Envlron. Sci. Technol. 1905, 19, 427-431
Studies on the Chlorination of Chlorolignins and Humic Acid Knut P. Krlngstad, Flllpe de Sousa, and Lars M. Stromberg" Swedish Forest Products Research Laboratory, Box 5604, S-114 86 Stockholm, Sweden
High relative molecular mass material (chlorolignins) in spent liquors from the chlorination and alkali extraction stages of the bleaching of softwood kraft pulp yields chlorinated phenolic and Ames test mutagenic compounds when treated with chlorine under conditions similar to those used in the disinfection of drinking water. Per unit weight of total organic carbon (TOC) the quantities are comparableto those formed in the chlorination of naturally occurring aquatic humic acid.
Introduction In the conventional bleaching of softwood kraft pulp some 70 kg of organic material/ton of pulp dissolves from the pulp into the bleaching liquors (1). By far the greater part of the material is dissolved in the first two of several consecutive bleaching stages. These are a chlorination and an alkaline extraction stage (C and Elstage, respectively). In recent years, available information on the chemical composition and biological effects of spent liquors from these bleaching stages has increased ( I ) . The dissolved material is chlorinated to a considerable degree and consists of a large number of compounds with a broad distribution of relative molecular masses and chemical natures. It has been well established that such liquors may exert acute toxic and genotoxic effects due to various chlorinated and nonchlorinated compounds of low relative molecular mass (I, 2). From an environmental point of view, one area which is not presently well understood concerns questions on the fate of the high relative molecular mass material (chlorolignins which constitute the greater part of the dissolved material) in the receiving waters. Recent investigations show that microorganisms will degrade chlorolignins (3, 4), and the observation that highly lipophilic chlorinated veratroles may form as metabolites in the biological degradation process is particularly interesting. Spent bleach liquors are often released into receiving waters used for the preparation of drinking water. The present paper describes studies on the behavior of chlorolignins when chlorinated under conditions similar to those used in the water disinfection process. Particular emphasis has been laid upon the possible formation of compounds known to be biologically active such as Ames test mutagenic and chlorophenolic compounds and therefore, undesirable from a human consumption point of view. For comparison identical studies have been carried out with aquatic humic acid as chlorination substrate since recent studies have shown that such material leads to biologically active compounds when it is chlorinated under drinking water disinfecting conditions (5-7). Experimental Section Spent Chlorination (C-Stage) and Alkali Extraction (El-Stage) Liquors. Industrially prepared spent chlorination liquor and alkali extraction liquor from the conventional bleaching of a softwood kraft pulp were used. Chlorination of the pulp (kappa number 36.0) was carried out at 3.0% pulp consistency with a charge of 6.3% C12, a temperature of 25 OC, and a reaction time of 1.5 h. The end pH of the chlorination liquor was 2.1 and the total 0013-936X/85/0919-0427$01.50/0
liquor volume per ton of pulp 78.2 m3. The alkali extraction liquor was prepared from the resulting chlorine bleached pulp at 12% pulp consistency with a charge of 2.6% NaOH, a temperature of 60 "C, and a reaction time of 2 h. The end pH of the liquor was 11.2 and the total liquor volume per ton of pulp 8.3 m3. Isolation of Material with High Relative Molecular Mass from the Chlorination (HM-C) and Alkali EXtraction (HM-E)Liquors. Ultrafiltration Procedure. Samples of the liquors from the two bleaching stages were subjected to ultrafiltration at the Department of Food Engineering at the University of Lund, Sweden. A Romicon PM 2-filter (cutoff 2000 dalton) with an area of 2.5 m2 was used with a pressure of 2 bar into and 0.65 bar out of the ultrafiltration module. The circulation flow was 5.7 m3/h. The filtration was carried out at a temperature of 10-13 "C; 120 L of C-stage liquor (prefiltered through a 34-pm filter) was pH adjusted to 5 with approximately 800 mL of 2 M NaOH. Thereafter, the liquor was subjected to ultrafiltration resulting in a final retentate volume of 24 L. The retentate was diluted with an equal amount of distilled water and then reconcentrated to the original volume by ultrafiltration. This procedure was repeated twice more. Finally, the pH of the concentrate was adjusted to the original level of 2.1 by adding approximately 100 mL of 1 M H2S04. The ultrafiltration of the E-stage liquor was carried out in a similar way: 50 L was pH adjusted to 7 by the addition of 410 mL of 1M H2S04and ultrafiltered to a final volume of 15 L. The retentate was washed 5 times, each time with 15 L of distilled water. The pH of the concentrate was kept at 7. All liquors were stored at -18 OC until used. Isolation of Humic Acid Material. An aquatic sample of humic acid material was isolated from a lake in the southern part of Sweden. The material was precipitated from the water by acidification with HC1 to pH 2.1 and worked up following a previously described method (8). Elemental analysis of the material revealed a C:H:O:N ratio of 1.001.08:0.42:0.10. The ash content was found to be 6.3%. The IR absorption spectrum showed absorption bands characteristic of humic acid material (9, 10). Chlorination. The ultrafiltered liquors were extracted continuously with ether (pro analysis) (May and Baker Ltd., Dagenham, England) for 24 h in order to remove any low relative molecular mass compounds still present. Prior to the extraction, the ultrafiltered HM-E liquor was diluted 3.3 times (i.e., to its original concentration) with distilled water, and the pH was adjusted to 4.0. Problems with emulsion-formation during the extraction procedure were thus considerably reduced. The ether present in the liquor after extraction was removed by using a rotary evaporator at a temperature of 25 OC. The pH of the liquor was thereafter adjusted to 7.0. The respective solutions were chlorinated immediately after extraction, four different C12/C ratios being used with each material, viz., 0.5:1, 1:1, 2:1, and 4:l (w/w). In the case of the bleaching liquors, 625 mL of the ether-extracted HM-C solution (correspondingto 3125 mL of C-stage liquor) or 130 mL of the ether-extracted HM-E solution (corresponding to the original concentration of E,-stage liquor) was diluted with distilled water (1150 mL total volume in each case). The HM-C solution was pH
0 1985 Amerlcan Chemical Society
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427
adjusted to 7.0 with 0.5 M NaOH. Phosphate buffer (approximately 500 mL) and chlorine water of the same pH were then added to give a total volume of 1700 mL in each case. The amount of HM-C or HM-E liquor was chosen so as to yield a final concentration of total organic carbon (TOC) of 73.5 mg/L. In the chlorination of humic acid, the material was dissolved in 60 mL of 0.01 M NaOH by stirring for 0.5 h. Thereafter, the pH of the solution was lowered to 7.0 by the addition of 2 M HC1. Phosphate buffer (approximately 25 mL) and chlorine water of the same pH were then added to give a total volume of 100 mL. The amount of humic acid was chosen to give a final concentration of TOC of 73.5 mg/L. All chlorinations were carried out at ambient temperature by using a reaction time of 2 h. The pHs of samples containing residual chlorine, HM-C (all chlorinations), HM-E ( 4 1 ratio), and humic acid ( 4 1 ratio), were lowered to 2.4 by the addition of 2 M HCl. The residual chlorine was thereafter removed by using a rotary evaporator at a temperature of approximately 25 "C. The pH of the samples was then adjusted to 7.0. Workup Procedure and Analytical Procedure. (1) Chlorinated Phenols. Samples of the reaction mixtures were acetylated according to a procedure outlined previously (11). The reagents included 2,6-dibromophenol (Fluka AG, Buchs AG), potassium carbonate (E. Merck), acetic anhydride (E. Merck), and n-hexane (E. Merck) and were all pro analysis. The volume of the reaction mixture used for the analysis was 20 mL for the humic acid and HM-C samples, respectively, and 10 mL for the HM-E reaction mixture. A K2C03(0.72 g/mL) buffer solution was added to the reaction mixtures (0.4 mL in the 20-mL samples and 0.2 mL in the 10-mL samples). Subsequently, 0.1 mL of a solution of 2,6-dibromophenol(O.93pg/mL) was added as internal standard. Each sample was then acetylated by the addition of 0.3 mL of acetic anhydride and shaken vigorously for 2 min. After being allowed to stand for another 2 min, the mixture was extracted with 5 mL of n-hexane (distilled prior to use) for approximately 2 min, and the phases were separated by centrifugation (5700 rpm for 5 rnin). The hexane phase was collected and washed with 5 mL of 0.1 M K2C03. After centrifugation for 3 min, the hexane phase was concentrated (rotary evaporator) to a final volume of about 0.3 mL. For the gas chromatographic analysis, 1.5 pL of the hexane extract was injected into a Carlo Erba gas chromatograph (Fractovap MOD 4100) equipped with an electron capture detector (ECD) (63Ni). Retention times and quantification data were measured with a Hewlett & Packard 3390 integrator. The column used was a DB-1 fused silica capillary column (30 m X 0.245 mm i.d.1 manufactured by J & W Scientific Inc. (Cordova, CA). The carrier gas flow rate (He) was 1mL/min (split ratio 1:50), and the column temperature was held constant at 180 "C. The temperatures of the injector and detector (auxilliary gas, nitrogen) were 250 and 320 "C, respectively. The quantification procedure was carried out according to the ECD gas chromatograms using the internal standard method (response factors related to 2,6-dibromophenol as internal standard). (2) Identification of Chlorinated Phenolic Compounds Formed in the Chlorination of Humic Acid. The gas chromatographic analysis of the reaction mixture from the chlorination of humic acid indicated that small quantities of chlorinated phenolic compounds were pres428
Environ. Sci. Technol., Vol. 19, No. 5, 1985
ent. In order to identify these, a specially prepared chlorination mixture was worked up and analyzed by gas chromatography-mass spectrometry (GC-MS). Humic acid was chlorinated (80 mg of TOC/L, total volume 4.0 L, C12:C ratio 2:l) according to the method described above. After a 2-h reaction time, the pH of the mixture was lowered to 3.0 by the addition of 1 M HC1. The mixture was then passed through a column (20 cm X 25 mm i.d.) containing XAD-2 sorption resin (Serva; 0.15-0.2 mm, research grade, Heidelberg, West Germany). Before use, the resin was purified by sequential solvent extractions with methanol, acetone, and dichloromethane according to a previously described method (12). The flow rate was 10 mL/min. Thereafter, the resin was extracted sequentially with acetone (50 mL) and dichloromethane (150 mL). The eluates were concentrated together to a final volume of approximately 5 mL. The extract was acetylated by the addition of 50 mL of 0.1 M K&03 buffer and 5 mL of acetic anhydride. After vigorous shaking for 2 min, the solution was extracted with 3 X 10 mL of hexane for 5 rnin each. To separate the phases, the mixtures were centrifuged (5700 rpm, 10 min). The combined hexane phases were washed with 30 mL of 0.1 M K2C03buffer and after centrifugation (5700 rpm, 5 min) dried with anhydrous NazS04and finally concentrated to a volume of 0.5 mL. Four microliters was used in the GC-MS analysis. The GC-MS investigation was carried out on a Carlo Erba (Fractovap MOD 2900) gas chromatograph-Finnigan mass spectrometer (3200 F) with an on-line computer system (6000). In this experiment a DB-1701 fused silica capillary column (30 m X 0.259 mm i.d.) manufactured by J & W Scientific Inc. (Cordova, CA) was used. The oven temperature was held constant at 200 OC, the transfer-line temperature at 300 OC, and the ion-source temperature at 150 OC. The mass spectra were all run with 70 eV. A complete identification based on mass spectral interpretation was made of 2,4-dichlorophenol acetate, 2,4,6-trichlorophenol acetate, 4,5-dichloroguaiacol acetate, 3,4,5-trichlorocatechol diacetate, 3,4,6-trichlorocatechol diacetate, and tetrachlorocatechol diacetate, the latter with MID technique with the instrument being locked on four mass fragments (246, 248, 250, 290). An additional dichloroguaiacol acetate and a dichlorocatechol diacetate were tentatively identified. The positions of the chlorine atoms could not be established due to the lack of reference compounds. (3) Ames Test Mutagenic Compounds. In the analysis for specific mutagenic compounds in the reaction mixtures from the chlorination of HM-C and HM-E, the pH of the solutions (1500 mL) was lowered to 3.5 by the addition of 2 M HC1, and the solutions were thereafter extracted continuously with ether for 30 h. After being dried with anhydrous Na2S04, the ether extracts were concentrated to approximately 3 mL on a rotary evaporator at a temperature of 20 "C. Thereafter, the extract was analyzed by the GC-MS (MID) technique as described previously (13). (4) Mutagenicity Tests. The reaction mixtures and ether extracts were tested for mutagenicity by using the Ames test as described previously (14, 15). Salmonella typhimurium TA 1535 was used as the test organism (without metabolic activation). Results and Discussion Chlorinated Phenolic Compounds from Chlorolignins. Chlorolignins HM-C and HM-E were chlorinated under conditions similar to those used in the drinking water chlorination process, and the formation of some chlorinated phenols, catechols, and guaiacols was studied
Table I. Quantitative Determination of Various Chlorophenolic Compounds in the Reaction Mixtures from the Chlorination of High Relative Molecular Mass Material (Chlorolignin) from the Alkali Extraction Liquor (HM-E)" C1,:C ratio (w/w) 0.5:l quantified compoundb~c
I
I1
I
4,5,6-trichloroguaiacol (6) tetrachloroguaiacol (9) 3,4,5-trichlorocatechol (7) tetrachlorocatechol (10)
I1
I
4: 1
I1
I
25.3 f 0.9 17.5 f 1.7 37.0 f 2.5 28.8 i 2.6 31.0 f 2.2 20.8 f 2.1 15.8 f 1.3 16.0 f 1.4 17.8 i 1.5 12.8 f 0.9 5.0 f 2.2 7.0 1.2 6.0 f 1.4 4.3 f 0.5 7.3 f 1.9 29.0 i 4.1 12.3 f 0.5 23.0 i 6.0 20.8 f 2.5 16.5 f 0.6 d 22.0 f 1.4 22.8 i 4.7 28.8 f 4.7 24.8 f 3.0 17.0 f 1.4 21.8 f 3.4 d 5.8 f 0.5 8.8 f 0.9 5.0 f 0.8 6.0 f 0.8 2.8 f 1.5 d 29.3 f 1.5 15.8 f 2.6 296.5 f 10.3 229.3 f 8.5 312.3 f 3.2 233.8 f 5.9 205.3 f 6.7 50.3 f 2.5 71.5 f 1.7 68.8 i 1.7 96.0 i 5.4 16.5 f 1.3 12.5 f 2.4 61.8 f 1.9
15.8 f 1.5 d 2,3,4,64etrachlorophenol(4) 13.8 f 0.5 17.3 f 1.7 3.3 f 1.5 6.3 f 0.5 pentachlorophenol (8) 12.3 f 1.5 10.0 i 1.4 3,4,5-trichloroguaicacol(5) 2,4,64richlorophenol (2)
2:l
1:l
*
I1 12.5 i 1.0 11.8 f 2.2 6.0 f 1.2 27.3 f 1.3 7.5 f 1.7 9.3 f 2.9 189.0 f 8.9 87.0 i 2.5
"The values are given as nanograms per milligram of organic carbon chlorinated and are mean values of four GC determinations (standard deviation n = 4). bFigures in parentheses refer to corresponding GC peaks in Figure 1. c4,5-Dichloroguaiacol was detected but not quantified due to the appearance of a double peak. dNot detectable.
(IS)
Figure 1. GC chromatogram of acetylated HM-E reaction mixture (CI,:C ratio 1: 1). Quantified compounds: (1) 2,4dlchlorophenol, (2) 2,4,6trichlorophenol, (3) (I. S.) internal standard, 2,6-dibromophenol, (4) 2,3,4,6-tetrachlorophenol, ( 5 ) 3,4,5-trlchloroguaiacol, (6) 4,5,6-trichloroguaiacol,(7) 3,4,5-trichlorocatechol, (8) pentachlorophenol,(9) tetrachloroguaiacol, and ( I O ) tetrachlorocatechol.
(see legend to Figure 1). This was done since recent investigations have shown that chlorophenolics may exert genetic activity when tested with tests other than the Ames test (16-1 8). The concentrations of chlorolignins used were higher than what may be expected after a raw water purification process involving flocculation and sedimentation. However, there is no reason to expect that flocculation and sedimentation will remove specific parts of the chlorolignins leading to a much different spectra of chlorination products formed. The concentrations were high in order to facilitate the detection of the various reaction products. In the reaction mixture from the chlorination of HM-C the only chlorinated phenolic compounds that could be detected were 2,4-dichlorophenol and 2,4,6-trichlorophenol, The quantities amounted to only 10-80 ng/mg of TOC chlorinated. Although previous investigations have shown that HM-C material has a low content of aromatic nuclei (14-231, this result is somewhat surprising and indicates that the major part of these is destroyed during the chlorination. In the HM-E reaction mixture, chlorinated phenols, guaiacols, and catechols were present as is evident in the typical chromatogram shown in Figure 1. Table I gives the quantitative data for the various compounds. Results from two independent experiments are included. As can be seen, the chlorinated catechols are dominating. The quantities increase with increasing C&:C ratio. This is a result of an increased depolymerizationand demethylation of the HM-E material (24). The quantities of chlorinated phenols and guaiacols present in the reaction mixtures are small and to a large degree independent of the C1,:C ratio.
With regard to the yields of the various products Table I reveals that in total this reaches a magnitude of about 0.5 pg/mg of TOC chlorinated. Since the TOC content of HM-E material is of the order of 45% (25), 0.5 pg/mg of TOC corresponds to about 0.23 pg of chlorinated phenolic compounds/mg of HM-E material. The aromatic content in this type of material has also been shown to be low (21,25) and amounts to about 0.5 kg/ton of pulp (15 pg/mg of HM-E) (25). Thus, the chlorinated phenolics formed here only constitute about 2 %, counted on a weight basis, of the aromatic content available for chlorination. This indicates that the aromatic nuclei to a large extent are destroyed to aliphatic degradation products. In comparison to the quantities of chlorinated phenolic compounds formed in the bleaching of pulp and present in the spent bleach liquors as such, the quantities formed here are low. Thus, in the conventional bleaching of softwood kraft pulp, chlorinated phenolic compounds are formed to the extent of 100-150 g/ton of pulp. In the present experiments, the amount of chlorinated phenolic compounds may be calculated to be equivalent to about 10-15 g/ton of pulp. Ames Test Mutagenic Compounds from Chlorolignins. As mentioned above, spent liquor from the chlorination of softwood kraft pulp exhibits mutagenic activity. A number of compounds have been identified as contributors to this property (13,15,26-29). The reaction mixtures from the chlorination of HM-C and HM-E materials were analyzed qualitatively and quantitatively with respect to some of the most important mutagenic compounds. From the obtained results listed in Table I1 it is apparent that the two particularly strong mutagens 2chloropropenal and 1,3-dichloroacetone are formed only in minor quantities. 2,3-Dichloropropanal could not be detected in the reaction mixtures. The various reaction mixtures or ether extracts of these were tested according to Ames test (Salmonella typhimurium TA 1535, without metabolic activation). It was not possible (even after considerable concentration of the samples) to obtain a 2-fold dose-related increase in the number of revertants over the background level in tests with acceptable survival rates of the test bacteria. This indicates, in accordance with the analytical findings, that in the reaction mixtures the contents of mutagenic compounds (acting on the bacteria strain used) are low. Chlorinated Phenolic Compounds from Humic Acid. In order to obtain an understanding of the environmental relevance of these findings, knowledge concerning the formation of mutagenic and chlorinated phenolic compounds in the chlorination of naturally occurring humic acid in raw water is helpful. We therefore chloriEnviron. Sci. Technol., Vol. 19, No. 5, 1985
429
Table 11. Quantitative Determination of Various Mutagens in the Reaction Mixtures from the Chlorination of Chlorolignins (HM-C and HM-E)" material chlorinated HM-C
HM-E
C1,:C ratio- (w/w) .,
I
quantified compound 2-chloropropenal 1,3-dichloroacetone 1,1,3,3-tetrachloroacetone pentachloroacetone hexachloroacetone 2-chloropropenal 1,3-dichloroacetone 1,1,3,3-tetrachloroacetone pentachloroacetone hexachloroacetone
0.5:l
I
I
I1
4:1
2:l
1:1
I1
I
I1
I
I1
b 2.9 f 1.5 380.0 f 8.6
b 6.9 f 2.2 298.7 f 7.5
b b 115.5 f 10.7
b b b b b b 95.8 f 7.4 241.8 i 12.6 206.0 f 6.0
b b 215.8 f 8.5
b 7.4 f 1.0 258.0 f 10.9
89.8 f 7.6 b b 8.3 f 0.4 364.0 f 5.1
73.8 f 4.5 587.8 f 19.6 533.3 f 9.0 b 56.5 f 5.8 26.8 f 5.9 b b b 6.7 f 0.7 20.4 f 2.8 19.1 f 1.0 213.8 i 4.9 631.5 f 11.9 484.3 f 11.2
807.8 f 20.1 119.3 f 13.7 2.7 f 1.2 16.8 f 1.8 486.0 f 6.6
897.0 f 12.5 857.0 f 25.6 91.8 i 6.2 180.5 i 7.4 3.4 i 0.7 4.3 f 0.5 14.1 f 1.9 15.7 f 0.4 406.0 f 6.2 600.3 f 5.9
176.0 f 6.9 27.8 f 3.8
163.3 h 7.6 855.5 f 14.7 839.8 f 13.5 2979.0 f 17.3 2545.0 f 19.5 2215.0 f 14.7 1563.3 f 34.6 69.3 f 3.5 393.5 f 8.7 19.9 f 1.0 75.5 f 5.2 316.5 f 7.6 415.5 f 3.4 393.3 f 7.2
726.3 f 12.1 176.3 f 5.2 4.1 f 0.4 24.9 f 3.1 443.0 f 6.6
DThevalues are given as nanogram per milligram of organic carbon chlorinated and are mean values of four GC-MS (MID) determinations (standard deviation n = 4). *Not detectable. Table 111. Quantitative Determination of Various Chlorophenolic Compounds in the Reaction Mixtures from t h e Chlorination of Aquatic Humic Acid"
C12:C ratio (w/w) 1:l
0.51 quantified compoundb~c
I
53.4 f 2.2 103.1 f 2.6 2,3,4,6-tetrachlorophenol(4) d 3,4,5-trichloroguaiacol (5) 6.3 f 0.5 4,5,6-trichloroguaiacol (6) d d 3,4,5-trichlorocatechol (7) tetrachlorocatechol (10) 2.3 f 0.5 2,4-dichlorophenol (1) 2,4,6-trichlorophenol (2)
I1 48.9 f 2.2 101.1 f 2.3 d 7.0 f 0.8 d d 2.3 f 0.5
I
2:l
I1
41
I
I
I1
48.9 f 1.6 58.7 f 2.2 90.6 f 1.3 92.3 f 3.9 73.6 f 6.7 145.7 f 2.2 130.4 f 3.3 281.6 f 6.2 226.3 k 9.6 22.4 f 0.6 d d 6.3 f 0.5 6.8 f 1.0 8.8 f 0.5 d d d d 5.0 & 0.8 d d 5.3 f 0.5 5.8 f 0.5 9.3 f 0.5 3.3 f 0.5 3.5 f 0.6 21.5 f 0.6 22.3 f 0.5 20.8 1.0 16.3 f 0.5 20.3 f 1.0 19.0 f 0.8 5.8 f 0.5 6.3 f 0.5
*
I1 75.7 f 4.7 21.6 f 1.6 8.8 f 0.9 5.5 f 0.6 10.3 f 0.5 21.8 f 0.9 21.3 f 0.9
"The values are given as nanograms per milligram of organic carbon chlorinated and are mean values of four GC determinations (standard deviation n = 4). bFigures in parentheses refer to corresponding GC peaks in Figure 2. c4,5-Dichloroguaiaco1was detected but not quantified due to the amearance of a double oeak. dNot detectable.
nated humic acid under conditions identical with those used in the chlorination of chlorolignins. As can be seen from the results given in Figure 2 and Table 111, all types of chlorinated phenols, catechols, and guaiacols were found. A complete identification based on mass spectral interpretation was made of most of these (see Experimental Section). However, the relative distribution of the compounds differs from that in the reaction mixtures from the chlorolignins. Characteristic for the humic acid mixture is the much larger quantities of 2,4-dichlorophenol and 2,4,6-trichlorophenol. The fact that chlorinated phenols are formed in the chlorination of humic acid materials was reported previously (30, 31). However, the formation of chlorinated catechols and guaiacols from aquatic humic acid has to our knowledge not been reported before. The formation of these compounds is not surprising since it was previously reported that the humic acid from natural waters contains guaiacol units (32-34). Ames Test Mutagenic Compounds from Humic Acid. With regard to the formation of mutagenic compounds we previously investigated the formation of such when chlorinating humic acid (9). In contrast to the reaction mixtures from the chlorination of chlorolignins, concentrated ether extracts of the 2 1 and 4:l C12:C ratio humic acid chlorination liquors showed significant mutagenic activity. This shows that the humic acid material seemed to be more prone to form Ames test T A 1535 mutagens than chlorolignins when both materials were chlorinated under identical conditions. The quantities found, on a weight per unit of TOC basis, were of the same 430
Environ. Sci. Technol., Vol. 19, No. 5, 1985
1 2 3
L
6
7
10
(IS.)
Figure 2. GC chromatogram of acetylated humic acid reaction mlxture (73.5 mg of TOC/L, CI,:C ratlo 2:l). Quantified compounds: see text to Figure 1.
order of magnitude as the quantities found in the reaction mixtures from the chlorination of chlorolignins described in Table 11. One exception was that 8-chloropropenal formed in somewhat higher quantities when chlorinating aquatic humic acid.
Conclusion Altogether the results show that chlorolignins will yield chlorinated phenolic and Ames test mutagenic compounds when chlorinated under conditions similar to those used in the drinking water disinfection process. On the basis of an equal TOC basis the quantities of the compounds are in the same order of magnitude as the quantities formed when chlorinating naturally occurring aquatic humic acid material.
Acknowledgment The research presented in this paper is part of a joint Nordic Research Project on “Reduction of Environmental Impact of Bleach Plant Effluents”. Registry No. 1,120-83-2; 2,88-06-2; 4,58-90-2; 5,57057-83-7; 6,2668-24-8; 7,56961-20-7; 8,8746-5;9,2539-17-5;10,1198-55-6;
chlorolignin, 8068-02-8;2-chloropropenal, 683-51-2;1,3-dichloroacetone, 534-07-6;1,1,3,3-tetrachloroacetone, 632-21-3; pentachloroacetone, 1768-31-6; hexachloroacetone, 116-16-5. Literature Cited (1) Kriigstad, K. P.; Lindstrom, K. Environ. Sci. Technol. 1984, 18,236A. (2) Kringstad, K. P.;Stockman, L. G.; Stromberg, L. M. J . Wood Chem. Technol. 1984,4 (3),389. (3) Neilson, A. H.; Allard, A.-S.; Hynning, P. A.; Remberger, M.; Landner, L. Environ. Microbiol. 1983,45,774, (4) Eriksson,K.-E.; Kolar, M.-C.; Ljungquist,P. 0.;Kringstad, K. P., submitted for publication in Environ. Sci. Technol. (5) Bull, R. J. Environ. Sci. Technol. 1982,16,554. (6) Meier, J. R.; Lingg, R. D.; Bull, R. J. Mutat. Res. 1983,118, 25. (7) Holmbom, B.; Kronberg, L. Laboratory of Forest Products
Chemistry, Academy of Turku, Turku, Finland, personal communication, 1984. (8) Christman, R. F.; Johnson, J. D.; Hass, J.-R.; Pfaender, F. K.; Liao, W. T.; Norwood, D. L.; Alexander,H. J. In “Water Chlorination: Environmental Impact and Health Effects”; Jolley, R. L., et al., Eds.; Ann Arbor Science: Ann Arbor, MI, 1978;Vol. 2,p 15. (9) Kringstad, K. P.; Ljungquist, P. 0.;de Sousa,E’.; Stromberg, L. M. Environ. Sci. Technol. 1983,17,553. (10) Hergert, H. L. In “Lignins: Occurrence, Formation, Structure and Reactions”;Sarkanen, K. V.; Ludwig, C. H., Eds.; Wiley-Interscience: New York, 1971. (11) Voss, R. H.; Wearing, J. T.; Wong, A. In “Advances in the Identificationand Analysis of Organic Pollutants in Water”; Keith, L. H., Ed.; Ann Arbor Science: Ann Arbor, MI, 1981; Vol. 2,p 1059. (12) Junk, G. A.; Richard, J. J.; Grieser, M. D.; Witiak, D.; Witiak, J. L.; Arguello, M. D.; Vick, R.; Svec, H. J.; Fritz, J. S.; Calder, G. V. J . Chromatgr. 1974,99,745. (13) Kringstad,K. P.; Ljungquist, P. 0.;de Sousa, F.; Stromberg, L. M. In “Water Chlorination: Environmental Impact and Health Effects”;Jolley, R. L., et al., E&.; &n Arbor Science:
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Received for review July 2,1984. Accepted November 27,1984. This work received financial support from the Nordic Fund for Technology and Industrial Development.
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