Determination of Genotoxic Pollution of Some Hospital Wastewater with Salmonella Ames Test

doi:10.4236/jwarp.2012.410100

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Journal of Water Resource and Protection, 2012, 4, 859-865

http://dx.doi.org/10.4236/jwarp.2012.410100 Published Online October 2012 (http://www.SciRP.org/journal/jwarp)

Determination of Genotoxic Pollution of Some Hospital

Wastewater with Salmonella Ames Test

Ali Rıza Atasoy1*, Engin Karakece2, Mustafa Petek3, Lokman Alpsoy3, Abdullah Kiran4

1Department of Microbiology, Faculty of Medicine, Sakarya University, Sakarya, Turkey

2Microbiology Laboratory, Sakarya Education and Research Hospital, Sakarya, Turkey

3Institute of Science and Engineering, Fatih University, Istanbul, Turkey

4Saruhanlı Food Vocational School, Celal Bayar University, Manisa, Turkey

Email: *[email protected]

Received August 6, 2012; revised September 3, 2012; accepted October 1, 2012

ABSTRACT

Wastewater of hospitals contains materials that would be a threat to alive. These water needs to be checked by a bio-

logical purification before leaving to nature. Hospital wastewater has differences than domestic waste because of espe-

cially blood, body waste, drugs, chemicals, medical device waste and radioactive materials. We aimed to determine

genotoxic effects of to tal pollution in ho spital wastewater on alive by Salmonella microsome test method. In this study,

we decided on three hospitals which weren’t checked as biological purification of waste. The samples were taken for six

1-week periods between March 2009 and June 2009. Mutagenite studies of samples taken from hospitals were made

with Salmonella typhimurium TA 98 and TA 100. Wastewater samples were evaporated. 27 different test materials

were prepared using DMSO, ethanol and acetone solvents, two different MGA plaques were used for each test material.

Each experiment was made for 3 times with known results of mutagens and we made it ready for “Ames” test method.

We had genotoxicity 50% in Istanbul University Medical Faculty Hospital, 56% in Haseki Hospital and 61% in Vakıf

Gureba Hospital. Accord ing to three hospitals result there are 9 positives, 9 negatives in DMSO; 9 positives, 9 negatives

in ethanol; 12 positives, 6 negatives in acetone. These values are totally 56%. Our results give important information

about mutagenic effect of total pollution in hospital wastewater. It is first time researched in Turkey that effect on DNA

of pollution is from hospital wastewaters. In prospective studies, it is necessary to use this system as a method to moni-

tor mutagenic genotoxic pollution in hospital wastewaters. These kinds of studies present applicability and importance

of our method because of placing in the literature. Method constitutes a new approach to check mutagenite of pollution

in hospital wastewater.

Keywords: Hospital Wastewater; Ames Test; TA 98; TA 100; Genotoxicity

1. Introduction

Medical waste is a byproduct of healthcare that includes

blood, chemicals, body parts, pharmaceuticals, medical

devices, and radioactive materials. Poor management of

health care waste may expose healthcare personal, waste

handlers, and the community to infectious agents, to toxic

materials. Medical waste material causes a large portion

of the diseases that develop due to poor waste man-

agement. Waste containing chemical substances e.g.,

laboratory chemicals, empty bottles of lab or pharmacy

chemicals, disinfectants that have expired or are no

longer needed; solvents, diagnostic kits, poisonous and

corrosive materials, and cleaning agents and others.

Unused liquids from radiotherapy or laboratory research;

contaminated glassware, packages, or absorbent paper;

urine and excreta from patients treated or tested with

unsealed radio nucleotides; sealed sources. Hospitals rep-

resent an incontestable release source of many chemicals

compounds in the aquatic environment due to laboratory

activity or medicine excretio n into wastewater [1].

Some of the substances found in wastewaters are

genotoxic and are suspected to be a possible cause of the

cancers observed in the last decades. Water genotoxicity

studies are of interest because epidemiologic investig-

ations have shown a link between genotoxic drinking

water intake and a rise in cancers [2]. The results of these

studies must, however, be interpreted with caution beca-

use the exposure to genotoxic water was only estimated

and not really measured. However, these results empha-

sized the importance of the determinatio n of water geno-

toxicity with an aim at controlling the population expo-

sure. Thus, the monitoring of water contamination for

potentially carcinogenic compounds represents a major

concern for human health. It is extremely difficult to

quantify the risk associated with these chemical pollut-

ants because they usually occur in concentrations too low

*Corresponding author.

A. R. ATASOY ET AL.

860

to allow analytical determin ation, and pu tative mutagens,

with few exceptions, have never even been identified.

These kinds of studies are used in order to confirm

mutagenic effect of total pollution of different sources on

alives or to investigate pollutions that come from only

one source [3-8] or to investigate materials that is poss-

ible to have mutagenic effects ([9,10]).

In this study we aimed to determine genotoxic effects

of total pollution in hospital wastewater on alive by

Salmonella microsome test method. This study is re-

searched first time in Turkey that effect on DNA of

pollution that is got from water samples from hospital

wastewaters.

2. Materials and Methods

2.1. Sample Preparation

Th ere are only few studies dealing with the hospital waste-

water genotoxicity [8,11-14]. Even if no standard fol-

lowed protocols for sample collection, sample proces-

sing, or selection of tests exist, and all the studies show

that the hospital wastewater could have a nontoxic poten-

tial.

The samples collected from Bezm-i Alem Valide

Sultan Vakıf Gureba Training and Research Hospital,

Haseki Training and Research Hospital, Istanbul Uni-

versity Medical Faculty (Capa Hospital). Hospitals were

very close and their sewage water goes to same purifier.

All samples collected three times to detect mutagenic

pollutants on living organisms. The samples were taken

for six 1-week periods between March 2009 and June

2009. Mutagenite studies of samples taken from hospitals

were made 3 times for 2 petri plaques for each sample

with TA 98 and TA 100. 27 different test materials and

DMSO, ethanol and acetone were prepared after waste

water samples that are taken from hospitals were eva-

porated. 2 different MGA plaques were used for each test

material. Each experiment was made for 3 times.

The Salmonella strains used in the test have different

mutations in various genes in the histidine operon; each

of these mutations is designed to be responsive to muta-

gens that act via different mechanisms. Additional muta-

tions were engineered into these strains to make them

more sensitive to a wide variety of substances. S. typhi-

murium TA 98 and TA 100 strains are used on Salmon-

ella/microsome mutagenite test system.

2.2. Control of Genetic Specialities of Strains

It was checked that test strains h ave original mutation s or

not according to safety of Salmonella/micro some muta-

jenite test system. The His character of the tester strains

is confirmed by demonstrating the histidine requirement

for growth on selective agar plates, Biotin is also re-

quired by all of the standard tester strains because of the

uvrB deletion which extend s through the bio gene.

Colonies that sensation characteristic was corrected

before were tested according to resistance for amphisiline.

The R-factor strains (TA 97, TA 98, TA 100 and TA 102)

should be tested routinely for the presence of the am-

picillin resistance factor because the plasmid is some-

what unstable and can be lost from the bacteria [15]. Spe-

cific regions of the pKMlOl DNA that are essential for

enhancement of UV and chemical mutagenesis, replica-

tion, and ampicillin resistance have been identified [16].

0,1 ml Samples that were got from night cultures of test

strains spread to plaqu es with nutrient agar for control of

RFA mutation. Disks that have 10 l crystal violet solu-

tion (1 mg/ml) were placed to the middle of plaques.

uvrB mutation avaibility was ch ecked with sensation test

to ultraviolet beams. Samples that were got with nightie

were planted as an only one colony. Only one colony that

increases after one night was planted to two plaques by

line test method. Test strains revert ant by itself that

causes to increase in situation with out histidine was mea-

sured as routine on mutajenite experiments and it was

mentioned one each plaque as number of bacteria that

revertant. Colonies of these bacteria can be seen easily

over a plant that shows a regular distribution. Each test

strain reverts with a distinctive frequency. In order to

find cytotoxic values for bacteria of samples that were

kept –20˚C in deepfreeze, samples that were prepared as

0.1 acceptable bacteria culture and most 0.1 ml were

added. Colony count was made after plaques were incu-

bated for one ni ght in 37 ˚C [17].

2.3. Expression of the Genotoxicity Results

In Salmonella micro some test system, it is necessary

colony number to be double at least to call a material as

mutagen. In order to simplify the reading of the results

we have classified the intensity of the genotoxic response

in three categories according to the tested concentration

and the significance level of the response. The three cate-

gories are: slightly (G1), moderately (G2) and strongly

(G3) genotoxic. We used SPSS 16.0, Independent- Sam-

ples T test method for control of data.

3. Result

Generally samples that are solved in acetone more

genotoxic than other in TA 98 and samples that are

solved in ethanol more genotoxic than other in TA 100.

Mutagenite results of solvents with DMSO, ethanol and

acetone is given on Table 1. Average value of 6 values

for each sample, standart deviation was found. Revertant

colony numbers results for positive mutagens (Sodium

Azide (1 .5 microgr am/plague) ): TA 98, 178 ± 32 and TA

100, 2348 ± 132 [18].

We examine result of 3 hospitals:

A. R. ATASOY ET AL.

861

Table 1. Genotoxicity range and average revertant colony result of samples and control group.

TA 98 TA 100

DMSO ETHANOL ACETONE DMSO ETHANOL ACETONE

Genotoxicity Genotoxicity Genotoxicity GenotoxicityGenotoxicity Genotoxicity

Hosp-

itals*

range Average range Average Range Average range Average range Average range Average

MARCH

1 47 - 63 54.83 49 - 63 46.33 50 - 70 74.5 262 - 326290.16302 - 374 291 256 - 352310.5

2 47 - 63 66 49 - 63 63.16 50 - 70 54.5 262 - 326318 302 - 374 344.66 256 - 352189.3

3 47 - 63 39.33 49 - 63 59.33 50 - 70 70.8 262 - 326254.33302 - 374 388.66 256 - 352274.1

4 27.5 27.83 30.16 147 168.83 152.1

MAY

1 50 - 74 38.83 46 - 74 40.16 52 - 72 57 255 - 323217 300 - 356 187.5 267 - 327301.6

2 50 - 74 42.83 46 - 74 39.33 52 - 72 49 255 - 323165.66300 - 356 187.5 267 - 327264.1

3 50 - 74 61.4 46 - 74 51.83 52 - 72 61.83255 - 323351 300 - 356 302.16 267 - 327421.5

4 30.83 30.33 30.66 144.5 163.83 148.5

JUNE

1 56 - 77 47.83 53 - 73 49.83 49 - 69 44 241 - 333168.33291 - 329 310.33 272 - 360261.5

2 56 - 77 60.16 53 - 73 28.5 49 - 69 49.16241 - 333289.33291 - 329 281 272 - 360181

3 56 - 77 50.5 53 - 73 46.16 49 - 69 42 241 - 333270.16291 - 329 271.66 272 - 360190

4 33.33 31.5 29 143.5 155.33 157.6

*Hospital: 1) Istanbul University Medical Faculty (Çapa Hospital); 2) Haseki Educational and Research Hospital; 3) Vakıf Gureba Educational and Research

Hospital; 4) Control group.

Çapa Hospital: Genotoxicity is found in each 3 sol-

vents in March in this hospital. Values are more on me-

dium and up levels. It has presented mutagenite in

wastewater of hospital especially when patient numbers

are more and when it is not rainy. In May, it was only

seen that mutagenite is available in samples that are

solved in acetone. But it is conspicuous that there are

many values on limits of mutagen ite. In June, mutagenite

values are seen only in ethanol. Decrease on number of

patients and being close of many values to mutagenite

made out this result. Fifty percent genotoxicity is deter-

minated as totally by Ames test.

Haseki Educational and Research Hospital: In all tests

that are made in this hospital genotoxicity values are

found in all expend one sample. In this sample, down

mutagenite is only 3 numbers less than limit with 264. In

May is samples that are solved in acetone values that are

close to down mutagenite values are found. In June

genotocity values are confirmed in samples that are

solved in DMSO and acetone. Fifty six percent genotox-

icity is determinated as totally b y Ames test.

Vakıf Gureba Educational and Research Hospital: In

this hospital positive results are obtained in samples that

are solved in acetone and ethanol in March. Very close

results to 39 and 254 down mutagenite values was ob-

tained in DMSO. All samples were positive in May.

Negative results have been obtained expect one sample

that is solved in DMSO in June. Sixty one percent

genotoxicity is determinated as totally by Ames test. 94 %

genotoxicity is determinated as to tally by T test. Accord-

ing to Ames test there are 9 positive, 9 negative in

DMSO; 9 positive, 9 negative in ethanol 12 positive, 6

negative in aceto ne. These values are totally 56%. Geno-

toxicity results are given Figures 1-3.

Generally results are 50% in Çapa, 56% in Haseki and

61% in Vakıf Gureba. At the same time it is attractive

that many values are close to mutagenite v alue. Especial-

ly during counting colonies, there may be same colonies

that are escape during counting operation. Results give

important information about mutagenic effect of total

pollution in hospital wastewater. According to three hos-

pitals result there are 9 positives, 9 negatives in DMSO;

9 positives, 9 negatives in ethanol; 12 positives, 6 nega-

tives in acetone. These values are totally 56%. General

A. R. ATASOY ET AL.

862

*TA 98; **TA 100.

Figure 1. Genotoxicity results in March (G0, not genotoxic; G1, slightly genotoxic; G2, moderately genotoxic; G3, strongly

genotoxic).

*TA 98; **TA 100.

Figure 2. Genotoxicity in May (G0, not genotoxic; G1, slightly genotoxic; G2, moderately genotoxic; G3, strongly genotoxic).

*TA 98; **TA 100.

Figure 3. Genotoxicity in June (G0, not genotox ic; G1, slightly genotoxic; G2, moderately genotoxic; G3, strongly genotoxic).

A. R. ATASOY ET AL. 863

genotoxicity results are given following figures. Gener-

ally samples that are solved in acetone more genotoxic

than other solvent in TA 98 and samples that are solved

in ethanol more genotoxic than other in TA 100.

4. Discussion

Wastewater of hospitals contain materials that would be

a threat for alive. These water needs to be checked by a

biological purification before leaving to nature. Hospital

wastewater has differences than domestic waste because

of especially blood, body waste, drugs, chemicals, me-

dical device waste and radioactive materials. Chemical or

biological agents analyses weren’t made cause this study

was planned to confirm total pollution of different

sources in hospital waste water three powerful solvent

materials “DMSO, ethanol and acetone” are used by

being inspired from studies in the literature as it is

explained detailed in method part [3,6,18,19]. It is

possible to pass different materials that these solvent

chemicals solved from bacteria cell structure and to test

by this way.

In present study, that are given Table 2, mutagenic

effects of total pollution in hospital wastewater is

researched by Salmonella micro some mutagenite test

system [6]. Ames method is a method that is used on

measuring mutagenic effect of different pollution sources

in water sources in the literature [20,21]. These kinds of

studies are used in order to confirm mutagenic effect of

total pollution of different sources on alive or to inves-

tigate pollutions th at come from only one sour ce [4-7,22]

or to investigate materials that is possible to have muta-

genic effects [9,10]. According to our result, all of the

hospital release mutagenic wastewater to sewage system.

March samples more genotoxic than other and June

samples generally not toxic because of patient number.

But it is conspicuou s that there are many values on limits

of mutagenite. In Çapa genotoxicity is found in each 3

solvents in March but it was only seen that mutagenite is

available in samples that are solved in acetone in May,

mutagenite values are seen only in ethanol in June. In

Haseki Hospital all tests that are made in this hospital

genotoxicity values are found in all expend one sample in

March. In May is samples that are solved in acetone. In

Vakıf Gureba Hospital positive results are obtained in

samples that are solved in acetone and ethanol in March.

It has presented mutagenite in wastewater of hospital

especially when patient numbers are more and when it is

not rainy.

In studies that are made in some countries, generally

some drugs and some pathogen bacteria are researched.

In some researching, total genotoxin was checked. In this

study, it is first time researched in Turkey that effect on

DNA of pollution that is got from water samples from

hospital wastewaters. Samples that are taken from 3

hospitals were investigated and total mutagenite was

checked in our study.

This study is researched first time in Turkey that effect

on DNA of pollution that is got from water samples from

hospital wastewaters. Mutagenic effect of pollution that

we do not evaluate, cause it is not for our aim, to use

adaptation method to mammal systems by adding of S9

system [18] maybe done as a part of routine scanning by

results of our study and th is will be an important stud y to

see effects and defect of pollution in Istanbul Bosphore.

Method constitutes a new approach to check mutagenite

of pollution in hospital wastewater. In prospective stud-

ies, it is necessary to use this system as a method to

monitor mutagenic genotoxic po llution in hospital waste-

waters. These kinds of studies present applicability and

importance of our method because of placing in the lit-

erature [19,23-25].

Table 2. Comparison of different genotoxicity studies on hospital wastewaters [21].

Atasoy et al.

Genotoxicity Studies Giuliani et al. (1996) Steger-Hartmann et al. (1997)Hartmann et al. (1999) Jolibois et al. (2003) 2009

Country Switzerland Switzerland Germany France Turkey

Number of samples 851 6 25 18 108

Sampling time (h) 1 24 24 10 2

Concentration method No Yes No No No

Genoto xic re sponse (%)13 50 56 55 56

Bioassay* 1 1 1,2 3 2

Studied period 1991 - 1992 1995 1992 - 1994 2001 2009

*: 1) umuC; 2) Salmonella test; 3) Salmonella fluctuation test.

A. R. ATASOY ET AL.

864

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