Organohalide Formation on Chlorination of Algal Extracellular Products Jan K. Wachter and Jullan B. Andelman"
Department of Industrial Environmental Health Sciences and the Center for Environmental Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 When certain chemical and physical parameters were controlled during chlorination of algal extracellular products (ECP), organohalide formation was modified. In general, decreases in temperature and contact time decreased the generation of purgeable (POX), nonpurgeable (NPOX), and total (TOX) organic halide. Decreases in pH increased NPOX but reduced POX and CHC& formation. Under most chlorinating concjitions, algal ECP generated more NPOX than POX and CHC13. The majority of NPOX formed was greater than 1000 daltons. The chloroform-generating potentials of algal ECP and algal biomass were similar but were lower than those reported by others for algal ECP, algal biomass, and humic substances. The organohalide-forming potentials of the blue-green alga studied tended to be higher than those for green algae.
Introduction Many water systems such as raw and finished water reservoirs are not covered, thus providing environments suitable for the growth and maintenance of microorganisms, including algae. Concentrations of algae as high as 4 X lo5 cells/mL have been found in finished drinking water reservoirs (1). Algae are known to excrete organic extracellular products (ECP) into water, either actively such as byproducts of metabolic activity or passively, for example, involving lysis due to cell death (2). Even though algae can be removed effectively via conventional water treatment practices such as flocculation, sedimentation, and filtration, their ECP may remain in the finished water. Also, algal ECP upon chlorination under laboratory conditions similar to those used during water treatment can act as precursors in chloroform- (CHC1,) generating reactions (3-5). The purpose of this investigation was to determine and compare the organohalide-formingpotentials of algal ECP derived from a number of algal strains under a variety of environmental conditions. Total organohalide (TOX), purgeable organohalide (POX), including chloroform, and nonpurgeable organohalide (NPOX) concentrations were determined. The latter parameter, NPOX, was characterized in greater detail, since recent studies have shown that nonpurgeable organics in various water samples upon chlorination had greater mutagenic potential than the organics present before chlorination (6). The results from these experiments could assist in the determination of the conditions under which organohalide generation could be minimized as well as the assessment of the contribution of algal ECP precursors, upon chlorination, to organohalide concentrations in water supplies. 0013-936X/84/0918-0811$01.50/0
ECP from three algal strains as well as a model ECP were selected for use in this study. Two green algae, Chlorella vulgaris and Chlorella pyrenoidosa, and one blue-green alga, Anabaena flos-aquae, were chosen because of their widespread Occurrence in reservoir water. Chlorophyll a was studied similarly as a model compound, since it is a common algal ECP. The ECP used in these chlorination experiments were isolated during the early stationary phase of algal growth to simulate ECP derived from an algal blooming episode.
Materials and Methods Chlorella vulgaris (UTEX No. 262), Chlorella pyrenoidosa (UTEX No. 26), and Anabaena flos-aquae (UTEX No. 1444) cultures were obtained from the Texas Culture Collection, Austin, TX (Richard C. Starr, Director), as algae maintained on proteose agar slants. To minimize possible organic contamination from the latter, aqueous stock algal solutions were used only after three successive generations of growth in fresh autoclaved aqueous media. The composition of the latter consisted of only inorganic ingredients (7). The water used in media preparation was house distilled and subsequently passed through a prefilter, an activated carbon filter, and two ion-exchange filters. It was autoclave sterilized and adjusted to pH 7.5 prior to use. Four to six large Pyrex (19-L capacity) bottles containing 15 L of sterilized media were used in the culturing of each alga. For each 19-L bottle containing algae, there was a corresponding control bottle consisting of only sterilized algal media exposed to the same laboratory environment as the algal cultures. The air flow into each bottle was approximately 150 mL/min using Teflon stoppers and tubing to minimize contamination. The air was filtered to remove particulates, water, oil, and bacteria. To grow the algae, a diurnal cycle with fluorescent lights was used which consisted of 12 h of light (65 ft-C/cm2) and 12 h of darkness at a temperature of 22.7 f 0.7 "C. When the cultures attained stationary growth, as evidenced by little or no change in growth with respect to time, they were harvested. A Teflon tube was inserted into the culture and control bottles, and their contents were gravity fed into a cream separator (De Lava1 gyrotester). The effluent was then passed through two glass microfiber filters (Whatman GF/D and GF/F, effective retentions being 2.7 and 0.7 pm, respectively). Water-soluble chlorophyll (obtained from Pfatz & Bauer) was added to autoclaved media at a concentration of approximately 5 mg/L and filtered for use in the chlorination studies. For each algal species the filtrates
0 1984 American Chemical Society
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from each 15-L culture and their controls were composited to minimize any differences (due to variations in growth cycle, aeration, light intensity, etc.) among the bottles. The filtrates were stored at 4 "C in a dark, cold room until time of chlorination. Following pH adjustment, no buffer was required to maintain pH during the subsequent chlorination studies. The solutions were chlorinated in 160-mL vials sealed without headspace by using Teflon-faced septa. A standardized stock solution of sodium hypochlorite (NaOC1) was used to chlorinate by injection through the Teflon septum at an initial concentration of 20 mg/L Clz, producing a free chlorine residual under all reaction conditions. The standard chlorination conditions used in this experiment were pH 7,24 "C, a contact time of 24 h, and exposure to light to simulate a typical environment of natural water systems and drinking water reservoirs undergoing chlorination. Parameters were also varied as follows while maintaining the others at standard conditions: pH, temperature, contact time, and light conditions. The free available chlorine concentration remaining at the conclusion of each experiment was determined in one bottle to assess the chlorine demand. All the remaining bottles were then quenched with excess sodium sulfite to eliminate the chlorine. The samples were stored in the dark at 4-7 "C prior to analysis. Purgeable organic halide (POX), total organic halide (TOX), nonpurgeable organic halide (NPOX) by calculation, chloroform (and other specific volatile chloro organics, if present), and total organic carbon (TOC) levels were measured in each group of chlorinated algal filtrate and control samples, as well as the unchlorinated algae and media control solutions. Results from the chlorinated controls were subtracted from those of chlorinated supernatant samples to obtain the final reported readings. On the average, chloroform and POX analyses were repeated four times, TOX analyses in triplicate, and TOC analyses in duplicate, per set of environmental conditions of each algal system investigated. The molecular size distribution of NPOX formed upon chlorination of algal and model ECP was determined by using size-exclusion high-performance liquid chromatography (HPLC), followed by TOX detection of the eluted fractions. Three liters of A. flos-aquae and C. pyrenoidosa filtrates and a 5 mg/L chlorophyll solution, as well as their controls, were adjusted to pH 7 and chlorinated at 20 mg/L Clz at 24 "C for 1day. The filtrates and their controls were then concentrated to between 30 and 45 mL by using rotary vacuum evaporation. Between 2 and 3 mL of each concentrate was injected via multiple runs onto a highperformance liquid chromatograph equipped with a sizeexclusion column. Four eluted fractions were collected for each concentrate, corresponding to >10000,10 000-1000, 1000-100, and 1oo0) organically combined chlorine, as well as those of Glaze et al. (24), who characterized the TOX-forming potentials of high molecular weight organics in lake water. Furthermore, it is not surprising that algal ECP precursors in NPOX-formingreactions were of high molecular weight, since many researchers have determined that phytoplankton release high molecular weight compounds, especially during the stationary phase of growth (25). Comparison of Results
Other studies that have attempted to document the effects of chlorination on algal ECP have dealt exclusively with trihalomethane (mainly chloroform) production. Chloroform percent yields, (CHC13as C/initial TOC before chlorination) X 100, obtained from two other studies which chlorinated algal ECP from a number of species at reaction conditions similar to those for standard chlorination in these experiments (pH 7,24 "C, reaction time 1day) were 0.18-0.38 (3) and 0.04-5.0 (41, while those determined in this investigation ranged from 0.019 to 0.058. Chloroform percent yields obtained on chlorination of algal biomass derived from a number of species have been reported as being 0.2-3.9 (41, 0.13-1.60 (3), and 0.10-0.17 (5), while those yields obtained on chlorination of humic and fulvic acids were, in general, at least 10 times higher than those reported here for algal ECP (26-29). In summary, the chloroform yields of the algal ECP generated in this experiment were lower than those reported for algal ECP, algal biomass, and humic substances reported by others. A comparison of the TOX- and NPOX-generating potentials of the algal ECP in this study with ECP and biomass from other algal systems was not possible since this data base does not exist. However, another indication of the tendency for an organic carbon matrix to incorporate chlorine is the ratio of the amount of chlorine incorporated into organic molecules (assessed via TOX measurements) to the amount of chlorine consumed (chlorine demand) in all of its reactions in water. Using this ratio, one can compare the magnitude of organohalide-forming reactions to that from the other reactions such as oxidation or chloramine formation. Jolley et al. (30)and other researchers have estimated that reactions (principally oxidation) other than organohalide-formingreactions account for 99% of the chlorine consumed in water treatment. However, the ratios, calculated from data in this study, show that organohalide-formingreactions account for a Environ. Scl. Technol., Vol. 18, No. 11, 1984
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Table IV. Organohalide-Forming Potentials of Cblorella vulgaris Biomass and ECP on a per Cell Basis algal system ECP biomass ECP plus biomass
TOC, mg/mL
3.2 X 1.9 X
cell concn, cells/mL
mg of TOC/cell
4.63 X lo6 2.91 X lo5"
6.9 X 6.6 X lo4
CHCl3, rg/mL
1.5 X 1.8 X
TOX, pg of Cl-/mL
pg of CHC13/ cell
pg of C1TOX/cell
9.8 X 1.7 X lo-'
3.2 x 10-9 6.2 X 6.5 X
5.8 x 10-7 6.0 x 10-7
2.1 x 10-8
"4.63 X lo6 cells/mL X 75 L/(11.91 X lo3 mg) X 10 mg/L = 2.91 X lo5 cells/mL, where 11.91 X lo3 mg was the weight of C.uulgaris biomass obtained upon filtering 75 L of algal culture and 10 mg/L biomass (1.9mg/L TOC) was the spiking concentration of biomass.
sizable portion of the chlorine consumed in the supernatant samples. The percentages of chlorine demand incorporated as organohalide upon chlorination of algal ECP under standard chlorinating conditions were the following: C. vulgaris, 9.3%; C. pyrenoidosa, 1.0%; A. flos-aquae, 15.8%; chlorophyll, 15.5%; the average being 10.4%. Furthermore, under certain conditions more than 50% of the chlorine demand was incorporated as organic halide (C.vulgaris at 7 "C; chlorophyll at 7 "C). The results here indicate that the majority of POX formed was chloroform, the only halogenated purgeable compound identified by using purge-and-trap chromatography. Other halogenated compounds contributing to the POX fraction may have decomposed or have been irreversibly retained on the liquid support phase. This explanation is supported by the work of Oliver (31),who showed that dihaloacetonitriles, which cannot be analyzed by using conventional purge-and-trap GC techniques, are formed upon chlorination of algae at a concentration approximately 10% of the generated trihalomethane concentration. In most cases, the POX concentrations generated upon chlorination of algal ECP were less than the NPOX levels. Others have shown similarly that upon chlorination of humic substances the amount of nonvolatile organic-bound chlorine generated exceeded the volatile fraction produced (32-34). These results could possibly be explained by the fact that the formation of POX (chloroform) from organic matter involved a number of time-dependent reactions involving very specific attacks by chlorine on a single carbon atom of a specific nature (given that the haloform reaction or a similar type of reaction was responsible for POX generation), in contrast to NPOX formation in which more numerous and mechanistically simpler reactions (such as chlorine addition to double bonds) could cause its formation. Organohalide-FormingPotentials of Algal Biomass and ECP on a per Cell Basis. The Chlorella vulgaris biomass generated during these ECP experiments was collected, ruptured, and chlorinated, as reported by Johnson (35). Upon chlorination of a ruptured C. vulgaris suspension (having an initial TOC of 1.9 mg/L) at pH 7, 24 "C, 20 mg/L C12,and 1-day reaction time, the following CHCl,, POX, NPOX, and TOX concentrations were obtained (in pg of Cl-/L): 16,16,152, and 168, respectively. The TOC remaining in solution after chlorination was 1.8 mg/L. On the basis of this information, the molar TOX/TOC and CHC1,-C/TOC ratios for algal biomass were calculated as being 0.032 and 1 X respectively. Under the same reaction conditions, it has been shown that the TOX/TOC and CHC1,-C/TOC ratios for C. vulgaris ECP were 0.011 and 4.9 X respectively (Table 11). These results indicate that, on a per carbon basis, the organohalide-formingpotentials between C. vulgaris ECP and biomass were similar (less than 1order of magnitude difference). However, this does not take into account the actual comparative excretions of algal biomass and ECP to the aquatic environment. Even though the potential to form chloro organics on a per carbon basis is similar 816
Envlron. Scl. Technol., Vol. 18, No. 11, 1984
between C. vulgaris biomass and ECP, the amount of carbon in solution having its origin as biomass or algal ECP may be quite different, and therefore, the contributions of each to organohalide formation may not be similar. Table IV lists the total organic halide and chloroformforming potentials of algal biomass and ECP on a per cell basis. As shown, it was calculated that 1.9 mg/L TOC (or 10 mg/L as C.vulgaris) of biomass was derived from an algal population of 2.91 X 105cells/mL. The concentration of ECP representing 3.2 mg/L TOC was derived from the concentration of algae occurring at harvest time (4.63 X lo6 cells/mL). The ratios of TOC/cell concentration for algal ECP and biomass are indicative of the comparative amount of extracellular carbon released and the amount of intracellular carbon contained per algal cell. On the basis of these ratios, it can be calculated that, on the average, an individual C. vulgaris cell upon attaining stationary phase of growth had excreted approximately 10% (6.9 X 10-1°/6.6 X lo4 X 100) of the amount of carbon contained inside an algal cell. Of course, one must also consider that the algal cells may be removed more readily than their ECP via conventional water treatment practices before cell lyses occur, thereby making the comparative contribution to organohalide formation of algal ECP higher than 10%. But if biomass removal does not occur, one can observe that 97% of the TOX generated (5.8 X 10-7/6.0 X lo-') and 95% of the CHC13generated (6.2 X 10-8/6.5 X would be derived from algal biomass. Included in Table IV are the estimates of the total potential of algal compounds (intra- and extracellular) on a per cell basis to form total organic halide (6.0 X pg of Cl-/cell) and chloroform (6.5 X 1a-Spg of CHCl,/cell). These values can perhaps be used as broad indicators of potential organohalide and chloroform formation in waters containing varying C. vulgaris populations, based on the fact that additional research by Johnson (35) and Wachter (20) indicated that biomass and ECP release are proportional to algal growth and that changes in TOX and CHC13 concentrations are generally proportional to changes in biomass and ECP concentrations. The alga C. vulgaris is of particular interest in this study due to the fact that it is the principal one occurring in many freshwater environments (36). By we of the above indicators and given that the average concentration of total phytoplankton found in treatment plant effluent, reservoir, and distribution systems in five water systems in the Pittsburgh area in 1978 was approximately 6000 cells/mL (36),the typical organohalide concentrations attributable to the chlorination of C. vulgaris biomass and ECP at standard chlorinating conditions would be less than 5 pg of Cl-/L of TOX and 1 pg/L CHC1,. Registry No. CHC13, 67-66-3; chlorophyll a, 479-61-8.
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and Health Effects”;Jolley, R. L.; Brungs, W. A.; Cumming, R. B., Eds.; Ann Arbor Science: Ann Arbor, MI, 1980;Vol. 3, pp 85-98. (35) Johnson, L. C. Master of Science Thesis, University of Pittsburgh, Pittsburgh, PA, 1982,pp 1-47. (36) Sykora, J. L.; Roche, R.; Kriess, F. L.; Barath, M. A.; Volk, D.; Coyne, J. T.; Jackim, R. R.; Keleti, G. “Water Quality in Open Finished Water Reservoirs-Allegheny County, Pennsylvania”; Graduate School of Public Health University of Pittsburgh, Pittsburgh, PA, 1980;pp 1-62.
Received for review June 10,1983,revised manuscript received December 27,1983. Accepted May 2,1984. This research was supported by the U.S. Environmental Protection Agency (EPA), Health EffectsResearch Laboratory, Cincinnati,OH, Cooperative Agreement CR-807365-03-3.W . Emile Coleman and Herbert Pahren were the EPA Project Officers. Some additional EPA support was provided under Cooperative Agreement CR-810543 between the EPA Office of Research and Development, Washington, DC, and the University of Pittsburgh Center for Environmental Epidemiology. This research has not been subjected to EPA peer and administrative review policy and therefore does not necessarily reflect views of the Agency, and no official endorsement should be inferred.
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