Toxicity of Tannary Eff

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Mutagenicity and genotoxicity of tannery effluents used for irrigation at

Kanpur, India

Mohammad Zubair Alam a,n, Shamim Ahmad b, Abdul Malik a, Masood Ahmad c

a Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202002, Indiab Microbiology Division, Institute of Ophthalmology, Faculty of Medicine, JN Medical College, Aligarh Muslim University, Aligarh 202002, Indiac Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India

a r t i c l e i n f o

Article history:Received 29 September 2009

Received in revised form

11 May 2010

Accepted 11 July 2010Available online 3 August 2010

Keywords:

Mutagenicity

Genotoxicity

Tannery effluent

Ames Salmonella test

XAD

GCMS

Dichloromethane

Solvent

a b s t r a c t

The tannery effluents at Kanpur (India) have been in use for irrigation since last many years, pollutingsoil directly while ground water and food crops indirectly. Gas chromatographymass spectrometric

analysis of the test samples revealed the presence of organic compounds including diisooctyl phthalate,

phenyl N-methylcarbamate, dibutyl phthalate, bis 2-methoxyethyl phthalate, and higher alkanes.

Tannery effluent extracts were prepared using XAD-4/8 resins, dichloromethane, chloroform, and

hexane and tested with AmesSalmonellatest and DNA repair-defectiveEscherichia coliK-12 mutants. In

the presence of XAD-concentrated tannery effluent, TA98 found to be the most sensitive strain in terms

of mutagenic index followed by TA97a whereas in terms of mutagenic potential TA102 was most

responsive. The extracts were also found genotoxic as determined in terms of survival ofE. coli K-12

mutants, suggesting the presence of DNA damaging compounds in the tannery effluents. In the light of

results, precautious use of tannery effluents for irrigation is suggested.

& 2010 Elsevier Inc. All rights reserved.

1. Introduction

The use of industrial or municipal wastewater in agriculture is

a common practice in many parts of the world (Sharma et al.,

2007). The major objectives of wastewater irrigation are that it

provides a reliable source of water supply to farmers and has the

beneficial aspects of adding valuable plant nutrients and organic

matter to the soil (Liu et al., 2005b; Horswell et al., 2003).

Untreated or partially treated wastewater can introduce a huge

amount of inorganic and organic contaminates into agricultural

lands (Wang and Tao, 1998). Hence, continual use of wastewater

over extended periods can exert adverse impacts on quality of soil

and plants grown on it (Madyiwa et al., 2002; Sinha et al., 2006).

Therefore, indiscriminate use of untreated wastewater can beconsidered as one of the significant sources of environmental

pollution that may affect the human health via crops and soil

(Wang and Tao, 1998; Butt et al., 2005). However, with careful

planning and management, the positive aspects of wastewater

irrigation can be achieved (WHO, 2006).

The Indian leather industry being a major contributor to the

national economy is unfortunately also one of the major polluters.

The leather processing units in India are more than 1900 out of

which 75% are in the small scale sector. The inherent nature of the

tanning process is such, that large quantities of water are

consumed (Khwaja et al., 2001). Around 30 litres of liquid effluent

is produced per kilogram of leather processed. Thus, a substantial

amount of effluent is discharged from tanneries, which affects the

aquatic life and makes the water hazardous for human consump-

tion. The composition of organic pollutants in tannery wastewater

is complex. Proteins, mainly collagen and their hydrolysis

products amino acids derived from the skin are predominant,

while others such as fats are in low concentrations. The most

important organics used in tanning of skin are tannins both

natural and synthetic, fatty aldehydes and quinones. Tanneries

also use compounds like aliphatic amines, non-ionic surfactants,

oils, and pigments. Most of these pollutants are in a soluble form,but a lot of them exist in suspension and only a few are colloids

(Ates et al., 1997; Cassano et al., 2001; Di Iaconi et al., 2002 ).

Pollutants can affect organisms at various levels of biological

organization, from molecular to community levels (Theodoraskis

et al., 2000). The composite effects of mixtures cannot be readily

assessed by way of analytic methods. Rather, toxicity is often

evaluated by means of tests like bacterial genotoxicity tests,

which do not require a priori knowledge of toxicant identity and/

or physicochemical properties. Several studies have been carried

out on industrial and domestic wastewater and have been found

genotoxic and mutagenic in various short-term test systems

(Houk, 1992). There are many assays for detecting mutagenicity

Contents lists available atScienceDirect

journal homepage: www .elsevier.com/locate/ecoenv

Ecotoxicology and Environmental Safety

0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.ecoenv.2010.07.009

n Corresponding author. Fax: +91 571 2703516.

E-mail address: [email protected] (M. Zubair Alam).

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and genotoxicity of surface waters, but the utilization of bioassays

with bacteria has proven to be very effective for monitoring

because these assays are sensitive, inexpensive, reliable, and can

be performed in a short period of time with relatively low cost.

Among the microbial bioassays, the Salmonella mutagenicity test

has been the most widely used for detecting mutagenicity in

surface waters. This test developed byAmes et al. (1975)is based

on the detection of histidine-independent revertants in selected

Salmonella strains after exposure to mutagens with or withoutadditional activating enzymes.

In previous study, we evaluated mutagenicity and genotoxicity

of agricultural soil irrigated with tannery effluent (Alam et al.,

2009). The objective of the present study was to determine

mutagenic and genotoxic activity of tannery effluents using two

different bioassays, namely Ames Salmonella/mammalian micro-

some test and survival of SOS defective Escherichia coli K-12

mutants. Mutagenicity of tannery effluents was tested with Ames

Salmonellatest whereas genotoxicity was determined using E. coli

K-12 wild type and mutant strains.

2. Materials and methods

2.1. Sample collection

Tannery effluent samples were collected from the outlet of Combined Effluent

Treatment Plant (CETP) at Jajmau, Kanpur, India. Samples (5 litres) were collected

twice, 3 months apart in neat and clean plastic container. The city of Kanpur

(881220E longitude and 261260 N latitude) located on the banks of River Ganges,

with a population of around 2.4 million, is a major industrial hub in Northern

India. Kanpur has large number of tanneries located in a cluster at Jajmau with an

estimated wastewater discharge of 5.88.8 million liters per day.

2.2. Preparation of tannery effluent samples

Tannery effluent samples were concentrated using XAD-4 and XAD-8 resins.

Prior to concentration, the effluent samples were filtered through two membrane

filters with pore size of 8 and 0.45 mm. Adsorption of organic constituents was

carried out using 1 litre of tannery effluent by passing it through a column packed

with equal mixture of XAD-4 and XAD-8 resins as described earlier ( Kool et al.,

1981; Wilcox and Willaimson, 1986). The adsorbed organic material was then

eluted with 20 ml of acetone (HPLC grade). The eluate was evaporated to dryness

and re-dissolved in 1 ml of dimethyl sulphoxide (DMSO) (SRL, India); filtered-

sterilized through 0.45 mm pore size filters and stored at 20 1C until testing was

complete.

Tannery effluents were also extracted separately with three different organic

solvents, namely dichloromethane (DCM), chloroform, and n-hexane (all HPLC

grade). Extraction of the effluent with a solvent was done in two parts using

500 ml effluent, which was shaken vigorously with 25 ml of the extraction solvent.

When solvent and waterlayers were separated, the solvent layer was collected in a

beaker. The process was repeated three times with fresh 25 ml extraction solvent.

In this way a total of 1 litre tannery effluent per solvent was extracted. The

extracted organic phase was evaporated at 40 1C under reduced pressure with the

help of a vacuum pump and re-dissolved in 1 ml of DMSO. These samples were

filtered through 0.45 mm membrane filter before they were used for mutagenicity

and genotoxicity testing.

2.3. Gas chromatographymass spectrometric analysis of sample

Gas chromatographymass spectroscopy (GCMS) analysis of tannery effluent

samples was performed using HewlettPackard model GCD-HP1800A equipped

with an HP-5 column (30 m long, 0.32 mm inner diameter, 0.32 mm film

thickness). An electron ionization detector was used in the instrument, with an

operating mass range 10425 atomic mass unit. Component identifications were

performed by comparing their mass spectra at particular retention indexes using

the National Institute of Standards and Technology (NIST) library. Extracts of

tannery wastewater for GCMS analysis was prepared with DCM. A combined

extract of chloroform and hexane was also analyzed. Dichloromethane and hexane

were used in GC as solvent for DCM extract whereas hexane was used for the

combined extract of chloroform and hexane. The temperature program was set as

initial temperature of 100250 1C at the rate of 10 1C/min hold time at 250 1C for

2 min, then to 270 1C at the rate of 30 1C/min, and remained isothermal at 270 1C

for 3 min. Helium was employed as carrier gas at the rate of 1 ml/min.

2.4. Bacteria

The characteristics ofSalmonella typhimuriumandE. coliK-12 strains used are

reported in Table 1. S. typhimurium strains kindly provided by Prof. T. Nohmi,

National Institute of Hygienic Sciences, Division of Genetics and Mutagenesis,

Tokyo, Japan, were maintained in frozen stocks and grown as described by Maron

and Ames (1983). Each strain was tested on the basis of associated genetic markers

raising it from a single colony from the master plate. The bacterial strains ofE. coli

K-12 were kindly supplied by Berlyn, M.K.B. (E. coli Genetic Stock Center, MCD

Biology Department, Yale University, New Haven, CT, USA).

2.5. Ames mutagenicity testing

The pre-incubation test was performed as described by Maron and Ames

(1983) with some minor modifications (Pagano and Zeiger, 1992). Five doses of

each tannery effluent extract, i.e., 5, 10, 15, 20, and 25 ml/plate were plated in

duplicate with 0.1 ml of the bacterial culture. These doses were equivalent to 5, 10,

15, 20, and 25 ml of the tannery effluent. After incubating the test sample and

bacterial culture for 30 min at 37 1 C, 2 ml top agar containing traces of histidine

and biotin was added and the contents were poured onto minimal glucose agar

plates. Plates were incubated at 37 1C for 48 h and scored. The experiment was

carried out twice and out of total four readings, best three were considered in

results. Negative and positive controls were included in each assay. The negative

plates had bacteria and solvent (DMSO) but no test sample. Methyl methane

sulfonate and sodium azide were used as positive controls. All the extracts were

also tested in the presence of the microsomal fraction, to which 20 ml of the S9

liver homogenate mix per plate was added. The results are expressed as the meannumber of revertants per plate. For each dose tested, the ratio of mean number of

revertants per plate with extract to the mean number of revertants per plate with

solvent control was calculated and this ratio is termed as mutagenic index. A

sample was considered mutagenic when it induced a 2-fold increase in the

number of revertant colonies over solvent control (Courty et al., 2004; Vargas

et al., 1995).

2.6. Treatment of E. coli K-12 strains with tannery effluent extracts

The SOS-defectiverecA, lexA, andpolAmutants ofE. coliK-12 as well as their

isogenic wild-type strains were harvested (1 ml) by centrifugation from

exponentially growing culture (13 108 viable counts/ml). The pellets so

Table 1

Characteristics ofS. typhimurium and E. coli K-12 strains.

Strain

designation

Relevant genetic markers Source

Ames tester strains

TA97a uvrB, hisD661, bio, rfa, R-factor-

plasmid-pkM101, frame shift

mutation at GC site

T. Nohmi, National Institute

of Hygienic Sciences, Division

of Genetics and Mutagenesis,

Tokyo, Japan

TA98 uvrB, hisD3052,bio,rfa, R-factor

plasmid-pkM101, frame shift

mutation at GC site

TA100 uvrB, hisG46, bio, rfa, R-factor

plasmid-pkM101, base-pair

substitution mutation at GC

site

TA102 rfa, R-factor plasmid-pkM101,

multicopy plasmid paQ1containinghisG428auxotrophic

marker andtetr, transition

mutation at AT site

TA104 uvrB, hisG428, rfa, R-factor

plasmid-pkM101, transition

mutation at AT site

E. coliK-12 strains

AB1157 Thi1, argE3, thr1, leuB6, proA2,

hisG4, lacY1, F , Strr, lsBerlyn, MKB E. coli Genetic

Stock Center, MCD Biology

Department, Yale University,

New Haven, CT, USA

AB2463 recA13, thi1, argE3, thr1, leuB6,

proA2, hisG4, F , Strr, ls

AB2494 lexA13, thi1, leuB6, proA2, hisG4,

metB, lacY1, F , Strr, ls

AB3027 polA20, thi1, thr1, leuB6, proA2,

lacY1, xthA14, hisG4, F , strr,

argE3

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obtained were suspended in 1 ml of 0.01 M MgSO 4solution and treated with 20 ml

of each tannery effluent extract. Samples were withdrawn at regular intervals,

suitably diluted and plated to assay the colony-forming ability. Plates were

incubated overnight at 37 1C. Solvent control was also run simultaneously.

3. Statistical analysis

3.1. Mutagenic potential

The mutagenic potential of the tannery effluent extracts were

calculated by the least squares regression method, based on the

linear portion of the doseresponse curve with various strains.

Moreover, the initial portion of the concentrationresponse curve

included the data on increasing revertants only (Watanabe et al.,

2003; Fatima and Ahmad, 2006).

3.2. ANOVA

To determine the statistical significance of the number ofhis +

revertants in the sample compared to the control, one-way

analysis of variance (ANOVA) was done at pr0.05.

4. Results

The mass spectra of fragments for the major peaks in the gas

chromatograms of different tannery effluent extracts at the

particular retention time were compared with the mass spectra

in the NIST library. The best matches are reported (Table 2).

Numbers of compounds identified in the DCM extract were higher

compared to the combined extract of chloroform and hexane. In

case of both the extracts, maximum percent area was covered by

the peaks that correspond to different phthalate compounds.

The results on mutagenicity of the different extracts of tannery

effluent toward Ames tester strains are presented in Tables 36.

All the extracts of tannery effluents showed maximum mutagenic

index with TA98 both in the absence and presence of S9 fraction.

In general, there was an increase in the number of reversion of

tester strains with increasing dose up to 20 ml equivalent/plate of

all the extracts, except, in few cases where increase in the number

of revertants were observed up to 25 ml equivalent/plate and

then begin to decline beyond this level. Among all the tester

strains, TA98 showed maximum mutagenic index of 9.1(+S9) and

8.9( S9) followed by TA97a with the XAD-concentrated tannery

effluent. The order of responsiveness of Ames tester strains, based

on mutagenic index in the presence and absence of S9 fraction, for

XAD-concentrate of tannery effluent was TA984TA97a4

TA1004TA1024TA104. In the presence of DCM extract, again

TA98 displayed highest response in terms of mutagenic indexboth in the absence (7.4) and presence (7.7) of S9 followed by

TA97a with mutagenic index of 4.7( S9) and 4.6(+S9).

The rate of reversion of tester strains was lower with

chloroform and hexane extracts of tannery effluent compared to

the XAD-concentrate and the DCM-extract. With the chloroform

extract, the highest mutagenic index was observed for TA98 (6.7)

in the absence of S9 followed by TA97a (3.0). But in the presence

of S9, decline in the mutagenic index was observed. A similar

Table 2

Compounds identified in tannery effluents using GCMS.

Extraction solvent Peak

no.

Retention

time (min)

Area

(%)

NIST library ID

Dichloromethane 1 6.19 1.14 Phenyl

N-methylcarbamate

2 6.28 0.21 Caprolactam

3 9.61 1.75 Octacosane

4 1 0.51 2.13 2, 6,1 0, 15- tetramethyl-

heptadecane5 11.29 1.90 Nonadecane

6 11 .41 1.02 2, 6,1 0, 14- tetramethyl-

hexadecane

7 12.08 1.77 Triacontane

8 12.67 0.47 Heptadecane

9 12.87 0.85 Tetracosane

10 13.64 0.99 Eicosane

11 14.39 0.59 9-methylnonadecane

12 15.12 0.66 Heptadecane

13 16.38 84.89 1,2-benzenedicarboxylic

acid, diisooctyl ester

(diisooctyl phthalate)

14 16.81 1.63 Dotriaconatne

C hlorofor m+hexane 1 6 .46 2.70 2-(2-hydroxy)- 2 propyl

cyclohexanol

2 11.13 8.54 Dibutyl phthalate

3 11.21 4.98 Tetratetracontane

4 11 .91 12.72 bis (2- metho xyethyl)

phthalate

5 12.01 9.58 Hexatriacontane

6 12.81 1.97 Heneicosane

7 13.58 1.25 Docosane

8 14.34 0.83 Tricosane

9 16.39 57.43 1,2-Benzenedicarboxylic

acid, diisooctyl ester

(diisooctyl phthalate)

Table 3

Evaluation of mutagenic activity with XAD concentrated tannery effluent by Ames Salmonella assay.

Ames strain S9 Control Number of his + revertants/plate LSD pr0.05

Dose (ml equivalent/plate)

5 10 15 20 25

TA97a 11177 299713 (2.7) 388717 (3.5) 465721 (4.2) 574726 (5.2) 591723 (5.3) 34.9

+ 9778 272714 (2.8) 349719 (3.6) 436718 (4.5) 514722 (5.3) 504729 (5.2) 34.3

TA98 2674 9677 (3.7) 14379 (5.5) 187712 (7.2) 231715 (8.9) 216715 (8.3) 18.1

+ 2373 7875 (3.4) 13177 (5.7) 170711 (7.4) 209714 (9.1) 207712 (9.0) 16.6

TA100 140715 252717 (1.8) 322713 (2.3) 406719 (2.9) 476723 (3.4) 462720 (3.3) 26.6

+ 14979 283716 (1.9) 387721 (2.6) 477721 (3.2) 521725 (3.5) 507727 (3.4) 17.1

TA102 254713 356715 (1.4) 432719 (1.7) 559722 (2.2) 711728 (2.8) 710728 (2.8) 37.3

+ 244712 342711 (1.4) 439714 (1.8) 610718 (2.5) 756724 (3.1) 732727 (3.0) 26.6

TA104 325717 358716 (1.1) 455719 (1.4) 520723 (1.6) 553720 (1.7) 618723 (1.9) 22.8

+ 330720 363714 (1.1) 429716 (1.3) 528719 (1.6) 594725 (1.8) 592721 (1.8) 30.9

Number of replicates (n) 3.

Control spontaneous revertants in presence of DMSO.

Values are mean7SD (with mutagenic index in parentheses).

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trend was also observed for TA97a and TA98 in presence of the

hexane extract of tannery effluent. The XAD-concentrated and,

DCM, chloroform and hexane extracts of tannery effluent were

non-mutagenic toward TA104 whereas hexane extract was

weakly mutagenic toward TA100.

The significance of the reversion of tester strains with

increasing doses was determined by one-way ANOVA. The

analysis shows that reversion of the tester strains increases

significantly (pr0.05) in comparison to the negative control with

increasing doses.

The mutagenic potentials of XAD-concentrate and DCM,

chloroform and hexane extracts of tannery effluent toward the

Ames tester strains are given in Table 7. XAD-concentrated

tannery effluent exhibited maximum mutagenic potential

Table 4

Evaluation of mutagenic activity of tannery effluent extracted with dichloromethane by Ames Salmonella assay.



5 10 15 20 25

TA97a 9775 213712 (2.2) 281716 (2.9) 359714 (3.7) 426721 (4.4) 456724 (4.7) 30.4

+ 9576 238714 (2.5) 294713 (3.1) 380720 (4.0) 437725 (4.6) 427726 (4.5) 30.6

TA98 2873 8779 (3.1) 134711 (4.8) 171710 (6.1) 207712 (7.4) 20478 (7.3) 15.9+ 2572 8877 (3.5) 12378 (4.9) 14877 (5.9) 192714 (7.7) 187711 (7.5) 14.2

TA100 157712 267714 (1.7) 345719 (2.2) 487726 (3.1) 565732 (3.6) 549731 (3.5) 40.1

+ 15877 237712 (1.5) 379716 (2.4) 474722 (3.0) 600734 (3.8) 585736 (3.7) 40.3

TA102 252713 403715 (1.6) 479714 (1.9) 554718 (2.2) 706732 (2.8) 655738 (2.6) 41.3

+ 242711 411717 (1.7) 484720 (2.0) 532725 (2.2) 702730 (2.9) 629733 (2.6) 41.7

TA104 328717 361718 (1.1) 394717 (1.2) 492721 (1.5) 590727 (1.8) 623731 (1.9) 28.5

+ 322714 354715 (1.1) 419713 (1.3) 515719 (1.7) 547724 (1.7) 580722 (1.8) 28.3




Table 5

Evaluation of mutagenic activity of tannery effluent extracted with chloroform by Ames Salmonella assay.

Ames Strain S9 Control Number of his

+

revertants/plate LSD pr

0.05Dose (ml equivalent/plate)

5 10 15 20 25

TA97a 10676 18777 (1.6) 24678 (2.1) 291710 (2.5) 338714 (2.9) 353716 (3.0) 19.4

+ 9878 13776 (1.4) 186711 (1.9) 225714 (2.3) 284713 (2.9) 255715 (2.6) 18.7

TA98 1973 4374 (1.7) 6774 (2.6) 9576 (3.8) 13179 (5.2) 16778 (6.7) 5.8

+ 2372 3273 (1.4) 5374 (2.3) 6974 (3.0) 8376 (3.6) 9475 (4.1) 5.8

TA100 156712 20274 (1.4) 249712 (1.8) 291717 (2.1) 349713 (2.5) 336718 (2.4) 20.4

+ 147714 235712 (1.6) 294717 (2.0) 353713 (2.4) 397718 (2.7) 382721 (2.6) 6.0

TA102 258717 346720 (1.3) 384718 (1.5) 437716 (1.7) 479715 (1.9) 509722 (2.0) 29.0

+ 251714 276711 (1.1) 351716 (1.4) 502723 (2.0) 588733 (2.3) 577735 (2.3) 42.7

TA104 329719 384715 (1.2) 422723 (1.3) 441724 (1.4) 397721 (1.2) 362718(1.1) 33.1

+ 337714 371713 (1.1) 405717 (1.2) 453722 (1.3) 441713 (1.3) 411717 (1.2) 28.1




Table 6

Evaluation of mutagenic activity of tannery effluent extracted with hexane by Ames Salmonella assay.



5 10 15 20 25

TA97a 10676 145 710 (1.3) 19277 (1.7) 237711 (2.1) 262710 (2.3) 213718 (1.9) 17.9

+ 9878 13179 (1.3) 17478 (1.8) 207711 (2.1) 216714 (2.2) 204712 (2.1) 18.4

TA98 1973 4074 (1.6) 6675 (2.6) 9078 (3.6) 12079 (4.8) 141711 (5.6) 12.6

+ 2372 4373 (1.9) 5576 (2.4) 8379 (3.6) 10578 (4.6) 11877 (5.1) 11.2

TA100 156712 172711 (1.2) 222713 (1.6) 268712 (1.9) 303716 (2.1) 278714 (2.0) 21.4

+ 147714 206713 (1.4) 250711 (1.7) 265718 (1.8) 323713 (2.2) 311716 (2.1) 22.5

TA102 258717 29579 (1.1) 342712 (1.3) 379715 (1.5) 413719 (1.6) 446715 (1.7) 23.5+ 251714 270715 (1.1) 301710 (1.2) 348714 (1.4) 427719 (1.7) 376722 (1.5) 27.5

TA104 329719 527719 (1.6) 559713 (1.7) 523716 (1.6) 469718 (1.5) 435717 (1.3) 27.4

+ 337714 506717 (1.5) 573732 (1.7) 567729 (1.7) 558719 (1.7) 550716 (1.6) 31.6






5/9

producing 25.8(+S9) and 22.3( S9) revertants/ml in TA102

whereas, 20.0(+S9) and 18.9( S9) revertants/ml against TA97a.

TA100 was most responsive when tested with the DCM extract of

tannery effluent with 22.4(+S9) and 20.7( S9) revertants/ml

equivalent followed by TA102 with 20.8( +S9) and

21.2( S9) revertants/ml equivalent of the tannery effluent. In

the presence of the chloroform extract, TA102 exhibited the

highest response with 18.0(+S9) revertants/ml equivalent

followed by TA100 with 12.4 revertants/ml equivalent in the

presence of the S9 fraction. A similar pattern was also observed

for the hexane extract of tannery effluent.

The extraction resin or solvents can be grouped in terms of

inducing mutagenic potential toward frame shift-type mutation-

detecting strains (TA97a and TA98) without the S9 fraction as

XAD4DCM4chloroform4hexane whereas in the presence of

the S9 fraction it was XAD4DCM4hexane4chloroform. Same

order of response was also seen in TA102 both in the presence and

the absence of the S9 and in TA104 in the presence of the S9.

TA100 which is a base-pair substitution-detecting strain exhibited

a different pattern toward resin/extraction solvents as it showed

22.4(+S9) and 20.7( S9) revertants/ml equivalent when tested

with the DCM extract followed by XAD concentrate, chloroform

and hexane extracts, respectively. A similar trend was also shown

by TA104 in the presence of S9.

Further, mutagenic potency of XAD-concentrated tannery

effluent was compared with DCM, chloroform, and hexane

extracts against each of the tester strains (Table 8).

XAD-concentrated tannery effluent exhibited percent increase in

the range of 5.231.3% over DCM extract either with or without S9

against all test strains except TA100 (+S9 and S9) and

TA104 ( S9). Similarly, an increase of mutagenic potential in

XAD-concentrate was observed ranging from 51.6% to 210.0% over

chloroform extract and 77.9% to 222.5% over hexane extract

against all the tested strains. With TA100, a decrease of

20.3% ( S9) and 16.1% (+S9) in the mutagenic potency of

XAD-concentrate was observed over DCM extract. Similarly,

decrease of 6.2% was observed in TA104 without S9 fraction.

The survival pattern in terms of colony forming ability ofE. coliK-12 (wild-type) as well as its isogenic mutant counterparts lexA,

recA, and polA in the presence of XAD concentrate and, DCM,

chloroform, and hexane extracts of tannery effluent is shown in

Fig. 1. The damage to the cells in the presence of XAD-concentrate

was found to be higher compared to the other extracts at

treatment of 20 ml/ml of culture. Among all the mutants, polA

exhibited the maximum decline and displayed a survival of 16.0%

in polA in the presence of XAD-concentrated sample after 4 h of

treatment. The lexA mutant exhibited survival of 27% whereas

recA mutant displayed 35% survival when treated with

XAD-concentrated sample for 4 h. In the presence of the DCM

extract, survival was 19% for polA, 25% for lexA, and 44% for recA

mutant. When the mutants were treated with the chloroform

extract, the survival was 40% in polA, 62% inlexA, and 53% inrecA

mutant. The hexane extract was found to be the least damaging to

all the mutants; here survival was 49% in polA, 55% in lexA, and

69% in recA mutant after 4 h of treatment. In contrast to the

mutant strains, no significant decline was seen in the survival ofE.

coli K-12 (wild-type).

5. Discussion

The Ames Salmonella test has been widely used to detect

mutagenic activity in complex environmental mixtures such as

river waters, lakes, industrial effluents, drinking water, and

hospital wastewater (Houk, 1992; Claxton et al., 1998; Jolibois

et al., 2003; Ohe et al., 2003). This test let the efficient detection of

trace amounts of organic genotoxic components either in raw

form or by their proper extraction/concentration using resins or

organic solvents, allowing the assay of equivalent volumes of

water samples which otherwise un-testable.

In wastewater or industrial effluents mutagenic potency canbe detected in non-concentrated samples (Czyz et al., 2002; Dizer

et al., 2002). However, mutagenic and genotoxic contaminants

usually present at such a low levels is difficult to be detected, and

therefore, some sort of extraction/concentration is required for

mutagenicity assessment of water samples (Umbuzeiro et al.,

2001; Courty et al., 2004). The identification of specific chemical

substances with genotoxic activity in untreated water, industrial

effluents, or soil is difficult because few compounds are present at

high concentrations. Moreover, most of the times genotoxic

activity cannot be attributed to specific compounds in the mixture

but rather to the set of properties and chemical interactions of the

sample as a whole (Mc George et al., 1983; Hartnik et al., 2007).

In the current study we initially tested raw tannery effluent

(not concentrated or extracted); however, such effluent failed to

Table 7

Influence of extraction solvents on mutagenic potential of tannery effluent towards S. typhimurium tester strains.

Solvent Number of revertants induced per ml equivalent of the tannery effluent in Ames tester strains

TA97a TA98 TA100 TA102 TA104

S9 +S9 S9 +S9 S9 +S9 S9 +S9 S9 +S9

XAD 18.9 20.0 10.0 9.3 16.5 18.8 22.3 25.8 12.1 14.3

Dichloromethane 14.4 16.5 8.8 7.9 20.7 22.4 21.2 20.8 12.9 11.2Chloroform 9.9 9.2 5.9 3.0 10.3 12.4 9.8 18.0 7.5 7.6

Hexane 8.1 6.2 5.0 3.9 7.8 8.2 7.6 8.6 6.8 7.1

Values were calculated using the data from Tables 25.

Table 8

Comparison of mutagenic potential of tannery effluent extracted with different

organic solvents/resin.

Strain S9 Percent variation in mutagenic potential

XAD: DCM XAD: c hlor oform XAD: he xan e

TA97a 31.3 90.9 133.3

+ 21.2 117.4 222.5

TA98

13.6 69.5 100.0+ 17.7 210.0 138.5

TA100 20.3 60.2 111.5

+ 16.1 51.6 129.3

TA102 5.2 127.6 193.4

+ 24.0 43.3 200.0

TA104 6.2 61.3 77.9

+ 27.7 88.2 101.4

Values are given as %.

Negative values indicate the percentage decrease in mutagenic potential.



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produce mutagenicity in tester strains up to a dose of 60 ml/plate,

but when we tested this raw effluent at higher dose volume

toxicity was observed instead of mutagenicity. This might be due

to the high concentration of trivalent chromium and other

inorganic constituents present in tannery effluent (Alkan et al.,

1996; Suvant et al., 1997; Vankova et al., 1999; Tisler et al., 2004 ).

The composition of organic pollutants in tannery wastewater

is complex. The most important organics used in tanning of skin

are natural and synthetic tannins, dyes, aliphatic amines, non-

ionic surfactants, sulphonated oils, fatty aldehydes and quinines,

to transform animal skin into an unalterable and imputrescible

product (United Nations Environment Program, 1991; Klinkow

et al., 1998). Mass spectroscopy combined with gas chromato-

graphy is the ideal detection method, and has been applied forcharacterization of the obtained extracts. Reemtsma and Jekel

(1997) used GCMS in the electron impact mode, for the

characterization of tannery wastewaters. Qualitative GCMS

analysis of the tannery effluent samples revealed the presence

of organic compounds like diisooctyl phthalate, phenyl

N-methylcarbamate, dibutyl phthalate, bis 2-methoxy ethyl

phthalate, etc (Table 2). The United States Environmental

Protection Agency (USEPA) and some of its international counter-

parts have classified most of the phthalic acid esters, such as

diethyl phthalate, benzyl butyl phthalate, di-n-butyl phthalate

and di-(2-ethyl hexyl) phthalate, as priority pollutants and

endocrine-disrupting compounds (Moore, 2000). The identifica-

tion of compounds in our study was based on comparison of the

mass spectra of the organic compounds in the extracted sample

with those in the NIST library. While the main organic

constituents that were present in high amounts in the fractions

could be identified, small amounts of other constituents could not

be identified by GCMS.

There are a large number of studies that implicated tanning

industry with a number of health hazards, including occupational

exposures (Battista et al., 1995; Mikoczy et al., 1996), water and

land contamination affecting crops, aquatic and terrestrial biota,

and humans (Barnhart, 1997), as well as acute toxicity in

Vibrio fisheri (Jochimsen and Jekel, 1997) and Daphnia magna

(Tisler et al., 2004). In a study, leachates derived from tannery

waste were analyzed for mutagenic activity using spot and plate

incorporation tests with Ames strains. The result suggested that

leachates from tannery wastes possess mutagenic properties(Singh et al., 2007).

The present study indicates an increase in the number of

revertant colonies with one or more Ames Salmonella strains in

the presence of the test samples. It was observed that the XAD

concentrated tannery effluent exhibited maximum response

toward Ames strains both in terms of mutagenic index and

mutagenic potential except TA100 (with and without S9)

and TA104 (without S9). TA98 (+S9) showed maximum response

in terms of mutagenic index but in terms of mutagenic potential

TA102 (+S9) was most sensitive when treated with the

XAD-concentrate. Adsorption on amberlite XAD resins is the

most commonly applied method for concentrating organic

substances from different kinds of surface waters, wastewater,

and industrial effluents. XAD resins generally adsorb a broad class

Time (h)

0

Percentsurvival

0

20

40

60

80

100

120

Perce

ntsurvival

0

20

40

60

80

100

120

Percentsurv

ival

0

20

40

60

80

100

120

Wild type (recA+,lexA+, polA+) recA- lexA- polA-

Percentsurvival

0

20

40

60

80

100

120

1 2 3 4

Time (h)

0 1 2 3 4

Time (h)

0 1 2 3 4

Time (h)

0 1 2 3 4

Fig. 1. DNA damaging activity in terms of survival ofE. coliK-12 strains in the presence of the tannery effluent extracted with (a) XAD, (b) dichloromethane, (c) chloroform,

and (d) hexane. The decline in survival of mutant strains was found significant at pr0.05compared to E. coli K-12 (wild type); number of replicates (n) 3.



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of mutagenic compounds, including polycyclic aromatic hydro-

carbons, arylamines, nitro-compounds, quinolines, anthraqui-

nones, etc (Reifferscheid et al., 1991; Galassi et al., 1992;

Hendriks et al., 1994). Using the XAD resin method, many positive

results were observed in those extracts which were otherwise not

mutagenic in bacterial mutagenicity assays (Helma et al., 1996;

Guzzella and Sora, 1998).

Kummrow et al. (2003) reported enhanced mutagenic sensi-

tivity with XAD-4 and blue rayon concentrated river watersamples under the influence from a dye processing plant

compared to the water from a reservoir not directly impacted

with industrial discharges.Pereira et al. (2007)studied mutagenic

activity in supply water in a state of Brazil and reported enhanced

mutagenic response in TA98 and TA100 strains after extraction

with XAD-4 resins compared to raw water. Several other workers

also reported enhanced mutagenic activity in the presence of

XAD, blue rayon, and blue chitin concentrated water samples

(Siddiqui and Ahmad, 2003; Aleem and Malik, 2005; Kataoka

et al., 2000; White and Rasmussen, 1998).

Our data are also indicative of the presence of dichloro-

methane, chloroform, and hexane soluble substances in tannery

effluents. The response observed in Ames strains in the presence

of dichloromethane extract was lower than XAD concentrate of

tannery effluent; even then the results are comparable to it.

Liquidliquid extraction with dichloromethane is the USEPA

method of choice for the analysis of acid or baseneutral organic

chemicals in water. Lippincott et al. (1990) reported that

extraction with dichloromethane was well suited for concentrat-

ing a sufficient amount of baseneutral trace organics for

chemical identification and for the Ames bioassay. Nielsen

(1992) considered dichloromethane as the best choice for

extractions of complex environmental mixtures. In a study on

mutagenicity of different type of soil, dichloromethane extract

induced highest level of mutagenicity than the other organic

solvents used like acetonitrile, hexane, acetone, and methanol

(Edenharder et al., 2000).Kwon et al. (2008)studied Kumho River

water (South Korea) after extraction with XAD-2 resin and

dichloromethane and reported extraordinarily high mutagenic

activity toward TA98 in both extracts.

In the present study, we have found the test effluent to be less

mutagenic in chloroform and hexane extracts compared with

XAD-concentrate and dichloromethane extract of tannery

effluent. In a very informative review on soil mutagens, White

and Claxton (2004) collected and statistically analyzed a large

number of published literatures on genotoxicity. They showed

that the average mutagenic potency of DCM extracts from soil

toward TA98 and TA100 is more than 20-fold greater than hexane

extracts.

In the present study, the test samples exhibited maximum

response in terms of mutagenic index with TA98 followed by

TA97a both in the presence and absence of metabolic activation

system. Courty et al., 2004 also reported greater sensitivity forTA98 than other Ames strains. Aleem and Malik (2005) and

Siddiqui and Ahmad (2003) reported that XAD concentrated

water samples from the River Yamuna, India, were remarkably

high for TA98 compared to TA100 both with and without S9. In

our study, TA102 was found to be the most sensitive in terms of

mutagenic potential toward XAD concentrated sample followed

by TA100, which was most responsive in dichloromethane extract

of tannery effluent. This difference in sensitivity based on

mutagenic index and mutagenic potential may be due to the

intrinsic property of the tester strains; TA102 produced nearly

250 spontaneous revertants, TA100 around 150 whereas TA98

produced only around 20 revertants spontaneously. Therefore, in

our study mutagenicity results are better represented by

mutagenic index and this criterion for a sample to be mutagenic

has also been adopted by other workers (Vargas et al., 1995;

Courty et al., 2004; Aleem and Malik, 2005).

Genotoxicity of XAD, DCM, chloroform, and hexane extracts of

the tannery effluent was determined by measuring survival in

terms of colony forming ability ofE. coli K-12 (wild-type) as well

as its isogenic mutant counterparts lexA, recA, and polA (Fig. 1).

The SOS response in E. coli results in the simultaneously induced

expression of more than 40 genes including recA, lexA, and polA

which occurs when cells are treated with DNA-damagingagents (De Henestrosa et al., 2000). The RecA protein of E. coli,

has several enzymatic activities, is required for homologous

recombination (Radding, 1985) and, in conjunction with the LexA

protein, serves to control a complex set of events which occurs

after cells are exposed to agents that damage DNA, the SOS

response. RecA protein is expressed at low levels under normal

growth conditions, but its synthesis is greatly increased after

SOS induction. It is known that the SOS response is dependent

on recA and lexA gene products, as well as on the presence of

single-stranded DNA (Walker, 1985; Strauss, 1989). The elevated

expression of these genes increases the capacity of cells for

DNA repair, damage tolerance, DNA replication, and mutagenesis

(Renzette et al., 2005). Since mutant strains do not permit

induction of the SOS system, the lack of SOS repair renders

such strains extremely sensitive to DNA-damaging agents.

(Kuzminov, 1999). TherecA,lexA, andpolAmutants ofE. coliwere

found to be sensitive to the test samples, suggesting damage to

the DNA of exposed cells as well as a role ofrecA+ ,lexA+ andpolA+

genes in coping with the hazardous effect of pollutants (Aleem

and Malik, 2003).

6. Conclusion

The present study confirmed that the tannery effluent contains

certain compounds having mutagenic and genotoxic activity.

Ames test is a suitable method to demonstrate the mutagenicity

of tannery effluents though there are some disadvantages also

associated with the Ames test and survival test of E. coli K-12

strains for environmental applications: (i) it requires sterilization

of the test sample to avoid bacterial contamination and (ii) the

wastewaters samples have to be extracted with organic solvents

or resins to detect traces of mutagenic pollutants. The extraction

procedure is an indispensable stage in the evaluation of

mutagenicity of such effluents using Ames test or other in vitro

assays. XAD resins were found to be the best concentration

method as the maximum response was observed in the tester

strains when tested with it followed by dichloromethane, chloro-

form and hexane extracts of tannery effluent. TA98 proves to be

the most sensitive in terms of mutagenic index in detecting

mutagens in extracts followed by TA97. In other words, tannery

wastewaters predominantly contained frame-shift mutagens.

Though it is difficult to predict actual mutagenic component insuch a complex effluent even then 1,2-benzenedicarboxylic acid

diisooctyl ester (diisooctyl phthalate), phenyl N-methylcarba-

mate, dibutyl phthalate, etc. can be suspected for mutagenic and

genotoxic activity of the tannery effluent extracts. Therefore,

complementary studies should be undertaken analytically in

order to identify and quantify the compounds responsible for the

genotoxicity. The findings of the present investigation point out

that the treatment carried out for tannery effluent is not as

efficient as it should be in removing hazardous organic con-

taminants from spent tannery water. Thus, better processes/

methods must be adopted for the treatment of complex effluents

originating from tanneries. In the light of our findings, it is

suggested that tannery effluents should be used precautiously for

irrigation of agricultural lands.



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Acknowledgments

Financial support from the Council of Scientific and Industrial

Research, File no. 24(0271)/04/EMR-II, Government of India, is

gratefully acknowledged. We also thank Dr. Vijaya S. Lakshmi of

SAIF at the Indian Institute of Technology, Bombay, for carrying

out GCMS analysis of samples.

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