeme in the ‘Virgin “Islands ‘can most readily become contaminated due to leakage ‘into the lines, cross connections; siphonage during periods of negative pressure, and insufficient: disinfection. Several questions have . been “raised by “the > public. concerning the quality of the water being distributed. In 1985 this was . particularly @ concern when 66 cases of typhoid were. diag- nosed and. confirmed at a “housing complex in St... Croix. Water ‘from the public distribution system was the suspected car rier. The immediate benefit ‘of the project is obvious. Analy- sis of water “at several points in the distribution Systeme... —- will assist in the identification of areas where water eon- tamination is more likely to occur. ~ This has been hindered in. the past when analysis with any similarity was performed on an. emergency basis and often by groups with particular self-interests. | This” investigation is ‘especially timely in that the distribution system in St. Thomas is currently undergoing major’ renovations. This project provides both before and after looks at .contamination in this system. The investi- gation provides information that can be be used in ‘planning similar renovations on the St. “John and st. Croix systems. Determination of quality profiles of the St. “Thomas distrib- “ution . system will also develop baseline data for monitoring the integrity of the system. For these reasons there is s need for a comprehensive systematic. survey of water quality ‘in the public distribution systems of the Virgin Islands. _ MATERIALS AND METHODS. Site Selection ; To help identify. the sample point locations, ‘a map of: the. potable w water distribution system was obtained, ‘then with the aid of the Department of Conservation and Cutural Affairs pergennel responsible for monitoring the quality of water in the “distribution systems, field trips were made to locate useable sample points. _ : In St. Thomas,- there are four main lines which consti- ture the distribution system (Figure De From these main lines extend the laterals which serves. the homes and busi- Snegeeas The heart of the. water supply system is a 2. 5 mil- Lion gallon per day desalination facility located at Krum | Bays “St. Thomas. “The jecalinated ¢ water is. s pumped firet to storage tanks, entene tte filter tanks, then passes through a “ghiovination” chanber, | From the chamber “dt enters” directly into the distribution system. ‘The first main Tine is aten inch line laid in 1975, — which travels ‘from Krum Bay west to the University (of the ‘Virgin Islands. 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Afet “i 4 was ters Bes ey seat fy a ay abt a iy & cores an a an Daf Ce ae ea on Saas sr oe oie aby eH) hea oo RS oh TH ots a seeds, asters eats, oe fats Le oe 49) ee as iE ates g.. ms a) set es + Hit HS rt eee oe ee Sie Hs rH} Hitt ay NS statis a fe, tr ofa ny +t nists Sait ter] oP ats Hy Se as we ae sats? ve ae at a 58 iM ats CASS} Pann HHH San cad ia, 38 Hy Ht Ht eeleb sty eats teks Ht seit sistetetate R23, sy aes ts sh "seco anh; Kk rote eo) pty or ahh tH So hs 4 eat butted abetaty| SHH Ee ‘ej 3 -4- tron pipe. The third main line is a ten inch line laid in 1963 | which services the French Town fishmarket, continues” under - the waterfront apron, services the main fire station, and branches into a dead end at the Boy Scouts of America Headquarters. The other branch of this line runs north, then east as far as Oswald Harris Court. The fourth and final main. line is a 24 inch line laid in 1976 which runs directly to a “ storage tank at the top of Raphune Hill. “this tank feeds the Estate (“Tutu area to the east. At the time of this study "there was no water flowing in this line which is activated ‘only as needed. ‘The other three lines are in continuous use. | gampling points which would provide samples” that were truely representative of the overall water quality of the ‘entire distribution system were selected, Samples were taken from the three active main lines, and from branches and ._- laterals originating off the main lines. Samples were. ob- tained as far away from the source as possible as well as the source itself, Nine ‘sampling points were selected on. the "old" distribution system and three on the "new" system. Of that twelve, two were controls. One was directly from Filter Tank #1, the "positive" control, and the other was from a tap. at the Public Works Department Motorpool, a point right after the water has been chlorinated, and the "negative" control. | Going | west, samples were taken from the Kirwan Terrace | “Fire Department, which is about 50 £t. off of the main line, and also at a tap at the Kirwan Terrace housing project maintenance building. | Going east, three samples were pulled directly off of the new line on. Harwood Highway and Main Street. Three other samples were pulled from laterals off of this main line. | One sample was taken each of the three from standpipes “an the Savan area of Charlotte Amalie. From the third main line, also going east, two samples sieve taken, one at the French Town fishmarket, and the second at the Boy Scouts of America Headquarters. | ‘One of the problems encountered was. locating points directly off of the distribution system. In many cases, a desirablé point would be eliminated because the water would first flow. into a cistern which also stored roof “top. har- vested rain water which made the sample not representative of. ‘the water quality in the lines.. During this initial phase of. locating sample points, when a point was Found that met the criterion of a) . coming off the distribution system b) not first entering’ a cistern c) being in an appropriate area, a sample was then collected for rough bacteriological background data. Total coliform, fecal coliform, and fecal streptococcus analysis were ‘run from each ‘acceptable point to determine if dilutions would have to be made once regular sampling began. Algo free and total residual chlorine levels were taken at this time. A: similar trip was made to. the island of ste John. The. gee John: distribution system is 's very small being. “about 0. 3 of a mile . Long. Only three. sampling points vere selected. They were the main storage tank at the southern end of. the system, the Agricultural Station, | just north of “the midpoint, and the (Office of the Administrator, which is the ‘northern “most accessable point. Sampling sites are shown in Figure 26 ‘St. Croix was also sampled. However,» ‘due to ‘time Limita- tions, only two mass sampling efforts could be done. During the initial visit, fifteen Samples were, collected. These “were taken ‘rom points extending from the (Pearl B. Larsen “school east of Christiansted, to La Grange which is. west of Prederiksted. At each sample point free and total residual chlorine levels. were made and. a sample collected, stored, and ‘returned ae Ste vhonee’ for t total coliform, fecal coliform, cand fecal streptococcus. analysis, with each sample being run in duplicate. The. second. visit. “involved the - pane “fifteen sample ipotnts, "Ge “wane aaatyeaai. put also included pH, téuperature and conductivity measurements. tn Figure 3, sampling points are shown. Collection Procedures Samples were collected in accordance to Standard Methods” ‘for the Examination of Water and Wastewater and Microbiologi- cal Methods for Monitoring the Environment. et bell wes sg, “OTANGTIISTG TOREM KXTOID "4g *ez emnbtg es 7 “dl of “4 “A “A ay ss od TZ on ss ey Cee ~9-- ea aoe. Ot One tit) ee eat atat ee) ‘38! ore oe oe ate "one sa oe oe is Oe a5 be) ay enh, ane ote ane Oo ‘ere ate or of ote’ St5 - ‘oe. on ee ae eos ree ot ero' a ‘ane, oe ors ‘are! eee "oa. are! ee. on ox "ono"! ora ooo ie) Oy oo oo of ao! oe SS Seek Patera ee re "ooo! note e a! ooo" oS oe ane "oe. Sch 5 oes “Sy “erate te" Ras eee eter ‘arora ee, ae "ata n we, "oa oe" aoe "er, ‘2! oe SS “ana arane eo Sea! 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The one exception was the sample for with either H or HNO ‘to a pH less than two depending on silica which was collected separately ina clean, thoroughly washed and rinsed polypropylene bottle. = ae “the microbiological samples were all collected in “one “Liter porosilicate glass bottles with ground glass’ stoppers. Prior to collection 0.8 mL of a 10% solution of sodium thio- “sulfate was added to each one liter bottle (giving a final concentration of 0.1 mL per 125 mL of sample) and the bottle was autoclaved. ee , , , | ‘once in the field, all the collection taps were flushed “for at least three minutes before the samples were collected. Once collected, the samples were immediately rushed back to the’ “Laboratory. - ~All microbiological analyses were begun within four hours of the time of collection, — “Chemistry ‘and Physical Analyses” The chemical parameters examined were temperature, free and total residual chlorine, apparent color, dissolved oxy- gen, turbidity, pH, conductivity, total solids, nitrate, ‘calcium, chloride, iron, silica, sodium, and magnesium. Temperature was ‘taken’ ‘using oa celsius degree . thermometer. aoe Free -and. total residual chlorine’ readings were done using the N,N-diethyl-p-phenylenediamine (DPD) colorimetric method. Color was done as apparent color using a “specto- photometric method. , Dissolved oxygen was measured using the azide modifica tion of the Winkler titration method... “Chloride was analyzed by the mercuric nitrate titration method. , Turbidity was measured by nephelometric using the HF, DRT 100 (HF instrument Co., Fredona, NY). . pH was done using the potentiometric system. We used two pH meters in this study the Hach 19,000 (Hach Chemical Co., — “Loveland, CO) and the Markson 90 (Markson Instruments, San Diego, CA) with sealed combination electrodes. Conductivity . was run using a Markson 15/16 conductivity meter (Markson Instruments). Total solids were ruri by evaporating enough sample at -103-105° C 80 that there was a aifisium difference of 5 mg between the initial weight and the final weight. "The amount of total solids were then calculated using the formula: “mg total solids/L = (A-B) x 1000 ; mo, a Sample volume, ml where A = Final weight (dish + residue in mg) B = Initial weight (dish in mg) Magnesium, sodium, calciumand total iron were analyzed by... atomic. absorption. With calcium the EDTA titrimetric = 12— method wae used; with total tren the 1.10 phenanthroline method ising spectrophotometry was used. ‘Nitrates and eilicates both were done by using the Hach DR-3. spectrophotometer (Hach Chemical Co.) which is part of ‘the Hach DREL-5 Field Laboratory Kit. Nitrates were analyzed by the cadmium reduction method and silicone by the heteropoly blue method. of , | Except for those analyses that used specific instruments such: as atomic absorption, temperature, conductivity, pH, residual chlorine, | turbidity -and total solids, all. other analyses “Were “done using the Hach DREL-5_ Field Laboratory Kit... While all of these Hach kits used prepackaged > "powder pillows" all the. analyses are based on methods. found in Standard Methods for the Examination of Water and Wastewater. Microbiological Analyses The microbiological parameteré examined were as stated ‘before, total coliform, fecal coliform, fecal streptococcus and standard plate count - heterotrophic plate. count. All are standard tests in determining water quality. In addi- tion, because of the typhoid outbreak on St. “Croix in 1985, attempts were. made. to isolate Salmonella “spp. “trom each ‘sample. : These five tests taken collectively yield information about the quality of the water, ‘and whether adequate disin- fection ig occurring, or if not, whether it might be prudent -=13- to ige other tests in conjunction with the stendard analyses to insure a safe supply of drinking water. As will be geen, all tests used are those in use throughout most water quality and public. health Laboratories. The one exception is our procedure for the qualitative isolation of Salmonella ‘sero- types. “Even here ‘the procedure is but a a modification of ‘two existing methodologies combined into one, and this. is nothing pore. than an extension of the total coliform procedure. This is important because with minimal additional training “and supplies, the procedure could be readily incorporated into the analysis of all. major problem supplies. In addition, it has the potential for rapid, wide, acceptance, because - with the- exception of the procedure ~. the methodologies are well : “known and universally accepted. ‘The total coliform has been the indicator organism of choice = for determining the bacteriological quality of drinking water ‘since 1914. The reason is it most - elosely approximates the, ideal indicator organisms. In recent years | however, the standard total coliform analysis has come under increasingly heavy scrutiny > because it has been “extensively reported in the literature that in many instances stressed coliforms fail to } be detected, - Indeed, “stressed or injured total” coliforms have become the rule rather than the. “excep- tion, and- now” many researchers have proposed either "new procedures, or new media,. whereby these stressed Or. “injured arom “coliforms can be detected. Tr is not the intent of this paper however to use either new procedures, or new media, “which are still in ‘the experimental phases. of teating; rather, it is the intent to use established methodologies to deter— nine the suitability of the water, , , | , The. two universally accepted techniques, for the - quanti- tative isolation of total coliforms are ‘the Most “Probable. Number (IPN) eechaique and the ‘Membrane Filtration (MF) technique. — the MPN technique is based upon ‘the: statistical probability of finding ‘x number -of total coliforms if ¥ ‘number of tubes are positive for ‘the production | of acid and gas in both. leuryl eryptose broth, and. brilliant green bile. 2%. The primary use of this technique ievouvde® those gamples "which tend to have either high ‘qurbidities, toxicity, or considered to be chlorinated effluents. The major drawbacks to , this procedure are: 7 ! ' 1) ‘The. results are statistical values and_ not a direct. count. , 2) The procedure tends. ). yield higher percentages of both false positives, and false negatives than. does the MF ~ fechnique. | - 3) It is “both “more costly ‘and time. consuming than the ME method. - A) It. requires “much more space. to do an ‘equivalent number of samples compared ‘to the MF method. . : . The MP technique is the preferred method because: 1) It yields a. ‘quantitative direct count of the total coliforms. present. : . -15- 2y It yields fewer false positives. and fewer false negatives. - 3) It is rapid and relatively inexpensive. 4) It takes up very little space to do a ' lot of samples. In. as much as the samples being analyzed were not either highly ‘turbid, -toxie or chlorinated. effluents and fot the reasons’ stated above, . all samples were protedsed by the MF technique. | | ‘The total coliform group includes. all the aerobic and facultative anaerobic, gram negative non- spore forming rod=- "shaped bacteria that ferment lactose in 24- 48 hours. at (35*C. tt includes the genera Escherichia coli, Citrobacter, Entero= _ bacter, and. Rlebeielia.. ‘In performing the total coliform analysis all gamples were run in duplicate. Each replicate involved. filtering. a 100 mi volume. of water through a 0. 45, mn type HA. filter (Millipore | Corp., Bedford, MA), and then “placing. the filter on m-Endo agar (Difco, Detroit, MI) - which waa contained in 50x9 mm pre-sterilized plastic, dic- posable petri dishes with tight fitting lids (Gelman, Ann Arbor, MI). These plates were then incubated at 35°C + ~—0.5°C for 24 hours. At the end of the 24 hour incubation ‘period the plates were examined and ‘the typical golden green metallic sheen colored colonies. were. counted, 7 These typical eckonies were. then subjected to. verification, — All. ada tara (ora nexinun of ten typical colonies) were. randomly picked. and placed in ‘single ‘strength lauryl tryptose broth (Difco) ae . “te : ; and® single strength brilliant green bile 2% _ (Difco) and | | incubated at 35°C + 0.5°C ‘for up to 48 houre. . ALL samples which showed production of acid and. gas in both media were considered to be positive for the presence of coliforms. “In the event only the lauryl tryptose broth tube was positive, a new tube of brilliant green bile 2% was innoculated from the positive tube and then incubated for up to. 48 hours. at 35°C + 0.5°C. If after this period of time the brilliant green bile (2% tube was still negative for the production of acid and gas, the colony was then listed as negative for the presence. ‘of total coliforms. , , If no typical colonies were seen then up to five non- typical or atypical red colonies were picked and subjected to the ‘same verification procedure. The final reported . “eolony count was adjusted accordingly. Analyses for fecal coliform were also run. 7 Fecal coli- form. are part. of the total coliform group but these grain ‘negative, non-spore fortiing rods which ferment lactose can grow at the elevated temperature of 44,5°C. + 022°C with che production of acid and gas in 24 hours and are frequently used as indicators of sewage treatment ‘plant 6 ficiency. Alternatively, they can be used to indicate contamination ‘from human sources. The primary fecal © coliform is Escherichia coli which is almost solely associated with man. “Their presence in potable water would indicate a supply that -17-.. may. have been contaminated by sewage seeping into the Lines, or by back siphonage from a cross connection. Because sewage seepage and /or cross connection dre thought to exist, the fecal coliform analysis was run. Like the total coliform, fecal coliform can be analyzed by either MPN or MF. methodologies. The MF procedure was again chosen, and again for the same basic reasons. Again. all samples were run as duplicates. Each replicate consisted of filtering a. 100 mL volume of water. through a-O. Jum type HC. filter “(wiltipore Corp.) as has been recommended for’ fecal. coliform analysis, and then placing. ‘the. filter on m- FC agar (Difco) £0 which Rosolic Acid has been added. The plates were then incubated at 44, 5° Cc + 0.2°C for 24 hours in a Blue. 7 waterbath. | At the. end of the. 24 hour incubation. period, ‘all typical blue colonies were counted. These blue colonies were then submitted to verification procédures. ALL, or up to a maxintin of ten, blue colonies. were verified, ' The colonies were first picked, and then inoculated into lauryl tryptose broth (Difco) and incubated at 35°C + 0.5°C for up. ‘to 48 hours. ALL positive tubes which showed the production of acid and gas within that period weré then transferred into EC broth (Difco) and incubated at 44.5°C + 0.2°6 for 24 hours. All tubes which showed the production of and gas at the end of that period were counted as verified fecal coli- forms. ~~ The : final | reported count was. then. adjusted accordingly. 7 18- The fecal streptococcus group are defined as those gram positive cocei belonging to the Lancefield's gtoups D-and Q.- The ‘normal habitat of the fecal streptococcus is the intes-° tinal ‘tract of man and other warm blooded animals. These “organisms too ‘are good indicators | of. fecal pollution. However unlike the fecal coliform, which comes piinariiy from man, fecal streptococci cotie primarily from “animale. _ When the fecal coliform count ois compared) to the fecal “streptococcus count, a ratio is established. This ratio can then be used to determine whether the source of the pollution is primarily from man enhanced sources = ‘such. as sewage - cr from animal sources ~ even as runoff fron animal feed lots or whether - “ait might be a mixture of both. | As. was” the case with both the ‘total ‘coliform and the fecal: coliform, the fecal streptococcus can be analyzed for. _ by either the MPN or MF techniques. ‘ Also, as were the-bades for both the total coliform, and fecal coliform, the fecal | gtreptocgecus” -y were analyzed by the MF technique. for the game... reasons as the total coliform | nd fecal ‘coliform. , As in the previous case > each sample was run as a = ‘duplicates each replicate. e nsisted of filtering 100m1_ ‘of sample through a 0. 45 ‘um “Type. 1A filter (MidLipore) which was, then _ placed on KF streptococcus Agar: (Difco) to. which. one ‘aillititer of ay sterile 12 solution of rre (2, 3, Ss “Triphenyl-— Qe tetrazolium. chloride) was added for each 100 mL of sterile. medium. The plates were then incubated at 35°C +.0.5°C far “48 hours. Tn an additional experiment, another exact set of dupii- cate plates using only loose fitting lids were run,. ‘They too were incubated -at 35°C + 0.5°C, but this time under a CO» ment was run to see if CO) incubation would significantiy increase the recovery of fecal streptococcus, ‘It is known that many Streptococci grow better under COp. In both cases, after the 48 hour incubation period, the | plates were examined and counted, Only the pink to dark red colonies were counted. Up. to ten colonies were then picked and. subjected to verification. The verification procedure N consisted of picking the pink to dark red colony to brain heart infusion agar (Difco) slants and to brain heart infu- “sion broth (Difco). After 24-48 hours of incubation at 35°C + 0.5°C the culture was tested for catalase activity, A Loopful of othe: growth from the brain heart. infusion agez slant was transferred to a clean slide and a few drops of. 3% hydrogen peroxide . (1995) ‘were added to the smear. Tf a positive reaction occurred (presence of bubbles) the tube was discarded az a noniustréptococcal species. Ifa negative test was recorded, a loopful from the brain heart infusion proth culture was transferred to fresh brain heart infusion broth =20-- and also ‘to a tube of brain heart. infusion broth” plus 40% bile. These tubes were then incubated at 44.5°C + 0.2°¢ and 35° C+ 0.5°C respectively. Growth in both tubes within 48 hours. was considered positive verification. Like “the ‘total coliforms and fecal coliforms, the finel fecal “atreptococeus count was adjusted accordingly. | The ‘standard plate count tells something about “the overall ‘treatment efficiency of whatever process is being. used. ‘While there are no current regulations in regards to the total number of organisms per milliliter of sample, the limit of 500/mL or less has been suggested as a bench mark figure. . However this figure of 500/mL is based upon growth “on a8 nutrient rich medium called plate count agar. In January of 1985 a new medium was reported in the literature® which 7 yields significantly higher “gounts (Reasoner | and Geldreich, 1985). The new medium instead of being a nutrient rich medium is a minimal nutrient agar called R2A. his. medium has rapidly gained acceptance in the field of Environ~ “mental Microbiology because less than one year after its formula was published, it became commercially available. The standard plate count has recently been changed to the hetero-. trophic plate count in the 16th edition of Standard Methods for the Examination of Water and Wastewater. Due to. the flux in this area, both media were used and run in. parallei. For -21- “purposes of this report the standard plate count will refer to those results obtained using plate count agar, The hetero~ trophic plate count will refer to those results .o o rt 05 fed “ (ru the using R2A. With both media, three dilutions wer: run on sample, and each dilution was run in duplicate. . The standard plate count using plate éount agar, (Difeo) can be obtained by using a pour plate technique, or a spread plate technique. It does not readily lend itself to a men brane filtration (MF) technique. The pour plate technique has several disadvantages that. prevent the recovery of the maximum number of organisms by whatever methodology for ‘determination of total bacteria, . The major drawbacks are ‘the user of tempered medium which. “may cause heat-shock to stressed bacteria, | and the use of a nutritionally rich medium - such as plate count agar 4 "which may likewise cause shock to starved bacteria. The “spread plate technique has two major advantages. The. primary advantage is to eliminate the heat-shock caused by the use of tempered agar’. The other major advantage Leo that all the colonies are on the surface of the agar where they can be readily seen and counted, ‘and can be easily. ‘distinguished from particulates and air bubbles. The one major disadvantage. lies in the fact” that . only relatively small volumes (0.1 ~ 1.0 ml) of samples can be used with this procedure depending on the absorbancy of the agar. In- spite ~22- of this drawback, it was felt thet the advantaged ‘outweighed: the disadvantages and the spread plate technique was chosen. | The heterotrophic plate count using R2A can be used as @ pour plate, spread plate, or as a membrane fiiter Me) “method. While it has. been reported that the spread plate ‘technique “gives better results than either the pour plate or ME techniques, it suffers from the threat of Mearry-off™ when using the ‘glass rod Or. “hockey stick", and aiso from spreaders on plates which are not perfectly dry. The disad- vantage of using a pour plate was seen, so while counts may not reach. the theoretical maximum with the MF method, nonetheless, the MF method was chosen for the reasons that it is relatively fast and inexpensive while still yielding: + good results. , , , , : The dilutions we used with both media were 1 m1, O.1 mi and 0.01 mL -. Later on when it became obvious that the heterotrophic platé, Counts’ we were seeing were higher than the ‘standard plate eountd: at the same dilutions, the i mi dilution was dropped and a 0.001 mL dilution added. The plate count agar was incubated at 35°C + 0.5°C for 48 hours, while the R2A was incubated at 30°C + 0.5°C for géven' days... After ‘the appropriate incubation period, all the plates ‘were counted.” The standard plate-count plates (100 x 15mm plates} were. counted with the aid of a ‘Quebec Darkfield colon; counter. © The “heterotrophic plate count plates (50 x 9 am) -23- wete counted with the aid of a 10-15 X binocular As has been stated earlier, an attempt was: isolate Salmonella spp. from éach potable water was a direct response to the 66 cases of typhoic St. Croix in 1985, in which the potable wat thought to be involved. The procedure is ‘a modi one: listed in Standard Methods for the Examination and Wastewater. The procedure followed is an-ext total coliform provedure. After the 24 hour ~ ‘total coliform plates were examined for typical and atypical total coliforms. A count of the typical green metallic sheen, colonies was made, and up to ten colonies were chen picked’ for verification. In, the event no typical colonies were “geen, “up to five atypical red colonies were picked and veri- fied to ensure that no false negatives were missed. At this point the membrane filter was aseptically removed from the “m-Endo. agar, and transferred to a clean, sterile, ampry 50 * 9 mm petri dish. To the petri dish which membrane filter, 10 mL of medium is added. consists of m-tetrathionate broth base (Difco) to been added a 2% iodine solution, 1:50,000 bri & green dye and 3 .mg of l-cystine per liter of the tetrathionate broth base.. The plate was then gently agitated, and Loopfuis of the media streaked out in duplicate on bi (Difco), brilliant green agat (Difce) and BLD. LD ho These = plates were “designated as TT1 (Tetrathionate) at 0" hours. The tetrathionate cultures, brilliant green agar, and _XLD agar plates were then. incubated for 24 hours. The bismuth sulfite agar. plates however were incubated at 35°C + 0.5°C for a total of 48 hours. After: the 24 hour incubation period, the qT1 cultures were removed, and 1 nl was aseptically transferred to a tube : containing new ‘tetrathionate proth. This tube was then designated as TT2. Plates of bismuth sulfite, brilliant green, and XLD were again streaked in duplicate, at 0. 24, and 48 hours. These plates then bore the designations T72/0, 192/24, or TT2/48. , , (After the. appropriate dneubation' time (24 hours for the brilliant green agar and XLD agar plates, and 48 hours “for the bismuth sulfite agar plates) the plates were ‘examined for both typical and atypical-colonies. An agar slant is created by pipetting a given volume of a molten agar into a test tube and tilting the test tube at an" angle to create an oblique surface apor which organisms ean grow. All slants will have two parte: The agar surface (air/agar interface) and the butt (solid agar/anderobic. zone) located at the bottom of the test tube. , Two types. of. colonies on the bismuth sulfite plates were: picked to dutvient: ager (Difco) slants. © One of these gole~ nies were the black and brownish-black colonies which were. “=25- surrounded ‘bya brownish-black zone exhibiting a “metallic sheen. The” other type. of colonies picked. were the flat or slightly raised green ones. The media, plate and “eblony color were recorded on each slant, On the brilliant green agar plates, pink~white opaque colonies. surrounded by brilliant red media or’ any ‘ted “eolo- nies. were picked to nutrient agar slants. On the XLD agar slants, red colonies, or red colonies with black centers were picked to nutrient agar slants. As before, media, plate and colony color were recorded on. each stant. ‘ALI nutrient agat slants were then incubated, ‘for 24 hours atl 35 °C + 0. 5 °C for 24. howrs. After the 24 hour period a “Loopful of the organism was | gtreaked out on MacConkey ager “(witeey.. nen lactose fermenting organisms (those that appear as colorless and translucent) were then picked “to, triple gugar iron eger (TSTA) slants (Difco). Those TSIA slants. ‘that gave an alkaline surface. and an acid or, acid and. gas butt along - with the production of hydrogen sulfide (CH) 8) were then submitted to the oxidase test, phenylalanine test, urease test , and ONPG test. If the reactions for these- four test were all negative, the culture was then inoculated into an API-20E strip. (Analytab Products Inc.) All strips which coded out as “Salmonella were then. reported out to the ‘appropriate authorities as PRESUMPTIVE for the presence of | Salmonella spp. with a culture submitted to public health for eonfirmation. — ~26—. RESULTS AND CONCLUSIONS The results of the chemical and physical analyses for! St. Thomas and St. John are summarized in Tables il and 2 while the microbiological results are summarized in Tables 3 and 4, The findings for St. Croix are presented in Tables 5 and 6. In Tables 1, 3 and 5 the data is presented chrono- logically “while in Tables 2, 4 and 6 the date is listed ac- cording to site. The Federal quality standards for drinking: water are listed in Table 7. | , All “water samples taken fell well within the limits set for color, dissolved oxygen, turbidity, pH, “conductivity, solids, nitrates, calcium, chlorides, silica, sodium, and magnesium in drinking water. Iron. did not fall within ~ drinking water limits. This is not suprising given the condi- tion of the distribution system. ‘Thus exiéept for iron, from the chemical standpoint, water distributed by public: pipe- _ lines -in the Virgin Islands apparently easily meets the Federal water quality standards for drinking water.. “The failings of the drinking water program lies not in the chemical realm, but rather in the microbiological realm. The microbiological problems stem from the almost total lack of free residual chlorine in the potable water distribution lines. This is of major concern because it is the free. residual chlorine which acts to check the spread of disease -27- ATELYWIXOUddY CGDEHD . NWHL SS41 [>] aTavTivAy LON. [¥/N]} daLoabad LON {an] {1 tstoawas 6° Le 9b ".0 7° OP. S8 6T ‘O2@T 88°0 Ley 98 z°0 0. ¥/N 9 L°L ye 8°90 820 €z2 anc ec. evge 8 6§ 650 L°S€ €°OL 0c 8tt 93°0. LOS 8 Z°0 U/N: L°9 Lek «CE 9°60 9°0 cz nar ze e°ge L£L°O0 s°Tte $°S9 gt “G°tt SS°0 18 OL ZO | W/N. 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ABR 28 ND 0: 1 0 0 5,300 140,000 72 JUN 2 ° ND mt 39 2 0 N/A 51,000 3 APR 21 ND 0 24 0 0 23,000.” 20,000,000 - 13 “MAY 27 ND 0 19 0 0. 5,300 TNTC - ra. APR-21. <0.2 0 ) 0 0 5 300° - 120 - T4 MAY 27. 0.4 0 3. 0 0 N/A TNTC - as) MAY 13 <0.2. 0 3 () 0 4 : 7100 os TS. JUN 17 ND 33 y 0 1 10 130 a Té. ‘APR 2100 «OND sO 9 0 0 3,600 35,000: - T6 | MAY 27 ‘ND 72 120 1 1 22,000 UpNTC - omy? MAY 13 ND 0 5 OO 1 100 “47,000 - 17. JUN 17 <0.2 2 0 0 50 1,200 = T8 MAY 2 woo «179 89° 6 0° 32,000 29,000 © - Ts... JUN’ 9 ND | 19." 1 “4 374,000. .. 240,000. - “4 org MAY (6 ND. 77 15°. O° 1 1,000 40,000 = 79 JUN 9 ND 0 30 2 66,000 610,000... - » P10 APR 28 ©. -ND 0 18 0 0 51,000 91,000 ~ - T10 JUN 2 9.2 0 ee 1 Q ND. 59 a 11. MAY 13 ND 0 8 0 0 130 37,000 Je Tll JUN 17 <0.2 0 0. 0 O° <10 30 - . "t 12 MAY 6 ND 184 328 Loe 1 32,000 40,000 - T12 gon-9 ND 2 a 0 6 520 75,000 - gl MAY 19 ND: 51 21000 | 6 B2 43,000 - 49,000. + Jl JUN 17 3 oO . 5 0 9 10 140 - 52 MAY 19 ND 1 >600 L 0 810 -y, 0008 © + 32° JUN 23 0.6 1 0 0 0 10 160° 2 33° 5/19/86 ND 1 - 600 “2 oa 2,000 410 - “33 6/23/86 0:8 0. 2 0 0 90 - 60 - SYMBOLS: {ND] NOT DETFCTED: {TNTC]. TOO NUMEROUS TO COUNT [<].LFSS THAN : ([>] (GRRATRR TRAN {+] PRESENT [=] -ABSFENP pmtj ~3]- . GILIILIG LON [ aN) xw2yo SN S887 BI ts 10aNAB t o 9 gee NC 18 9°6 Te Of NNE «STIX 0 Q 6IZ aN ‘aN 0z ya. Of Of NOE FTX , 0 EL ' aN an OL 6°9 62 «OL NME —- «ETX St 0 It eat. ° aN. ° an 89 29. «62 «(«OE NAL | OUT “tT 0 L Ze. z70> dN 99 079 «2 «(COE NOE OTT T o- 0 0 2*0> 2*0> OfT 9°9 9€ OE NNE. ° OTX t t tt 692.0 NON OL te ve 0€ Nac 6X Q 0 0 0 aN "aN o€2 . “LOT Ee. -0£ NAL BX L 0° oz”. z- N aN’ el $9. S€ Of NMC | LX T a) € 0z aN aN 18 9*L 9. 0 ne 9X 0 0 T t aN aN PL 9°L Of Oe NOE SX t i) ot eyt. ON aN 8 «TTL: SE. OE NAL yx “9 “9 € s aN GN opt = $6. £€ OE NOL eX: 0 0. 0 o- aN aN oft . “f6 €€. O€ NMG = 2x 0 0 91 ot a aN Q6t 94 O€ Of.NME = TX 0 0 8 0 aN aN “$T.uav . SIX 8 0 92 0 z*0> aN St wav PTX 0 0 g 0 20> aN ST Yay GTX 0 0. € "9 z70> aN -GT Maw 21K: ) ot et 0° Ort | 9°0 St Yay TTX 9 0 it z g't OT. $tWav OTK T 0 T ) r°0 v0. SU wav 6x y 0 00z “02 aN aN St wav 8X. ) Q ) 0 v0. Z°0 ST Mav “ 0 0 z 0 z°0 z°0> ‘ST Maw OX z Q Zz 0 -%"0> aN St wav SX 0 ‘) zz 0 20>. aN ST Wav) PX 0 0 ) 0 aN aN ST ¥av™ eX 0 0. € t z*0> aN ST ¥av 2X 0 0 0: 0 2709 an ST ¥av 1X TW OOT/ TH. 00T/ Tw oot/ . TW oOT/ © .T/bu 1 /bu cuo/soyw - 2° ‘LaggS © WOUEII0D aSOLWT. WOTTON -T2. - =~ ZLI8 Wwogd qwoaa HON WLOL TWLOL . 9aud ano ud 4WaL aiva alan XTOID “aS Zoy- seskeuy TeotsdAud ; “Gg eTqeL pue TeOTHOTOTGOzTW Ewe) SITNSOY TeoTboTouoxyo xud Nyy ssa7 [>] aguoaiga LON [aN] *STORWAs t 0 9 “YEE. aN aN 1a g’6 Te OE NAL SIX. 0. 0. 8 0 “aN aN ST ad GTX 0 0 6tz € aN an . oz v8. Of -0€ NAL TX 8 o 92 0 @0>- aN -.GT-udwv #TX y 0° €L Y ON ~ aN OL 6°9. 6% - OE NOE sOETX 0 o 5 0 z*09 aN a St wav £IX, St O° tt est aN aN. 89 zo 62 COE NAL CTX 0 0 £ 0 20> aN. oo “ST wav zIX T 0 L zel. z*0> “NS 99 09. «@e .. 0€ NaC TIX 0 ot Et 0 Ot 970. “ST aw tlk. 1 0 O° “0 z°0> z"02 oft | °8'9) «9 ~- «OE NMG OT 9° Q Lt z s°T ort . St Nav OTX. - T ot tt 692 aN aN OL. T's ve o€ Nac 6X t- 0 t 0 v0 "0 - ST wav 6x. 0 go 0 0: aN aN "get utot’ zoe NAL aX * Q 00z oz aN an: aa "Gt day eX T Q 0z A ON ‘aN tL s79 SEE NA LX 0 o- 0 0 0 z*0 a GT Wave ix t 0 € oz. aN aN icy gv 98... Of NOL 9X Oo 0. z 9 z°0 z*0> ~§t Wav 9X 0 0 t I an’ aN” lL 9°s. 08. 0. NOE sx z 0 z 0. z*0> -aN ST wav SX T 0 Ot. €PT aN” dN. 8 TL St Of NOC- +X 0 0 zz 0. z°0> aN ST ua¥ 9X “9 0 € 5 “aN: - aN “OvT- 6°6 €€ . Of NOL £Xx 0 0- 0 0 aN aN St ua¥ eX 0 0. ‘9 0 an aN et L6)©= se Ne x. 0 0 E Tt 20>. aN GT waw 2X 0 0 Ot a “aN an O6T - gL «Ok OE NOL. 1X" 7) 0 0 0 -&"0> aN st wav 1 Tu Qot/zed tw OOT/ TH OOT/ TH OOT/__- TH DOT/ T/bu guo/soys "aauzs WHOdTTOD 3SOLOW1 . WHOdTTOD —_-TD ID ne a “gars qvogd IYO" = “NON TWLOL = TYLOL agua axoo nd) awat gawd a1anvs. eats o3 ‘BuTpr000V. pednoz9. XTOID- ag TOF sosiTeuy TeoTsdua pue TeoTbOTOTqor0 TW FO, SI TNSoY ‘9 OTIeL -33- ‘Table 7. U.S. E.P.A. Physical-Chemical Standards for Drinking Water Free Residual Chlorine Color. Dissolved Oxygen . Turbidity pH Conductivity Total Dissolved Solids. ‘Nitrate Calcium Magnesium Sodium. Chlorides Iron , Silica 23 4- Maximum legaus concentration 0.2 mg/L 15 color units 3 mg/L 4 NTU | 6.5 - 8.5 ph units 50 Af MHOS : , 500 mg/L 10 mg/L 250. mg/L 100 mg/L 250 mg/L 250 mg/L 0.3 mg/L _UNK causing organisms. It. has been shown that without free residual chlorine, the: risk of having ‘a viable pathogen “present is indreased significantly. “(Kutcha, J, M. et. al. 1983; Burke, V., et. al. 1984; Keswick, B. H., et. al. 1985; Geldteich, E. E., et. al. 1978; Craun, G. F. et. al. 1978; McCabe, L. 3. 19785 Natural Resources Council 1977; American ‘Public | Health “Association 1985). The fact . that almost _ without exception all the samples taken showed standard plate ‘counts in excess of 500 total organisms per milliliter proves - beyond a doubt that there is inadequate disinfection occuring. throughout the distribution lines, even though the majority of the samples were negative for the presence of the standard total coliform and fecal coliform bacteria. ‘Since it appears. “that there is inadequate disinfection occurring in the lines, “it is no real surprise when on occasion, a total coliform or | tue is found. | Nor was it surprising to find after a lateral ‘line. to one of the local housing projects had broken in two places, that the numbers of total coliform, “fecal coliform, and fecal streptococcus, had increased significantly “and | - that once “the line was fixed the numbers, dropped” back to ‘normal levels... , The value of the total coliform analysis has been shown — time and time again (McFeters,G. A. et. al. 1978; Craun, G. ‘F. 1978; Allen, M. J. and Geldreich, E. EB. 19783; National -35- Research Council 19773. American Public Health Association 1985; Bolt, T. L. 1973; LeChevallier, M. W. et. al. 1983; McFeters, G. A. et. al. 1986) even though questions have arisen about its use as the sole source in determining water quality. This study supports its validation. .In short, if total coliforms are found in any significant “numbers “other potential pathogens might be found. This study also supports the. conclusions found in other studies which suggest that if total coliforms are absent, the water is safe to drink when in reality it is. not. Both situations were poignantly realized during the investigation of the st. John . potable water lines: At the main storage tank no free residual chlo- rine was detected, thus it was not surprising £6 find. a total ‘coliform count of 51 with more than 1000 other -non-lactose fermenting Organisms present, a fecal coliform count of. six, a streptococcal count of 91, and a standard plate count Of 4300. - Again, it was also not a great surprise given these numbers to end up isolating a Salmonella species. Less than a quarter mile away at the Agricultural Sta- tion another sample was taken. Here, after a total coliform count of. one in the presence of around 700 other non-lactose fermenting organisms, a fecal coliform count of one, a Fecal. streptococcol count of 34, and a standard plate count of 810 was obtained. “While contaminated, it is not overly so in ‘comparison to other samples. From this point it was very surprising to isolate a Salmonella species. (=36- 7 Taken from individual standpoints, assuming that “the samples had not been taken together, but alone, and that had microbial analysis been limit d to the standard total coli- ‘form analysis alone to determine the suitability of the: water for drinking purposes, the main storage tank which showed 51 total coliforms would have failed to pass acceptable levels. Contrastly, the other sample taken at the Agricultural Sta- tion which showed only one total coliform would have met existing minimal water quality standards when. in facet - health hazard was. present. , , Several conclusions can be drawn from this study. The “basic conclusions are: 1) %If-no free residual chlorine levels:are found assume the probability of finding high num-_ bers of non-lactose. fermenting organisms to be high, thus do a standard plate count. 2). Just because a water ‘supply shows only. one | oe total coliform, or ‘no total coliform, and | ‘the amount of. background growth (the “non— ' lactose fermenting organisms) is high . and- there is an absence of any free residual - chlorine, then the supply should not. be assumed safe. : 3) Montlhy > analysis for fecal: coliform and fecal streptococcus should be: performed in addition to the. total coliform and. standard plate count if the lack. of the free residual chlorine levels. are found on..a continuous — basis from any given point. . Alternatively, both analyses should be performed as part of. ‘the check sample . “if the previous ‘sample showed more than one total coliform. 237+ 4) I£ the sample showed unusually high numbers of total coliforms - more than 16 -—- and. an unusually high standard plate count - greater than 1000 - then in addition to the total coliform, fecal coliform, fecal streptococ- cus, and standard plate count, do an analysis for Salmonella. The key to assuming a safe supply of drinking water is to insure that there is an adequate amount of free residual chlorine available in the lines at all times for disinfection - purposes. . , , ‘If free residual chlorine levels cannot either be tain- -tained or assured on a continous basis, then the alternative -is . to more closely monitor the bacteriological quality of that water.” But this would both be more costly and time. consuming. “only in these ways can we be relatively certain “that the water supply is safe to drink. From an unbiased point of view it would be a lot easier to properly chlorinate the supply in the first place, and if aeéeasary install supplemental automatic chlorine injectors throughout the distribution network to prevent over chlorina- tion in ‘one particular area or under chlorination in another. —38- ‘American Public Health Association. 1985... Standa Bott, REFERENCES. Allen, Martin J. and Edwin E. Geldreich Jr. (1978, Evaluating the microbial quality of potable waters, p. 3-11 In Charles W. Hendricks (Ed.) Evaluation: of the microbiology standards for drinking water O38. Environmental Protection Agency, Washington, Dec . ard methods for the examination of water and wastewater, 16th. ed. American Public Health Association, Washington, D.C. T. L. 1973. Bacteria and. the assessment of water quality , p. 11-75. In ASTM STP528. Biological. methods for the assessment of water quality. American Society for Testing and Materials, Washington, D.C. : “Burke, Valerie, Jennifer Robinson, Michael Gracey, Dennis Peterson, Norman Meyer, and Vernon Haley. 1984, “Isolation of Aeromonas spp. from an unchlorinated domestic water supply. Appl. Environ, Microbiol. - A483 23 369-370. , Graun, G. F. 1978. Impact. of the coliform standard on the transmission of disease, p..21-35. In Charles W. Hendricks (Ed.) Evaluation of the microbiology standard for drinking water. U.S. Environmental ‘Protection Agency, Washington, D.C, Geldreich, EsE., MJ. Allen, and RH. ‘Taylor. 1978 -Intereferences to coliform detection in potable water stpplies, p. 13-20 In Charles W. Hendricks (Ed.) Evaluation of the microbiology standard for ‘drinking water. U. S. Environmental Protection Agency, Washington, D.C. Keswick, Bruce T., Terry K. Satterwhite, Philip C. Johnson, Herbert L. DuPont, Sandy L. Secor, Jo-Ann Bitsura, G. William Gary, and John C. Hoff. 1985. Inactivation of Norwalk virus in drinking water by chlorine. Appl. Environ. Microbiol. .50:261264. -Kuchta, John M., Stanley J. States, Ann M. McNamara, Robert M. Wadowsky, and Robert B. Yee. 1893... - Susceptibility of Legionella pneumophila to chlorine in tap water. Appl. Environ. Microbiol. 46:53:1134- 1139. ~39-