Genotoxicity of influents and effluents of the wastewater treatment plant

8

Pergamon

War. Sci. Tech. Vol. 34, No. 7-8, pp. 9-14,1996. Copyright © 1996 fA WQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved. 0273-1223/96 $15'00 + 0'00

PH: S0273-1223(96)00716-0

GENOTOXICITY OF INFLUENTS AND EFFLUENTS OF THE WASTEWATER

TREATMENT PLANT

M. Filipic* and M. J. Toman** * Institute of Public Health, Grabloviceva 44,61000 Ljubljana, Solvenia ** University of Ljubljana, Biotechnical Faculty, Department ofBiology,

Vecna pot 111, Ljubljana, Slovenia

ABSTRACT Wastewaters are treated through different processes of wastewater treatment procedures. Nevertheless they often contain mutagens especially when the proportion of industrial wastewater in comparison to municipal wastewater is high. In this study we evaluated mutagenic potential of influents and effluents from a wastewater treatment plant that is processing both industrial and domestic wastewater. The mutagenicity of XAD-2 extracts of influent and effluent was evaluated by means of Ames test using S. typhimurium strains TAIOO and TA98 in the presence and in the absence of metabolic activation. Extracts that were mutagenic to strain TA98 without metabolic activation were suspected to contain nitropolyaromatic hydrocarbons (nitro-PAHs). To confirm this hypothesis they were tested with nitroreductase (TA98NR) and O-acetiltransferase (T A9811 ,8DNP ~ deficient derivatives of strain TA98, that are resistant to nitropolyaromatic hydrocarbons. The nitroreductase deficient strain TA98NR was less sensitive to the mutagenic extracts of influent than the parent strain TA98. The O-acetiltransferase deficient strain TA9811 ,8DNP6 was resistant to the mutagenic extracts of influent. The mutagenic extracts of the effluent were nearly equally mutagenic to the parent strain TA98 and both deficient strains TA98NR and TA9811,8DNP 6. On the basis of the responses of nitroreductase deficient strains on the influent and effluent it was concluded that the influent contained nitro-PAHs. These were not removed or inactivated during the biological treatment of the wastewater, but activated to the final nucleophylic form able to induce mutations strain TA98 and in its nitroreductase deficient derivatives TA98NR and TA9811,8DNP 6. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd.

KEYWORDS Mutagenicity; municipal wastewaters; nitropolyaromatic hydrocarbons; Salmonella/microsome test. INTRODUCTION Introduction of the mutagens into the environment represents a substantial threat to the affected populations including human beings. It is known that through wastewaters, that represent the most abundant way of waste discharges, mutagens are introduced into water flows. Even though wastewaters are treated through different processes of wastewater treatments they often contain mutagens especially when the proportion of industrial wastewater in comparison to municipal wastewater is high (Meier and Bishop, 1985, Meier et al., 1987). The fact is that, even if biological wastewater treatments purify the wastewater and retain large 9

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M. FlLIPIC and M. J. TOMAN

amounts of organic material (the chemical and biochemical oxygen demand values are within the statutary limits), numerous potentially mutagenic substances produced by different industries are refractory to the aerobic biodegradation and create concern about potential health effects to both humans and other components of the ecosystem (i.e. fishes). The aim of our study was to evaluate mutagenic potential of influents and effluents from the wastewater treatment plant that is processing both industrial and domestic wastewater. The organic matter was extracted from the samples by adsorption to XAD-2 resin and subsequent elution with appropriate solvents. ~he mutagenicity of the XAD-2 extracts was evaluated by means of Ames test using S. typhimurium strams TA I00 to detect base pair substitution mutagens and TA98 to detect frameshift mutagens in the presence and in the absence of metabolic activation (Maron and Ames 1983). The profiles of mutagenic activities of neutral and acid XAD-2 extracts of the samples were compared to identify types of mutagens that are refractory to the aerobic biological treatment. Extracts that were mutagenic to strain TA98 without metabolic activation were suspected to contain nitropolyaromatic hydrocarbons. To confirm this hypothesis they were tested with TA98NR, a nitroreductase and TA98/1 ,8DNP6, an O-acetiltransferase deficient strains of S. typhimurium, that are resistant to nitropolyaromatic hydrocarbons. MATERIALS AND METHODS Sample preparation: 10 I of the samples was collected continuously through 12 hours at the inflow and outflow from the municipal wastewater treatment plant. Samples were filtered through a glass filter and for the testing in nonconcentrated form also through membrane filter with 0.22 Jlm pore size. Sample extraction: The extraction of dissolved organics was performed by adsorption on XAD-2 resin (Sigma), volume 20 cm3 (the diameter of the column was 1.4 cm, the height of the column was 13 cm) using 10 I of water. The extraction was performed sequentially at two different pH values. Filtered raw water was at the first step adsorbed at pH of the raw sample, that was in all cases between 7.2 and 8.9. The adsorbed organic matter was sequentially eluted with 200 ml of acetone (neutral acetone extract - NA) and with 200 ml of dichloromethane (neutral dichloromethane extract - ND). After passing the column the sample was collected, acidified with 5M HCI to pH2 and allowed to pass the second column. After sequential elution with acetone and dichloromethane this extract are designated as acid acetone extract - AA and acid dichloromethane extract - AD, respectively. The eluates were evaporated to a small volume at 40°C under reduced pressure and finally to dryness under a nitrogen stream. The dry residue was dissolved in 1 ml of dimethylsulphoxide (DMSO) and than diluted with distilled water to a volume of 10 ml (1000 times concentrate of the original water sample). Bacterial strains: The S. typhimurium strains used were TA100, TA98 and the derivatives of TA98 capable of detecting nitroarenes and aminoarenes: TA98NR and TA981l ,8DNP 6' Strain TA98NR is deficient in the classical nitroreductase that is necessary to induce the mutagenicity of typical nitroarenes (Rosenkranz and Speck, 1975, 1976). Strain TA98/1 ,8DNP6 is deficient in O-acetiltransferase necessary for the induction of mutagenicity of nitroarenes and aminoarenes (McCoy et al., 1981). Mutagenicity testing: Mutagenicity of NA, AA, ND and AD extracts and the mutagenicity of a mixture of the four extracts (1: 1:1:1) of influent and effluent samples was assayed by plate incorporation procedure (Maron and Ames, 1983) with 90 minutes of liquid preincubation. The preincubation mixture consisted of 100 JlI of bacterial suspension, 200 JlI of appropriately diluted sample extract and 500 JlI of phosphate buffer or 500 JlI of S9-mix. This mixture was preincubated on the rotary shaker for 90 minutes at 37°C. At the end of the preincubation period 2 JlI of 0.6% top agar containing biotin and limited quantity of histidine was added, gently mixed and poured on to minimal agar plates. The plates were incubated for 48 hours at 37°C. The concentrations of the extracts ranged from 3 to 100 JlI per plate (corresponding to 3 to 100 JlI equivalent of original sample per plate) depending on the toxicity of the extract for the tester strains.

Genotoxicity of influents and effluents

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As a metabolic activation post-mitohondrial fraction (S9 fraction) prepared from rat liver induced with Arochlor 1254 was used, according to Maron and Ames (1983). Prior to the use S9-mix was prepared: S9 10%, nicotine amid adenine dinucleotide (NADP) 4 mM, glucose-6-phosphate 5 mM, sodium phosphate pH 7.4 100 mM, KCI 33 mM and MgCl2 8 mM. The mutagenic activity is expressed as EC2 which was defined as the concentration of the sample inducing the doubling of the number of spontaneous revertants. EC2 was calculated from the linear part of the log-log dose response curve. On the figures mutagenic profiles of the extracts are shown in log dose units (LDU = log (1000/EC2)). RESULTS AND DISCUSSION The mutagenic profiles of extracts of influent and effluent samples are shown in Figure 1. More detailed discussion on mutagenic profiles of water and wastewater samples is given in Filipic and Toman (1996). The mutagenic activity for strain TA98 without metabolic activation was present in the nonconcentrated influent sample, its ND extract and the mixture of the extracts. In the effluent this activity was detected in NA and AA extracts and in the mixture of the extracts.

o o

TA98 -S9 TA98+S9 • TAIOO -S9 • TAIOO+S9

INFLUENT 1E+03

1E+02

~ ~

I

I 1E+01 I

i 1E+OO

NA

M

ND

MIXT.

AA

ND

MIXT.

EFFLUENT 1E+03

1E+02 /

1E+01

1E+OO"----"'-'

NA

Figure 1. Mutagenic profiles of NA, AA and NO extracts and the mixture of the extracts of the influent and effluent water sample from wastewater treatment plant.

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M. FILIPIC and M. J TOMAN

12

Extracts that were directly mutagenic in strain TA98 were further tested with strains TA98NR and TA98/1,8DNP6 to elucidate the chemical properties of the mutagens present in the ND fraction of the influent and NA and AA fractions of the effluent. The results are shown in Figures 2 and 3. ~------

-- -

--,------~~-----

----...,.-

•

MIXTURE OF EXTRACTS

NDEXTRACT

7t

• TA98 - - C - . TA98 NR

I

6+!

-

5t

TA98 1.8DNP6

.. -2

:f I

G

A-

2+I

2

I

1

t

i

o ,..~.....",'__'I_=b.-~ o 20 40

.tt-------a I

!

I

60

80

100

cone. ml ekv.lpl

O~~~~~·~-~z~:_~

o

20

40

60

cone. ml ekv.lpl

I

----_._------

I

Figure 2. Induction of revertants in S. typhimurium strains TA98, TA98NR and TA98II,8DNP6 by ND extract and the mixture of extracts of the influent. (G is the ratio between number of revertants induced in the presence of the sample and the number of revertants induced on the control plates.)

NAEXTRACT 4

AAEXTRACT

1

2

1

I I

:j

3t G

l

MIXTURE OF EXTRACTS

,t

2

1

1.

I i

I "

0 0

0&------+-----

50

100 i

cone. ml ekv.lpl I

o

50

100

cone. ml ekv.lp._l---L

0

0

--+-

I

I

20

40

60

I

co~c. ml~

Figure 3. Induction of revertants in S. typhimurium strains TA98, TA98NR and TA98II,8DNP6 by ND extract and the mixture of extracts of the effluent. (G is the ratio between number of revertants induced in the presence of the sample and the number of revertants induced on the control plates.)

The ND extract of the influent sample was highly mutagenic for strain TA98. The mutagenic activity for the strain TA98NR was significantly lower and strain TA98/l ,8DNP() was resistant to ND extract of influent. The mixture of the extracts induced mutations in strain TA98 while in deficient strains no mutations were induced. The overall mutagenic activity of the mixture of the extracts was lower than the mutagenic activity of ND extracts which was expected as in the mixture the mutagenic ND extract represented only one fourth

Genotoxicity of influents and effluents

13

of the total volume. These results indicate that the direct mutagenic activity for the strain TA98 might be mostly due to the presence of nitro-PAHs. Of the extracts of the effluent, NA and AA extracts and the mixture of the extracts were mutagenic for strain TA98. We assume that nitro-PAHs that were isolated from the influent sample only in ND extract were metabolically transformed due to the microbial enzymatic activity of the activated sludge. The consequence was that their polarity was changed so that they were soluble in acetone. From Figure I it can be seen that NA and AA extracts of the effluent were mutagenic for strain TA98, while ND extract of the effluent was inactive. NA and AA and the mixture of the extracts of the effluent were mutagenic also for nitroreductase (TA98NR) and O-acethyltransferase (TA98/1 ,8DNP6) deficient strains (Figure 3). This can mean that nitro-PAHs detected in the ND extract of the influent were not removed or inactivated but metabolically transformed to final nucleophilic form able to induce mutations in strain TA98 and in nitroreductase and 0• acetyltransferase deficient strains TA98NR and TA98/1 ,8DNP 6, respectively. The most important step in metabolic activation of nitro-PAHs is the reduction of the aromatic nitro compound to hydroxylamine that is able to react with DNA molecule (Compadre et ai., 1990). It is known that many bacterial strains have nitroreductase activity so they are able to activate nitro-PAHs. In anoxic conditions that are not uncommon in biological wastewater treatment plants the reduction will be evenly facilitated. Many authors reported higher mutagenic activity of effluents from biological municipal wastewater treatment plants comparing to influents for strain TA98 (Denkhaus et ai., 1981; Meier and Bishop, 1985; Meier et ai., 1987; Fracasso et ai., 1992; Doereger et ai., 1992). That means, that certain frame shift mutagens that are obviously rather common contaminants are not inactivated by the biological treatment. Our assumption is that nitro-PAHs that are widespread contaminants can be responsible for a large part of the so frequently observed mutagenic activity of influents and effluents of wastewater treatment plants. CONCLUSION Our results are indicating that the direct frame shift mutations inducing mutagenic activity of the influent were most probably due to certain types of nitro-PAHs that induced mutations in strain TA98 but not in nitroreductase and O-acetyltransferase deficient derivatives. During the biological treatment of the wastewater, microorganisms present in the activated sludge were not able to remove or inactivate mutagenic nitro-PAHs. In fact they only reduced nitro-PAHs to the final nucleophilic form able to induce frame shift mutations in strain TA98 and in both nitroreductase and 0• acety1transferase deficient derivatives. Further chemical and biological analysis of mutagenic extracts are needed to confirm our assumptions. REFERENCES Compadre, R. L. L., Debnath, A K., Schusterman, A 1. and Hansh, C. (1990). LUMO energies and hydrophobicity as determinants of mutagenicity by nitroaromatic compounds in Salmonella typhimurium. Environ. Mol. Mutag., 14, 44-55. Denkhaus, R., Grabow, W. O. K. and Prozesky, O. W. (1981). Removal of mutagenic compounds in a wastewater reclamation system evaluated by means of the Ames salmonella/microsome assay. Prog. Wat. Tech., 13(1),571-589. Doereger, J. D., Meier, J. R., Dobbs, R. A, Johnson, R. D. and Ankley, G. T. (1992). Toxicity reduction evaluation at a municipal wastewater treatment plant using mutagenicity as an endpoint. Arch. Environ. Contam. Toxicol., 22, 384-388. Filipic, M. and Toman, M. J. (1996). Ecotoxicological studies using modified Ames bioassay. Wat. Sci. Tech., 34(7-8), 1-7 (this issue). Fracasso, M. E., Leone, R., Brunello, F., Monastra, C., Tezza F. and Storti, P. V. (1993). Mutagenic activity in wastewater concentrates from dye plants. Mutat. Res. 298, 91-95. Maron, B. and Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutat. Res., 113, 173-212. McCoy, E. C. and Rozenkranz, H. S. (1982). Cigarette smoking may yield nitroarenes. Cancer Lett., 15,913. McCoy, E. c., Rosenkranz, H. S. and Mermelstein, R. (1981). Evidence for existence of a family of bacterial nitroreductases capable of activating nitriated polycyclics to mutagens. Environ. Mutagen., 3, 421-427.

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Meier. 1. R. and Bishop. D. F. (1985). Evaluation of conventional treatment processes for removal of mutagenic activity from municipal wastewaters. J. Water. Pol/Ufo Control Fed., 59. 999-1005. Meier. J. R.. Blazak. F. W.• Riccio. E. S.• Stewart. B. E.. Bishop. D. F. and Codie, L. W. (1987). Genotoxic properties of municipal waste waters in Ohio. Areh. Environ. Toxieo/.. 16, 671-680. Rosenkranz. H. S. and Speck. W. T. (1975). Mutagenicity of metronidazole: Activation by mammalian liver microsomes. Biochem. Biophys. Res. Commun. 66, 520-525. Rosenkranz. H. S. and Speck.. W. T. (1976). Activation of nitrofurantoin to a mutagen by rat liver nitroreductase. Biochem. Biophys. Res. Commun, 25, 1555-1556.