United States Environmental Protection Agency Municipal Environmental Research Laboratory Cincinnati OH 45268 Research and Development EPA-600/S2-84-005 Jan. 1984 4MER& Project Summary Alternative Water Disinfection Schemes for Reduced Trihalomethane Formation Volume II. Algae as Precursors for Triha/omethanes in Chlorinated Drinking Water Kathryn F. Briley, Robert F. Williams, and Charles A. Sorber Three species of algae (Anabaena cylindrica, Scenedesmus quadricauda, and Pediastrum boryanum) were inves- tigated for their trihalomethane (THM) formation potential in water treated with chlorine. Algae were cultured and the cells (algal biomass) were separated from the extracellular products (ECPs) at several points along the normal growth curves of each species for a separate study of their contributions as THM precursors. The cells were resus- pended in organic- and chlorine-demand- free water, and the cells and ECPs were then separately dosed with three chlo- rine concentrations. The THMs formed after 1 and 24 hr of chlorine contact time were analyzed by the gas-sparging technique and gas chromatography. For each point examined along the growth curve, growth was monitored by both cell counts and fluorometric assay of chlorophyll-a. Correlation of the algae growth period and THM production was observed. Furthermore, significant levels of THM were produced from both the ECPs and the isolated algal cells of all three species when dosed with chlorine. As expected, the THM levels formed were related to the free chlorine residual and to the TOC levels observed. These findings suggest that THMs may be partially reduced by observation and control of the natural phytoplankton communities in the wa- ter sources for domestic water supplies. This Project Summary was developed by EPA's Municipal Environmental Re- search Laboratory. Cincinnati. OH. to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Introduction Chlorination of surface waters gener- ally results in the formation of trihalo- methanes (THMs). These compounds result from a reaction or series of reac- tions of chlorine with natural organic precursor material present in the source water. The exact nature of the precursor material is not clear, though humic and fulvic acids and algae have been impli- cated as contributors. Chloroform (CHCI3) is the THM found in greatest quantities in finished waters, but dibromochlorometh- ane (CHBr2CI), bromodichloromethane (CHBrCI2), and bromoform (CHBr3) are commonly found in varying quantities as ------- well. The U.S. Environmental Protection Agency (EPA) has established a maximum contaminant level of 0.10 mg/Lfor THMs in finished waters for communities with populations of 10,000 or more. Attempts to reduce total trihalomethanes (TTHMs) below the established levels in an eco- nomical way are hampered by the lack of a complete understanding of the nature of the THM precursors. Identification of THM precursors would allow establish- ment of techniques to remove them selectively. The purpose of this study was to examine a class of potential THM pre- cursors in surface waters. Specifically, investigations were made of the contribu- tion of algal cells and extracellular meta- bolic products (ECPs) from algae to the production of THMs. This information may be useful for developing alternative disinfection procedures that can allow reduced levels of THMs in finished water and still maintain effective water disin- fection. Experimental Procedures To learn the extent to which algal cells and ECPs might contribute to THM forma- tion, three algal species were grown in the laboratory under controlled condi- tions. The cells were separated from the ECPs, each fraction was chlorinated, and THM concentrations were measured. The latter were compared with algal cell concentrations in the original suspension to indicate the ability of algae to cause THM problems. The algal species chosen for study were nonaxenic Anabaena cylindrica, Pediastrumboryanum, and Scenedesmus quadricauda. These species were select- ed for study because they are easy to grow in the laboratory and because they are abundant in South Central Texas lakes. The algae were grown using Bold* basal pH 6.6 medium cultured in Erle- meyer flasks with metal snap-on caps that permitted some air flow to the cultures without allowing contamination. The cultures were further aerated by frequent shaking. The algae were sub- jected to 16 hr of light and 8 hr of dark each day on a constant schedule with a light source that ranged from 275 to 450 foot-candles. The temperature for algal growth was maintained between 23° and 25°C. For each experiment in this study, algae from a stock culture with a known number of cells/mL were inoculated into "Mention of trade names or commercial products does not constitute endorsement for use. 2 L of freshly prepared medium to make a final dilution of approximately 103 cells/ mL. The algae were then cultured until the growth phase desired for THM analy- sis was reached. The growth phases desired for THM precursor studies were determined by cell count techniques and chlorophyll-a measurements. These growth-monitor- ing techniques allowed algal growth curves to be generated. From these curves, the times chosen for THM pre- cursor examination were selected with the intention of encompassing the five basic growth phases (lag, early expo- nential, late exponential, early stationary, and late stationary) of each species employed. The death phase was not examined. The times chosen for experi- mentation along the growth curve of Anabaena were days 0, 2, 4, 7, 10, 14, and 21. Day 0 encompassed the brief, if any, lag phase; days 2, 4, and 7 were during the exponential growth phase, and day 10 was transitional between the late exponential and early stationary phases. Days 14 and 21 were during the station- ary phase. Similarly, for Pediastrum, days 0,2,7,10,15,21, and 28 were chosen for study, and Scenedesmus experiments were conducted on days 0, 2,4, 8,11,15, and 21 of the growth cycle. At the chosen time, a 270-mL aliquot was removed from the 2-L experimental culture. From this aliquot, 20 mL was used for growth-monitoring measure- ments done by cell enumeration and fluorometric assay of the chlorophyll-a. Duplicate fluorometric chlorophyll-a meas- urements and cell counts were made on the algae each time THM experiments were performed. The chlorophyll-a measurement was •made using a fluorometer with a 5-60 excitation filter and a 2-28 emission filter. Chlorophyll-a was extracted as described in Standard Methods (Standard Methods for the Examination of Water and Waste- water, 14th edition; 1975; Amer. Public Health Assoc., Amer. Water Works Assoc., Water Pollution Control Federation; pp. 1030-1032) for the spectrophotometric procedure. A calibration curve for the fluorometer was constructed using com- mercially obtained chlorophyll-a. The calibration curve obtained had a correla- tion coefficient (R2) of 0.993 determined by regression analysis. Reference sam- ples were prepared by extraction in the same manner without algae, and these readings were subtracted from the fluo- rescence readings before chlorophyll-a calculations. Replicate measurements were performed on each organism. Cell count measurements were made along the algal growth curves using a • mechanical counter with an aperture tube of 100-jUm diameter. The instrument was calibrated using pollen and glass spheres of known volumes, and the optimal sensitivity settings for measuring cell counts for each algal species were determined. Commercially obtained elec- trolyte solution was used for making the cell dilutions and corresponding control dilutions. The reference reading values made using medium and diluent alone were subtracted before cell count calcula- tion. The algae were dispersed by vigorous shaking with glass beads before cell counts were performed. After the 20-mL volume had been removed for the growth measurement procedures, the remaining 250 mL was used for the THM precursor experiments. The cells and their corresponding extra- cellular products were separated by centri- fugation at 2400 to 2600 rpm (1050 to 1250 x g) for 20 min. The ECPs and medium (supernatant) were then poured off and vacuum filtered using a 0.45-yum (47-mm) membrane filter. The filtered ECPs were transferred to a clean flask and refrigerated at 4°C in the dark overnight or until chlorine addition. Be- fore chlorination, the ECPs were warmed to room temperature. The pellet formed , after centrifugation (algal biomass) was washed twice with 0.9 percent sterile sodium chloride, resuspended, and recen- trifuged. The supernatant was then dis- carded. The washed cells were resus- pended in 250 mLof organic-free, chlorine- demand-free water. Approximately 10~4 moles of phosphate buffer (filtered through a 0.45-/jm membrane filter) were added to the resuspended cells for pH control. The resuspended cell or ECP aliquots were then divided into three 80- mL samples, measured for pH and temper- ature, and immediately dosed with the desired amount of chlorine (for the ECPs, 7, 24, 33 mg/L; and for the cells, 1.5 and 5.9 mg/L). After 1 hr of chlorine contact time, the pH, temperature, and chlorine residuals (free and total) were again measured in duplicate. Samples were obtained for TOC, TKN, and NH3-N deter- minations. A THM sample (1-hr contact time) was collected in a vial containing 0.3 to 1.0 mLof 0.01 N sodium thiosulfate (according to chlorine dose) to quench the reaction (instantaneous THM) and sealed with a Teflon-faced septum that allowed no headspace. A second THM sample was stored at room temperature in a Teflon-sealed vial with no headspace for 24 hr and then quenched with sodium ( ------- thiosulfate (terminal THM). After the sodium thiosulfate addition, the THM samples were all stored at 4°C until analysis. All vials were warmed to room temperature before THM analysis. THM samples were measured using the gas- sparging technique developed by Bellar and Lichtenberg (Bellar, T. A., and J. J. Lichtenberg; 1974; Determining Volatile Organics at Microgram-per-Litre Levels by Gas Chromatography; J. Amer. Water Works Assoc., 66(12J:739) and all anal- yses were performed on a Varian 3700 gas chromatograph equipped with a flame ionization detector system and a reporting integrator. A microprocedure for residual chlorine determinations (free and total) was de- veloped because of the sample size available for analysis. The leuco crystal violet technique for chlorine residual was modified to use a 5-mL sample (a total of 10 ml for both free and total chlorine) in both the presence and absence of the Bold basal medium. The reference tech- nique was amperometric titration. The absorbance of 592 nm of the leuco crystal violet color development was monitored. Calibration curves were constructed for free and total available chlorine residual determinations in both chlorine-demand- free water and in the Bold basal medium. Chlorine-demand-free water was used in the resuspension of the algal cells for THM precursor studies, and the Bold basal medium was used for the extra- cellular product THM studies. The free and total chlorine residuals were meas- ured after a 1-hr chlorine contact time for both the water and the medium calibra- tion curves. The calibration curves ob- tained for the chlorine-demand-free water had correlation coefficients (R2) of 0.995 and 0.991, respectively. Results Increasing cell count was directly re- lated to the amount of chlorophyll-a produced by the cells. Linear regression analyses for each algal species were obtained by averaging each day's chloro- phyll-a replicate measurements and com- paring these with the average of that day's cell count replicate measurements. These analyses yielded correlation co- efficients (R2) for chlorophyll-a to cell count of 0.952 for Anabaena, 0.955 for Scenedesmus. and 0.989 forPediastrum. The three species exhibited different growth curve shapes and growth rates. After chlorine was added to the cell and ECP test samples of Anabaena, THM production was monitored at contact times of 1 and 24 hr (Table 1). The pH for Table 1. Comparison of THM Production from Three Species (ECPs and Cells) Day in Growth Cycle (days) 7* 21 Contact Chlorine Time (hr) Dose 1 1 24 24 1 1 24 24 Low High Low Medium Medium High Low Medium Anabaena Cells ECPs 315.6 392.8 408.6 400.3 138.0 392.8 308.3 549.2 29.6 45.4 64.6 258.9 73.1 149.5 57.2 497.0+ THM, ug/L Pediastrum Scenedesmus Cells ECPs Cells ECPs 252.1 424.0 97.3 343.4 481.8 31.9 119.6 59.7 944.6 167.4 466. 1 <0. 1 120.8 333. 1 124.7+ 359.7 157.1 <0.1 7. 1 <0. 1 285.6 <0.1 333.3 <0. 1 397.9 37.8 321.5 782.8 47.3 13.9 553.0 539.7 *Eight days for Scenedesmus. +Onlyhigh chlorine dose data available. all measured data points ranged between 6.5 and 7.4 for the ECPs and 6.4 and 7.6 for the cells. The temperature ranged between 21 ° and 24°C for all Anabaena measurements. Typically, TTHM produc- tion increased slightly and then fell as the stationary phase of growth was reached. The free chlorine residual was low during the early portions of the growth curve, indicating a high chlorine demand. After 6 days, however, a free residual persisted, indicating a change in the type and/or degree of ECP produced. THM production increased with an ill-defined maximum near the late exponential growth phase, followed by a slight decrease in amount of THM. This type of pattern was also observed for the other chlorine doses. The drop off in THM level was not due to chlorine limitations alone, since a free residual (0.4 mg/L) persisted for the 32.5-mg/L dose and a similar decrease was noted. Results of chlorinating the isolated Anabaena cells paralleled those observed for the ECPs. A free chlorine residual was maintained throughout the growth cycle except in 1(3? case of the lowest dose (at days in the growth cycle greater than 10). The Scenedesmus cells exhibited a pH range of 6.2 to 7.4, and the pH of the ECPs ranged from 6.4 to 7.0. The temperature for all Scenedesmus measurements ranged from 24° to 25°C. The Scene- desmus cells after both 1 and 24 hr of chlorine contact time showed high chloro- form production immediately after initia- tion of the experiment. After the initially high levels, the chloroform levels dropped until approximately day 4 of growth, at which time a peak in chloroform produc- tion was observed (days 4 to 10). After this peak, the chloroform levels dropped to less than the detection limits toward the last day examined along the growth curve (day 21). A comparison of 7- to 8- day versus 21 -day THMFP values appears in Table 2. The Scenedesmus ECPs at a 1-hr contact time produced chloroform levels in generally the same pattern as that described for the cells. For the Scenedesmus ECPs after 24 hr of chlorine contact time, maximum chloroform produc- tion occurred somewhat later in the growth curve (approximately days 11 to 21) than that observed for the cells of the ECPs at the 1 -hr contact time. The Pediastrum cells had a pH range of 6.5 to 7, and the pH of the ECPs ranged from 6.5 to 7.9. For the Pediastrum cells and ECPs, the temperature ranged be- tween 23° to 25°C. After both 1 - and 24- hr contact times, the Pediastrum cells showed two chloroform production peaks. The chloroform levels were initially low during the beginning of the growth curve. Maximum chloroform production was reached between days 7 and 10 of growth. After day 10, the chloroform levels dropped and then began to increase again until the second chloroform maxi- mum was reached between days 15 and 21. After the second peaks in chloroform production, the amount of chloroform dropped to low levels toward the latter part of the growth curve. Chloroform production from the Pediastrum ECPs for both chlorine contact times followed the same general pattern as that of the Pediastrum cells. The chlorine doses employed for the Scenedesmus ECPs and cells were com- parable and generally sufficient to main- tain a residual at 1 hr of contact time. With both Scenedesmus cells and ECPs, a free chlorine residual was present for all but the lowest chlorine doses after 1 hr of chlorine contact time, even during the latter portions of the growth curve. Thus, the decline in chloroform levels toward ------- the end of the growth curves for the cells and the ECPs was not due to chlorine limitations. This result is general for all three species. Discussion The THM levels produced with the chlorination of algae depended on the species (Anabaena. Pediastrum, orScene- desmus). the age of the culture, the chlorine dose, and the substance chlori- nated cells versus ECPs). THM production was quite variable. Nevertheless, the results can be presented to facilitate comparison by calculating the THM for- mation potential (THMFP) of cells and ECPs per million algal cells in suspension. The THMFP (//g/106 cells) for the cells and ECPs of each of the algal species is listed in Table 2 for two time points in the growth cycle. The chlorine doses chosen for representation were 23.7 mg/L for the ECPs and 5.0 mg/L for the cells. The range of values depends on the algal species and the position of the algae in its growth cycle, with greater THM formation occurring in the exponential growth phase; but the values do not vary greatly between chlorine contact times of 1 and 24 hr. A bloom of these three species would produce 0.2 to 120 prg/L THM per 106 cells. The ECPs produced by 108cells of the species studied would produce an additional 0.1 to 50 tig of THM. These values are approximate and vary with growth condition and chlorine dose, but algal sources can clearly contribute sig- nificantly to the overall THM production in a treatment facility. The amount of algae-related THM precursor in water can be influenced by factors other than the number of algae present in the water. In an undisturbed natural water, both cells and ECPs would be present and would contribute to THM formation. In a water treated with algacide to control algal blooms, some cells would be present along with ECPs and a quantity of disintegrating cell material. At a water treatment plant that practiced coagulation and filtration for algae removal and then chlorinated the filtrate, the cells would be removed from the water before chlorina- tion, but the ECPs would probably pass through the plant and react with chlorine to produce THMs. The higher chlorine doses and the longer chlorine contact time (24 hr) for the cells and ECPs of both Pediastrum and Scenedesmus produced lower chloroform levels than expected (i.e., compared with the chloroform levels produced by the lower chlorine doses and by the 1-hr contact time). The reason for this obser- vation has not been established, but possibly organohalide compounds other than haloforms were formed, given that these species had higher chlorine doses and a longer chlorine contact time, and may have different precursor compounds from Anabaena. These factors imply different mechanisms of action. The concentrations of bromodichloro- methane, dibromochloromethane, and bromoform for the Pediastrum and Scenedesmus cells and ECPs were gener- ally below the detection limits, except for a few isolated instances in which the bromine-containing volatile concentra- tions were disparately high when com- pared with the chloroform levels. These results were probably caused by contami- nants rather than by the compounds of interest. Thus for Scenedesmus and Pediastrum, only the chloroform levels were used in the data analysis. The technique used for the Anabaena THM analyses gave no bromoform results, and the dibromochloromethane measure- ments were questionable. For this reason, the total THM data given here for Anabaena actually reflect the concentra- tions of chloroform and bromodichloro- methane only. The results presented demonstrate that high THM concentrations are pro- duced from both algal biomass and metab- olites. The significance of these results suggests that THM may be partially reduced by observing and controlling the natural phytoplankton communities in the water source for domestic water Table 2. THMFP for the Three Algal Species (ECPs and Cells) with a Medium Chlorine Dose Time in Growth Contact Cycle (days) Time (hr) 7* 24 21 24 21 1 Anabaena Cells ECPs 3.6 0.8 0.2 2.3 0.7 0.1 THMFP I tig /1W Cells) Pediastrum Cells ECPs 122.6 5.7 45.7 44.5 31.6 14.9 Scenedesmus Cells ECPs 21.6 <.001 <.001 47.1 6.6 0.6 'Eight days for Scenedesmus. supplies. Clearly, humic or fulvic pre- cursors or both contribute to THM produc- tion upon chlorination; but this source can only account for a portion of the THMs produced. To date, no complete mass balance has been possible to deter- mine all the precursor molecules that produce THMs. This problem is further complicated by the incomplete yields of THMs that model compounds produce. A significant amount of algal biomass is unlikely to survive through the coagula- tion and filtration steps of a well-operated water treatment plant; however, ECPs may persist if they are not destroyed by bacteria or removed in the coagulation process. Furthermore, algae can pass through the coagulation and filtration steps if they are present in large numbers or if the treatment plant is not being optimally managed. THM precursors (ECPs and cells as well as humic and fulvic acids) will be present at the point of prechlorination (chlorination before co- agulation), a process used at many sur- face water treatment plants. For this reason, the presence of such materials may allow the THM levels to be greater than those formed if coagulation and filtration are performed before chlorina- tion. Conclusions 1. Significant THM levels (10 to 950 /ug/L) were produced from both the resuspended cells and the ECPs at various stages of the growth curves of Anabaena, Pediastrum, and Scene- desmus. The Anabaena cells provided as many or more THMs as did the Anabaena ECPs. The Scenedesmus ECPs yielded more chloroform than the corresponding algal biomass. For Pediastrum, the chloroform provided by the ECPs upon chlorination was comparable to the chloroform levels provided by the Pediastrum eel Is. The THM concentrations from all three algal species were comparable with THM yields obtained in other studies evaluating humic and fulvic acids. 2. THM production from the Anabaena ECPs increased as the stationary growth phase was approached. The THM production from the Anabaena cells also followed this trend. 3. Chloroform production from the Scenedesmus cells and ECPs was initially somewhat high, then de- clined until the late exponential or early stationary growth phase, at ------- which point maximum chloroform production occurred. 4. The Pediastrum cells and ECPs pro- vided two chloroform production peaks during the growth period ex- amined. Both chloroform production peaks occurred during the period of growth considered to be the late exponential or early stationary growth phase. 5. THM production by the chlorination olAnabaena, Pediastrum, and Scene- desmus cells and ECPs was related to the total organic carbon (TOC) pres- ent, the cell count (growth phase), and the chlorine residual after 1 hr of chlorine contact time. 6. The ECP chlorine residuals of all three organisms increased with cul- ture age, indicating a change in type and/or degree of ECPs produced. An alternative explanation is that a chlorine-demanding component of the medium may have been assim- ilated by the cells. 7. Because cell count is related to THM production and to chlorophyll-a con- centration along the growth curve, a relationship between chlorophyll-a and THM production for the algal cells is inferred. The ECPs do not have significant levels of chlorophyll- a, indicating that because THMs are produced from both the cells and the ECPs, more than one THM precursor must be involved. arbitrarily be abandoned. Monitoring algal conditions can help minimize THMs and simultaneously help maxi- mize finished water quality through the proper choice of chlorination conditions. Kathryn F. Briley, Robert F. Williams, and Charles A. Sorber are with the University of Texas, San Antonio, TX 78285. Gary S. Logsdon is the EPA Project Officer (see below). The complete report, entitled "Alternative Water Disinfection Schemes for Reduced Trihalomethane Formation: Volume II. Algae as Precursors for Tri- halomethanes in Chlorinated Drinking Water," (Order No. PB84-129 006; Cost: $11.50, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Municipal Environmental Research Laboratory U.S. Environmental Protection Agency Cincinnati. OH 45268 Recommendations 1. Since this study demonstrated that both algal biomass and metabolites from algae produce high THM con- centrations, THM should be reduced by proper choice of the position of chlorination in a water treatment plant. Coagulation and filtration of surface waters should precede chlori- nation to reduce THM levels in fin- ished waters. Such treatment before chlorination also reduces other THM precursors (organic and humic) that may be present. 2. Algal blooming should be monitored and controlled so that appropriate water treatment procedures can be adopted for the source water condi- tions. Prechlorination is an advan- tageous procedure at many water treatment plants and should not ------- United States Environmental Protection Agency Official Business Penalty for Private Use $300 Center for Environmental Research Information Cincinnati OH 45260 ;;--, -~Ti ;',,', ,« IL 60601 i, U.S. GOVERNMENT PRINTING OFFICE: 1984-759-102/844 ------- |