Environ. Sci. Technol. 1985, 19, 422-426

High-Performance Size Exclusion Chromatography of Chlorinated Natural Humic Water and Mutagenicity Studies Using the Microscale Fluctuation Assay

Georg Becher,*?+ Georg E. Carlberg, Egll T. GJesslng,s Jan K. Hongslo,t and Silvano Monarca§

Center for Industrial Research, P.O. Box 350, Blindern, 0314 Oslo 3, Norway

High-performance size exclusion chromatography (HPSEC) was applied to the fractionation of a natural humic water sample before and after chlorination. By use of intermediate ionic strength eluents, reasonably good resolution was obtained. After chlorination the amount of dissolved organic carbon decreased in early eluting fractions and increased in the later fractions. This implies that chlorination resulted in degradation of high molecular weight humic material to compounds with intermediate and low molecular weight. Bound organic chlorine was found in all fractions. Mutagenic activity of the HPSEC fractions was determined by using the microscale bacterial fluctuation assay. Mutagenic activity was found in the last three fractions containing substances at intermediate and low molecular weight.

Introduction Humic substances are among the most widely distrib-

uted organic material on earth and comprise a major part of the organic matter in natural waters (1, 2). Aquatic humic substances are polar, very complex products of terrestrial origin. They are probably innocuous by them- selves a t the level a t which they occur in surface waters. However, several studies have shown that they may be precursors to potentially harmful compounds when chlo- rination is performed during drinking water disinfection (3-7). There is now great evidence that chlorination of humic waters leads to the formation of compounds with direct-acting mutagenicity in the Salmonella/microsome assay (Ames test) (3-7). Recently, it has been demon- strated that the formation of mutagenic activity is related to the concentration of both humic matter and chlorine (5).

Low molecular weight compounds such as trichloro- methane, chlorinated organic acids, phenols, ketones, and chloroacetonitriles have been identified by gas chroma- tography/mass spectrometry (GC/MS) as chlorination byproducts of aquatic humus (8-10). However, recent studies show that these compounds can account for only a minor part of the total organic bound halogen (TOX) present (11,12). This indicates that some of the organi- cally bound halogen occurs in unidentified higher molec- ular weight compounds not amenable to GC/MS analysis. It has been pointed out by Glaze and Peyton (13) that if small molecules such as chloroform are formed by the action of chlorine on macromolecular humic substances, chlorine-containing species intermediate in molecular weight must surely exist. Very little is known about the chemical character and the toxicology of these intermediate chlorination products.

t Present address: Toxicological Department, National Institute

*Norwegian Institute for Water Research, Blindern, Oslo 3,

*Present address: Istituto di Igiene, University of Perugia, 06100

of Public Health, Geitmyrsveien 75, 0462 Oslo 4, Norway.

Norway.

Perugia, Italy.

Table I. Characterization of the Natural Humic Water Used in the Experimentsa

parameter results

PH 4.5 color, mg of Pt/L 122

total nitrogen, pg/L 200 total phosphate, pg/L 5

conductivity, pS/cm 33 dissolved organic carbon (DOC), mg/L 17.5

Mean values for the last 20 years.

Therefore, our objectives have been, first, to fractionate chlorinated natural humic water by high-performance size exclusion and, second to determine the mutagenic activity in these fractions by using bacterial short-term mutagen- icity tests. The investigations were performed on a heavily colored water sample (120 mg of Pt/L) collected from the outlet of a small, unpolluted marsh area.

Experimental Sectian Sample Preparation. Water samples were collected

from the outlet of a marsh area (Hellerudmyra). The catchment was small and without influence from human activity. The dissolved organic matter from this particular sampling station has been extensively studied during the last two decades, showing that it predominantly consists of humic substances (14). Mean values of some parameters for the last 20 years are shown in Table I. Samples were filtered through 0.45-pm filters and stored in the dark at 4 OC before further treatment.

The unbuffered water samples were chlorinated by adding chlorine water to a final concentration of 10 mg of Cl/L. The reaction mixture was allowed to stand for 5 days in sealed bottles in the dark at ambient temperature ensuring complete reaction between humic material and chlorine. The total organic bound halogen was determined to be 1.33 mg/L. Both unchlorinated and chlorinated samples were concentrated 50-fold by rotary evaporation under reduced pressure at 35 OC to yield a dissolved or- ganic carbon concentration of 626 and 669 mg/L, respec- tively. For biological testing, samples were concentrated 100-fold. The effect of concentration on the distribution of humic and fulvic acids was not determined. However, the chromatographic pattern obtained by HPSEC was not significantly affected by a 50-fold concentration. The solutions were filtered through a 0.45-pm Millex HA filter (Millipore Corp., Bedford, MA) before high performance liquid chromatography (HPLC) separation.

High-Performance Size Exclusion Chromatogra- phy (HPSEC). HPSEC was carried out at ambient tem- peratures on a Waters HPLC system (Waters Associates), consisting of a Model 6000 A pump, a Model U6K injector, a Model 440 absorbance detector operating at 280 nm, and a Model 730 data module. The column used was a 7.5 X 600 mm Ultropac column packed with TSK-G 3000SW (LKB, Bromma, Sweden) and a 7.5 X 75 mm precolumn with the same packing material. The void volume (V, =

422 Environ. Sci. Technol., Vol. 19, No. 5, 1985 0013-936X/85/0919-0422$01.50/0 0 1985 American Chemical Society

11.3 mL) and the total permeation volume (Vo + Vi = 26.4 mL) were determined with Blue Dextran and water, re- spectively.

Recovery of humic material was determined by com- paring total UV peak areas for identical samples passing through and bypassing the column.

Separation of humic substances was performed with a 0.02 M KHZPO4 buffer, pH 6.5, a t a flow rate of 1.0 mL min-l. Injection volumes ranged from 10 pL for analytical separations to 200 pL for preparative separations from which fractions were collected. Carbon yields throughout the fractionation were monitored by measuring total or- ganic carbon (TOC) using the persulfate oxidation tech- nique with gas chromatographic detection of COZ. Total organic halogen (TOX) was determined by the activated carbon adsorption/microcoulometric method using a Dohrman DX-20 analyzer.

Ultrafiltration. Ultrafiltrations were performed with a 400-mL Amicon cell (Amicon B.V., The Netherlands) using the following Amicon ultrdilters: YM 2, YM 10, and YM 30, with nominal molecular weight cutoff levels of 1000, 10 000, and 30 000, respectively.

The samples were fractionated in the following manner: 0.5 L of the natural humic water sample was placed in the cell containing the ultrafilter with highest molecular weight cutoff. The sample was concentrated to approximately 10 mL by applying a pressure with highly purified nitrogen. The concentrate was diluted twice with 50 mL of distilled water and reconcentrated. The final concentrate was transferred quantitatively to a 104mL measuring flask. The filtrate was concentrated in the same manner by using the YM 10 and the YM 2 filter subsequently. The final filtrate (M, <low) was concentrated to 10 mL by rotary evaporation under reduced pressure at 35 "C.

Microscale Fluctuation Test and Ames Test. The microscale bacterial fluctuation assay, as described by Gatehouse (15,16), was chosen for detecting mutagenicity in the chlorinated water sample before and after HPSEC separation became of its high sensitivity and the possibility to incorporate water samples in the assay (17, 18). S. typhimurium strain TAlOO developed by Ames et al. (19) was used for its sensitivity to direct-acting mutagens produced by chlorination of humic acids and natural water (5, 6).

In this test the bacteria were incubated with the sample in liquid medium (about 20 mL) and dispensed in 96 microwells of a disposable microtiter tray (200 pL/tray). The number of wells in which mutations have occurred was scored after 3 and 4 days by adding a pH indicator and tested for significant increase in comparison with blank controls by the x2 method (20). Positive controls (sodium azide) were concurrently tested.

For comparison, the Ames test (19) was performed with the TAlDO strain without metabolic activation.

Before HPSEC separation the 100-fold concentrated chlorinated water sample was membrane sterilized and tested at increasing doses in the fluctuation test and Ames test by incorporating up to 1 mL per assay (equivalent to 100 mL of original water). Each of the seven fractions obtained by HPSEC separation of 0.4 mL of concentrated water (equivalent to 40 mL of the original water sample) were membrane sterilized and totally incorporated in the fluctuation test by replacing a portion of liquid medium with the fraction. Direct incorporation of the fractions obtained from larger volumes of concentrated chlorinated water sample was impossible because the maximum vol- ume to be incorporated in the assay is about 18 mL. To overcome this limitation Sep-Pak CIS cartridges (Waters

,

I - 0 5 10 15 20 25 Vc[rnl]

Figure 1. Influence of mobile phase ionic strength on the elution of the natural humic water.

Associates, Milford, MA) were used to concentrate frac- tions derived from 0.8 mL of concentrated chlorinated water (equivalent to 80 mL of original water), and the Sep-Pak eluates were incorporated in the test (21).

Sep-Pak cartridges were activated with 5 mL of CH30H and flushed with 5 mL of distilled water. The fractions obtained from 0.8 mL of concentrated chlorinated water were acidified to pH 2 and passed through the cartridges at a 10 mL/min flow with a syringe. Adsorbed compounds were eluted with 5 mL of CH,OH, and the solvent was evaporated at 37 OC with a gentle stream of nitrogen. The residues were dissolved in 200 pL of dimethyl sulfoxide (MezSO) and tested in the fluctuation assay.

Results and Discussion The

macromolecular nature of aquatic humic substances makes these compounds suitable for separation and characteri- zation by size exclusion chromatography. Molecular size fractionations of humic substances have mainly been carried out with soft gels such as Sephadex and Bio-Gel (14,22). Major disadvantages are the poor resolution and long analysis time. Recent advances in HPLC column technology have made high efficiency, high speed size fractionations of water-soluble polymers possible (23-25). However, only a few papers have been reported on the application of HPSEC to humic substances (26-28). In this study we used a rigid, microparticulate packing based on porous silica with a hydrophilic bonded phase. The standard calibration curve for globular proteins using 0.1 M phosphate buffer a t pH 6.5 as mobile phase was found to be linear from about 12 000 to 500 000 daltons.

Several investigations have shown that due to gelsolute interactions, elution of polyelectrolytes depends on eluent parameters such as ionic strength, pH, and addition of organic solvent (25, 29). Figure 1 shows that the ionic strength of the mobile phase has a strong effect on the elution of humic material from the column. Apparently, low ionic strength results in decreased elution volume, probably due to charge repulsion between the solutes and the packing material (28,29). Retarded elution is observed for the high ionic strength mobile phase (0.02 M phosphate buffer + 0.1 M sodium sulfate) which has been ascribed to the adsorptive interaction of aromatic parts of the so- lutes with the stationary phase (29). The importance of these extraneous factors on size separation of humic sub- stances has recently been pointed out (28). From various eluents tested, 20 mM phosphate buffer gave best resolu- tion of the aquatic humus studied. The average recovery of humic material from the column using this mobile phase was 99% for three determinations which suggests that humic substances are not irreversibly adsorbed onto the packing material.

Separation of Humic Matter by HPSEC.

Environ. Sci. Technol., Vol. 19, No. 5 , 1985 423

Flgure 2. Size exclusion chromatogram of 50 X concentrated natural humic water before (solid line) and after (broken line) chlorination. The fractions collected (1-7) are Indicated.

t

0 5 10 15 20 25 30

Flguro 3. Size exclusion chromatograms of collected fractions from the chlorinated natural humic water superimposed on original chro- matogram.

Table 11. Size Exclusion Data for Fractions of Unchlorinated Natural Humic Water

apparent M, at peak maximum

frac- ve globular tions (peak max) dextransn proteins

1 11.3 b b 2 18.5 16 800 65 000 3 19.6 12 800 45 000 4 20.4 10 000 34 000 5 21.1 7 900 28 000 6 21.9 5 800 21 000 7 23.0 3 300 14 000

Calculated by using the dextran molecular weight calibration curve of Frigon et al. (30). bExcluded material.

Seven fractions were collected by semipreparative chromatography of both unchlorinated (solid line) and chlorinated (broken line) samples as shown in Figure 2. Fraction 1 contains totally excluded material possibly consisting of large aggregates formed by self-association of humic substances. Apparently, UV absorbance has shifted toward longer elution times after chlorination, in- dicating an increase in the amount of lower molecular weight compounds. Figure 3 shows chromatograms of the reinjected fractions of the unchlorinated sample superim- posed on the original chromatogram. The figure demon- strates the reasonably good separation efficiency of the column and the high reproducibility of the method. Table I1 shows the size exclusion data for the seven fractions of the unchlorinated sample. The apparent molecular weight a t peak maximum of the individual fractions certainly depends on the standard used, and the molecular weight data are only rough estimates. The results obtained from calibration with dextrans (30), however, compare well with the molecular weight distribution of aquatic humus as determined by Glaze and co-workers (12, 13). Table I11 shows the characteristics of the seven fractions of both unchlorinated and chlorinated samples. After chlorination DOC levels are shifted significantly to longer elution times, i.e., DOC decreases in the earlier fractions (2-4) and in- creases in the later fractions (5-7). The implication is that

Table 111. Characteristics of Unchlorinated and Chlorinated Natural Humic Water Fractionated by HPSEC

DOC chlorinated (mg/L) (chi)/ TOX/

frac- unchlo- chlo- DOC TOX, DOC, tion rinated rinated (unchl) pg/L 70

1 0.79 0.68 0.86 54 7.9 2 1.59 1.32 0.83 37 2.8 3 3.19 2.02 0.63 193 9.6 4 1.25 0.80 0.64 42 5.3 5 1.96 2.26 1.15 121 5.4 6 2.08 2.69 1.29 111 4.1 7 2.82 3.51 1.25 132 3.8

sum 13.6 13.3 690 original 12.9 13.4 1330

sample

4 I O r n I O n _ 6 * C l O 1 s I h l I"! 01,

1000-1010~ .. 0:03-10030

- c l c 3 0

1 - > > O O C O

El

5 I0 I5 20 25 30 ve h0

Flgure 4. Size exclusion chromatograms of the ultrafiltration fractions of chlorinated natural humic water superimposed on original chroma- togram.

chlorination resulted in a degradation of humic substances with high molecular weight to intermediates with a lower molecular weight. The results indicate that the separation of humic material under the conditions used is to a great extent based on size exclusion.

As is seen in Table I11 organic bound chlorine is found in all fractions; i.e., it is distributed over the whole mo- lecular weight range of the humic material. The TOX values normalized to dissolved organic carbon (DOC) levels (last column in Table 111) show that the amount of chlorine per carbon atom is similar in all fractions. While the total DOC is recovered quantitatively after HPLC fractionation, the TOX recovery is only about 50% (Table 111). The reason for this is presently not understood. During later repetition of the analysis, serious experimental problems occurred. Combustion of the activated carbon with the adsorbed organohalogen compounds from several of the HPSEC fractions resulted in immediate base-line drift, preventing the quantitative determination of TOX.

Comparison of HPSEC and Ultrafiltration. Another method that has been shown to be a helpful tool in frac- tionating humic material into molecular size classes is ultrafiltration using selectively permeable membranes. The method is simple and rapid, and it may be operated to result in concentrated samples. Therefore, it seems well suited to isolate larger quantities of fractionated chlori- nated humic substances needed for biological testing.

Four fractions were obtained from the ultrafiltration of chlorinated brook water containing substances with an apparent molecular weight higher than 30 000, between 10000 and 30000, between 1000 and 10000, and lower than 1000. The four fractions were all adjusted to 10 mL and analyzed by HPSEC.

The chromatograms are shown in Figure 4 superimposed on the chromatogram of the original chlorinated water sample. There is very little UV-adsorbing material in the fraction above 30000 daltons. However, 60% of the total UV absorbance is found in the filtrate from the membrane with nominal molecular weight cutoff of 1000. Elution of

424 Environ. Sci. Technol., Vol. 19, No. 5, 1985

Table IV. Ultrafiltration of Unchlorinated and Chlorinated Natural Humic Water

DOC (mgC/L) nominal unchlo- chlo-

size range rinated rinated

>30 000 0.28 0.59 30 000-10 000 0.38 1.92 10 000-1000 5.92 0.79 <loo0 3.38 6.90

sum 10.0 10.2

chlorinated TOX/

TOX, DOC, mg/L %

43 7.3 95 4.9 42 5.3

712 10.3

892

Table V. Ames Test on 100-fold Concentrated Natural Humic Water after Chlorination

volume of original TA100(-S9) humic water, mutagenicity,

sample mL/plate revertants/plate"

chlorinated 100 495a.b humic water 50 824"

25 512" 10 386

control 0 227 sodium azide 1 C 802"

"Values considered positive (%fold increase in the number of revertants per plate over control value). Toxic effect. Units: wg/plate.

this fraction from the size exclusion column begins a t rather high molecular weight ranges. On the basis of the dextran standard calibration, the maximum is observed at 7900 daltons. It has also been observed by others (31) that larger molecules than expected permeate to a great extent through the Amicon YM 2 membrane used in this study. Table IV shows the characteristics of the four fractions obtained from ultrafiltration of chlorinated and unchlorinated natural humic water. Again, a shift toward lower molecular weight compounds is observed after chlorination. In this case, however, 80% of the total or- ganic halogen is found in the final filtrate. As HPSEC gave narrower molecular weight fractions compared to ultra- filtration, mutagenicity testing was performed on HPSEC fractions only.

Mutagenicity Tests on Chloriaated Humic Water. Direct-acting mutagens were found to be present in the concentrated chlorinated humic water as previously re- ported for samples from humic acid chlorination (5). Ames test with TAlOO strain showed highly positive and dose- related results starting from the dose of 25 mLJplate (Table V). At a 100 mL/plate dose the revertants per plate values decreased, showing a toxic effect. Specific mutagenic activity calculated by least-squares regression analysis of the linear portion of the dose-response curve showed very high values (11 590 net revertants/L).

Meier et al. (5) also found extremely high mutagenic activities, with TAl00 and TAB8 strains without metabolic activation, in chlorinated solutions of humic acids. These genotoxic activities were predominantly due to nonvolatile compounds.

The microscale fluctuation test on the same sample gave dose-related mutagenic responses (Table VI), starting from lower doses than in the Ames test (5 mL/assay). This confirms the higher sensitivity of this test in comparison with the Ames test (18,21). Therefore, only the fluctuation test was used for detecting mutagens in the HPSEC fractions of humic water. Moreover, the fluctuation test can incorporate much higher volumes of liquid samples than the Ames test: up to 90% of the 20 mL of liquid

Table VI. Microscale Fluctuation Test on 100-fold Concentrated Natural Humic Water after Chlorination

TA100(-S9) volume of original mutagenicity,

humic water, no. of positive sample mL/assay wells per 96

chlorinated 100.0 94" humic water 50.0 79"

10.0 31" 5.0 20b 2.5 14 1.0 11

control 0 7 sodium azide l0OC 35"

"Significance was calculated by using x 2 analysis (20): p < Significance was calculated by using x2 analysis (20): p < 0.001.

0.01. "Units: nelassav.

Table VII. Fluctuation Test on HPSEC Fraction Derived from 40 and 80 mL of Chlorinated Natural Humic Water and Directly Incorporated in the Assay

TA100(-S9) mutagenicity,

no. of positive wells per 96

fraction 40-mL 80 mL no." sample sampled

1 6 13 2 6 9 3 5 9 4 6 16 5 6 17c 6 32b 546 7 10 32b

control 10 10e sodium azide 22b 26b

(100 ng/assay)

,,See Figure 3. (20): p < 0.001. p < 0.05. dAfter Sep-Pak concentration. eSep-Pak blank.

medium of fluctuation test can be replaced with a liquid sample, while only 1-2 mL can be incorporated in the Ames test.

Fluctuation Test on HPSEC Fractions. The possi- bility to incorporate high volumes of aqueous samples in the fluctuation test made this test particularly suitable for testing HPSEC fractions without preliminary concentra- tion steps, However, for several fractions concentration on Sep-Pak C18 columns was performed to increase the sensitivity of the test in the detection of the mutagenic fractions. These C18 bonded-phase columns have previ- ously shown promising results for the concentration of organics (32-35) and mutagens (21) from water samples.

Table VI1 shows the mutagenic activity from testing the total fractions obtained by HPSEC of 0.4 mL of concen- trated chlorinated water (equivalent to 40 mL of original sample). Only fraction 6 at lower molecular weights presented significant (p < 0.001) mutagenic activity. From fractionation of 0.8 mL of concentrated chlorinated water it was possible to detect significant mutagenic activity (p < 0.001) also in fraction 7 and slight mutagenic activity 0, < 0.05) in fraction 5 (Table VIr). (Sep-Pak concen- tration of the fractions was performed to be able to in- corporate the whole fractions in the assay.)

The results show that the chlorination of a natural humic water sample with relatively low TOC concentration not uncommon in fresh water used for drinking water supply has led to the formation of nonvolatile mutagens.

Environ. Sci. Technol., Vol. 19, No. 5, 1985 425

Significance was calculated by using x 2 analysis Significance calculated by using x 2 analysis (20):

The HPSEC fractions that showed mutagenic activity accounted for about two-thirds of the TOC of the original sample and about half of the TOX. The mutagenic activity was found to be associated with substances of intermediate and low molecular weight, probably chlorinated degrada- tion products of macromolecular humic material. Recently, we have found that although only about 20% of the or- ganochlorine compounds in the effluent from a pulp bleachery had nominal molecular weight below 1000, this fraction contained all the mutagenic compounds. Fallon and Fliermans (36) using fractionation 9f chlorinated fresh water by ultrafiltration observed that dissolved organic material with an apparent molecular weight smaller than 2000 was primarily responsible for the formation of di- rect-acting mutagens.

Hitherto, little is known about the chemical nature of the compounds responsible for the mutagenic activity of chlorinated humic water. As the concern over potential human health hazards of these chlorination byproducts is increasing, further chemical and toxicological studies are needed.

Acknowledgments

We thank G. Tveten and A. Kringstad for skillful technical assistance. We also thank L. Berglind for the chlorination of the natural humic water samples.

Registry No. H,O, 7732-18-5.

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Received for review June 4,1984. Accepted November 26, 1984. This work has been presented in part at the First International Meeting of the International Humic Substances Society, Estes Park, CO, Aug 16-23, 1983.

426 Environ. Sci. Technol., Vol. 19, No. 5, 1985