Literature Cited (1) Phadke, M. S., Tiao, G. C., Grupe, M. R., Liu, S. T., Fortney, W., Wu, S. C., “Los Angeles Aerometric Data on Oxides of Nitrogen”,
Tech. Rep. No. 395, Dept. of Stat., University of Wisconsin, Madison, Wis., 1974. (2) Phadke, M. S., Tiao, G. C., Grupe, M. R., Wu, S. C., Krug, A. G., Liu, S. T., “Los Angeles Aerometric Data on Sulphur Dioxide, Particulate Matter and Sulphate”, Tech. Rep. No. 410, Dept. of Stat., University of Wisconsin, Madison, Wis., 1975. (3) Tiao, G. C., Box, G.E.P., Grupe, M. R.;Liu, S. T., Hillmer, S., Wei, W. C., Hamming, W. J., “Los Angeles Aerometric Ozone Data”, Tech. Rep. No. 346, Dept. of Stat., University of Wisconsin, Madison, Wis., 1973. (4) Tiao, G. C., Phadke, M. S., Grupe, M. R., Hillmer, S., Liu, S. T., Fortney, W., “Los Angeles Aerometric Carbon Monoxide Data”, Tech. Rep. No. 377, Dept.of Stat., University of Wisconsin, Madison, Wis., 1974. (5) Box, G.E.P., Tiao, G. C., J. Am. Stat. Assoc., 70,70-9 (1975). (6) Tiao, G. C., Box, G.E.P., Hamming,W. J., J.Air Pollution Control
Assoc., 25,260-8 (1975). (7) Tiao. G. C.. Box G.E.P.. Hamming. W. J.. ibid.. DD 1129-36. (8) “1974 Profile of Air Pollution Cor&ol”, p 45, Cob& of Los Angeles Air Pollution Control District, Los Angeles, Calif., 1974. (9) Chass, R. L., Hamming, W. J., Dickinson, J. E., MacBeth, W. G., “Los Angeles Photochemical Smog-Past, Present and Future,” p 8, Int. Conf. on Automobile Pollution, Toronto, Ont., Canada, 1972. (10) Jenkins, G. M., Watts, D. G., “Spectral Analysis and Its Applications”, Holden-Day, San Francisco, Calif., 1968. (11) DraDer, N. R.. Smith. H., “Amlied Regression Analvsis”. “ ,.D 263. Wiley,-NewYork, N.Y., 1966. (12) Box, G.E.P.,Jenkins, G. M.. “Time Series Analvsis Forecastine and Control”, Holden-Day, San Francisco, Calif.,-1970. I .
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Received for review April 11, 1977. Accepted October 14, 1977. Research supported in part by a grant from the American Petroleum Institute and in part by the U.S. Environmental Protection Agency under Contract No. 68-02-2261.
Production of Cobalamins During Activated Sewage Sludge Treatment Robert A. Beck‘’ and John J. Brink Department of Biology, Clark University, Worcester, Mass. 0 16 10
Benzyl alcohol extractable cobalamins in sludge sampled from three stages of a conventional activated sludge facility were quantitated using high-pressure liquid chromatographic (HPLC) analysis. T h e HPLC technique utilized a reversephase column and a detector at 550 nm. The mean HPLC cobalamin concentration levels for primary stage sludge, secondary stage activated sludge, and tertiary stage nitrification sludge were respectively calculated to be 3.79,9.75, and 4.75 pglg of dried sludge. For a n annual study period, the cobalamin concentration in secondary stage activated sludge ranged from 1.87 t o 27.08 pglg of dried sludge. Observed increases in the cobalamin concentration levels coincided with elevated environmental temperatures and depressed oxygen levels in activated sludge. ~~
Inorganic nutrient levels of phosphates, nitrates, and other inorganic compounds d o not explain the responses of many varieties of phytoplankton t o their environments. In recent years it has become apparent that concentration levels of vitamin BIZ (cyanocobalamin) and its cobalamin analogs may be intrinsically involved in the population dynamics and nutritional biochemistry of both phytoplankton and dinoflagellates. Environmental cobalamin concentration levels originate in natural waters from benthic bacteria (1-4). Although little is known about the seasonal availability of cobalamins in natural waters, several nanogramsfliter may have a profound effect on growth and succession patterns of phytoplankton and dinoflagellate populations (5-7). Exclusive of cobalamin production by benthic bacteria, activated sludge and sewage discharged from sewage treatment facilities probably represent the single most important point source of cobalamins released into freshwater and coastal marine environments. Cobalamin concentration levels in sewage sludge have ranged from 3.5 to 22.0 pg/g of sludge (dry weight) (8-13). However, there is a paucity of recent information concerning the cobalamin levels generated throughout the course of
conventional activated sewage sludge treatment stages in addition t o the seasonal productivity of cobalamins in activated sludge. Furthermore, any currently available information related to quantitative estimates of cobalamin concentration levels in sewage sludge has employed bioassays (8-13). In many cases, bioassays do provide credible estimates of environmental cobalamin concentration levels. However, in chemically complex samples such as activated sludge and sewage, the validity of bioassays may be obfuscated by the response of the bioassay organism to one or more compounds other than cobalamins such as thymidine, deoxyribosides, and methionine (8, 14). In view of the potential environmental significance of cobalamins as well as difficulties incurred in the application of bioanalytical techniques t o the quantitative assessments of cobalamins in chemically complex samples, a method for high-pressure liquid chromatographic (HPLC) analysis of benzyl alcohol extractable cobalamins has been developed
Present address, Department of Chemistry and Food Science, Framingham State College, Framingham, Mass. 01701. 0013-936X/78/0912-0435$01 .OO/O
0 1978 American Chemical Society
(15).
T h e investigation outlined in this paper details results of HPLC cobalamin assays on sludge obtained from three stages of a conventional activated sludge facility in addition to the annual cobalamin productivity levels observed in the secondary aeration treatment stage.
Experimental The concentration levels of cobalamins occurrent in various stages of a n activated sewage sludge treatment process were studied a t the Easterly Marlborough Water Pollution Control Facility in Marlborough, Mass. Designed on the basis of a n average sewage flow rate of 5.5 million gal/day, this modern treatment plant utilizes a two-stage activated sludge system for biochemical oxidation demand (BOD) reduction and the conversion of ammonia to nitrates. Chemical precipitation and coagulation of phosphorus are achieved by using alum. Under normal operating conditions, the treatment facility removes in excess of 90% of the incoming load of suspended solids and BOD. Sludge Sampling Techniques. To evaluate the cobalamin concentration levels in sludge samples from the primary stage settling tank, secondary stage mechanical aeration tank, and the mechanically aerated tertiary nitrification stage, multiple Volume 12, Number 4, April 1978 435
Table 1. Cobalamin Content of Sludge Sampled from Various Activated Sludge Treatment Stages Sampling date
Treatment dam
Samples assayeda
Mean cobalamin concn f 2 SD)
3/17/75 Primary 6 1.93 f 0.36 3/17/75 Secondary 8 3.45 f 0.70 3/17/75 Tertiary 11 3.60 f 0.33 7/7/75 Primary 8 5.65 f 2.42 7/7/75 Secondary 4 16.05 f 1.21 7/7/75 Tertiary 5 5.90 f 1.69 a Duplicatebenzyl alcohol extractionswere performedon each sludge sample, and extractablecobalamins were quantitated on the basis of quadruplicate HFLC assays.
Table II. Cobalamin Content of Sludge Sampled from Various Activated Sludge Treatment Stages Sampllng date
Mean cobalamin concn (fig4 f 2 S W a
Sampling date
Mean cobalamin concn (ag/o f 2 S W a
17.01 f 5.30 1.87 f 0.40 8/7/75 1/23/75 8/21/75 14.88 f 3.97 2/6/75 2.00 f 0.51 12.25 f 6.97 2.37 f 0.29 9/4/75 2120175 13.26 f 0.35 2.01 f 0.65 9/18/75 3/6/75 1012175 10.83 f 3.10 3120175 2.86 f 0.21 13.11 f 3.66 4.03 f 1.13 10/16/75 4/3/75 9.25 f 3.10 5.61 f 1.96 10/30/75 41 17/75 11/11/75 7.26 f 3.56 5.41 f 0.52 511175 7.03 f 2.80 5.84 f 0.88 11112175 5/15/75 5/29/75 6.44 f 0.35 11/13/75 6.97 f 3.50 5.88 f 1.47 7.85 f 0.71 11/27/75 6/12/75 6.48 f 0.48 19.02 f 5.40 12/11/75 6/26/75 12/25/75 3.61 f 0.25 7110175 16.05 f 1.21 1/8/76 4.36 f 0.86 7/24/75 27.08 f 7.52 Mean cobalamin concentration levels reported are for quadruplicate assays.
1-gal samples of fresh microbiological floc and sludge obtained from the respective treatment stages were permitted to settle for 20 min. The sedimented sludge was concentrated by siphoning the supernatant liquor from the settled sludge. The weight of solids for each of the sludge samples was determined by drying triplicate aliquots of the sludge samples at 103 "C for 12 h. On the basis of water loss and the remaining solids weight, the percent dry weight of total solids was calculated for the entire volume of the sludge sample being assayed. In addition to sampling sludge from the three stages cited above, sludge samples were also obtained on a biweekly basis for a period of a year (January 1975-January 1976) from the secondary stage mechanical aeration tank. Cobalamin Assay Protocol. Sodium nitrite and sodium cyanide were respectively mixed with a sludge sample of known volume and known weight of sludge solids in a ratio of 050.2 g/100 mL. The pH was adjusted to 4.0 with acetic acid. This procedure and the following steps preceding the lyophilization of aqueous cobalamin extracts must be carried out under a well-ventilated hood. The sample was boiled for 5 min, and if necessary, octanol was used as a defoaming agent. Zinc acetate dihydrate was dissolved in the pH 4.0 solution or slurry to a final concentration of 10%w/v. The pH was adjusted to 8.5 using sodium hydroxide (50% w/v). The zinc hydroxide floc was separated from the clarified liquor using suction filtration. The filtrate was mixed with sodium sulfate (20% w/v) and then extracted three times with one tenth volume of benzyl alcohol. One-half volume of chloroform was 436
Environmental Science & Technology
added to the combined benzyl alcohol extracts. The resulting solvent phase was extracted three times with one-tenth volume of water. Emulsions generated during the course of the previous extraction steps were easily eliminated by centrifuging the respective benzyl alcohol or aqueous phase a t 2500 x G for 10 min. The combined aqueous extracts were lyophilized to dryness and reconstituted to a known volume with water. The resulting amber-colored extract was passed through a 0.9 X 14.0 cm column packed with 7.0 cm of WN-3 activated alumina (Sigma Chemical Co., St. Louis, Mo.) overlayed with 7.0 cm of Sephadex G-10 (Pharmacia Fine Chemical Co., Piscataway, N.J.) and eluted with water. One-half milliliter fractions were collected, and the combined pink cobalamin fractions were lyophilized to dryness. One-tenth milliliter of water was added to the lyophilized cobalamin extract. The sample was quantitated by injecting 10-20 pL into a Tracor 3200 liquid chromatograph. A 1220 X 2.1 mm RPV-reverse-phase column was employed with 0.01 M potassium dihydrogen phosphate-methanol (3:l) as a carrier solvent. The flow rate for the carrier solvent was 0.5 mL/min. Cobalamins were detected a t 550 nm with a Beckman K-24/25 spectrophotometer equipped with an 18-pL flow cell. T o assess the significance of cobalamin concentrations observed in secondary stage activated sludge from the perspective of operational plant parameters, the primary, secondary, and tertiary stage biochemical oxidation demand (BOD), phosphate, pH and ammonia concentration levels as well as dissolved oxygen (DO) levels, and temperatures were recorded on a daily basis from official plant records. Mean values were respectively recorded for each biweekly sampling period.
Results Cobalamin concentration levels in both primary stage sludge and sludge samples from secondary and tertiary treatment stages of the activated sludge process were assayed during stable periods of plant operation during winter and summer months. The data obtained during the course of these studies are outlined in Table I. Table I1 details the results of biweekly cobalamin assays obtained for secondary stage activated sludge over a one-year study period (January 1975January 1976).
Discussion Cyanocobalamin bioassays by Hoover et al. (11) reported that primary treated sewage sludge typically contained cobalamin concentrations of 1.5 pg/g of sludge, whereas sludge generated in the secondary aeration stage displayed concentrations of 15.0 pg/g. The results for March cobalamin assays in this study indicated mean concentrations of 1.93 f 0.36 pg/g of sludge in the primary stage and 3.45 f 0.70 Mg/g for sludge generated in the secondary treatment stage (Table I). The July studies indicated concentration levels of 5.65 f 2.42 and 16.05 f 1.21 pg/g, respectively. The current study supports the results of Hoover et al. ( I I ) since cobalamins in sludge sampled from the secondary aeration stage were present in higher concentration levels than those calculated for sludge sampled from the primary stage. Contrary to the studies of Hoover et al. ( 1 1 )reporting that secondary sludge has a cobalamin concentration 10 times that existing in primary sludge, a doubling or tripling of cobalamin concentrations seems to be more realistic for the present study. The mean cobalamin concentration levels for sludge sampled from the tertiary nitrification stage during March and
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July were respectively calculated to be 3.60 f 0.33 and 5.90 f 1.69 pg/g. Therefore, regardless of the sampling date, the cobalamin levels of the tertiary sludge samples were as high as, or greater than, the cobalamin levels of sludge samples obtained from the primary stage. The productivity of cobalamins generated in secondary sludge ranged from a low concentration of 1.87 f 0.40 pg/g on January 23 to a high of 27.08 f 7.52 pg/g on July 24 (Table 11). Although Neujahr (9, 10) has reported cobalamin concentration levels in sewage sludge as high as 19.0-22.0 pg/g, the concentration levels determined in this study are higher than any previously reported. An examination of all operational plant parameters recorded during this study suggested that both elevated primary stage effluent BOD levels and environmental temperatures as well as low secondary stage DO levels coincided with high cobalamin concentrations observed in activated sludge (Figures 1-3). From the perspective of these apparent correlations, the influence of environmental temperature may be the most significant factor regulating the normal cobalamin productivity levels of activated sludge since high temperatures not only decrease the DO level of the activated sludge but also accelerate the rate of oxygen utilization by bacteria involved in biochemical oxidation processes. Based on these considerations, it is speculated that methanogenic anaerobic bacteria populations, rich in cobalamin concentration levels (16-18), may be encouraged during periods of high environmental temperatures and associated low DO levels in activated sludge. Although it is tempting to directly implicate the coincident rise in primary stage effluent BOD levels with the productivity of cobalamins in activated sludge (Figure l),it is doubtful that this factor supersedes the importance of environmental temperature in the overall regulation of DO concentrations in activated sludge. However, it could contribute to the further depression of DO concentrations in secondary stage activated sludge already resulting from high environmental temperatures.
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Figure 1. Cobalamin productivity levels in activated sludge compared with primary stage effluent biochemical oxidation demand
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Figure 2. Cobalamin productivity levels in activated sludge compared with activated sludge temperatures
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Figure 3. Cobalamin productivity levels in activated sludge compared with dissolved oxygen levels in activated sludge
On the basis of this study, the cobalamin concentrations assayed certainly varied within the activated sludge itself, since only sedimented activated sludge was used for Cobalamin determinations and all assays were based on weights of uniformly dried sludge samples prior to analysis. Furthermore, although the seasonal cobalamin productivity was measured Volume 12, Number 4, April 1978
437
on a biweekly basis, the results obtained do not merely represent random variations on an annual basis. Early pilot studies prior to the study reported here have shown the daily variations of cobalamin concentration levels in activated sludge to be minimal. The typical variability for cobalamin concentration levels on a daily basis is exemplified by a set of assays carried out over a three-day period from November 11-13 (Table 11).
Acknowledgment Appreciation is expressed to John Hartley and John Harrington of the Easterly Marlborough Water Pollution Control Facility, Marlborough, Mass., for their assistance and cooperation in supplying operational plant parameter data during the course of this study. Literature Cited (1) Burkholder, P. R., Burkholder, L. M., P. R. Bull. Mar. Sci., Gulf Carribbean, 8,201-23 (1958). (2) Burkholder, P. R., Burkholder, L. M., Linnol. Oceanogr., 1,202-8 (1956). (3) Provasoli, L., “Algal Nutrition and Eutrophication,” in “Eutrophication: Causes, Consequences, Correctives,” Symp. Proc. Nat. Acad. Sci., Washington, D.C., 1969.
(4) Starr, T., Jones, M. E., Martinez, D., Limnol. Oceanogr., 2,114-9 (1957). (5) Daislev. K. W.. Nature. 180.1042-3 11957). (6) Droop: M. R., Ibid., pp’1041-2. (7) Cairns. J., Lanza. G. R.. Parker. B. C.. Proc. Acad. Nat. Sci. Philadelphia, 124 (5),79-127 (1972). ’ (8) Smith, E. L., “Vitamin BIZ,”Wiley, New York, N.Y., 1969. (9) Neujahr, H. Y., Acta Chem. Scand., 9,622-30 (1955). (10) Neujahr, H. Y., “On Vitamin BIZ Factors in Sewage Sludge,” Almquist Wiksells, Uppsala, Sweden, 1950. (11) Hoover, S. R., Jasewicz, L.,Pepinsky, J. B., Porges, N.,Sewage Ind. Wastes, 24, 38-44 (1952). (12) Hoover, S. R., Jasewicz, L., Porges, N., Science, 114, 213 (1951). (13) Miner. C. S., Wolnak, B., U S . Patent 2.646.386 (1953). (14) Shive, W., Ravel, J. M., Harding, M. M., J . Biol. Chem., 176, 991-2 (1948). (15) Beck, R. A., Brink, J. J., Enuiron. Sci. Technol., 10 (2), 173-5 ~I
(1 ~ 976) - -_,..
(16) Metzler, D. E., “Biochemistry,” pp 509-11, Academic Press, New York, N.Y., 1977. (17) McBride, B. C., Wolf, R. S., Biochemistry, 10 (12), 2317-24 (1971). (18) Sebrell, W. H., Harris, R. S., “The Vitamins,” Vol 11, pp 119-259, Academic Press, New York, N.Y., 1968.
Received for reuiew May 20, 1977. Accepted October 17, 1977
Evaluation of Direct Aqueous Injection Method for Analysis of Chloroform in Drinking Water Frederic K. Pfaender” and Robert B. Jonas Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, N.C. 27514
Alan A. Stevens and Leown Moore Municipal Environmental Research Laboratory, Environmental Research Center, Environmental Protection Agency, Cincinnati, Ohio 45268
J. Ronald Hass National Institute of Environmental Health Sciences, Research Triangle Park, N.C. 27709
A direct aqueous injection (DAI) technique, employing a bypass value to vent water and electron capture detector, was compared with the purge method for chloroform measurement in drinking water. The DAI method gave consistently higher values for chloroform than the purge method. Comparable results were obtained if the DAI value after a 30-min purge was subtracted from the before-purging value. The nature of the residual measured by DAI after purging was investigated and shown to be due to nonpurgeable intermediates that decompose within the injection port of the gas chromatograph to chloroform. The residual varied depending on the source of the water sample examined and the specific configuration of the chromatograph employed. The results indicated the need for caution in the interpretation of chloroform and other trihalomethane values, especially haloform potentials, generated by DAI.
The discovery of chloroform and other trihalomethanes in the drinking water of 79 of the 80 U S . cities examined in the recent EPA National Organics Reconnaissance Survey (1) has generated great interest in volatile organics in water and their mechanisms of formation as a result of water chlorination, as well as potential methods for their removal. Chloroform has been measured by head space analysis ( 2 )and by purging the chloroform from water with an inert gas and trapping in a 438
Environmental Science & Technology
short column packed with a porous polymer, from which it is thermally eluted into a gas chromatograph ( 3 ) . Solvent extraction procedures have also been proposed (4-6). Recently, several reports have suggested that chloroform can be measured by direct injection of water into a gas chromatograph (GC) or gas chromatograph/mass spectrometer (GC/MS), eliminating the need for handling volatile materials and halogen specific special detectors (7-9). When Nicholson and Meresz (10, 11) measured chloroform by both the direct aqueous injection (DAI) method and the purge method, they detected considerably higher amounts of chloroform by the DAI method. The difference was attributed to nonpurgeable chlorinated compounds, formed as a result of chlorination processes, that supposedly decomposed in the heated injection port of the gas chromatograph to yield chloroform. These results raised significant questions about the usefulness of the DAI method in the analysis of chloroform or other trihalomethanes that occur in waters disinfected by chlorination. It was the intent of the research reported here to compare the DAI and purge methods for chloroform analysis using waters from several sources and to further investigate the cause of any discrepancies that arise in the results obtained by the two procedures.
Experimental Sample Collecting and Handling. Water samples exchanged between UNC-Chapel Hill and EPA-Cincinnati were
0013-936X/78/0912-0438$01 .OO/O
0 1978 American Chemical Society
