Environ Monit Assess (2015) 187:4180 DOI 10.1007/s10661-014-4180-0
Monitoring hospital wastewaters for their probable genotoxicity and mutagenicity Pratibha Sharma & N. Mathur & A. Singh & M. Sogani & P. Bhatnagar & R. Atri & S. Pareek
Received: 17 April 2014 / Accepted: 19 November 2014 # Springer International Publishing Switzerland 2014
Abstract Cancer is a leading cause of death worldwide. Excluding the genetic factors, environmental factors, mainly the pollutants, have been implicated in the causation of the majority of cancers. Wastewater originated from health-care sectors such as hospitals may carry vast amounts of carcinogenic and genotoxic chemicals to surface waters or any other source of drinking water, if discharged untreated. Humans get exposed to such contaminants through a variety of ways including drinking water. The aim of the present study was, thus, to monitor the genotoxic and mutagenic potential of wastewaters from three big hospitals located in Jaipur (Rajasthan), India. One of them was operating an effluent treatment plant (ETP) for treatment of its wastewater and therefore both the untreated and treated effluents from this hospital were studied for their genotoxicity. Two short-term bacterial bioassays namely the Salmonella fluctuation assay and the SOS chromotest were used for the purpose. Results of fluctuation assay revealed the highly genotoxic nature of all untreated effluent samples with P. Sharma (*) : N. Mathur : A. Singh : R. Atri : S. Pareek Environmental Molecular Microbiology Lab, Department of Zoology, University of Rajasthan, Jaipur, Rajasthan 302004, India e-mail: [email protected] M. Sogani Department of Biotechnology, Jaipur Engineering College and Research Centre, Jaipur, Rajasthan 302022, India P. Bhatnagar Department of Life Sciences, IIS University, Jaipur, Rajasthan 302020, India
mutagenicity ratios (MR) up to 23.13±0.18 and 42.25± 0.35 as measured with Salmonella typhimurium strains TA98 and TA100, respectively. As determined with the chromotest, all untreated effluents produced significant induction factors (IF) ranging from 3.29±1.11 to 13.35 ±3.58 at higher concentrations. In contrast, treated effluent samples were found to be slightly genotoxic in fluctuation test only with an MR = 3.75 ± 0.35 for TA100 at 10 % concentration. Overall, the results indicated that proper treatment of hospital wastewaters may render the effluents safe for disposal contrary to the untreated ones, possessing high genotoxic potential. Keywords Water pollution . Hospital wastewaters . Genotoxicity . Mutagenicity . Short-term bacterial assays
Introduction Cancer is a leading cause of death worldwide, accounting for 7.6 million deaths (around 13 % of all deaths) in 2008 and about 70 % of all cancer deaths in 2008 occurred in low- and middle-income countries (WHO 2013). In India, the International Agency for Research on Cancer estimated indirectly that about 635,000 people died from cancer in 2008, representing about 8 % of all estimated global cancer deaths and about 6 % of all deaths in India (Ferlay et al. 2010). The absolute number of cancer deaths in India is projected to increase because of population growth and increasing life expectancy. Many known or suspected carcinogens first identified through studies of industrial, commercial, and
4180, Page 2 of 9
agricultural occupational exposures have since found their way into the soil, air, and water, resulting in the spread of these contaminants through environmental pollution. The line between occupational and environmental contaminants is fine and often difficult to demarcate. People from disadvantaged populations are more likely to be employed in occupations with higher levels of exposure (e.g., mining, manufacturing, agriculture, certain service sector occupations like health-care industries) and to live in more highly contaminated communities (Reuben 2010). Following occupational exposure, large nonoccupational population also gets exposed to such contaminants due to their careless release into the environment. Environmental pollution caused by potential carcinogens has become a great threat to the existence of living organisms including human beings. Wastewater contaminated with potentially carcinogenic and genotoxic substances is one of the main issues in this context of the pollution. The concern becomes more intense if the wastewater comes from health-care industry (comprising mainly hospitals) as hospital effluents are heavily loaded with toxic chemical substances such as antitumor agents, antibiotics, and organohalogen compounds in addition to pathogenic microorganisms, pharmaceuticals partially metabolized, and radioactive elements (Emmanuel et al. 2002). Many of these chemical compounds are found to show resistance towards normal municipal wastewater treatment, and thus wastewater from hospital origin released from municipal wastewater treatment plants (WWTP) can exert detrimental effects on environmental entities. In fact, some of the substances found in wastewaters are genotoxic and are suspected to be a possible cause of cancer observed in the last decades (Jolibois and Guerbet 2006). Concerns regarding the exposure of humans are mainly associated with exposure through drinking water produced from contaminated surface water (Pauwels and Verstraete 2006). In some countries (e.g., Japan, China, Greece, USA) wastewater from big hospitals is pretreated on-site (Kosma et al. 2010; Liu et al. 2010; Pauwels and Verstraete 2006), whereas in most of the low- and middle-income developing countries, including India, it is connected directly to a municipal sewer and treated at the municipal WWTP (Gupta et al. 2009). Thus, the aim of the present study was to evaluate the possible genotoxic effects of final effluents from three major hospitals (one of which is having an on-site effluent
Environ Monit Assess (2015) 187:4180
treatment plant) located in Jaipur, Rajasthan (biggest state of India, in terms of area). Also, the efficacy of the effluent treatment plant was investigated for its role in the reduction of genotoxicity of waste effluents. Two bioassays viz. Salmonella fluctuation assay and SOS chromotest were used to investigate the genotoxicity of these effluents. The genotoxic effects detected by the Salmonella fluctuation test include at least two different molecular mechanisms: base-pair substitution mutation (TA 100 positive) and frameshift mutation caused by nucleotide insertion or deletion (TA 98 positive). This assay is particularly well adapted to detecting mutagenicity in water samples due to its greater sensitivity than the classical Ames test/Salmonella microsome assay (Monarca et al. 1985). The SOS chromotest successfully detects primary DNAdamaging agents on Escherichia coli. These two tests are not equivalent, but may complement each other in order to broaden mutagen detection capacity (Rosenkranz et al. 1999).
Materials and methods Sampling of wastewaters For this work, the samples were collected on July 4 and 5, 2012, and November 10 and 11, 2012, i.e., sampling was repeated at two consecutive days in a month. Samples were collected from main sewers of the following hospital sites situated in different areas of Jaipur (Rajasthan), India: Sawai Man Singh Hospital (SMS) Sawai Man Singh Hospital is the biggest government hospital in Rajasthan. There are 43 wards with a total of 1563 beds. Samples from this hospital were collected from the main sewer as the hospital does not have any wastewater treatment plant to treat its wastewater before releasing it into the municipal sewerage system. S. K. Soni Hospital Soni Hospital is the most frequented private hospital for Critical Care in Rajasthan, with over 300 ICU beds to handle all kinds of trauma and emergency cases. S.K. Soni hospital does not have an effluent treatment plant, but all the liquid waste is disinfected by sodium
Environ Monit Assess (2015) 187:4180
hypochlorite solution before being released into the municipal sewer. Different concentrations of hyposolution are used for disinfection of different wastes. For instance, the microbiology laboratory uses 1 % sodium hypochlorite solution, while for cytotoxic waste a 5 % hyposolution is preferred. For cleaning of floor and wash rooms also, chlorination is done. From this hospital, samples were taken from the main sewer. Santokba Durlabhji Memorial Hospital (SDMH) cum Medical Research Institute SDMH is the most modern hospital of its kind in the state of Rajasthan. It has a bed capacity of 310 including 31 ICC/CCU beds and 161 beds for special care of neonatals. This hospital has a functional effluent treatment plant encompassing provision for filtration and thereafter proper chlorination of effluent. The samples were collected both before and after treatment. Untreated sample was taken from the sewer of the hospital, where the entire water from the hospital is collected before being treated. Treated wastewater was collected from the outlet from where it comes out of the treatment plant and is then used for irrigating the gardens of the hospital. Brief plan of effluent treatment plant (ETP) at SDMH The ETP operation in SDMH is based on the conventional process to treat the hospital wastewater that is activated sludge process. Wastewater from the whole hospital premise is collected into a ‘collection tank’ or ‘sewage tank.’ The wastewater from the collection tank is then propelled into a ‘reactor tank’ by submersible pumps. In the reactor tank, air is supplied through an air blower to provide oxygen for the aerobic microbes. After a specific retention time, the sewage is carried into a ‘settler tank’ where the solids settle and are separated from treated wastewater. The process of sludge settling is enhanced by the addition of ‘alum’ [hydrated potassium aluminum sulfate {KAl(SO4)2·12H2O}]. It works as a sludge dewatering agent and helps in the flocculation of solids. The settled sludge is pumped back into the reactor tank to maintain specific microbes’ density in incoming wastewater, and after two to three cycles, the sludge is removed from the system and waste sludge is then deposited into the soil (landfills) after ozonization. The clear overflow is then pumped into a ‘balance tank’ or ‘level tank’ where chlorination of treated wastewater is done following filtration. Different doses of
Page 3 of 9, 4180
sodium hypochlorite are provided in the balance tank to ensure proper disinfection of treated effluent. The volume of wastewater being treated in the ETP of SDMH is expected to be 50,000 L per day.
Sample collection Wastewater samples from hospitals were collected in precleaned, sterilized glass bottles. Samples were taken during maximal activity periods, usually 8:00 am– 6:00 pm, in hospitals. All samples were stored at 4 °C until testing. These samples were then tested for their mutagenic and genotoxic potential in their crude state without being concentrated.
Bioassays Salmonella fluctuation assay The fluctuation test on S. typhimurium is a version in liquid medium of the widely used Ames test. The tester strains of S. typhimurium viz. TA98 and TA100 were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTEC), Chandigarh, India. The fluctuation test was performed as described by Legault et al. (1994). The samples were analyzed with and without hepatic S9 fraction. Introduction of mammalian liver enzymes into a prokaryotic system incorporates the aspect of mammalian metabolism into the in vitro test. Uninduced Swiss albino mice were used to prepare the standard S9 mixture. It was prepared according to the protocol described by Maron and Ames (1983). To a volume of 2.5 mL medium consisting of Davis-Mingioli salts (5.5×), D-glucose (400 mg/mL), D-biotin (0.1 mg/ mL), L-histidine (1 mg/mL), and bromocresol purple (2 mg/mL), 20 μL cultures grown overnight in Oxoid broth and 17.5 mL sterile ultrapure water containing 0.2 mL sample (1 % sample concentration) or 2 mL sample (10 % sample concentration) were added. For assays with metabolic activation, 2 mL sterile ultrapure water was replaced by 2 mL S9 mix. A 200-μL volume of the mixture was dispensed into 96-well microtiter plates. The plates were sealed in plastic bags and incubated at 37 °C for 3 to 5 days. Sodium azide (5 ng/mL in fluctuation medium) was used as positive control and sterile distilled water was used as negative control.
4180, Page 4 of 9
All reagents used in the assay were of analytical grade, supplied by Himedia Laboratories Limited and Sigma-Aldrich (Mumbai, India). SOS chromotest This test was performed using SOS Chromotest basic kit ver. 6.4 supplied by Environmental Bio-detection Products Inc. (EBPI), Canada. The SOS Chromotest kit is based on a novel genetically engineered E. coli, which measures the primary response of a cell to genetic damage. This kit utilizes the cell’s own mechanisms for the detection of genotoxicity. All living cells have developed a sensitive system for the detection of lesions in their genetic material so that a complex enzymatic system—the SOS repair system—can be activated to repair the damage. Once a lesion has been detected, an SOS promoter is induced to start the transcription of the SOS genes. In chromotest, the SOS repair system of the bacterium is attached to a β-galactosidase gene (βgal) (lac Z), and the synthesis of the enzyme, upon activation of SOS promoter, was observed visually by the addition of a chromogen and/or analytically using a microtiter plate reader (spectrophotometric measurement) at 620 nm. Cell viability or cytotoxicity was monitored through the activity of phosphatase alkaline (PAL) at 405 nm. The sample concentrations were expressed as the percentage of sample volume contained in a particular well of 96-well microtitre plate. A range of sample concentrations (100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 %; by making serial dilution in a particular column of the plate) was tested using dimethyl sulfoxide (DMSO) as a diluent. DMSO was taken as negative control, and 4nitroquinoline-1-oxide (4-/NQOO) was used as positive control (10 μg/mL).
Environ Monit Assess (2015) 187:4180
plates. The results were expressed as a mutagenicity ratio (MR; the number of positive wells in treated plates/number of positive wells in the negative control plates) and were an average of two experiments (±SD). Three categories slightly, moderately, and strongly genotoxic could be classified according to the tested concentration and significance level of exposure. A sample was considered strongly genotoxic if it was mutagenic at a significance level P
Monitoring hospital wastewaters for their probable genotoxicity and mutagenicity Pratibha Sharma & N. Mathur & A. Singh & M. Sogani & P. Bhatnagar & R. Atri & S. Pareek
Received: 17 April 2014 / Accepted: 19 November 2014 # Springer International Publishing Switzerland 2014
Abstract Cancer is a leading cause of death worldwide. Excluding the genetic factors, environmental factors, mainly the pollutants, have been implicated in the causation of the majority of cancers. Wastewater originated from health-care sectors such as hospitals may carry vast amounts of carcinogenic and genotoxic chemicals to surface waters or any other source of drinking water, if discharged untreated. Humans get exposed to such contaminants through a variety of ways including drinking water. The aim of the present study was, thus, to monitor the genotoxic and mutagenic potential of wastewaters from three big hospitals located in Jaipur (Rajasthan), India. One of them was operating an effluent treatment plant (ETP) for treatment of its wastewater and therefore both the untreated and treated effluents from this hospital were studied for their genotoxicity. Two short-term bacterial bioassays namely the Salmonella fluctuation assay and the SOS chromotest were used for the purpose. Results of fluctuation assay revealed the highly genotoxic nature of all untreated effluent samples with P. Sharma (*) : N. Mathur : A. Singh : R. Atri : S. Pareek Environmental Molecular Microbiology Lab, Department of Zoology, University of Rajasthan, Jaipur, Rajasthan 302004, India e-mail: [email protected] M. Sogani Department of Biotechnology, Jaipur Engineering College and Research Centre, Jaipur, Rajasthan 302022, India P. Bhatnagar Department of Life Sciences, IIS University, Jaipur, Rajasthan 302020, India
mutagenicity ratios (MR) up to 23.13±0.18 and 42.25± 0.35 as measured with Salmonella typhimurium strains TA98 and TA100, respectively. As determined with the chromotest, all untreated effluents produced significant induction factors (IF) ranging from 3.29±1.11 to 13.35 ±3.58 at higher concentrations. In contrast, treated effluent samples were found to be slightly genotoxic in fluctuation test only with an MR = 3.75 ± 0.35 for TA100 at 10 % concentration. Overall, the results indicated that proper treatment of hospital wastewaters may render the effluents safe for disposal contrary to the untreated ones, possessing high genotoxic potential. Keywords Water pollution . Hospital wastewaters . Genotoxicity . Mutagenicity . Short-term bacterial assays
Introduction Cancer is a leading cause of death worldwide, accounting for 7.6 million deaths (around 13 % of all deaths) in 2008 and about 70 % of all cancer deaths in 2008 occurred in low- and middle-income countries (WHO 2013). In India, the International Agency for Research on Cancer estimated indirectly that about 635,000 people died from cancer in 2008, representing about 8 % of all estimated global cancer deaths and about 6 % of all deaths in India (Ferlay et al. 2010). The absolute number of cancer deaths in India is projected to increase because of population growth and increasing life expectancy. Many known or suspected carcinogens first identified through studies of industrial, commercial, and
4180, Page 2 of 9
agricultural occupational exposures have since found their way into the soil, air, and water, resulting in the spread of these contaminants through environmental pollution. The line between occupational and environmental contaminants is fine and often difficult to demarcate. People from disadvantaged populations are more likely to be employed in occupations with higher levels of exposure (e.g., mining, manufacturing, agriculture, certain service sector occupations like health-care industries) and to live in more highly contaminated communities (Reuben 2010). Following occupational exposure, large nonoccupational population also gets exposed to such contaminants due to their careless release into the environment. Environmental pollution caused by potential carcinogens has become a great threat to the existence of living organisms including human beings. Wastewater contaminated with potentially carcinogenic and genotoxic substances is one of the main issues in this context of the pollution. The concern becomes more intense if the wastewater comes from health-care industry (comprising mainly hospitals) as hospital effluents are heavily loaded with toxic chemical substances such as antitumor agents, antibiotics, and organohalogen compounds in addition to pathogenic microorganisms, pharmaceuticals partially metabolized, and radioactive elements (Emmanuel et al. 2002). Many of these chemical compounds are found to show resistance towards normal municipal wastewater treatment, and thus wastewater from hospital origin released from municipal wastewater treatment plants (WWTP) can exert detrimental effects on environmental entities. In fact, some of the substances found in wastewaters are genotoxic and are suspected to be a possible cause of cancer observed in the last decades (Jolibois and Guerbet 2006). Concerns regarding the exposure of humans are mainly associated with exposure through drinking water produced from contaminated surface water (Pauwels and Verstraete 2006). In some countries (e.g., Japan, China, Greece, USA) wastewater from big hospitals is pretreated on-site (Kosma et al. 2010; Liu et al. 2010; Pauwels and Verstraete 2006), whereas in most of the low- and middle-income developing countries, including India, it is connected directly to a municipal sewer and treated at the municipal WWTP (Gupta et al. 2009). Thus, the aim of the present study was to evaluate the possible genotoxic effects of final effluents from three major hospitals (one of which is having an on-site effluent
Environ Monit Assess (2015) 187:4180
treatment plant) located in Jaipur, Rajasthan (biggest state of India, in terms of area). Also, the efficacy of the effluent treatment plant was investigated for its role in the reduction of genotoxicity of waste effluents. Two bioassays viz. Salmonella fluctuation assay and SOS chromotest were used to investigate the genotoxicity of these effluents. The genotoxic effects detected by the Salmonella fluctuation test include at least two different molecular mechanisms: base-pair substitution mutation (TA 100 positive) and frameshift mutation caused by nucleotide insertion or deletion (TA 98 positive). This assay is particularly well adapted to detecting mutagenicity in water samples due to its greater sensitivity than the classical Ames test/Salmonella microsome assay (Monarca et al. 1985). The SOS chromotest successfully detects primary DNAdamaging agents on Escherichia coli. These two tests are not equivalent, but may complement each other in order to broaden mutagen detection capacity (Rosenkranz et al. 1999).
Materials and methods Sampling of wastewaters For this work, the samples were collected on July 4 and 5, 2012, and November 10 and 11, 2012, i.e., sampling was repeated at two consecutive days in a month. Samples were collected from main sewers of the following hospital sites situated in different areas of Jaipur (Rajasthan), India: Sawai Man Singh Hospital (SMS) Sawai Man Singh Hospital is the biggest government hospital in Rajasthan. There are 43 wards with a total of 1563 beds. Samples from this hospital were collected from the main sewer as the hospital does not have any wastewater treatment plant to treat its wastewater before releasing it into the municipal sewerage system. S. K. Soni Hospital Soni Hospital is the most frequented private hospital for Critical Care in Rajasthan, with over 300 ICU beds to handle all kinds of trauma and emergency cases. S.K. Soni hospital does not have an effluent treatment plant, but all the liquid waste is disinfected by sodium
Environ Monit Assess (2015) 187:4180
hypochlorite solution before being released into the municipal sewer. Different concentrations of hyposolution are used for disinfection of different wastes. For instance, the microbiology laboratory uses 1 % sodium hypochlorite solution, while for cytotoxic waste a 5 % hyposolution is preferred. For cleaning of floor and wash rooms also, chlorination is done. From this hospital, samples were taken from the main sewer. Santokba Durlabhji Memorial Hospital (SDMH) cum Medical Research Institute SDMH is the most modern hospital of its kind in the state of Rajasthan. It has a bed capacity of 310 including 31 ICC/CCU beds and 161 beds for special care of neonatals. This hospital has a functional effluent treatment plant encompassing provision for filtration and thereafter proper chlorination of effluent. The samples were collected both before and after treatment. Untreated sample was taken from the sewer of the hospital, where the entire water from the hospital is collected before being treated. Treated wastewater was collected from the outlet from where it comes out of the treatment plant and is then used for irrigating the gardens of the hospital. Brief plan of effluent treatment plant (ETP) at SDMH The ETP operation in SDMH is based on the conventional process to treat the hospital wastewater that is activated sludge process. Wastewater from the whole hospital premise is collected into a ‘collection tank’ or ‘sewage tank.’ The wastewater from the collection tank is then propelled into a ‘reactor tank’ by submersible pumps. In the reactor tank, air is supplied through an air blower to provide oxygen for the aerobic microbes. After a specific retention time, the sewage is carried into a ‘settler tank’ where the solids settle and are separated from treated wastewater. The process of sludge settling is enhanced by the addition of ‘alum’ [hydrated potassium aluminum sulfate {KAl(SO4)2·12H2O}]. It works as a sludge dewatering agent and helps in the flocculation of solids. The settled sludge is pumped back into the reactor tank to maintain specific microbes’ density in incoming wastewater, and after two to three cycles, the sludge is removed from the system and waste sludge is then deposited into the soil (landfills) after ozonization. The clear overflow is then pumped into a ‘balance tank’ or ‘level tank’ where chlorination of treated wastewater is done following filtration. Different doses of
Page 3 of 9, 4180
sodium hypochlorite are provided in the balance tank to ensure proper disinfection of treated effluent. The volume of wastewater being treated in the ETP of SDMH is expected to be 50,000 L per day.
Sample collection Wastewater samples from hospitals were collected in precleaned, sterilized glass bottles. Samples were taken during maximal activity periods, usually 8:00 am– 6:00 pm, in hospitals. All samples were stored at 4 °C until testing. These samples were then tested for their mutagenic and genotoxic potential in their crude state without being concentrated.
Bioassays Salmonella fluctuation assay The fluctuation test on S. typhimurium is a version in liquid medium of the widely used Ames test. The tester strains of S. typhimurium viz. TA98 and TA100 were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTEC), Chandigarh, India. The fluctuation test was performed as described by Legault et al. (1994). The samples were analyzed with and without hepatic S9 fraction. Introduction of mammalian liver enzymes into a prokaryotic system incorporates the aspect of mammalian metabolism into the in vitro test. Uninduced Swiss albino mice were used to prepare the standard S9 mixture. It was prepared according to the protocol described by Maron and Ames (1983). To a volume of 2.5 mL medium consisting of Davis-Mingioli salts (5.5×), D-glucose (400 mg/mL), D-biotin (0.1 mg/ mL), L-histidine (1 mg/mL), and bromocresol purple (2 mg/mL), 20 μL cultures grown overnight in Oxoid broth and 17.5 mL sterile ultrapure water containing 0.2 mL sample (1 % sample concentration) or 2 mL sample (10 % sample concentration) were added. For assays with metabolic activation, 2 mL sterile ultrapure water was replaced by 2 mL S9 mix. A 200-μL volume of the mixture was dispensed into 96-well microtiter plates. The plates were sealed in plastic bags and incubated at 37 °C for 3 to 5 days. Sodium azide (5 ng/mL in fluctuation medium) was used as positive control and sterile distilled water was used as negative control.
4180, Page 4 of 9
All reagents used in the assay were of analytical grade, supplied by Himedia Laboratories Limited and Sigma-Aldrich (Mumbai, India). SOS chromotest This test was performed using SOS Chromotest basic kit ver. 6.4 supplied by Environmental Bio-detection Products Inc. (EBPI), Canada. The SOS Chromotest kit is based on a novel genetically engineered E. coli, which measures the primary response of a cell to genetic damage. This kit utilizes the cell’s own mechanisms for the detection of genotoxicity. All living cells have developed a sensitive system for the detection of lesions in their genetic material so that a complex enzymatic system—the SOS repair system—can be activated to repair the damage. Once a lesion has been detected, an SOS promoter is induced to start the transcription of the SOS genes. In chromotest, the SOS repair system of the bacterium is attached to a β-galactosidase gene (βgal) (lac Z), and the synthesis of the enzyme, upon activation of SOS promoter, was observed visually by the addition of a chromogen and/or analytically using a microtiter plate reader (spectrophotometric measurement) at 620 nm. Cell viability or cytotoxicity was monitored through the activity of phosphatase alkaline (PAL) at 405 nm. The sample concentrations were expressed as the percentage of sample volume contained in a particular well of 96-well microtitre plate. A range of sample concentrations (100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 %; by making serial dilution in a particular column of the plate) was tested using dimethyl sulfoxide (DMSO) as a diluent. DMSO was taken as negative control, and 4nitroquinoline-1-oxide (4-/NQOO) was used as positive control (10 μg/mL).
Environ Monit Assess (2015) 187:4180
plates. The results were expressed as a mutagenicity ratio (MR; the number of positive wells in treated plates/number of positive wells in the negative control plates) and were an average of two experiments (±SD). Three categories slightly, moderately, and strongly genotoxic could be classified according to the tested concentration and significance level of exposure. A sample was considered strongly genotoxic if it was mutagenic at a significance level P
