The Analytical Approach
Irwin H. Suffet Department of Chemistry Environmental Studies Institute Drexel University Philadelphia, Pa. 19104
Edited by Claude A. Lucchesi
Patrick R. Cairo Research and Development Philadelphia Water Department Philadelphia, Pa. 19107
Cooling Tower Reservoir on Roof
AC System
1A
Water Tanks
Drinking ( Fountain 18th Floor Carbon Filter 17th Floor \
In Search of the Cause of Legionnaires' Disease
Ice
1 Γ*
9th Floor
Machine Lobby
Filter Cleaning Room Broad Street Figure 1. Schematic diagram of water system of convention center hotel
Transmission of disease occurs through a vector, e.g., insect, water, soil, air, or food. The cause (etiologic agent) and the vector of Legionnaires' disease, which occurred in Philadel phia, July 21-24, 1976, were heing sought by the Center for Disease Con trol (CDC) (Atlanta, Ga.) and the Philadelphia Health Department. Yet, three weeks after the outbreak of the disease, no agent or vector had been found. Theories abounded! Symptoms of Legionnaires' disease closely resembled those of pneumonia. T h u s , to separate victims from back ground cases of pneumonia that would normally develop in a city the size of Philadelphia, CDC selected both clini cal and epidemiologic characteristics. Persons were classified as victims of the disease if they possessed fever and chest x-ray evidence of pneumonia or a temperature greater than 102 °F and a cough. To further narrow the defini tion, they had to have attended the American Legion Convention or have visited the convention center hotel be tween -July 1 and the onset of illness. 0003-2700/78/0350-875AS01.00/0 © 1978 American Chemical Society
Statistical analysis of 182 suspected cases ultimately revealed that a typi cal victim became ill seven days after arriving in Philadelphia and had a temperature rise to 102-105 °F. Since these symptoms could be produced by numerous types of microorganisms and toxins, the investigation was broadened to include all possible envi ronmental factors. A cross-connection survey of the convention center's water system, con ducted on August 8, 1976, showed many violations. Either the wastewa ter or the air conditioning system could have contaminated the hotel drinking water system. A cross-con nection could bring toxic substances into the water supply or perhaps bring organic compounds that would react in a synergistic manner with com pounds commonly found in part-perbillion quantities in drinking water. Also, possibly, an agent from a crossconnection, such as dichromate (an antifouling chemical in the air condi tioning system), could change a set of trace organics in some unknown fash
ion into toxic substances. In addition to helping victims of Legionnaires' disease, protection of the people re maining in the hotel was also a major concern. Would there be a subsequent outbreak? Were cross-connections or toxic compounds involved? The Phila delphia Water Department, asked to investigate the environmental systems of the hotel, reviewed with health agencies their findings and learned t h a t a thorough evaluation of the trace organics in the hotel drinking water system had not been undertaken. Thus, an effort in cooperation with Drexel University was initiated im mediately to study the drinking water and the air conditioning systems. How to study water entering and circulating in a 50-year-old hotel was a major problem. Drinking water en tering the hotel could come from two treatment plants, each obtaining its water from different surface water supplies, the Schuylkill and Delaware Rivers. T h e two water sources then in termix within the distribution system and enter the hotel through two water
ANALYTICAL CHEMISTRY. VOL. 50. NO. 9. AUGUST 1978 · 875 A
mains. Where should samples be taken? How could representative samples be obtained? What isolation methods were readily available, and which should be used? How could the source of water entering the hotel be determined? How could a comparison of analyses performed at different locations be made? It should be emphasized t h a t a comprehensive trace organic survey of any site in a complex water distribution system had never been accomplished. No protocol or standard methods existed for this type of study, and adaptation of laboratory methods to field locations was necessary. T h e effect of chlorination and detention time on the trace organic content of a drinking water has only recently been studied and still is not fully understood. Data from many water treatment plants indicate that each drinking water may be unique in composition and chemical reactivity. Studies of collected samples show that even treated water is of ever-changing quality. Recent studies at one of Philadelphia's Water T r e a t m e n t Plants showed a variation in trace organic content during a week of continuous composite sampling. The chlorinated material also has been shown to change with time. In fact, the rate of change may be different for each sample. Furthermore, at a specific location in a complex water distribution system, such as the one servicing the hotel, the water may be comprised of a changing mixture of river sources since water flow is controlled by consumption.
Sample Site Selection With limited time available, the choice of sampling sites was extremely important. To ensure t h a t no possible contamination source would be missed, sampling locations were chosen to follow the path of the water through the hotel distribution system (Figure 1) from one water main (IB) to the tanks on the 19th floor (1C, ID) and then down through the other floors (9th and lobby, I F and 1G). All these samples were collected simultaneously on 8/25/76. In addition, a cross-connection with the air conditioning system (IE) on the 19th floor was sampled on 8/25/76. Two special sites at the input and output water from a drinking water fountain outside of a ballroom on the 18th floor (2E, 2F) and at an ice machine on the 2nd floor (3E, 3F) were sampled along with the cooling and condenser water from the air conditioning system on 8/27/76. T h e second water main (1A) also was sampled on 8/27/76. These sites were representative of the location where attendees at the Legionnaires' conference could obtain water that might be a vector of the disease. Representative Samples and Choice of Isolation Method No sample sites are ideal, no isolation method is totally efficient, and no method of obtaining a representative sample is time tested. Consequently, one must make choices based upon the state-of-the-art at that time and the pragmatic considerations of manpower and equipment available.
Figure 2. On-line sample collection and isolation system as operated in screen washing room located in subbasement of hotel Walnut Street water main (2A) sample collected at this point. Continuous composite sample after dechlorination and pH adjustment fed continuously to on-line MRR-XAD-2 macroreticular resin sampler; at overflow, sample collected for subsequent continuous liquid-liquid extraction 876 A ·
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1978
T h e investigation reported here has enabled the development of an analytical protocol for the study of future emergency water problems. T h e decision was made to utilize a composite sampling method to obtain representative samples throughout the hotel's distribution system. Eighthour composite samples were collected during midweek working days. Macroreticular resin (MRR) XAD-2 and continuous liquid-liquid extraction (CLLE) were the isolation methods used. They have been used successfully in identification of trace organics in drinking water in Philadelphia since 1975 (1). T h e M R R and C L L E equipment was operational, and the manpower was experienced in its use. These isolation methods are useful for analysis of the "neutral"-type organics, boiling between 40 and 230 °C. Figure 2 shows the sampling apparatus at a location in the hotel's subbasement where a water main sample (2A) was collected. Comparison of Samples Collected at Different Locations A gas chromatographic "profile" method was used to compare samples (2, 3), and selected samples were run by G C / M S to identify the components of these samples. T h e analytical approach used to study the trace organics present in the hotel's water system is shown in Figure 3. Of prime importance is the GC profile evaluation in which the question of possible contamination may be considered. A GC profile is a fingerprint of a complex group of trace organics present in the extracted water and constitutes an information pattern of the sample. By plotting peak area or height proportional to a standard on a relative retention time scale, many GC profiles can be compared to obtain an understanding of the spatial changes in the hotel's water system. A unique GC profile computer program was used to study the difference in the trace organics between samples. It must be emphasized that differences between GC profiles can be caused by different compounds or different relative concentrations of a mixture of compounds in a sample. GC profiles are used to observe peak changes and to aid in the selection of samples to best utilize severely limited GC/MS analysis time. A cursory evaluation of the GC profiles of the organics present through the distribution system showed it to be extremely useful in demonstrating differences between samples. When G C / M S was used to identify a compound, its identity was considered confirmed if it matched the mass spect r u m of a pure reference compound and if its relative retention time on a 20% SE-30 packed column was the
Drinking Water
SAMPLE SOURCE Representative Sample
TECHNIQUE
Special Sites 8-hour composite
Isolation Method
MRR-CLLE
Analysis
Programmed Temperature GC Packed SE-30
lii£U13ïa
Comparison of Samples
Capillary OV-101
Computer Reconstructed GC Profiles 9th floor sample
•«MMrfWHMJH
Air Conditioning System
GC/MS
Selected
retention time windows of other samples
(1 ) A general similarity among GC profiles at different drinking water sampling locations (including special sites) was observed. (2) No compound thought to be a cause of Legionnaires' Disease was found one month after the outbreak.
Figure 3. Analytical approach for identification of trace organics
same as the reference compound. In addition, tentative identifications were made where reference com pounds were not commercially avail able but the mass spectrum of a peak matched uniquely characteristic liter ature mass spectra. When needed, computer-assisted interpretation was provided by Cornell University's Mass Spectral Identification System P B M and S T I R S .
Was There a Difference Between Samples? A detailed GC profile analysis was performed on the organics in the drinking water samples and air condi tioning system. Figure 4 shows "spiked" GC profiles of samples ob tained at various locations by the M R R sampling method. Each spike represents a GC peak, with the height
Internal Standards Solvent Blank Set 2 ( 2 0 0 — - 1 ) After Filter (2F) Before Filter (2E) Lobby (1G) Ninth Floor (1F) 19th Floor (1E)
Water Tank (1C) Broad St. Main (1B) Walnut St. Main (2A) Queen Lane Drinking Water (9/13/76)
Relative Retention Time Figure 4. GC profiles on SE-30 of drinking water samples from convention center hotel Water samples collected by MRR-XAD-2 macroreticular resin column over 8-h period on 8/23/76 and 8/27/76. Sample 1 from one of the water treatment plants that feeds water to the hotel. Sample 2 collected on 8/27/76 and listed as 2A to show it was not collected on 8/23/76. V Missing in 9th floor, present in sample. * Missing in sample, present in 9th floor. •& Minor peak in carbon filter effluent, not in influent Peak lowered in intensity in effluent of carbon filter as compared to influent to carbon filter. Internal standards were 2-ethyl-1-hexanol (RRT: 0.78) dibutyl phthalate (2.00). All samples eluted with 200 mL ether evaporated to 1 mL
of the peak representative of the size of the original GC peak—either small, medium, or large (relative heights on the GC peak profiles are respectively, 0.2, 0.5, and 1.0 units). A reproducibil ity of ±0.015 R R T units was obtained during this study. Two phenomena were observed in drinking water samples with these spiked profiles: • A general similarity among GC profiles of M R R samples at different drinking water sampling locations (in cluding special sites) is obvious. T h e CLLE samples were very similar to the MRR samples except for concen tration differences (CLLE collection volumes were around 30 L at a 10:1 water:solvent ratio, whereas MRR vol umes were around 100 L). • Minute changes could be seen less distinctly. Comparison with the origi nal chromatograms showed t h a t some times shoulders on peaks could be seen, and at other times these were obscured by large peaks. This is dem onstrated more clearly by simulated 3-D profiles, with peak heights and widths of the profile actually mea sured (Figure 5). T h e CLLE samples from the air conditioning system were the only samples to appear quite different from the drinking water system. With an equivalent sample volume, more and higher GC peaks of different R T T ' s were observed.
GC/MS Identification of Trace Organics in the Drinking Water and Air Conditioning System T h e 9th floor samples were chosen as reference GC profiles for the hotel drinking water system since they con tained the most GC peaks of the larg est concentration. T h u s , it was rea soned t h a t if a compound that could have caused Legionnaires' disease was in the drinking water, it would be found in this sample or in a sample where a GC peak was missing from the profile of the 9th floor ( φ Figure 4). A detailed GC/MS analysis was completed with a 6% SE-30 column on the 9th floor MRR and CLLE sam ples and on selected samples at R R T ' s where GC peaks were missing from the 9th floor MRR sample. Also, a complete GC/MS analysis was run on the MRR samples for the drinking water fountain with the carbon filter. T h e carbon filter had been installed on this fountain for an unknown peri od of time. Table I shows G C / M S re sults for the 9th floor MRR and CLLE samples. GC/MS analysis of the se lected samples where GC peaks were missing from the 9th floor sample (Figure 4) showed that R R T shifts due to changes in relative concentrations
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST
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Figure 5. GC profiles of drinking water samples taken before and after a carbon filter on a drinking water fountain Chromatograms same as sample numbers 8 and 9 from Figure 4. Before sample plotted as 1st, 3rd, and 4th chromatograms, respectively
of mixtures of compounds eluting as single peaks was the primary reason for differences. Benzene (19th floor) and a branched C8 alkene (19th and lobby) were the only compounds in these samples that were not found in the 9th floor sample. Two possibilities are t h a t they were obscured by a larger peak or were added in water distribution. GC/MS of these CLLE samples added further confirmation of these components. Other CLLE samples were too dilute to allow G C / M S analysis. G C / M S analysis of the trace organ-
ics present in the carbon filter influent showed a set of compounds which was similar to those found in the drinking water system (Table I). Two new compounds were observed: phenylacetic acid and l,2:3,5-di-0-isopropylideneD-xylofuranose. This may be accounted for by the fact t h a t the influent sample to the carbon filter was collected two days after the drinking water samples. G C / M S analysis of the trace organics present in the carbon filter effluent showed the same compounds that were present in the influent except for the addition of trichloroethyl-
ene, diethyl phthalate, 2-dichlorobenzene isomers, dichloroacetonitrile, toluene, a dichloropropene isomer, and a C9 branched hydrocarbon. These compounds have previously been observed in Philadelphia's drinking water and are apparently being displaced by others t h a t are adsorbed. Most of the compounds found in the hotel drinking water system have been identified in drinking waters throughout the United States and are also part of the variable complement of trace organics found in Philadelphia's drinking water. Thus, it was evident that these could not be the cause of Legionnaires' disease. 1,1-Dichloroethane, a C5 alkene, methyl bromide, and phenanthrene or anthracene are compounds t h a t had never been isolated in any other Philadelphia drinking water sample, but a search of the toxicological significance of these compounds indicated that these should not have led to the disease. Table I shows the G C / M S analysis of the air conditioning system. Diethyl sulfate is an additional compound to the drinking water sample mixture of compounds. Since this compound was not found in any of the drinking water samples, contamination caused by a cross-connection at the time of sampling is unlikely. Once again, no obvious solution to Legionnaires' disease was found. Capillary C o l u m n G C
T h e decision was made to select several samples and run capillary column GC. Its high resolving power would be used to detect minute differences between samples. Portions of
Relative Retention Time Figure 6. Portions of capillary GC profiles on OV-101 of drinking water samples from convention center hotel These chromatograms from same samples as numbers 4, 5, 6, 8, and 9 from Figure 4. V Missing in 9th floor, present in sample. * Missing in sample, present in 9th floor.(v)lf peaks are very large, they are circled
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Table I. Compounds Identified by GC/MS in Bellevue-Stratford Samples Taken by Macroreticular Resin (MRR) and by Continuous Liquid-Liquid Extraction ( C L L E ) ' drinking water 9th floor (MRR)
HALOGENATED bromoform carbon tetrachloride chloroform dibromochloromethane dichlorobromomethane dichlorobenzene isomer tetrachloroethylene trichloroacetone dichloropropene isomers dichloroacetonitrile chloro-methyl-butene isomers 1,1,1 -trichloroethane 1,1 -dichloroethane 1,2-dichloroethane methyl bromide AROMATIC acetophenone benzaldehyde ethyl benzene or xylene isomers toluene m- or p-tolunitrile dimethyl or ethyl phenol isomer dimethyl or ethyl benzaldehyde isomers' C5-benzene isomer phenanthrene or anthracene MISCELLANEOUS 2,3:4,6-di- O-isopropy lidene- L-sorbof uranose dibutyl phthalate tributyl phosphate dihydroactinidiolide C5 alkene C6 branched hydrocarbon C7-9 branched hydrocarbons ethanol acetone diethyl sulfate trlethyl phosphate TENTATIVES ethyl sorbate 1 (2 or 4-chlorophenyl) ethanol 1,1,2-trichloroethane
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air conditioning system chill condenser (CLLE) (CLLE)
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