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
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.
C
opyright © 2012 SciRes. JWARP

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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