Environ. Scl. Technol. lQ88, 22, 1419-1425
Factors Limiting Success of Inoculation To Enhance Biodegradation of Low Concentrations of Organic Chemicals Baqar R. Zaldi,+ Yoshltaka Murakaml,t:and Martin Alexander" Institute for Comparative and Environmental Toxicology and Department of Agronomy, Cornell University, Ithaca, New York 14853
rn Corynebacterium sp. added to lake water rapidly mineralized 100 pg and 1.0 mg of p-nitrophenol (PNP)/L but acted very slowly on the substrate present at 26 pg/L. The rate and extent of mineralization of the lowest PNP concentration in Beebe Lake water varied according to the time the sample was taken and were directly related to rainfall, and presumably runoff, in the watershed. The addition of high concentrations of inorganic P or N to water samples collected after a drought period, during which mineralization by the bacterium was slow, enhanced PNP decomposition. Mineralization in Cayuga Lake water was increased slightly by 10 mg of K2HP04/L,but the enhancement was marked by 100 mg/L. The stimulation was a response to P and not K. Glucose stimulated PNP mineralization in samples from Beebe and Cayuga Lakes, and K2HP04further increased the rate and extent of the transformation. The addition of either of two eucaryotic inhibitors increased the rate of Corynebacterium sp. growth in lake water amended with 26 pg of PNP/L but decreased the rate of mineralization. We suggest that the successful use of bacterial inocula to destroy low levels of organic pollutants in natural waters requires more information on environmental factors affecting their activity or isolates that are able to grow rapidly at the low substrate levels. Introduction Several factors may affect the rate of microbial degradation of organic pollutants. Among these factors are concentration of the organic chemical (1, 2) and also of inorganic nutrients (3, 4). Concentration of the organic substrate may affect its susceptibility to microbial attack, and the ability of microorganisms to metabolize organic molecules at high but not low concentrations may explain the presence of low levels of certain organic compounds in natural environments (1). The concentration of inorganic nutrients may be important because many natural environments have low levels of such nutrients. Although members of the indigenous microbial community may be able to grow readily at low concentrations of limiting nutrients (5), bacteria selected to be used as inocula may not have such abilities. Several attempts have been made to stimulate the degradation of organic pollutants by bacterial inoculation. However, most of the studies designed to promote biodegradation by inoculation have simply been reported as successes (6)or failures (7,8),and no attention was given to identifying the reasons for failure. In contrast, Brunner et al. (9) reported that the mineralization of polychlorinated biphenyls in soil inoculated with Acinetobacter sp. was limited because of the lack of a suitable carbon source for the bacterium, and Goldstein et al. (10) provided evidence that limited movement of an inoculum added to the soil surface restricted the extent of mineralization of 'Present address: Department of Marine Sciences, University of Puerto Rico, Mayaguez, PR 00708. t Present address: Osaka Gas Co. Ltd., 6-19-9 Torishima, Konohana-ku, Osaka 554, Japan 0013-936X188/0922-1419$01.50/0
2,4-dichlorophenol by Pseudomonas sp. It has also been suggested that toxins present in pollutant-containing environments or active grazing by protozoa may suppress the activity of bacterial inocula designed to destroy organic pollutants (IO). The present study was designed to investigate some of the factors limiting the success of inocula added to lake water in order to bring about biodegradation. The test chemical was p-nitrophenol (PNP). However, when it became evident that the biodegradation rate at low concentrations was less than predicted from a linear extrapolation of the rates at somewhat higher concentrations, attention was focused on the factors affecting mineralization of low concentrations of PNP. Materials and Methods The culture was a strain of Corynebacterium sp. originally obtained from Beebe Lake, Ithaca, NY. An antibiotic-resistant mutant of Corynebacterium sp. was obtained by growing the bacterium in Trypticase soy broth continuing increasing concentrations of streptomycin, kasugamycin, and spectinomycin. Cell suspensions derived from these cultures were plated on Trypticase soy agar containing 100 mg of streptomycin, 10 mg of kasugamycin, and 25 mg of spectinomycin/L. The population size of this bacterium was determined by the drop plate technique (11) on 0.3% Trypticase soy agar containing the three antibiotics at the indicated concentrations, and triplicate counts were made of each dilution. The plates were incubated at 29 "C for 48-72 h before counting. Lake water was collected from Beebe and Cayuga Lakes, Ithaca, NY. The samples were used within 1 h of collection. In some instances, samples of lake water were sterilized by passage through 0.2-pm pore-size membrane filters (Sybron Corp., Rochester, NY). Analysis of Beebe Lake water before and after a rain in 1986 showed the presence of 2.0 and 6.6 mg of nitrate N, 10 pg of ammonium N, and less than 10 pg of total P/L, respectively. To measure mineralization, duplicate 25-mL samples of inorganic salts solution or lake water were placed in Teflon-lined screw-capped 200-mL bottles. Washed cells from 24-h-old cultures grown in salts solution amended with 10 mg of PNP/L were added to give initial cell densities of approximately 104-105cells/mL. Because it is the convention in microbiology, cell counts are given as numbers per milliliter. The salts solution contained 0.8 g of K2HPO4, 0.2 g of KH2P04,10 mg of FeC13, and 0.1 g each of ",NOs, CaC12.7H20,and MgS04 per liter of distilled water. All bottles received similar amounts of 14C-labeled compounds (1000-2900 dpm/mL), but the final substrate concentration was varied by adding different quantities of unlabeled compounds. The liquids were incubated at 29 "C on a rotary shaker operating at 120 rpm. At regular intervals, 1.0-mL samples were transferred from the bottles to 9-mL plastic vials, and the liquids were acidified with 0.2 mL of 1 M H2S04. Compressed air was bubbled through the liquid for 5 min to drive off C02, and then 7 mL of Liquiscint (National Diagnostics, Inc., Somerville, NJ) was added to 1.0 mL of each of the acidified samples. The radioactivity was counted with a liquid scintillation
0 1988 American Chemlcal Society
Environ. Sci. Technol., Vol. 22, No. 12, 1988
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100 UNNOCULATED
8C
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6C
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8
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Figure 1. Mineralization of 1.0 mg of PNP/L in uninoculated Cayuga Lake water and 26 pg, 100 pg, and 1.0 mg of PNP/L in water Inoculated with Corynebacterium sp.
counter (Model LS 7500; Beckman Instruments, Inc., Irvine, CA). To measure PNP uptake, between 1 X lo3 and 1 X lo4 Corynebacterium sp. cells/mL were added to 25 mL of filtered lake water containing 26 pg of labeled PNP (1.1 GBq/mmol) with and without 100 mg of K2HP04/L. Duplicate samples (1.0 mL) were taken at various time intervals in a 24-h period and filtered through polycarbonate membranes (0.2-pm pore diameter; Nuclepore Corp., Pleasanton, CA). Before and after addition of the samples, the filters were washed with 2 mL of 0.1 M Tris, pH 4.7. The washed filters were placed in 7 mL of Liquiscint, and the radioactivity was then counted. Controls with heat-killed cells indicated that a negligible amount of [14C]PNPwas retained on the filters during filtration. p-Nitr~[U-'~C] phenol (1.1 GBq/mmol; 98 % radiopurity) was obtained from California Bionuclear Corp., Sun Valley, CA. Antibiotics were obtained from Sigma Chemical Co., St. Louis, MO. All glassware was soaked overnight in concentrated sulfuric acid containing 15 g of Nochromix/L (Godax Laboratories, New York, NY), followed by rinsing in doubly distilled water. Results At a concentration of 1.0 mg/L, PNP was not mineralized in lake water in 7 days (Figure 1). Identical tests showed that PNP was not mineralized at 26 and 100 pg/L (data not shown). However, when Corynebacterium sp. was inoculated into Cayuga Lake water containing 1.0 mg or 100 pg of PNP/L, extensive mineralization was evident after 1 day, and nearly all of the substrate carbon was mineralized in 2 days. On the other hand, the bacterium only slowly converted 26 pg of PNP/L to volatile products, and even after 6 days, only 24% of the carbon was mineralized. Although the percentage of PNP that was min1420 Environ. Sci. Technol., Vol. 22, No. 12, 1988
A F U 16
2
4
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DAYS Figure 2. Mineralization of 26 pg of PNP/L by Corynebacterlum sp. inoculated into Beebe Lake water collected at various times in 1986.
eralized differed at the three substrate concentrations, the actual amounts of C remaining at the two lower substrate concentrations were quite similar. Because of the unexpectedly low extent of mineralization at the lowest substrate concentration, studies were conducted of factors affecting the transformation at these low levels. The mineralization of 26 pg of PNP/L by Corynebacterium sp. was measured in Beebe Lake water collected at various times of the year. In the week immediately preceding the February 11, March 3, April 12, April 16, April 22, and May 12,1986 sampling dates, 2.01,0.13, 3.15, 3.94, 1.57, and 0.00 cm of precipitation fell in the watershed, as measured in Ithaca, NY. Inoculation of the bacterium into lake water collected after the heavy rains in February and April resulted in extensive mineralization of PNP in 6 days, whereas only -10% of the chemical was mineralized in Beebe Lake water collected after the prolonged dry period in May (Figure 2). The extent of mineralization was also small after the light precipitation in the week preceding the March 3 sample. No mineralization was evident in uninoculated lake water in 7 days. To test whether the differences in PNP mineralization resulted from differences in the levels of N and P in the lake water, the bacterium was inoculated into filter-sterilized and nonsterile Beebe Lake water collected on May 12, 1986 during a prolonged dry period. After 4 days of incubation, at which time no mineralization of 26 pg of PNP/L had been observed, the triplicate 25-mL samples were divided to give six 12.5-mLsubsamples. Duplicates of the subsamples were amended with either 0.1 g of NH4N0,/L, 1.0 g of K2HP04/L,or nothing. Within 2 days after the addition of phosphate, 67% of the PNP was mineralized by the bacterium added to filter-sterilizedlake water, but little or no mineralization was detected in samples not amended with phosphate (Figure 3). The addition of phosphate to inoculated nonsterile lake water also enhanced the degradation of PNP as compared to water with no added P. The addition of inorganic N to filter-sterilized and nonsterile lake water inoculated with the isolate also enhanced PNP mineralization as compared to the comparable waters not receiving supplemental N.
IOC
I00
80
P w
26 UG/L,STERLE
60
a 0
i8
*
UG / L , NONSTERLE
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STERILE t P
20 28 U G / L .NONSTERLE
I
I
I
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Figure 3. Effect of the addition at 4 days of 1.O g of K2HP04or 0.1 g of NH,NO,/L on the mineralization of 26 pg of PNP/L by Corynebacterium sp. inoculated Into sterile and nonsteriie lake water collected after a prolonged dry period.
It is noteworthy that the stimulation could be achieved by adding either nutrient. Tests were also conducted with samples of Beebe Lake water collected on April 16, 1986 after 2 days of heavy rains. The water was amended with 10 or 26 pg of PNP/L, and some samples were sterilized by filtration before inoculation. Addition of the organism to sterile lake water containing 10 pg of PNP/L resulted in slow mineralization, and only 34% was mineralized in 12 days (Figure 4). The transformation was apparently linear with time. The percentage mineralization was somewhat more rapid at a PNP concentration of 26 pg/L, but much of the difference resulted from a more rapid percentage conversion in the first 2 days. Mineralization was more rapid and more extensive at both concentrations if Corynebacterium sp. was added to nonsterile lake water. No mineralization was evident in 6 days in the uninoculated nonsterile lake water collected after the heavy rains. At day 12, when 60 and 34% of PNP at 26 and 10 pg/L, respectively, had been mineralized in the sterilized lake water inoculated with the bacterium, the addition of 1 g of K2HP04/Lled to rapid mineralization of PNP at both substrate concentrations. A study was then conducted to determine whether mineralization in Cayuga Lake water would also be enhanced by supplemental P and N. In a sample of Cayuga Lake water taken on July 29,1987, no PNP mineralization was observed in 7 days. The addition of Corynebacterium sp. at an initial density of 6 X lo3 cells/mL to lake water amended with 10 pg of PNP/L resulted in slow mineralization of PNP; the cell density increased within the first day under these conditions, but the numbers never became large (Figure 5). The addition of 10 mg of K2HP04or NH,N03/L individually did not enhance growth or PNP degradation. In lake water amended with 100 mg of K2HP04 or NH4N03/L,the bacterium mineralized PNP
0
4
8
12
DAYS Figure 4. Mineralization of 26 and 10 pg of PNP/L by Corynebacterium sp. inoculated into nonsterile and sterile lake water collected after heavy rains. K,HPO, (1.O g/L) was added to some samples at day 12.
rapidly and extensively, and its population size was also increased. The addition of both K2HP04and ",NO3 at 100 mg/L resulted in an even greater enhancement of PNP degradation. Addition of K2HP04caused a pH change of 0.02 unit, so that the effect was not a result of a change in pH. The difference in the stimulation of mineralization between the higher N and P concentrations was statistically significant ( P < 0.05), as was the difference in water with N and N plus P (P < 0.01). However, because of the differences between the duplicate samples analyzed, the stimulation of mineralization by N in P-amended water was not statistically significant. To determine the effect of phosphate on PNP uptake by Corynebacterium sp., duplicate samples of lake water were amended with 26 pg of PNP/L with and without 100 mg of K2HP04/L,and they were sterilized by filtration and inoculated with the bacterium. At regular intervals, samples of the suspension were passed through 0.2-pm filters, and the radioactivity associated with the cells was measured. The effect of phosphate on PNP uptake was marked (Figure 6). PNP uptake by cells in phosphatesupplemented lake water was consistently greater than in unamended lake water. Moreover, the rate of uptake was biphasic in supplemented and unsupplemented lake water. To determine whether the enhancement of PNP degradation by K,HP04 was an effect of P or K, 100 mg of K2HP04or Na2HP04/Lwas added to separate lake water samples that had been amended with 26 pg of PNP/L. Corynebacterium sp. was introduced at an initial density of 1.5 X lo4 cells/mL. In lake water not supplemented with phosphate, the cell number initially declined and then increased somewhat before falling again (Figure 7). Under these conditions, little PNP was mineralized in 5 days. In water to which was added either of the two salts, Corynebacterium sp. grew extensively,and its population density exceeded 106/mL before falling. The addition of K2HPOl Environ. Sci. Technoi., Vol. 22, No. 12, 1988
1421
K2HP0,,
100
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N H q N 4 , 100 MG K,HPO~,loo MG
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c
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NHqN03, 10 MG
A 100
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Flgure 6. Incorporation of PNP (26 pg/L) by Cotynebacterium sp. added to filter-sterlllzed lake water with and without 100 mg of K,HPO,/L. 40
100 MG)
K2HP0,,
NH4N03, 100 MG
/h
20 B I
I
2
4
DAYS Flgure 5. Growth of Cotynebacterlumsp. (A) and PNP mineralization (B) In Cayuga Lake water amended with 10 Mg of PNP and 0, 10, or 100 mg of K2HP0, and NH,NO,/L.
or Na2HP04markedly increased the extent of PNP mineralization. A study was conducted to determine whether an additional carbon source would stimulate the degradation of 26 pg of P N P / L by Corynebacterium sp. in Beebe Lake water. The amendments were either 10 mg of glucose/L, 100 mg of K2HP04/L,glucose and phosphate, or nothing. As in previous tests, mineralization was slow with no supplemental nutrients, and the Corynebacterium sp. population initially increased and then declined (Figure 8). However, the bacterium mineralized -70% of PNP in 3 days in lake water amended either with glucose or K2HP04,and the amendments also increased the population size, which still declined abruptly. However, when lake water was amended with both glucose and K2HP04, Corynebacterium sp. mineralized 82% of the PNP in 3 days, and the initial population increase was even more marked. Determinations of mineralization were made more frequently than bacterial counts in this study; hence, it is not certain when the decline in cell numbers began, but presumably it started after the PNP supply was exhausted. The effect of glucose was also tested in samples of Cayuga Lake water inoculated with 9 X lo4cells/mL. The concentration of sugar was 100 mg/L. Under these conditions, the degradation of 26 pg of PNP/L was again enhanced by additions of the sugar. For example, the 1422 Envlron. Sci. Technol., Vol. 22, No. 12, 1988
h No ADDITON A
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Figure 7. Growth of Corynebacteriumsp. (A) and mineralizationof 26 Mg of PNP/L (B) in unamended Cayuga Lake water or in water amended with 100 mg of Na2HP04or K,HP04/L.
percentages of PNP mineralized at 29 h were 25 and 44% in the absence and presence of glucose, and the percentages at 149 h were 61 and 90%. Interestingly, however, glucose
6 KiHPO&NYSTATN
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i
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00 75 50
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E
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ap
25
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I
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Flgure 8. Effect of glucose, K2HP04,or both on growth (A) and mlneraiization (e) by corynebacteriumsp. of 26 pg of PNP/L in lake water.
did not increase the Corynebacterium sp. population in Cayuga Lake water. PNP was not mineralized in 21 days in uninoculated water amended with 200 mg of glucose/L. A study was conducted to determine the possible effect of eucaryotes on the activity of the inoculated bacterium. For this purpose, Corynebacterium sp. was added to a sample of Cayuga Lake water in which no mineralization of 26 pg of PNP/L was detected in 7 days. Samples of the water were also amended with either 100 mg of KzHP04/L, 30 mg of nystatin/L, KzHP04and nystatin, or nothing. Nystatin was added to the water without a solvent. As expected, K,HP04 stimulated the rate of PNP mineralization, and it also increased the rate of growth of Corynebacterium sp. (Figure 9). Nystatin with or without KzHP04increased the length of the initial period of slow mineralization, so that less was converted to COz at each sampling time. Addition of the inhibitor also resulted in faster growth of Corynebacterium sp. It is noteworthy that the initial rapid rise in numbers was not accompanied by PNP mineralization and that the density of Corynebacterium sp. was far greater than predicted from the amount of PNP added if it is assumed that one cell is generated from 1pg of added substrate. When tested in sterile lake water containing 1 mg of PNP/L and inoculated with Corynebacterium sp., nystatin was found to have no effect on growth of the bacterium or PNP mineralization. The influence of eucaryotes was also investigated by adding amphotericin B (without a solvent) to Cayuga Lake water amended with 26 pg of PNP/L. The samples also received either 100 mg of amphotericin B/L, 100 mg of K2HP04/L, amphotericin B and K2HP04, or nothing. Phosphate again enhanced the rate of Corynebacterium sp. growth, although it increased the extent but not the rate of mineralization (Figure 10). Amphotericin B with or without added phosphate almost completely prevented mineralization for more than 4 days. Nevertheless, Cor-
-
3 2
4
6
DAYS
Flgure 9. Effect of K,HP04, nystatin, or both on growth of Corynebacterium sp. (A) and mineralization (B) of 26 pg of PNP/L in Cayuga Lake water.
ynebacterium sp. grew faster in lake water amended with amphotericin B, whether phosphate was added or not; this indicates that the compound is not toxic to the bacterium. In uninoculated lake water, mineralization was not detected during the first 4 days. Discussion The purpose of this study was not to determine the factors affecting biodegradation of organic compounds in natural ecosystems, because a large literature already exists that shows the significance of concentration of inorganic nutrients, season of year, levels of total C, and concentration of the test compound. The purpose was to establish some of the factors that limit the success of inoculation to stimulate biodegradation, especially because few investigations have been carried out to determine why inoculation often fails (10). Corynebacterium sp. readily mineralized 100 pg and 1.0 mg of PNP/L when inoculated into lake water. However, the organism mineralized lower concentrations of the substrate at a rate far slower than would be expected if the rate was assumed to be a linear function of concentration. Such slow rates of destruction of organic compounds have been reported previously for a pseudomonad (12) and Salmonella typhimurium (13),and they may reflect the growth of bacteria at concentrations of the carbon source near the threshold for growth (14).Because such low levels are characteristic of many organic pollutants found in natural waters, some of the factors that affect the transformation at these trace concentrations were investigated. The finding that PNP mineralization was markedly affected by the time of sampling was unexpected. It is Environ. Sci. Technol., Vol. 22, No. 12, 1988
1423
4 100
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3 .
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AMPHOTERICIN
Y
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t
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DAYS Figure 10. The influence of K,HPO, and amphotericin B on growth of Corynebacteriumsp. (A) and mineralizationof 26 pg of PNP/L (e) in Cayuga Lake water.
known that biodegradation may occur in samples of water taken at one but not another time of year (15). The effect of season could result from the sporadic presence of microorganisms able to metabolize the compound of interest or the influence of temperature fluctuations in nature, but such explanations cannot account for our findings because active bacteria were added to all water samples and the temperature of incubation was constant. However, a comparison of the rates with the intensity of rainfall in the watershed suggested that the critical factor was inorganic nutrients because the large amounts of rainfall in these watersheds cause considerable runoff of soil materials into edjacent streams and ultimately into the lakes; nevertheless, rainfall brought clay, organic matter, and other nutrients into the lakes, so that it is not possible to conclude that the effect is indeed an influence of inorganic nutrients per se. The hypothesis that an inadequate supply of inorganic nutrients was responsible for the slow biodegradation rates in lake water samples taken at certain times was supported by results showing that added P and N enhanced the mineralization of low PNP concentrations and the growth of Corynebacterium sp. The data also show that more PNP was taken up by Corynebacterium sp. in sterile lake water when the water was supplemented with phosphate. Others have shown that additions of these two elements to samples from natural environments enhance decomposition of organic compounds (3,16),but it was not shown that such additions might stimulate the degradation of such low concentrations of synthetic molecules nor that they enhance development of a strain used for inoculation. However, 1424
Environ. Sci. Technoi., Voi. 22, No. 12, 1988
Law and Button (17) noted that amino acids reduced the threshold concentration for glucose utilization by a coryneform bacterium. It is not surprising that bacterial activity was stimulated by additions of either N or P because it has long been known that the growth of indigenous or added microorganisms in lake water responds to single, separate additions of N and P (18-20). It may seem anomalous that such high phosphate levels were needed to bring about the stimulation, at least if one assumes that the P need of microorganisms should be determined by the amount of organic C in the water. However, if the K, for phosphate, i.e., the concentration to support a growth rate of half the maximum, is high, the level of this anion necessary to support maximum rates of proliferation would be high regardless of the C concentration. Nevertheless, a high K,for phosphate and a low K, for the C source is still surprising. It is also possible that the bacterium is especially affected by cation imbalance during the degradation of low substrate concentrations and that the high P levels act by making some of the cations less available. This would not explain the stimulation by high N levels, however. It is unlikely that the response to large amounts of added P resulted from contamination of the P salt, because the finding of the same response to K2HP04and Na2HP04would then require that the two P sources have the same contaminant and in similar amounts. When two C sources are available at high concentrations, bacteria in pure culture usually first metabolize the substrate that supports the highest rate of growth (13, 21). However, under conditions of C deficiency, bacteria are capable of using two organic substrates simultaneously (13, 22). The data presented indicate that not only does glucose not reduce or delay PNP mineralization but the sugar enhanced PNP degradation. The stimulation may result from the larger populations of Corynebacterium sp. resulting from addition of glucose. The inhibition of PNP mineralization by nystatin or amphotericin B is noteworthy because these compounds affect the membranes of eucaryotes, causing the leakage of cell constituents (23). The enhanced growth of Corynebacterium sp. in the lake water containing either of these two antibiotics confirms that they did not affect the bacterium but also suggests that grazing by protozoa may result in a small population of the PNP degrader. It is not clear, however, why mineralization of the nitro compound was inhibited by the antibiotics since the bacterium multiplied rapidly. Although P cycling in the water would be diminished if protozoan feeding on the bacterial biomass was suppressed, the greater growth of Corynebacterium sp. under these conditions suggests that the P constraint was eased. It is possible that more C became available to the bacterium as a result of lysis of the protozoa and this C, in contrast with glucose, caused a delay in metabolism of the aromatic substrate. The effect of eucaryotic inhibitors in suppressing PNP mineralization in inoculated lake water is in contrast to the reduction in the acclimation phase when indigenous populations are mineralizing the same compound (24). It is clear that more information is needed to define the factors affecting the biodegradation of low concentrations of synthetic substrates that are rapidly transformed at higher concentrations. Until such information is available or organisms readily able to metabolize the low levels are obtained, inoculation to enhance biodegradation of chemicals in polluted natural waters often will not give satisfactory results. Registry No. PNP, 100-02-7;Na2HP04,7558-79-4;glucose,
Environ. Sci. Technol. 1988, 22, 1425-1429
Schmidt, S. K.; Alexander, M.; Shuler, M. L. J . Theor. Biol.
50-99-7;nystatin, 1400-61-9;amphotericin B, 1397-89-3;ammonium nitrate, 6484-52-2.
1985,114, 1-8.
Yordy, J. R.; Alexander, M. Appl. Environ. Microbiol. 1980,
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39, 559-565.
Jobson, A.; McLaughlin, M.; Cook, F. D.; Westlake, D. W. S. Appl. Microbiol. 1974, 27, 166-171. Law, A. T.; Button, D. K. J. Bacteriol. 1977,129,115-123. Wurtzbaugh, W. A.; Horne, A. J. Can. J. Fish. Aquat. Sei.
1979,37,1211-1216.
Jannasch, H. W. Limnol. Oceanogr. 1967, 12, 264-271. Atlas, R. M.; Bartha, R. Environ. Sei. Technol. 1973, 7,
1983, 40, 1419-1428.
538-541.
MOSS,B. J . Ecol. 1973,61, 193-211. Maloney, T. E.; Miller, W. E.; Shiroyama,T. Spec. Symp.
Lewis, D. L.; Kollig, H. P.; Hodson, R. E. Appl. Environ. Microblol. 1986,51, 598-603. Veldkamp, H.; Jannasch, H. W. J. Appl. Chem. Biotechrwl.
Am. SOC.Limnol. Oceanogr. 1972,1, 134-140. Harder, W.; Dijkhuizen, L. Annu. Rev. Microbiol. 1983,37,
1972,22, 105-123.
1-23.
Subba-Rao,R. V.; Rubin, H. E.; Alexander, M. Appl. Environ. Microbiol. 1982, 43, 1139-1150. Lehtomaki, M.; Niemala, S. Ambio 1975, 4, 126-129. Tagger, S.; Branchi, A.; Julliard, M.; LePetit, J.; ROUX, B. Mar. Biol. (Berlin) 1983, 78, 13-20. Brunner, W.; Sutherland, F. H.; Focht, D. D. J . Environ. Qual. 1985,14, 324-328. Goldstein, R. M.; Mallory, L. M.; Alexander, M. Appl. Environ. Microbiol. 1985, 50, 977-983. Hoben, H. J.; Somasegaran, P. Appl. Enuiron. Microbiol. 1982,44, 1246-1247.
Boethling, R. S.;Alexander, M. Environ. Sei. Technol. 1979, 13,989-991.
Schmidt, S. K.; Alexander, M. Appl. Environ. Microbiol. 1985,49,822-827.
Harder, W.; Dijkhuizen, L. Philos. Trans. R. SOC.London, B. 1982,297,459-479. Lyr, H. In Plant Disease; Horsfall, J. G., Cowling, E. B.,
Eds.; Academic: New York, 1977; Vol. 1, pp 239-261. Wiggins, B. A.; Jones, S. H.; Alexander, M. Appl. Environ.
Microbiol. 1987, 53, 791-796. Received for review September 3, 1987. Revised manuscript received March 10,1988. Accepted June 9,1988. This research was supported by funds provided by Public Health Service Training Grant ES-07052 from the Division of Environmental Health Sciences, National Institutes of Health, and by the Army Research Office.
Isolation and Identification of Reaction Products Arising from the Chlorination of Cytosine in Aqueous Solution Gllllan L. Reynolds, Helen1 A. Filaderll, Alun E. McIntyre, Nlgel J. D. Graham, and Roger Perry*
Public Health and Water Resource Engineering Section, Department of Civil Engineering, Imperial College, University of London, London SW7 2BU England
The reaction between free available chlorine and the pyrimidine base cytosine has been studied under controlled conditions of pH and chlorine dose. Reaction mixtures were separated and analyzed by reversed-phase highperformance liquid chromatography. Eluant fractions corresponding to the major UV-absorbing reaction products were further analyzed by field desorption mass spectrometry and nuclear magnetic resonance spectroscopy. The reaction products identified were 1-, 3-, and 5-chlorocytosine and 3,5-dichlorocytosine. 1- and 3chlorocytosine were formed in high yields at both neutral to low and high pH values with a formation minimum at pH 9.0. 5-Chlorocytosine and 3,5-dichlorocytosine were produced in highest yields under alkaline conditions with a progressive decrease in production as pH decreased. The aromatic reaction products identified were formed in highest yields at [chlorine]:[cytosine] molar ratios of 2-3. Nonaromatic reaction products became increasingly predominant with increase in the initial [chlorine]:[cytosine] molar ratio. Introduction Increased awareness of the possible relationship between human disease and environmental pollutants has resulted in the necessity for the measurement and characterization of both natural and synthetic organic compounds in raw and potable waters. To date, the majority of concern over organic contaminants in potable water has centered on the volatile compounds, particularly trihalomethanes, for which quantitative analytical methodology has been developed (I). This 0013-936X/88/0922-1425$01.50/0
concern has been reflected in the promulgation of water quality standards (2). However, it is now accepted that only 20% of the organic matter present in water is volatile, and interest is currently being directed toward the nonvolatile organic fraction and in particular to the identification of those compounds resulting from the chlorination of raw and waste waters (3). Among some of the more biologically active nonvolatile organic compounds that have been identified in raw and potable waters are the N-heterocyclic purine and pyrimidine bases (41, which are the fundamental building blocks from which nucleic acids are constructed. Early chlorination studies involving purines and pyrimidines were carried out in order to elucidate the mechanisms of microbial and viral inactivation during the disinfection process (5-7). Current interest in the chlorination reaction of these bases follows the display of mutagenic activity (8) and the toxicity of some chlorinated base derivatives (9). General agreement on the relative reactivity of purines and pyrimidines toward chlorination has previously been found: the purine bases adenine, guanine, and xanthine tend to be resistant to attack by aqueous chlorine (IO), while the pyrimidine bases uracil, cytosine, and thymine appear to be significantly more reactive and undergo halogen substitution reactions or ring cleavage (6,7,11-15). However, the substrate and/or disinfectant concentrations used in these studies have generally been far higher than those encountered under typical water-treatment conditions, and the reactions occurring and products formed may differ from those found at lower concentrations (16). In addition, reactions have been monitored by nonspecific
0 1988 American Chemical Society
Environ. Sci. Technol., Vol. 22, No. 12, 1988
1425
