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Concentration, Fractionation, and Characterization of Organic Mutagens in Drinking Water H. J. Kool , C. F. van Kreijl, and M.Verlaan-deVries 1

National Institute of Public Health and Environmental Hygiene, P.O. Box 150, 2260 AD LEIDSCHENDAM, The Netherlands A combination of Amberlite XAD-4 and XAD-8 resins is very suitable for concentrating organic mutagens (Ames test positive) in drinking water. Fractionation of these mutagenic organic drinking water concentrates with the aid of Sephadex LH20 revealed that organic mutagens showed a molecular weight in the range of 100-300. The organic mutagens were able to induce chromosomal aberrations in CHO cells. Furthermore, nitro organics, in part, were shown to be responsible for mutagenic activity in organic concentrates prepared from chlorinated drinking water in The Netherlands. Finally, results strongly indicate that nitro organics (halogenated or not) are introduced and/or activated in drinking water after a chlorine treatment. THE

PRESENCE OF ORGANIC CONSTITUENTS IN DRINKING WATER

has

been known for many years because these substances were found to influence the taste, color, and odor of drinking waters (J). The organic constituents consist of compounds of both natural and industrial origin. The natural ones compose the major portion and include mainly undefined fulvic and humic acids (2). For the industrial ones, most attention has been paid so far to the volatile nonpolar compounds. In part, this situation is due to analytical (technical) restrictions and to the growing awareness (3, 4) that volatile halogenated hydrocarbons are introduced as a result of a chlorine treatment. To date, hundreds of organic constituents, including several known mutagens and carcinogens, have been identified in drinking water in many countries in the world, but these organics usually are present Current address: Stichting Waterlaboratorium-Oost, Terborgseweg 138, 7005 BD Doetinchem, The Netherlands 1

0065-2393/87/0214/0605$06.00/0 © 1987 American Chemical Society

Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986.

606

O R G A N I C P O L L U T A N T S IN W A T E R

below the mierogram-per-liter level (J, 5-7). In addition, the nonpurgeable fraction, which composes 90-95% of the total organics in the water, has not been identified (6, 8) because this fraction cannot be readily volatilized for its subsequent separation and identification b y gas chromatographic-mass spectrometric ( G C - M S ) analysis. Thus, the majority of the organics in drinking water have not been identified, and this situation is probably also true for the organic mutagens (9, JO). Therefore, advances are being made to combine analytical procedures and biological testing to separate the biological active fraction. This separation w i l l permit the isolation of bioactive subfractions, w h i c h w e hope w i l l lead to the identification of the bioactive compounds. Coleman et al. (11) investigated what k i n d of organic compounds could be identified in a mutagenic concentrate of Cincinnati tap water. M o r e than 700 organic compounds could be detected in an Ames test positive concentrate, and 460 of these could be identified. This result shows that a more sophisticated coupled bioassay-chemical fractionation procedure, w h i c h w i l l separate the organics not responsible for mutagenic activity f r o m the organic mutagens, is necessary to identify the biological active organics in a complex mixture. Recently Tabor and Loper (12) carried out initial partitioning b y l i q u i d - l i q u i d extractions, followed b y repeated high-performance liquid chromatography ( H P L C ) , for separation into smaller subfractions. Active subfractions (positive in the Ames test) were analyzed b y G C - M S and consequently for peak identification. Preliminary results obtained with a carbon-chloroform extract of tap water processed in 1962 f r o m an Ohio River source showed that a polychlorinated aliphatic ether was responsible for the mutagenic activity. M o r e recently, the structure of this mutagenic compound has been identified as 3-(2-chloroethoxy)-l,2-dichloropropene (13). The investigation of the organic mutagens in drinking water in The Netherlands is n o w moving along similar lines, and results of this approach w i l l be presented and discussed in this chapter.

Materials and Methods X A D Resins. Amberlite X A D - 2 , -4 and -8 were obtained f r o m Serva G m b H , Heidelberg, Federal Republic of Germany. The resins were purified b y repeated Soxhlet extraction for 16 h in (consecutively) methanol, ethyl ether, acetonitrile, and again methanol. A subsample (column packed) of the resin was then eluted with ethyl ether, and the eluate was checked for purity b y means of G C analysis (no detectable impurities). The resins were stored in methanol at room temperature. Bacterial Strains. Salmonella typhimunum T A 9 8 and TA100 (14, 15), as well as both nitroreductase-deficient strains T A 9 8 N R " and T A 1 0 0 N R - (16), were used.


29.

607

Organic Mutagens in Drinking Water

K O O L E T AL.

X A D Procedure. For the concentration of organic constituents, 150-10,000 L of drinking water was collected per sample. Adsorption of the organic constituents on the X A D resins was carried out as described b y Junk et al. (17). Glass (25 X 1.5 cm) and stainless steel (60 X 2.5 cm) columns were packed v/ith 20 c m and 250 c m of X A D - 2 , respectively, or similar amounts of a 1:1 mixture of X A D - 4 and X A D - 8 (XAD-4/8) as indicated. The columns were washed successively before use with 4 b e d volumes of methanol, acetone, methanol, dimethyl sulfoxide ( D M S O ) , and distilled water. F o r about 7 X l O ^ f o l d con centration, 150 L of the water was passed over columns containing 20 c m of X A D at a f l o w rate of maximal 4 bed volumes/min and at a —21-mL constant temperature of 15 °C. Elution of the adsorbed organic constituents was carried out with a volume of either D M S O or acetone (>1 b e d volume, neutral fraction). In some cases, the X A D filtrate was collected after passing the X A D column adjusted to p H 2 with H C I and readsorbed on X A D - 4 / 8 . Subsequent elution was carried out with D M S O or acetone (acid fraction). F o r about 1 X 10 -fold concentration, four samples of 40,000 L of water were passed over eight columns con taining 250 c m of X A D per column at a f l o w rate of maximal 4 b e d volumes/min. Elution of the adsorbed organic constituents was carried out with 350 m L of acetone per column (neutral fraction). The acetone in the drinking water concentrates was removed b y rotary evaporation under reduced pressure at 30 °C. The remaining aqueous sample, about 200 m L , was subsequently extracted three times with 200 m L of ethyl ether. The ether extracts of all columns were pooled, the ether was removed b y rotary evaporation under reduced pressure at 30 °C, and the dry residue was dissolved in 2-3 m L of isopropyl alcohol. 3

3

3

e

3

Freeze-drying. F o r a 7000-fold concentration, 70 L of drinking water was lyophilized in a Virtus Unitrap II. The dried residue was then divided into equal weights and packed into two columns (25 X 1.5 cm) with a sintered glass filter. The organic material was eluted consecu tively with acetone, ether, and D M S O . The ether in the ether eluate was removed b y rotary evaporation, and the dried residue was dissolved in D M S O . The D M S O concentrates were sterilized b y filtration over a 0.2-μηι Teflon filter (Millipore). The acetone and D M S O concentrates were tested in the Ames test.

Salmonella Mutagenicity Test (Ames Test). The methods of bac terial culture, the verification of genetic markers, and the plate incor poration assay were essentially the same as described previously (14, 15). Petri dishes (90 mm) containing about 20 m L of 1.22 N o b l e agar in minimal V o g e l Bonner M e d i u m Ε supplied with excess biotine and


608


22 bactodextrose (Difco) were used. They were seeded with 3 m L of molten top agar (45 °C) to which the following were added consecu tively: 0.1 m L of nutrient broth culture of the bacterial tester strain (containing about 5 Χ 10 bacteria/mL) and 0.5 m L of S9 mix (as indicated). Rat liver S9, induced b y Aroclor 1254, was obtained f r o m Litton Bionetics. In the S - G mix, 0.075 m L of liver homogenate was added per milliliter of mix. The organic water concentrates were tested in the Ames test in three- to fivefold, and the deviation of the mean in the figures was usually less than 202. The results were considered significant when a t w o f o l d increase above the background and a dose-response effect were observed. Routine controls were included to check for the presence of histidine and other growth-stimulating substances or possible effects in the sample. First, 0.5 m L of each D M S O concentrate was plated out i n the absence of histidine in the top agar and then compared with the normal spontaneous background level. Second, as an internal control, a fixed amount of test mutagen (nitrofurazone) was dissolved in 0.50 m L of each concentrate and tested for possible differences in the mutagenic response. Finally, as a control for the concentration procedures, similar con centrates (7 Χ 10 fold) of tap water (The Hague) were assayed for mutagenic activity. This control was always found negative. 8

3

Cells, Treatment, and Chromosome Analysis. About 4 Χ 10 C H O - K 1 cells (Flow Laboratories, Scotland) were seeded into Ham's F10 m e d i u m (Flow) supplemented w i t h 102 newborn calf serum ( F l o w ) . These cells were incubated at 37 °C and 52 C 0 in 25-cm tissue culture flasks. Twenty-four hours later, the cells were exposed for 1 h at 37 °C to a maximum of 50 μ\^ of drinking water concentrate (neutral fraction) in a total volume of 3 m L of Ham's F10, without serum. As a positive control, 4-methoxyaniline, dissolved in D M S O , was used. After treatment, the exposure mixture was removed and cells were grown for 18 h at 37 °C in m e d i u m plus serum. Then, 1 μg of colcemid (Gibco) was added per tissue culture flask, and cells were incubated for another 2 h at 37 °C. The collection of the cells b y trypsinization, their fixation, and the staining of chromosomes with Giemsa were carried out a c c o r d i n g to standard procedures (18). A t least 50 metaphases were screened for chromosomal aberrations per treatment group. 5

2

3

C h e m i c a l Analyses. Organic halogens of drinking water con centrates were analyzed b y microcoulometry (19) b y direct injection of 100 μία of the organic concentrate.


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KOOL E T AL.


609

Fractionation of Drinking Water Concentrates on Sephadex LH20. A glass column (height 80 c m , i . d . 2.5 cm) was packed with Sephadex L H 2 0 i n isopropyl alcohol. About 3 m L of an acetone or D M S O - X A D concentrate of drinking water (neutral fraction) was layered on the column, and subsequent gel filtration was performed upside d o w n b y using isopropyl alcohol as the solvent. Fractions of 6 m L were collected, and the absorbance at 263 n m was measured. When the first peak was eluted (in general, about 90-100 fractions), the solvent was changed to dioxane/water (7:3 v/v), and A 2 6 3 absorption was mea sured again. When no more UV-absorbable material was eluted from the column (usually after 150 fractions), the isopropyl alcohol fractions were pooled together and the dioxane/water fractions were pooled in two subfractions, as indicated. Isopropyl alcohol and dioxane were removed b y rotary evaporation under reduced pressure at 30-40 °C and 50-60 °C, respectively, and the d r y residues were dissolved in 5 m L of acetone and stored at —20 °C prior to mutagenicity testing, if indicated. Molecular Weight Determination on Sephadex LH20. The glass column (height 40 c m , i . d . 1 cm) was packed with Sephadex L H 2 0 in dioxane/water (7:3) as described previously (20). About 1.0 m L of a D M S O - X A D concentrate of drinking water was layered on the column, and subsequent gel filtration was performed b y using dioxane/water (7:3) as the solvent. Fractions of 1 m L were collected with an automatic fraction collector. After measuring the absorbance at 263 n m , the fractions were pooled, fivefold diluted in water, reconcentrated on X A D - 4 / 8 (bed volume of 4 m L ) , and eluted with 5 m L of D M S O . The concentrate was stored at —20 °C prior to mutagenicity testing. Calibra tion of the column was performed b y using two colored markers, namely, vitamin B12 (MW 1355) and nitrofurazone ( M W 198). H P L C and Isolation of Mutagenic Fractions. Analytical and semipreparative reverse-phase H P L C separations were performed by using a water-to-acetonitrile linear gradient (12). Separations were carried out on a Hewlett Packard M o d e l 10084 Β equipped with an automatic sampling device, a solvent programmer, a variable absorbance detector, and an automatically steered fraction collector. The instrument was fitted with a 3.9-mm X 30-cm prepacked analytical column of 10-μπι silica particles bonded with octadecylsilane (Bondapack-Cm) for ana lytical scale. F o r semipreparative scale separations, the H P L C was fitted with a 7.8-mm X 30-cm prepacked column packed with 10-μπι silica particles bonded with octadecylsilane. Samples for H P L C were injected at volumes of 20 /uL (flow rate 1 mL/min) and 80 μΙ> (flow rate 4 m L / m i n ) , and the absorption was measured at 254 n m . Fractions


610


or subfractions were pooled as indicated, and after reconcentration b y ether extraction, they were assayed for mutagenicity i n the Ames test.

Results E f f e c t of Resin T y p e . T h e use of the nonpolar X A D resins for the concentration of organic mutagens f r o m drinking water has been k n o w n for many years (9, JO). As indicated b y results of W e b b and Rossum (21, 22), an equal mixture of X A D - 4 and X A D - 8 is most effective for concentrating a broad spectrum of organic compounds for subsequent G C - M S analyses. Yamasaki and Ames (23), however, found X A D - 2 to be superior to X A D - 4 during the testing of several model compounds. B y using acetone, w e have compared the mutagenic activity of organic concentrates of drinking waters obtained b y adsorption on either X A D - 2 or X A D - 4 / 8 . Figure 1 shows a representative example i n which X A D - 4 / 8 showed a somewhat higher direct mutagenic activity i n the Salmonella mutagenicity assay with strain T A 9 8 , whereas hardly any difference was observed i n promutagenic activity with this strain. Neither direct nor promutagenic activity with strain TA100 was significantly different with both resins (not shown).

XAD-4/8

TA 9 8 < 100

TA98+S9

200 XAD- 4/8 XAD-2

Q. CO

150

Z

ûE

50

100

UJ

>

CONTROL

UJ

50 0

10.25

la:50

ο

I0.25

h0.50

CONCENTRATE (ml)

Figure 1. Effect of resin type on the mutagenic activity of drinking water concentrates in the Ames test. The sampling, 7000-fold concentration with either XAD-2 or XAD-4/8, DMSO elution (20 mL, neutral fraction), and subsequent mutagenicity testing were as described in Materials and Methods. Simûar concentrates of The Hague tap water were used as controls. Each point represents the average of four pfotes, and 0.50 mL of concentrate corresponds to 3.5 L of water per plate.


29.

K O O L E T AL.


611

Comparison of the Mutagenic Activity in Lyophilized and X A D Concentrated Drinking Water. Because of the selective nature of the X A D concentration procedure, only a small fraction of the organic constituents present in drinking water eventually ends up in the organic concentrate, as has been shown with surface water (24). Therefore, the mutagenic activity of a lyophilized drinking water concentrate, containing about 80% of the original D O C (value of 5 m g C / L in the dried residue), was compared with the activity of a similar X A D - 4 / 8 concentrate. The results obtained in Figure 2 show that despite the l o w recovery of the total organics, the X A D method is at least as effective as the freeze-drying technique in concentrating organic mutagens f r o m drinking water. Furthermore, it was shown that acetone was very effective in eluting the organic mutagens. The lyophilized concentrates became toxic for the bacteria above 0.25 m L , an effect that was also observed in the internal control (see Material and Methods). Some P h y s i c a l - C h e m i c a l Properties of Organic Mutagens in D r i n k ing Water. Investigations of many mutagenic drinking waters in The Netherlands have shown previously (25-28) the presence of another class of organic mutagens (acid fraction) after readsorption of the acidified X A D filtrate on a second X A D - 4 / 8 column. It was also shown previously (25) that ethyl ether elution of X A D - 4 / 8 , which is widely used for analytical purposes such as G C - M S , yielded only a minor part of the mutagenic activity. Subsequent elution with acetone, however, eluted the major part of the activity. These results indicate that (1) the major part of the organic mutagens is composed of the somewhat more polar and less volatile organics and (2) the organics already identified b y G C - M S in these types of drinking waters with X A D - 4 / 8 ether elution are, in general, not identical to the organic mutagens (25). The organic mutagens concentrated b y using the X A D procedure proved not to be gas chromatographable because in a routine preparative G C procedure, less than 108! of the mutagenic activity (Ames test) could be recovered (not shown). T o find out whether the heating step in the G C analysis may have caused this poor recovery, an experiment was set up in w h i c h a mutagenic drinking water concentrate was heated to 250 °C; this experiment was similar to our routine G C analysis that uses a heated injection system. The results, as depicted in Figure 3, show that after heating a mutagenic drinking water concentrate, mutagenic activity of strain T A 9 8 and TA100 is completely lost. This finding indicates that the organic mutagens in the concentrate showed thermolabile properties under our G C conditions. Fractionating and Molecular Weight Determination. Another approach to obtain more information on the nature of the responsible


612


TA 9 8

TA98 + S9 XAD/ACETONE XAD/ACETONE FD/ACETONE

FD/ACETONE

FD/ETHER F D/DMSO

CONCENTRATE (ml) TA 1 0 0

TA100 + S 9 XAD/ACETONE

FD/ACETONE

FD/ETMER FD/DMSO

CONCENTRATE (ml)

Figure 2. Comparison of mutagenic activity in lyophilized and XADconcentrated drinking water. The sampling; 7000-fold concentration with either XAD-4/8 (XAD) or freeze-drying (FD); XAD-4/8 elution with acetone (neutral fraction); freeze-drying elution successively with acetone, ether, and DMSO; and subsequent mutagenicity testing with strains TA98 and TA100 were as described in Materials and Methods. Each point represents the average of three plates, and 0.2 mL of concentrate corresponds to 1.4 L of water per plate. mutagens is to apply thin-layer chromatography (TLC) and H P L C on concentrates of drinking water and look for possible fractionation of the activity. Previous results with T L C showed that the mutagenic activity is found predominantly in one distinct zone (25, 26). Experiments using gel filtration on Sephadex LH20 according to the methods of Concin et al. (20) and designed to obtain information Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986.

29.

KOOL E TAL.

613


on the molecular weight of the organic mutagens showed for one drinking water concentrate that the organic mutagens present in the neutral fraction h a d a molecular weight range of 100-300 (26). T o see whether this result w o u l d also hold for X A D concentrates prepared f r o m other chlorinated drinking waters, experiments shown in Figures 4 and 5 were performed. T h e results obtained with t w o drinking water concentrates prepared f r o m Meuse Hiver water (Figure 4) and Rhine River water (Figure 5) confirmed our previous results: the bulk of the organic mutagens have a molecular weight in the range of 100-300 because the majority of the activity in drinking water (Figure 4) is detected in the molecular weight range of nitrofurazone ( M W 198). Also, after a chlorine treatment (1.5 m g / L of C l ; Figure 4), the mutagenic activity strongly increased. Furthermore, the organic mutagens formed after this chlorine treatment are in the same molecular 2

Figure 3. Effect of heating on the activity of a mutagenic drinking water concentrate. A mutagenic XAD-4/8 acetone concentrate (neutral fraction) was heated to 250 °C. The total heating time was 1 h. After this period, the organic residue was dissolved in acetone and retested in the Salmonella mutagenicity test. Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986.

614


D- S9 • S9

SPONTANEOUS REVERTANTS A : BEFORE CHLORINATION Β : AFTER CHLORINATION

7.0

Î

6.0 Η

LOG MW

[A (MW =10000)

Î

_3 (MW . 1000) _2 (MW = 100) J

Concentration, Fractionation, and Characterization ... - ACS Publications

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