Trihalomethanes from the Chlorination of Aquatic Algae Barry G. Oliver" and David B. Shindler National Water Research Institute, Canada Centre for Inland Waters, Burlington, Ontario, Canada L7R 4A6
Algae can be potent trihalomethane (THM) precursors under chlorination conditions used for drinking water purification and disinfection. Chlorination of seven species of cultured algae and a few natural samples showed that T H M production is strongly dependent on pH and chlorine concentration. Species-dependent changes in T H M production were also apparent. Fractionation and extraction experiments showed that THMs formed from algal chlorination do not come from a specific cellular component or substance. Algae removal from raw water prior to chlorine disinfection should reduce the T H M concentrations in finished drinking water. Introduction
The widespread occurrence of trihalomethanes (THMs) in chlorinated drinking waters is well documented (1-5). In many instances, humic materials appear to be the major organic precursors to THMs ( 6 - I O ) , but other naturally-occurring organic matter, such as algae ( I I ) , tannic acid (12), and nitrogen-containing compounds (13)also produce THMs on chlorination. Hoehn et al. (11)showed that summer T H M concentrations in finished chlorinated water from a Virginia reservoir correlated well with chlorophyll-a concentrationstrongly implicating algae as an important source of T H M precursor for this water supply. Also, cells and extracellular products of the blue-green alga Anabaena cylindrica have been shown to produce THMs on chlorination (14). This paper reports the T H M concentrations produced by the chlorination of various types of commonly-occurring aquatic algae under varying conditions of pH and chlorine dose. The T H M yield from chlorination of algal cellular components separated by fractionation and extraction is considered in the context of identification of possible specific T H M precursors. The implications of algal T H M production in terms of common water treatment chlorination practices are briefly considered. Experimental Section
Chlorination and Analysis Procedures. Mixtures of algae, buffer, and chlorine were prepared in volumetric flasks and transferred for the reaction to 160-mL headspace-free serum vials with crimp tops. Two mixtures of each algal culture or sample were prepared, and each mixture was run in duplicate with a blank. A vial which contained sufficient NazSz03 to quench the chlorine served as a blank. During reaction, temperature was maintained a t 20 f 0.01 "C. Phosphate buffer (0.05 M) was used to maintain the pH: pH 7 was chosen to represent typical water treatment reaction conditions, and pH 11was used to yield the maximum T H M concentration for a given reaction time, since intermediates are rapidly hydrolyzed to THMs a t this high p H (13).Postreaction T H M concentrations for the replicate mixtures agreed to within f 5 % . Chlorine in the form of NaOCl was added at sufficiently high concentrations to maintain a large chlorine residual throughout the experiments. The minimum chlorine residual we measured was 2 mg/L for a 96-h pH 7 reaction a t a chlorine-to-algae ratio of 3:l. Samples usually consisted of algal suspensions added in volumes to achieve appropriate final carbon concentrations in chlorination reaction mixtures. When cells or cell fragments were concentrated by filtration under reduced pressure onto 27- or 47-mm diameter Whatman GF/F glass fiber disks (disks 1502
Environmental Science & Technology
were rinsed with 500 mL of distilled water, dried, then combusted a t 450 OC for 4 h before use), the entire disk was ground, homogenized, and weighed so that a specific quantity of material could be added to reaction mixtures. When cells on glass fiber filters were treated with acetone, the extracts were pipetted into individual serum vials. The acetone was evaporated with a stream of prepurified nitrogen gas. Care was taken to ensure that all acetone was removed before addition of chlorine to the reaction mixtures. T H M analysis was performed by using pentane extraction, gas-chromatographic separation, and electron-capture detection (15). Reproducibility was 55%. Because bromide concentrations were very low in the cultures, the only T H M found in significant concentration was chloroform (CHC13). Negligible amounts of THMs were formed from chlorination of unused GF/F filters or unused algal medium. Algal Cultures. Seven strains of cultured algae were used in the experiments (Table I) along with two natural samples. Cultures of Anabaena oscillarioides, a filamentous blue-green algae, contained a few bacteria (less than lo4 mL-l) which could not be removed from the cultures, but the rest, including a small unicellular blue-green alga, Anacystis nidulans, two strains of the green alga Scenedesmus, another green alga Selenastrum capricornutum, and the two Navicula diatom strains, were cultured without bacterial contaminants. All cultures were grown in Chu no. 10 medium (18) slightly modified by the addition of 160 pg L-l of FeC13,2 mg L-' of NazEDTA, and 4.84 mg L-l of tris(hydroxymethy1)aminomethane; the trace element mix associated with medium G-11 (19)was also added. Nitrate was omitted from the Chu no. 10 formulation for the growth of Anabaena, as this strain obtains its nitrogen requirements by fixing atmospheric Nz. Light was supplied continuously to the stationary culture flasks a t 100 yeinsteins m-2 s-l; growth temperature was 20 f 1 OC. Algal concentrations in reaction mixtures are expressed in milligrams of dry weight per liter; corresponding measured algal carbon concentrations are also occasionally stated. Both of these quantities were determined from filtered material. Chemical characteristics of the cultured algae are shown in Table I. The NaOH extracts were also analyzed for organic carbon by using a Beckman Model 915A carbon analyzer to within *IO%. Results and Discussion
THM Yields from the Chlorination of Algae. The effects of chlorine dose and reaction time on the chloroform (CHC13) yields from Anabaena oscillarioides at pH 7 and 11are shown in Figures 1and 2. Reaction is about 3-4 times more rapid a t pH 11than at pH 7 (Figure l),and nonlinear with time at the higher pH. The 24-h CHC13 yield a t pH 11 increases rapidly with chlorine dose up to a weight ratio of chlorine to algae of -1O:l then levels off (Figure 2). A t pH 7, however, the yield is nearly maximum a t a 4:l ratio and does not increase very much as the chlorine concentration is increased further. These results differ from those obtained from the chlorination of aquatic humic material. Under the same conditions, the CHC13 concentration from humics changed by less than a factor of 2 between pH 7 and 11,and the CHC13 concentration increased rapidly for the first few hours of reaction but increased a t a much slower rate at reaction times longer than 24 h (20). Although chlorination reactions are complex, it appears that the mechanisms of reaction with cellular carbon
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@ 1980 American Chemical Society
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Figure 2. Effect of chlorine dose on CHCI3 production from Anabaena oscillarioides. Reaction conditions: algae, 3.6 mg dry weight/L; T = 20 OC; reaction time = 24 h. TIME (HOURS)
Figure 1. Time-course of production of CHCI3 from chlorination of Anabaena oscillarioides. Reaction conditions: chlorine, 10.3 mg/L; algae, 3.6 mg dry weight/L; T = 20 OC.
Table II. Chloroform Yields (pg CHCI3/mg material) from the Chlorination of Algaea PH 7 algae
Table 1. Algal Species and Chemical Composition organic carbon, a
chlorophyll a, mug
carotenolds, mg/g
9.0
17.2 8.4 8.9 12.6 13.0 8.6 2.5
3.6 3.3 2.2 3.7 3.6 3.1 1.3
algae
%
organic nitrogen, a %
Anabaena oscillarioides A nacystis nidulans Scenedesmus quadricauda Scenedesmus basiliensis Selanastrum capricornutum Navicula minima Navicula pelliculosa
43.2 30.6 38.1 34.7 42.7 25.0 34.7
6.3 3.9 2.9 3.4 2.6 2.5
Anabaena oscillarioides Anacystis nidulans Scenedesmus quadricauda Scenedesmus basiliensis Selanastrum capricornutum Navicula minima Navicula pelliculosa Thomson Lake algae Lake Ontario Cladophora water fulvic acid
low ratiob
high ratio8
3.4 2.8 3.0 2.5 3.4 1.9 2.4 3.2 1.4
7.3 4.4 6.0 4.8 4.2 3.5 5.0
pH 11 low high ratiob ratioc
11 16 11 12 17 7.9 15
47 24 24 27 31 27 38
5.3 17.0
29
Determined from analysis of filtered material by using a Hewlett-Packard CHN analyser (&5%) expressed as a percentage of dry weight. Determined by the Me2SO-spectrophotometric technique ( 16). Determined by resonance Raman spectroscopy ( 17).
Reaction conditions: [C12] = 10 mg/L ; T = 20 'C; reaction time = 24 h. Low ratio: [C12]/[algae] = 1.5. High ratio: [Cle]/[algae] = 10. From ref. 20.
compounds differ from those with humic materials. At any rate, algae seem to be potent precursors for T H M production. The CHC13 yields from laboratory cultures of seven common species of algae are shown in Table 11. Species differences in CHC13 yields are apparent. Also, the CHCl3 yields doubled when the chlorine-to-algae ratio was increased about one order of magnitude, and the yields increased three- to sixfold when the pH was raised from 7 to 11. Results obtained from chlorination of two natural samples are also shown. The Thompson Lake algae sample, consisting mainly of blue-green algae, yielded approximately the same amount of CHC13 as the Anabaena oscillarioides and Anacystis nidulans cultures. A Lake Ontario sample of Cladophora yielded less CHCl3 than the laboratory cultures (see Table 11).Cladophora, possibly because of its fibrous nature, seemed resistant to chlorine. Other algae in the study were completely decolorized by the chlorine within a few hours, whereas a green clump of seemingly unchanged Cladophora was clearly visible, even after 24 h. For comparison, the CHCl3 produced from aquatic fulvic acid (20) under the same reaction conditions is shown in Table
11. The 24-h CHC13 yields for the algae are close to the humic material yields at pH 11but are somewhat lower a t pH 7. Thus the importance of algae and humic substances as T H M precursors in water supplies will largely depend on their relative concentration and on water treatment methods. Many utilities use the following drinking water treatment sequence: (1) prechlorination; ( 2 ) coagulation and settling; (3) filtration; (4) postchlorination. Since most algae would be removed by coagulation, settling, and filtration, the elimination or reduction of prechlorination (while, of course, maintaining proper treatment practices) should reduce T H M production from algal material, as has also been suggested by Singer (21) for organic THM precursors. Previous studies (9,lO) indicated that if no prechlorination were carried out, coagulation and settling followed by filtration would also remove some of the humic substances, thus further reducing the concentrations of potential T H M precursors. Algal Fractionation to Identify Major THM Precursors. T o find out whether any single compound or class of compounds was responsible for T H M production from chlorinated algae, we used the following techniques to separate the algae into components:
a
a
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Table 111. Chloroform Yieldsa ( % ) from Fractionated Algal Components % In filtrate or extract
YO in method
residue
1 1 2 3 3 3
90 80 85 90 88 82 98 44 71 56 23
Anabaena oscillarioides Scenedesmus basiliensis Anabaena oscillarioides Anabaena oscillarioides Anacystis nidulans Scenedesmus basiliensis Anabaena oscillarioides Anabaena oscillarioides Scenedesmus basiliensis Selanastrum capricornutum Navicula pelliculosa
4 5 5 5 5
10 20 15 10 12 18
2 56 29
44 77
Chlorinationconditions: [algae] 1 mg/L; [chlorine] = 10 mg/L; reaction time = 24 h: pH 7: T = 20 OC. See text for fractionation methods: (1)separation of cells from extracellular medium: (2) cell disruption and separation of soluble intracellular materials from cell debris: (3)acetone extraction: (4) hexane extraction; (5)NaOH extraction.
Table IV. CHC13 Yields (pg CHCI3/mg TOCa) for NaOH Algal Extracts and Remaining Residues pn 7 algae
Anabaena oscillarioides Scenedesmus quadricauda Scenedesmus basiliensis Navicula Pelleculosa
pn 11
filtrate
residue
filtrate
25 11 12 18
21 13 10 10
112 61 58 96
residue
99 65 78 79
TOC = total organic carbon.
(1) filtration through a Whatman GF/F glass fiber filter which separated cells from dissolved and colloidal extracellular substances; (2) mechanical cell breakage with a French pressure cell (American Instruments Co., Silver Springs, MD), followed by 1-min ultrasound treatment (the released soluble intracellular materials were separated from the broken cells and cell debris by centrifugation at 15000 g); (3) acetone extraction of algae by grinding in 90% acetone (the extracted material was separated from debris by filtration through a 0.2-pm Nuclepore polycarbonate membrane filter); (4) hexane extraction by grinding, followed by Nuclepore filtration (see technique 3 above); and (5) NaOH extraction by stirring algae for 1-2 h in 0.1 M NaOH solution (with 1-min ultrasound treatment a t the beginning and end of extraction), followed by filtration through a Nuclepore filter. CHCla yields were measured from chlorination of the cells, cell fragments, filtrates, or extracts. The results of chlorination at pH 7 are shown in Table 111. Similar results (not shown) were obtained at pH 11. Technique 1 separated cells from extracellular products produced by algal growth, and technique 2 released soluble intracellular materials. Neither the extracellular products nor the intracellular materials accounted for more than 20% of the total CHC13 production. The relative importance of extracellular or intracellular materials as THM precursors probably varies with growth and physiological conditions, but the majority of precursors appear to be associated with algal cells or cell fragments. The acetone extraction procedure, no. 3, efficiently removes chlorophyll-a and related pigments from algae ( 2 2 ) .Chlo1504
Environmental Science & Technology
rination of acetone extracts resulted in formation of only a small portion of the total cellular CHCl3 yield (Table 111). Material balance measurements showed that, for S c e n e d e s mus basiliensis, 13%(by weight) of the culture was extracted by the acetone. Since this acetone fraction yielded 18%of the CHC13, the extracted material produced only -1.5 times the CHCla per unit weight compared to whole cells. Although Morris and Baum (13)have shown that chlorination of chlorophyll-a produced CHCl3, our data indicate that the algal pigments are not particularly important THM precursors. In fact, little correlation between chlorophyll-a concentration in the cultured algae (Table I) and their CHC13 yields (Table 11) is apparent (linear correlation coefficients: low ratio pH 7,0.61; high ratio pH 7,0.44;low ratio pH 11,-0.17; high ratio pH 11,0.32). Extraction of Anabaena oscillarioides with the nonpolar solvent hexane recovered very little (only 2%) of the THM precursor material (Table 111). Treatment with 0.1 M NaOH, a technique commonly employed for humic material extraction ( 2 3 ) ,recovered much higher but variable concentrations of the THM precursors. We interpret the variation between species in the THM yields as caused by differences in the efficiency of NaOH as an extractant for algal organic carbon. The CHC13 yields per unit weight organic carbon in the extracts and in the residues (Table I V ) were found to be virtually identical for each of the four algal species used in these experiments. The results from all five extraction and separation experiments indicate that no single compound or specific cell component appears responsible for T H M production from chlorinated algae. While minor differences in T H M yield per unit weight of material are evident, many organic components of the algae appear to be capable of producing THMs on chlorination. Reduction of THMs produced from algae would, then, depend upon removal of algal cells before chlorination. During periods when algal populations in reservoirs are large, and especially during and immediately after algal blooms, such removal would appear to be able to significantly lower T H M concentrations in finished drinking water. Acknowledgment
We thank Karen Bothen and Donna Nuttley for their technical assistance. L i t e r a t u r e Cited (1) Rook, J. J. Water Treat. Exam. 1974,23, 234. (2) Symons, J. M.; Bellar, T. A.; Carswell, J. K.; De Marco, J.; Kropp, K. L.; Robeck, G. G.; Seeger, D. R.; Slocum, C. J.; Smith, B. L.; Stevens, A. A. J . Am. Water Works Assoc. 1975,67, 634. (3) Foley, P. D.; Missingham, G. A. J . Am. Water Works Assoc. 1976, 68, 195. (4) Health and Welfare Canada, “National Survey for Halomethanes in Drinking Water,” Report No. 77-EHD-9 (1977). (5) Trussell, A. R.; Cromer, J. L.; Umphres, M. D. Water Chlorination: Environ. Impact Health Eff., Proc. Conf., 3, in press. (6) Rook, J. J. J . Am. Water Works Assoc. 1976,68, 168. ( 7 ) Rook, J. J. Enuiron. Sei. Technol. 1977,11, 478. (8) Stevens, A. A.; Slocum, C. J.; Seeger, D. R.; Robeck, G. G. J. Am. Water Works Assoc. 1976,68, 615. (9) Young, J . S.; Singer, P. C. J . Am. Water Works Assoc. 1979, 71, 87. (10) Oliver, B. G.; Lawrence, J. J . Am. Water Works Assoc. 1979,71, 161. (11) Hoehn, R. C.; Randall, C. W.; Goode, R. P.; Shaffer, P. T. B. Water Chlorination:Enuiron. Impact Health Eff., F’roc. Conf. 1977 1978,2, 519. (12) Youssefi, M.; Zenchelsky, S. T.; Faust, S. D. J . Enuiron. Sci. Health, Part A 1978,13, 629. (13) Morris, J. C.; Baum, B. Water Chlorination: Enuiron. Impact Health Eff., Proc. Conf., 1977 1978,2, 29. (14) Briley, K. F.; Williams, R. F.; Longley, K. E.; Sorber, C. A. Water Chlorination: Enuiron. Impact Health Eff., Proc. Conf., 3, in press. (15) Richard J. J.; Junk, G. A. J . Am. Water Works Assoc. 1977,69, 62.
Burnison, B. K. Can. J . Fish. Ag. Sci. 1980,37, 729. Hoskins, L. C.; Alexander, V. Anal. Chem. 1977,49, 695. Nichols. H. W. In "Phvcoloaical Methods," Stein, J. R. Ed.; Cambridge University Press: London, 1973; p '20. (19) Stainer, R. Y.; Junisawa, R.; Mandel, M.; Cohen-Bazire, G. Hactcriol. Reo. 1971, 35, 171. (20) Oliver, B. G. Water Chlorination: Enuiron. Impact Health E//., Proc. Con/. 3, in press. ( 2 1 ) Singer, P. C., Department of Environmental Science and En-
gineering, University of North Carolina, Chapel Hill, NC, personal communication, 1980. (22) Kerr. J. D.: Suhha Rao Monoer. Oceanoer. Methods 1966., 1., 65. (23) Aiken, G. R.; Thurman, E. M.; Malcolm, R. L.; Walton, H. F. Anal. Chem. 1979,52, 1799.
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Receioed for reuiew April 8, 1980. Accepted July 24,1980.
Isolation and Identification of a Direct-Acting Mutagen in Diesel-Exhaust Particulates Stephen M. Rappaport," Yi Y. Wang, and Eddie T. Wei Department of Biomedical and Environmental Health Sciences, School of Public Health, University of California, Berkeley, California 94720
Robert Sawyer Department of Mechanical Engineering, University of California, Berkeley, California 94720
Bruce E. Watkins and Henry Rapoport Department of Chemistry, University of California, Berkeley, California 94720
Diesel-exhaust particulates contain chemicals which are directly mutagenic in the Ames test. This indicates that the mutagens are not among those classes of mutagenic compounds associated with soot, nor are they among those classes of unstable compounds which are currently known to be directly mutagenic. Reported herein are the isolation, identification, and synthesis of one direct-acting mutagen, pyrene3,4-dicarboxylic acid anhydride, from a sample of dieselexhaust particulates. Although this compound is only weakly mutagenic in the Ames test (220 net TA 98 revertantdmg), it is speculated that it is but one of a class of mutagenic dicarboxylic acid anhydrides of various polynuclear aromatic hydrocarbons in diesel exhausts. Particulate matter emitted from vehicular engines contains organic compounds, some of which are carcinogens (1-5). The classes of carcinogens identified thus far include unsubstituted polynuclear aromatic hydrocarbons (PAH), amino-substituted PAH, and nitrogen heterocyclic compounds. These carcinogens and their analogues are active in the Ames Salmonellalmicrosome mutagen bioassay (6, 7). Their activity is only observed, however, after the addition of mammalian enzymes which convert them to mutagenic forms. Recent investigations have shown that vehicular engine exhausts contain other organic compounds which are directly mutagenic in the Ames test, Le., mammalian enzymes are not required for activity to be observed (8-10) though activation by bacterial enzymes may be involved (8).These compounds, which contribute the majority of the mutagenic activity, have not been identified. I t is suspected that some are stable in the environment since direct mutagenic activity has been observed in air-pollution particulates (of vehicular origin) collected over 15 yr ago (8). We infer from these findings that the direct-acting mutagens in engine-exhaust particulates may represent a new and hitherto unrecognized class of environmental toxins. This inference is based in part upon the apparent stabilities of these mutagens after they are formed. Thus far, most compounds (9) known to be directly mutagenic are alkylating agents which are unstable in the environment since they react rapidly with commonly encountered nucleophiles such as water. This is not the case for direct-acting mutagens in engine exhausts. 0013-936X/80/0914-1505$01.00/0
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A previous paper (9)suggested the futility of trying to assess the hazard that unidentified direct-acting mutagens from engine exhausts may pose to human health. Thus, it is important that the chemical identities of these mutagens be determined. This will shed light not only upon potential health hazards but also upon the environmental fates of these substances and upon their formation and control. Mutagens which have been identified can then be tested in mammalian systems to determine whether they, indeed, have deleterious effects on more complex organisms. The mutagens can also be measured in the environment and during the combustion process, so that their production can be related to the designs and performance of engines. We report here the characterization of one direct-acting mutagen, pyrene-3,4-dicarboxylic acid anhydride (PDAA), which was isolated from a sample of diesel-exhaust-particulate matter. Although this compound is a weak mutagen in the Ames test, we speculate that it may be but one of a class of mutagenic dicarboxylic acid anhydrides of PAH in engine exhausts. Experimental Section Generation and Collection of Diesel-Exhaust Particulates. Exhaust-particulate samples were collected from an experimental diesel engine in the Department of Mechanical Engineering, University of California, Berkeley. The engine, of a type often found in heavy-duty trucks, was mounted on an Eddy-Current dynamometer to simulate loaded operation. Pertinent specifications and operating conditions are given in Table I. Exhaust emissions were drawn through a stainless steel probe located in the center line of the exhaust pipe. Particulate matter was collected on high-efficiency glass-fiber filters a t 63-66 "C. Two types of filters were used. Forty rectangular 20.3 X 25.4 cm filters (Type A B , Gelman Instrument Co., Ann Arbor, MI) were used for -30 min each to collect a total of 20 g of particulate matter. Two pleated HEPA filters (Type 7040-L-N2N2-BBD, 47.0 X 17.5 cm, Flanders Filters Inc., Washington, NC) were used for 18 h each to collect an estimated 20 g of sample. Since the HEPA filters were contained in plywood frames, weight estimates of these samples were based upon weighings of equal areas of clean filter media and samples.
1980 American Chemical Society
Volume 14, Number 12; December 1980
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