Evaluation of industrial, hospital and domestic wastewater genotoxicity with the Salmonella fluctuation test and the SOS chromotest

Mutation Research 565 (2005) 151–162

Evaluation of industrial, hospital and domestic wastewater genotoxicity with the Salmonella fluctuation test and the SOS chromotest B. Joliboisa,b,∗ , M. Guerbeta b

a Laboratoire de Toxicologie, Facult´ e de M´edecine et de Pharmacie, 22 Bd. Gambetta, F-76183 Rouen Cedex, France Service Hygi`ene Hospitali`ere, Centre Hospitalier de Compi`egne, 8 Avenue Henri Adnot, F-60321 Compi`egne Cedex, France

Received 14 June 2004; received in revised form 22 September 2004; accepted 19 October 2004

Abstract An evaluation of the genotoxic potential of different wastewaters collected in the Rouen area was performed with the SOS chromotest (on Escherichia coli PQ37) and the Salmonella fluctuation test on Salmonella typhimurium strains TA98, TA100 and TA102 with or without metabolic activation. The samples were taken during two 1-week periods, one in January and one in April 2003. Six sites were selected for wastewater sampling in order to allow a comparative study between an area of mixed discharge (industrial, hospital and domestic) and an area of primarily domestic discharge. Out of a total of 71 daytime samples tested, 46 (65%) were positive in at least one assay: 22 samples out of 33 in January (67%), and 24 samples out of 38 in April (63%). The two genotoxicity tests have different sensitivities. Indeed, the Salmonella fluctuation test allowed the detection of 56% of the samples as genotoxic in January (18 out of 33), and 63% in April (24 out of 38) while the SOS chromotest allowed the detection of 18% of the samples as genotoxic, whatever the sampling period. The samples collected in domestic wastewater are at least as genotoxic as the samples collected in mixed wastewater. The possible source of the detected genotoxicity (industrial, hospital or domestic) is discussed. The results of this study show that the different types of wastewaters present a genotoxic risk. Additional studies should be undertaken in the analytical field in order to try to identify and quantify the compounds responsible for the genotoxicity. This difficult task will be necessary in order to identify the sources of toxicants and thus to take preventive and/or curative measures to limit the toxicity of the wastewater. © 2004 Elsevier B.V. All rights reserved. Keywords: Genotoxicity; Salmonella fluctuation test; SOS chromotest; Wastewater

1. Introduction ∗

Corresponding author. Tel.: +33 3 44 23 66 28. E-mail address: [email protected] (B. Jolibois). 1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.10.006

Human and industrial activities are at the origin of the discharge of multiple chemical substances in the

152

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

environment and are the main causes of environmental pollution [1]. For example, reviews of the literature have reported the fate of some pharmaceutical compounds as well as their occurrence and effects in the aquatic environment [2,3]. Some of these substances 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 epidemiological investigations have shown a link between genotoxic drinking water intake and a rise in cancer cases [4–6]. The results of these studies must, however, be interpreted with caution because the exposure to genotoxic water was only estimated and not really measured. However, these results emphasized the importance of the determination of water genotoxicity with an aim at controlling exposure of the population. Thus, the monitoring of water contamination for potentially carcinogenic compounds represents a major concern for human health. It is a particularly difficult task because the genotoxic compounds likely to be found in the aquatic environment have a large chemical diversity and can come from various sources, like hospital, industrial or domestic discharges. In a previous study [7] based on wastewater samples collected at the Rouen University Hospital (Rouen, France), we detected a large number of genotoxic samples by using the SOS chromotest and the Salmonella fluctuation test. In order to have an overall view of the genotoxic pollution in the Rouen wastewater network, we chose to broaden our investigation to sampling sites located in industrial and domestic areas. In the present study, in addition to the Rouen University Hospital, five other sampling sites were selected in order to allow a comparative study between an area of mixed discharge (industrial, hospital and domestic) and an area of primarily domestic discharge. Genotoxicity was studied by a combination of two tests: the SOS chromotest and the Salmonella fluctuation test on strains TA98, TA100 and TA102 with or without metabolic activation (S9). Ten liter wastewater samples were taken proportionally over time by an autosampler during a 24 h period for two 1-week periods in January and April 2003. The samples were used un-concentrated in the Salmonella test or 10-fold concentrated in the SOS chromotest. The Salmonella test has been extensively used for the evaluation of genotoxicity in numerous types of waters, e.g. tap water [8], sewage treatmentplant effluent [9], urban or hospital wastewater [10,11],

surface water [12], groundwater [13] or river water [14,15]. In this study, we used the Salmonella fluctuation test with Salmonella typhimurium, which is a liquid version of the widely used Salmonella test. The genotoxic effects detected by the Salmonella fluctuation test include at least two different molecular mechanisms: base-pair substitution mutation (TA100- and TA102-positive) and frame-shift mutation caused by nucleotide insertion or deletion (TA98-positive). This assay is particularly well adapted for detecting mutagenicity in water samples due to its greater sensitivity than the classical Salmonella test [16]. Moreover, this test allows the incorporation of larger sample volumes in the assay and thus the detection of genotoxic compounds at lower concentrations, without requiring a concentrating method. As no extraction method exists that recovers all relevant substances from the sample in equal proportion [17], the Salmonella fluctuation test has the advantages of an extraction method without its drawbacks. The second genotoxicity test used was the SOS chromotest, which allows the detection of primary DNA-damaging agents in Escherichia coli. This test is used less often than the Salmonella test for the evaluation of aquatic genotoxicity, but it allowed others to study the genotoxicity of river water [18,19], or hospital wastewater [7]. These two tests are not equal but complement each other [20] and have been jointly selected in order to broaden the detection capacity and to evaluate the overall genotoxic risk present in the wastewaters. The aim of this work was to confirm the previous results obtained in the Rouen University Hospital wastewater study [7], and to detect possible genotoxic activity in the Rouen wastewater network. The possible source of the detected genotoxicity (industrial, hospital or domestic) will be discussed.

2. Materials and methods 2.1. Description of the sampling sites The sampling sites are described below and presented in Fig. 1. Site 1: samples are collected from the main sewer of the Rouen University Hospital (2600 beds, 1500–2000 m3 wastewater daily).

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

153

Fig. 1. Sampling sites located in the Rouen area: (1) Rouen University Hospital wastewaters; (2) Rouen wastewater network; (3) influent of the “Petit Quevilly” wastewater treatment plant; (4) effluent of the “Petit Quevilly” wastewater treatment plant; (5) influent of the “Grand Couronne” wastewater treatment plant; (6) effluent of the “Grand Couronne” wastewater treatment plant.

Site 2: samples are collected in a point of the Rouen wastewater network where the Rouen University Hospital effluents are mixed with urban or industrial effluents before going to the wastewater treatment plant of the city of Petit Quevilly located on the other bank of the Seine river. Sites 3 and 4: the wastewater treatment plant (WTP) located in the city of Petit Quevilly is the end point of the course of Rouen mixed wastewater (hospital, industrial, and domestic). Among the hundred industries connected to this treatment plant there are 2 laundries, 2 dyeing factories, and 13 medical centers (among which Rouen University Hospital is the largest). This wastewater treatment plant serves the wastewater needs of approximately 550,000 people and processes 85,000 m3 wastewater daily. Samples are collected both on the influent (site 3) and on the effluent (site 4) sides of this treatment plant in order to check the efficiency of the treatment in removing the genotoxicity potentially present in the influent. Sites 5 and 6: the Grand Couronne wastewater treatment plant is located in the Rouen area. This treatment plant was selected because it processes only domestic wastewater. Indeed, in this area, no industries and no hospitals are present. This wastewater treatment plant serves the wastewater needs of approximately

20,000 people and processes 4800 m3 wastewater daily. For technical reasons only five samples were collected weekly both in WTP influents (site 5) and effluents (site 6). The fifth sample corresponds to a Friday–Saturday–Sunday mixed sample. 2.2. Sample collection Ten-liter wastewater samples were collected proportionally over time by an autosampler during a 24 h period (Isco 3700 for hospital wastewater samples, and Endress-Hauserau for the other sites). The samples were taken continuously for 1 week in January (from 13th to 19th), and for another week in April (from March 31st to April 6th 2003). The samples were partitioned into aliquots in polyethylene bottles and stored for up to 7 days at −25 ◦ C until tested. Before the genotoxicity assays the samples were filtered through cellulose acetate filters (0.45 ␮m pore size, Sartorius Minisart, Germany). 2.3. Salmonella fluctuation test The tester strain TA98, TA100 and TA102 were a gift from Dr. V. Andr´e (Laboratoire de Canc´erologie Exp´erimentale, Centre Franc¸ois Baclesse, Caen,

154

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

France). The Salmonella fluctuation test is a liquid version of the Salmonella mutagenicity test usually performed in agar plates [21]. The January and April samples were tested without metabolic activation using strains TA98, TA100 and TA102. Indeed, White and Rasmussen [1] indicated that the putative genotoxins present in surface water or municipal wastewater are primarily direct-acting (with no need of metabolic activation to be genotoxic). However, many researchers have also noted substantial increases in the genotoxic activity of surface water [22,23] and municipal wastewater [24] upon addition of a metabolic activation mixture. Thus, the April samples were also tested with metabolic activation on strains TA98 and TA100. The fluctuation test was developed by Green et al. [25] and modified by Gatehouse [26] in a microtiter version. We performed this test as described in Legault et al. [27]. To a volume of 2.5 mL of 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 ␮L of an overnight culture grown in Oxoid broth and 17.5 mL of sterile ultra-pure water containing 0.2 mL of sample (1% sample concentration), 2 mL of sample (10% sample concentration) or 4 mL of sample (20% sample concentration) were added. For the assays with metabolic activation, 2 mL of sterile ultra-pure water were replaced by 2 mL of S9 mix. A 200 ␮L volume of the mixture was dispensed into 96-well microtitre plates. The plates were sealed in plastic bags and incubated at 37 ◦ C for 3–5 days. Mitomycin C (1 ng/mL in fluctuation medium), sodium azide (5 ng/mL in fluctuation medium) and 2-nitrofluorene (50 ng/mL in fluctuation medium) were used as positive controls without metabolic activation for strains TA102, TA100 and TA98, respectively. 2-Aminofluorene (20 ng/mL in fluctuation medium) was used as a positive control with metabolic activation for strains TA100 and TA98, and sterile ultra-pure water as a negative control. All yellow, partially yellow or turbid wells were considered positive, and all purple wells were scored as negative. Chi-square analysis [28] was used for statistical evaluation of the treated plates versus the control plates. A sample is considered mutagenic when there is a significant increase of the number of positive wells in treated plates over the negative control plates. The results are expressed as mutagenicity ratio (MR = number of positive wells in treated plates/number of positive

wells in the negative control plates) and are an average of two experiments (±S.D.). 2.4. SOS chromotest The tester strain E. coli PQ37 was kindly provided by M. Hofnung (Institut Pasteur, Paris, France). The SOS chromotest was performed without metabolic activation as described by Quillardet and Hofnung [29], with modifications provided by Mersch-Sundermann et al. [30] and Kevekordes et al. [31]. A 600 ␮L volume of an appropriate overnight culture dilution was added to a tube containing 20 ␮L sample, and incubated with agitation for 2.5 h at 37 ◦ C. Beta-galactosidase (␤gal) and alkaline phosphatase activity (PAL) were then determined using O-nitrophenyl-␤-d-galactopyranoside and p-nitrophenyl phosphate disodium as substrates, respectively. Absorption was measured at 405 nm using a reference solution with no bacteria. The samples (20 ␮L) were tested as neat samples and as 10-fold concentrates (concentrated by means of a Speedvac® concentrator). The sample concentrations are expressed as percentage of the sample volume contained in the medium (3.3 and 33%, respectively). The genotoxic activity for a concentration c of the sample is expressed in the ratio Rc = ␤gal/PAL, where ␤gal represents the ␤-galactosidase activity and PAL the alkaline phosphatase activity. The induction factor (IF) for a concentration c of the sample is defined as Rc /R0 , where R0 is the ratio measured in the solvent control (sterile ultra-pure water). The criterion to consider a sample positive in the SOS chromotest differs between authors [32–34]. We chose to consider a sample as an SOS repair-system inducer when the IF is higher than 1.5, the ␤-galactosidase activity is significantly increased compared with the solvent control, and the result is reproducible. All results are expressed as mean of three experiments (±S.D.). Sterile ultra-pure water is used as a negative control and 4-nitroquinoline1-oxide as a positive control (2.5 ␮g/mL).

3. Results The results of the genotoxicity tests on the January and April 2003 samples are summarized in Table 1 for the SOS chromotest, in Tables 2 and 3 for the Salmonella fluctuation test without metabolic activa-

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

tion, and in Table 4 for the Salmonella fluctuation test with metabolic activation. Out of a total of 71 daytime samples tested, 46 (65%) were positive in at least one assay: 22 samples out of 33 in January (67%) and 24 samples out of 38 in April (63%). The distribution of the genotoxic response was different between sites, but constant between sampling periods. Thus, the percentage of genotoxic samples in at least one assay was 100% for sites 1 and 5, 71% for site 3, and 0% for site 4, whatever the sampling period. An increase of the number of genotoxic responses between the sampling periods was only observed for site 2 (71–100%). No genotoxic activity was detected in April for site 6. The two genotoxicity tests showed different responses. Indeed, the Salmonella fluctuation test allowed the detection of 56% of the samples as genotoxic in January (18 out of 33), and 63% in April (24 out of 38), while the SOS chromotest allowed the detection of 18% of the samples as genotoxic, whatever the sampling period. It must be emphasized that the SOS chromotest allowed detection of genotoxic samples only for the Rouen University Hospital (site 1), for which 13 samples out of 14 (93%) were genotoxic. For the Salmonella fluctuation test, the order of strain sensitivity based on the number of positive responses was: 51% for TA102 −S9 (36 out of 71), 18% for TA98 +S9 (7 out of 38), 11% for TA100 −S9 (8 out of 71), 4% for TA98 −S9 (3 out of 71) and 0% for TA100 +S9 (0 out of 38). A summary of the results obtained according to sampling-site characteristics is presented in Table 5. Samples collected at sites 1–3 are classified as mixed wastewater (hospital, industrial and domestic), samples collected at site 5 as domestic wastewater. Sites 4 and 6, corresponding to wastewater treatment plant effluent are not included in this comparison. Even if the number of samples tested between these two kinds of site is different, a large number of genotoxic responses are observed at both sites. Domestic wastewaters are at least as genotoxic as mixed wastewaters, because the percentage of genotoxic responses in at least one assay is similar (100 and 88%, respectively). A positive response in the SOS chromotest is only observed for the mixed wastewater. In the Salmonella fluctuation test, whatever the wastewater, the decreasing order of strain sensitivity is: TA102 −S9, TA98 +S9, TA100 −S9 and TA98 −S9 (only for mixed wastewater).

155

4. Discussion An evaluation of the genotoxicity of the different wastewaters collected in the Rouen area was performed with the SOS Chromotest and the Salmonella fluctuation test over two 1-week periods in 2003. These tests showed that the wastewaters present a genotoxic effect. Indeed, out of a total of 71 samples tested, 46 samples (65%) are positive in at least one assay. If we consider first the hospital wastewater (site 1), all of 14 samples tested from this site are positive in at least one assay. It is difficult to compare these results with other studies, because many parameters can influence the genotoxicity test response (composition of the sample, hospital size and its degree of activity, nature of the medicines used in treatments, nature of the genotoxicity tests, etc.) and there are only few hospital wastewater studies in the literature. In our previous study on the Rouen University Hospital wastewater, we observed fewer genotoxic samples (55%) using the same tests [7]. Even if samples are of a different origin, studies of Steger-Hartmann et al. [35] on the umuC test, and Hartmann et al. [11] on the umuC test, the Salmonella test and the V79 chromosomal aberration assay exhibited 50 and 56% genotoxic samples, respectively. In the present study, 10 samples (71%) were positive in the Salmonella fluctuation test (Tables 2–4). In our previous study [7], 50% of the samples (9 out of 18) were genotoxic in strains TA98 and TA100 without metabolic activation, whereas Hartmann et al. [11] found only 8% of their samples positive (2 out of 25) in the same strains. Even if samples are of different origin, the use in our study of greater sample volumes leading to detection of lower concentrations of genotoxic compounds could explain the difference observed with the data of Hartmann et al. [11]. This difference could also be explained by the use of strain TA102. Indeed, this strain was the most sensitive and detected 9 genotoxic samples out of 14 (64%). Strain TA 102 is very sensitive because the histidine mutation has been introduced into a multi-copy plasmid (pAQ1) of which approximately thirty copies are present, and not on the bacterial chromosome like in other strains [36]. Strain TA102 detects a variety of oxidative mutagens (like fluoroquinolone), and many anticancer drugs (mitomycin, adriamycin, bleomycin, daunomycin, etc.), which are not or poorly detected with the other strains.

156

Table 1 Results of the SOS chromotest c (%) January 2003

IF

Site 2 S.D.

IF

Site 3 S.D. IF

Site 4 S.D. IF

Site 5 S.D. IF

Site 1 S.D. IF

Site 2 S.D.

IF

Site 3 S.D. IF

Site 4 S.D. IF

Site 5 S.D. IF

Site 6 S.D. IF

S.D.

Monday

3.3 33

1.12 0.07 1.54 0.08

1.15 0.09 1.33 0.04

0.91 0.02 1.03 0.05

1.02 0.03 1.05 0.20

1.28 0.09 1.01 0.27

1.13 0.04 2.03 0.04

0.93 0.01 1.15 0.01

0.89 0.01 1.15 0.03

0.76 0.08 0.96 0.07

0.64 0.03 0.75 0.05

0.87 0.01 0.96 0.00

Tuesday

3.3 33

1.00 0.09 1.70 0.11

1.06 0.08 1.36 0.07

1.00 0.09 1.09 0.10

1.05 0.07 1.07 0.25

1.38 0.19 0.91 0.18

1.13 0.01 1.79 0.29

0.93 0.02 1.13 0.01

0.80 0.02 1.09 0.09

0.76 0.00 0.99 0.01

0.68 0.05 0.82 0.02

0.81 0.03 1.05 0.05

Wednesday

3.3 33

1.02 0.10 1.42 0.05

1.04 0.06 1.18 0.12

1.00 0.04 1.08 0.07

1.10 0.06 1.12 0.04

1.37 0.16 1.06 0.22

0.92 0.08 1.56 0.12

0.92 0.01 1.18 0.02

0.87 0.18 1.15 0.08

0.73 0.03 1.00 0.11

0.69 0.00 0.87 0.01

0.76 0.02 0.95 0.01

Thursday

3.3 33

1.04 0.03 1.55 0.08

0.89 0.04 1.20 0.07

1.03 0.07 1.11 0.13

0.88 0.26 0.99 0.04

1.44 0.24 1.27 0.23

1.14 0.28 2.03 0.08

0.90 0.04 1.19 0.16

0.82 0.05 1.14 0.04

0.76 0.00 1.00 0.03

0.70 0.02 0.87 0.01

0.77 0.04 0.97 0.01

Fridaya

3.3 33

1.08 0.08 1.78 0.25

0.98 0.10 1.24 0.03

1.03 0.06 1.04 0.12

0.96 0.02 1.10 0.02

1.47 0.14 1.18 0.18

1.11 0.03 2.15 0.07

0.96 0.03 1.27 0.14

0.68 0.04 0.99 0.01

0.85 0.60 1.09 0.09

0.82 0.05 1.03 0.02

0.80 0.05 1.02 0.05

Saturday

3.3 33

1.03 0.09 1.52 0.16

0.98 0.09 1.15 0.01

1.04 0.09 1.06 0.08

0.95 0.08 1.15 0.07

1.13 0.07 1.82 0.05

1.01 0.01 1.26 0.04

0.71 0.03 1.18 0.14

0.52 0.02 0.70 0.03

Sunday

3.3 33

1.07 0.03 1.51 0.03

1.00 0.09 1.27 0.05

0.99 0.08 0.98 0.03

0.94 0.02 1.09 0.05

1.18 0.20 2.02 0.22

0.97 0.01 1.42 0.03

0.79 0.08 1.11 0.01

0.64 0,00 0.71 0.03

6/7 (86%)

0/7 (0%)

0/7 (0%)

0/7 (0%)

0/7 (0%)

0/7 (0%)

0/7 (0%)

0/5 (0%)

0/5 (0%)

Proportion of genotoxic samples

0/5 (0%)

7/7 (100%)

Genotoxic samples are indicated in bold letters. A sample is considered as genotoxic if the IF is higher than 1.5, the ␤-galactosidase activity is significantly increased compared with the solvent control, and the result is reproducible; c: tested concentration; IF: induction factor; S.D.: standard deviation. a For the sites 5 and 6, the Friday sample corresponds to a Friday–Saturday–Sunday mixed sample.

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

Site 1

April 2003

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

157

Table 2 Results of the Salmonella fluctuation test without metabolic activation on January samples c (%)

Monday

1 10 20

Site 1 TA98 MR 0.50 0.50 0.00

Tuesday

1 10 20

0.50 0.00 0.00

0.71 0.00 0.00

1.00 2.25 1.50

1.06 1.06 0.35

1.15 0.71 0.09

0.17 0.17 0.12

1.00 0.00 0.00

0.71 0.00 0.00

1.89 1.33 1.56

0.00* 0.16 0.63

1.19 0.91 0.75

0.11 0.05 0.06

0.50 0.00 0.00

0.71 0.00 0.00

0.88 0.63 1.33

0.06 0.06 0.00

1.10 0.62 0.69

0.03 0.12 0.01

Wednesday

1 10 20

1.50 1.00 0.00

2.12 0.00 0.00

0.88 1.25 1.13

0.53 0.35 0.18

1.32 0.80 0.26

0.07* 0.03 0.36

0.25 0.00 0.00

0.35 0.00 0.00

1.89 1.00 1.61

0.16* 0.16 0.08

1.05 0.82 0.84

0.00 0.15 0.12

0.50 0.00 0.00

0.71 0.00 0.00

0.83 0.58 1.21

0.12 0.12 0.18

1.18 0.69 0.90

0.01** 0.15 0.12

Thursday

1 10 20

0.50 0.00 0.00

0.71 0.00 0.00

0.88 1.00 2.00

0.53 0.00 0.71

1.09 1.05 0.39

0.12 0.14 0.55

0.50 0.00 0.00

0.71 0.00 0.00

1.89 0.94 1.67

0.31* 0.24 0.31

0.91 1.05 1.15

0.17 0.03 0.02

1.00 0.00 0.00

0.00 0.00 0.00

1.00 0.75 1.58

0.12 0.00 0.00

1.10 0.68 0.60

0.09* 0.02 0.04

Fridaya

1 10 20

1.50 0.50 0.00

2.12 0.71 0.00

1.00 1.38 1.63

0.35 0.18 0.53

0.96 0.90 0.33

0.16 0.07 0.47

0.25 0.00 0.25

0.35 0.00 0.35

1.11 1.11 1.22

0.16 0.16 0.31

0.96 0.91 0.94

0.23 0.17 0.05

1.00 0.00 0.00

0.00 0.00 0.00

0.67 0.71 1.25

0.35 0.18 0.59

1.09 0.61 0.67

0.09* 0.00 0.06

Saturday

1 10 20

0.00 0.00 0.00

0.00 0.00 0.00

0.88 1.25 0.75

0.18 0.00 0.00

1.06 0.84 0.27

0.05 0.09 0.38

0.25 0.00 0.00

0.35 0.00 0.00

1.61 1.11 1.22

0.24 0.16 0.16

0.92 0.98 1.24

0.09 0.05 0.23*

1.00 0.00 0.00

0.00 0.00 0.00

0.96 0.79 1.04

0.06 0.06 0.29

1.05 0.70 0.75

0.02 0.15 0.02

Sunday

1 10 20

2.00 0.00 0.50

0.00 0.00 0.71

1.25 0.88 1.13

0.35 0.18 0.88

1.29 0.96 0.15

0.10* 0.02 0.21

0.00 0.00 0.00

0.00 0.00 0.00

1.33 0.72 1.22

0.47 0.24 0.00

1.08 1.13 1.25

0.12 0.17 0.06*

0.50 0.00 0.00

0.71 0.00 0.00

1.00 1.04 1.17

0.12 0.41 0.24

1.16 0.81 0.82

0.01** 0.27 0.06

Proportion of genotoxic samples

S.D. 0.71 0.71 0.00

TA100 MR 1.38 1.75 2.88

0/7 (0%)

S.D. 0.18 0.35 0.08*

TA102 MR 1.00 0.79 0.39

1/7 (14%) 3/7 (43%)

S.D. 0.07 0.16 0.55

Site 2 TA98 MR 0.00 0.25 0.00

2/7 (29%)

S.D. 0.00 0.35 0.00

TA100 MR 1.17 1.44 1.33

0/7 (0%)

S.D. 0.08 0.16 0.00

TA102 MR 1.02 0.91 1.14

3/7 (43%) 5/7 (71%)

S.D. 0.26 0.14 0.15

Site 3 TA98 MR 0.50 0.50 0.00

S.D. 0.71 0.71 0.00

TA100 MR 0.63 1.00 1.71

S.D. 0.29 0.00 0.29*

TA102 MR 1.08 0.64 0.71

S.D. 0.05 0.01 0.02

2/7 (29%)

0/7 (0%)

1/7 (14%) 5/7 (71%)

4/7 (57%)

Monday

1 10 20

Site 4 TA98 MR 0.50 0.00 0.00

Tuesday

1 10 20

1.00 0.00 0.50

0.00 0.00 0.71

0.58 1.25 0.92

0.12 0.82 0.35

1.03 0.31 0.38

0.02 0.09 0.11

0.00 0.00 0.00

0.00 0.00 0.00

1.14 1.86 2.29

0.61 0.20 0.20*

0.98 1.30 1.54

0.08 0.14* 0.11***

Wednesday

1 10 20

0.50 0.00 0.00

0.71 0.00 0.00

1.00 0.83 0.75

0.00 0.71 0.12

0.94 0.37 0.23

0.02 0.09 0.02

0.25 0.00 0.00

0.35 0.00 0.00

1.36 1.71 1.50

0.10 0.61 0.71

0.89 1.39 1.49

0.05 0.02** 0.02***

Thursday

1 10 20

0.50 0.50 0.00

0.71 0.71 0.00

1.67 1.25 1.33

0.47 0.35 0.00

0.83 0.45 0.23

0.26 0.15 0.07

0.25 0.00 0.00

0.35 0.00 0.00

1.79 1.14 1.57

0.71 0.20 0.40

1.04 1.30 1.35

0.08 0.02* 0.03**

Fridaya

1 10 20

0.50 0.00 0.00

0.71 0.00 0.00

0.92 0.92 1.67

0.12 0.35 0.47

0.89 0.46 0.23

0.04 0.13 0.02

0.00 0.00 0.00

0.00 0.00 0.00

1.50 1.21 1.50

0.30 0.51 0.10

1.26 1.34 1.22

0.02* 0.02** 0.02

Saturday

1 10 20

1.50 0.00 0.00

0.71 0.00 0.00

1.67 1.17 0.58

0.47 0.00 0.12

0.83 0.34 0.20

0.09 0.04 0.02

Sunday

1 10 20

0.50 0.00 0.00

0.71 0.00 0.00

0.92 0.58 1.08

0.59 0.12 0.35

0.92 0.58 0.32

0.17 0.13 0.07

Proportion of genotoxic samples

S.D. 0.71 0.00 0.00

TA100 MR 1.25 0.75 1.17

0/7 (0%)

S.D. 0.82 0.12 0.24

TA102 MR 0.97 0.32 0.25

0/7 (0%) 0/7 (0%)

S.D. 0.07 0.02 0.04

Site 5 TA98 MR 0.25 0.00 0.25

S.D. 0.35 0.00 0.35

TA100 MR 1.79 1.86 2.07

S.D. 0.10 0.40 0.30*

TA102 MR 1.14 1.47 1.43

S.D. 0.06 0.08*** 0.02**

0/7 (0%)

0/5 (0%)

2/5 (40%) 5/5 (100%)

Genotoxic samples are indicated in bold letters; c: tested concentration; MR: mutagenicity ratio; S.D.: standard deviation. a For site 5, the Friday sample corresponds to a Friday–Saturday–Sunday mixed sample. ∗ p < 0.05. ∗∗ p < 0.01. ∗∗∗ p < 0.001.

5/5 (100%)

158

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

Table 3 Results of the Salmonella fluctuation test without metabolic activation on April samples c (%)

Site 1

Site 2

TA98

TA100

TA102

Site 3

TA98

TA100

TA102

TA98

TA100

TA102

Monday

1 10 20

MR 0.75 0.25 0.75

Tuesday

1 10 20

0.75 0.00 0.25

0.35 0.00 0.35

0.58 0.67 1.58

0.12 0.71 0.35

1.20 0.56 0.46

0.03** 0.03 0.11

0.75 3.50 9.00

1.06 0.00* 1.41***

0.75 0.58 0.50

0.59 0.82 0.71

1.27 0.89 1.01

0.01*** 0.09 0.13

2.25 0.00 0.25

1.06 0.00 0.35

1.33 0.22 0.22

1.57 0.31 0.00

0.81 0.82 0.85

0.02 0.12 0.08

Wednesday

1 10 20

1.25 0.00 0.00

0.35 0.00 0.00

1.00 0.50 0.83

0.47 0.24 0.47

1.16 0.62 0.50

0.03* 0.02 0.12

0.75 0.75 0.25

1.06 0.35 0.35

0.25 0.42 0.17

0.12 0.12 0.00

1.22 0.85 0.85

0.08*** 0.22 0.02

0.25 0.25 0.50

0.35 0.35 0.71

1.00 0.56 0.78

0.79 0.16 0.16

1.31 0.88 0.92

0.15* 0.06 0.06

Thursday

1 10 20

1.75 0.75 0.25

0.35 0.35 0.35

1.33 0.92 1.58

0.24 0.12 0.12

1.22 0.89 1.03

0.02** 0.08 0.07

2.00 1.25 0.00

0.00 1.06 0.00

0.83 0.50 0.17

0.00 0.24 0.24

1.23 0.79 0.82

0.02*** 0.22 0.12

1.00 1.00 0.25

0.00 0.00 0.35

1.56 0.78 0.89

0.00 0.16 0.31

1.76 0.94 0.97

0.06*** 0.02 0.06

Fridaya

1 10 20

0.75 3.75 2.25

1.06 1.06* 1.06

0.67 0.50 0.17

0.24 0.24 0.00

1.30 0.72 0.52

0.03*** 0.06 0.04

0.75 0.25 0.00

0.35 0.35 0.00

0.42 0.25 0.17

0.35 0.35 0.00

1.25 0.72 0.58

0.06*** 0.05 0.10

1.50 0.50 0.00

0.71 0.71 0.00

1.11 0.44 0.11

0.00 0.00 0.16

1.65 0.78 0.13

0.20*** 0.05 0.06

Saturday

1 10 20

1.00 0.50 0.25

0.71 0.00 0.35

0.58 0.42 0.83

0.35 0.35 0.24

1.22 0.64 0.46

0.02** 0.04 0.07

1.00 0.00 0.25

0.71 0.00 0.35

1.00 0.08 0.00

0.00 0.12 0.00

1.18 0.30 0.13

0.05** 0.01 0.09

0.50 0.25 0.25

0.00 0.35 0.35

1.44 0.11 0.22

0.79 0.16 0.00

1.27 0.73 0.32

0.03* 0.00 0.03

Sunday

1 10 20

0.75 0.25 0.25

0.35 0.35 0.35

0.50 0.33 1.00

0.24 0.24 0.47

1.23 0.55 0.40

0.01** 0.02 0.08

0.00 0.25 0.50

0.00 0.35 0.71

0.58 0.08 0.08

0.35 0.12 0.12

1.18 0.41 0.23

0.00* 0.18 0.03

1.50 2.00 5.25

0.71 0.71 0.35**

1.00 2.44 4.67

0.16 0.00* 0.31***

1.54 1.71 1.96

0.05*** 0.11*** 0.06***

Proportion of genotoxic samples

S.D. 0.35 0.35 0.35

MR 0.83 0.67 0.67

S.D. 0.24 0.24 0.00

MR 1.14 0.60 0.40

S.D. 0.09* 0.13 0.05

MR 0.25 0.75 0.50

S.D. 0.35 0.35 0.71

MR 0.75 0.42 0.25

S.D. 0.12 0.35 0.12

MR 1.23 0.74 0.79

S.D. 0.03*** 0.03 0.00

MR 1.75 1.25 1.00

S.D. 0.35 0.35 1.41

MR 1.00 0.89 0.89

S.D. 0.79 0.00 0.31

MR 0.96 0.80 0.74

S.D. 0.08 0.09 0.02

1/7 (14%)

0/7 (0%) 7/7 (100%)

7/7 (100%)

Site 4

1/7 (14%)

0/7 (0%) 7/7 (100%)

7/7 (100%)

Site 5

TA98

TA100

TA102

1/7 (14%)

1/7 (14%) 5/7 (71%)

5/7 (71%)

Site 6

TA98

TA100

TA102

TA98

TA100

TA102

Monday

1 10 20

MR 0.50 0.75 3.25

Tuesday

1 10 20

1.00 0.75 1.00

0.00 1.06 0.71

1.11 0.11 0.44

0.00 0.16 0.31

1.02 0.75 0.48

0.08 0.09 0.08

0.25 1.00 0.25

0.35 0.71 0.35

2.00 0.40 0.40

1.13 0.00 0.57

1.29 1.39 1.39

0.03 0.03 0.38

0.75 0.50 0.75

0.35 0.71 0.35

1.60 0.40 1.20

0.57 0.57 0.57

1.00 0.78 0.46

0.24 0.28 0.17

Wednesday

1 10 20

0.75 0.50 0.75

0.35 0.71 0.35

1.11 0.00 0.67

0.31 0.00 0.63

0.85 0.60 0.45

0.11 0.06 0.00

0.75 0.00 0.50

1.06 0.00 0.71

2.60 0.00 0.20

0.28 0.00 0.28

0.95 1.59 1.37

0.03 0.17* 0.07

1.25 0.25 0.00

0.35 0.35 0.00

1.80 0.40 0.80

0.28 0.00 1.13

1.22 0.59 0.59

0.07 0.07 0.00

Thursday

1 10 20

0.75 1.25 1.00

0.35 0.35 0.71

0.78 0.11 0.44

0.47 0.16 0.63

0.92 0.75 0.54

0.09 0.03 0.06

1.00 0.50 0.75

0.71 0.00 0.35

0.80 0.20 0.00

0.57 0.28 0.00

1.17 1.41 1.83

0.14 0.07 0.31**

0.50 0.25 0.50

0.00 0.35 0.00

2.20 0.00 0.80

0.28 0.00 0.00

1.29 0.98 0.61

0.03 0.28 0.10

Fridaya

1 10 20

1.00 0.50 0.75

0.71 0.71 1.06

1.00 0.22 0.22

0.16 0.00 0.31

1.14 0.63 0.30

0.00 0.11 0.06

0.25 0.50 1.25

0.35 0.71 1.06

1.80 1.40 1.80

0.28 1.41 0.28

1.54 1.51 1.90

0.10* 0.28* 0.14**

0.75 0.25 0.00

1.06 0.35 0.00

0.60 0.40 1.00

0.85 0.57 0.28

1.12 0.68 0.46

0.14 0.21 0.03

Saturday

1 10 20

0.75 0.75 0.00

1.06 0.35 0.00

0.67 0.00 0.56

0.00 0.00 0.16

1.04 0.53 0.44

0.05 0.08 0.08

Sunday

1 10 20

1.75 1.50 1.25

0.35 0.00 0.35

1.22 0.78 1.00

0.47 0.47 0.79

0.96 0.40 0.26

0.38 0.20 0.06

Proportion of genotoxic samples

S.D. 0.71 0.35 0.35

MR 0.78 0.11 1.00

S.D. 0.47 0.16 0.16

MR 0.97 0.74 0.40

S.D. 0.15 0.11 0.02

MR 1.25 0.25 0.75

S.D. 0.35 0.35 0.35

MR 1.20 0.00 0.00

S.D. 1.13 0.00 0.00

MR 1.51 1.32 0.80

S.D. 0.00* 0.14 0.10

MR 1.50 0.25 1.25

S.D. 0.71 0.35 0.35

MR 1.80 0.20 1.40

S.D. 1.41 0.28 0.28

MR 1.34 0.63 0.34

S.D. 0.03 0.21 0.07

0/7 (0%)

0/7 (0%) 0/7 (0%)

0/7 (0%)

0/5 (0%)

0/5 (0%) 4/5 (80%)

4/5 (80%)

0/5 (0%)

Genotoxic samples are indicated in bold letters; c: tested concentration; MR: mutagenicity ratio; S.D.: standard deviation. a For sites 5 and 6, the Friday sample corresponds to a Friday–Saturday–Sunday mixed sample. ∗ p < 0.05. ∗∗ p < 0.01. ∗∗∗ p < 0.001.

0/5 (0%) 0/5 (0%)

0/5 (0%)

0/5 (0%) 2/5 (40%) 0/7 (0%) 2/7 (29%) 2/7 (29%) 1/7 (14%)

Genotoxic samples are indicated in bold letters (p < 0.05); c: tested concentration; MR: mutagenicity ratio; S.D.: standard deviation. a For sites 5 and 6, the Friday sample corresponds to a Friday–Saturday–Sunday mixed sample.

TA100

0/5 (0%) 0/5 (0%) 0/5 (0%) 2/5 (40%) 0/7 (0%) 0/7 (0%) 0/7 (0%) 2/7 (29%) 0/7 (0%) 2/7 (29%) 0/7 (0%) Proportion of genotoxic samples

Monday Tuesday Wednesday Thursday Fridaya Saturday Sunday

1 1 1 1 1 1 1

1/7 (14%)

MR 1.00 0.97 1.18 1.08 1.05 MR 0.52 0.70 0.87 1.00 0.83 MR 1.18 1.21 1.03 1.13 1.11 MR 1.04 0.96 1.22 0.74 0.52 0.87 0.52 MR 1.11 1.03 1.18 0.97 0.84 0.95 1.08 MR 1.00 0.91 1.00 0.83 1.04 0.83 0.91 MR 0.88 1.13 1.75 1.75 1.50 1.31 0.69 S.D. 0.00 2.12 0.71 0.00 0.71 0.71 1.41

S.D. 0.25 0.18 0.31 0.00 0.12 0.06 0.25

MR 1.21 0.82 0.87 1.03 1.21 1.03 1.13 MR 0.96 0.91 0.91 0.61 1.13 0.83 0.87 MR 3.00 1.50 1.50 5.00 0.50 1.50 2.00

S.D. 0.07 0.04 0.19 0.26 0.07 0.19 0.04

MR 1.04 1.09 0.65 1.00 0.70 1.09 0.96

S.D. 0.37 0.18 0.06 0.18 0.00 0.43 0.12

Site 3

TA98 TA100

Site 2

TA98 TA100

Site 1

TA98

c (%)

Table 4 Results of the Salmonella fluctuation test with metabolic activation on April samples

S.D. 0.18 0.18 0.18 0.00 0.53 0.27 0.09

TA100

S.D. 0.31 0.18 0.06 0.43 0.12 0.18 0.06

Site 4

TA98

S.D. 0.07 0.04 0.04 0.11 0.15 0.15 0.04

TA100

S.D. 0.25 0.00 0.25 0.31 0.12 0.25 0.00

Site 5

TA98

S.D. 0.11 0.07 0.04 0.04 0.07

TA100

S.D. 0.25 0.00 0.12 0.31 0.06

Site 6

TA98

S.D. 0.07 0.04 0.04 0.11 0.00

MR 0.78 0.96 0.70 0.65 0.74

S.D. 0.25 0.37 0.25 0.68 0.31

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

159

The use of metabolic activation (S9) with strains TA98 and TA100 to test the April samples (Table 4) does not enhance the strain sensitivity. Indeed, one positive response was observed both with and without S9 in strain TA98 and no positive response in strain TA100 ±S9. These results are in agreement with those of Hartmann et al. [11], who showed that hospital wastewater genotoxicity in the umuC test was independent of the use of S9 metabolic activation. Similarly, White and Rasmussen [1] indicated that the putative genotoxins in both surface waters and municipal wastewaters are primarily direct-acting, i.e. S9 addition does not enhance the response. The SOS chromotest (Table 1) allows the detection of 13 genotoxic samples out of 14 (93%), whereas Giuliani et al. [37] with the umuC test (similar to the SOS chromotest) found 13% of the samples genotoxic. This difference could be explained by the use of 10-fold concentrated samples in our study or by the different origin of the wastewaters. In addition to hospital wastewater, five other wastewater sampling sites were studied in an area of mixed discharge (hospital, industrial and domestic: sites 1, 2, 3 and 4) and an area of primarily domestic discharge (sites 5 and 6). It must be emphasized that the SOS chromotest allowed the detection of genotoxic samples only in hospital wastewater (site 1). Similar results were described by Giuliani et al. [37]. Indeed, the genotoxic activity of hospital wastewater has not been detected in samples taken from the influent of the wastewater treatment plant that receives the hospital wastewater. This hospital specificity could be explained by the hospital’s use and discharge of primary DNAdamaging compounds. These compounds could be either of hospital nature or used in greater quantity at the hospital level than at the urban level. Among the numerous compounds used in hospitals, ciprofloxacin could be one of the causative agents. This compound has been identified by Hartmann et al. [38,11] as the source of genotoxicity in German hospital wastewater. Even if ciprofloxacin is also available for ambulatory treatment, it can be assumed that the domestic discharges are less important than the hospital discharges. However, other compounds could also explain this hospital specificity. At other sampling sites, the dilution of hospital wastewater by industrial or domestic wastewater leads to a reduction of compound concentrations below the threshold causing an effect on the SOS chromotest.

160

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

Table 5 Comparison of the number of positive responses by the nature of the sampling sites TA100 +S9

TA102 −S9

SOS

Global

Mixed wastewater (hospital–industrial–domestic) (21 samples) January 0 (0%) – 5 (24%) April 3 (14%) 5 (24%) 1 (5%) Global 3 (7%) 5 (24%) 6 (12%)

TA98 −S9

– 0 (0%) 0 (0%)

8 (38%) 19 (90%) 27 (64%)

6 (29%) 7 (33%) 13 (31%)

17 (81%) 19 (90%) 36 (88%)

Domestic wastewater (5 samples) January 0 (0%) April 0 (0%) Global 0 (0%)

– 0 (0%) 0 (0%)

5 (100%) 4 (80%) 9 (90%)

0 (0%) 0 (0%) 0 (0%)

5 (100%) 5 (100%) 5 (100%)

TA98 +S9

– 2 (40%) 2 (40%)

TA100 −S9

2 (40%) 0 (0%) 2 (20%)

−S9: without metabolic activation; + S9: with metabolic activation.

The genotoxicity detected in the Rouen wastewater network cannot be solely ascribed to the wastewater discharge of the Rouen University Hospital. If this hospital were the single source of genotoxicity, the hospital wastewater dilution in the Rouen wastewater network should result either in the absence of a positive response at sites 2 or 3, or in a positive response, but of a weaker intensity in the same strains and for the same day as at the hospital, which was not the case in our study. As the hospital is not the only source of the genotoxicity detected by the Salmonella test in the Rouen wastewater network, other causes must be considered, like industrial or domestic discharges. Indeed, in the flow to the wastewater treatment plant (site 3), the hospital wastewater is mixed with domestic and industrial wastewater, which can be sources of genotoxic contamination. Some studies have suggested that municipal wastewater genotoxicity could be proportional to the industrial contribution [39–41]. However, attempts to demonstrate a statistical association between these two parameters have been unsuccessful [42]. A review by Houk [43] has demonstrated the noteworthy genotoxic potency of wastewater from chemical industries, pulp and paper mills, and metal and petroleum refineries. In addition, White and Rasmussen [1] showed that over 90% of the genotoxic load for the Montreal Urban Community was from nonindustrial origin. They have also analysed data of ten studies that used the Salmonella test to measure the genotoxicity of surface waters in European, Japanese and South African rivers. An excellent relationship was found between mutagenicity in strain TA98 (expressed in mg benzo(a)pyrene equivalent) and population to discharge ratio. These data reinforce the role of nonindustrial emissions in determining surface water geno-

toxicity. The results obtained for the influent of the Grand Couronne wastewater treatment plant (site 5), which processes domestic wastewater, showed that domestic wastewater plays an important role in the overall genotoxicity. Indeed, results obtained in January with the influent of the wastewater treatment plant located in a domestic area (site 5) are similar to those obtained in an area of mixed discharge (site 3). In order to compare different types of wastewater, we have collected samples that are the influents (sites 3 and 5) and effluents (sites 4 and 6) of two wastewater treatment plants. The tests show that during the two sampling periods, a genotoxic potential was found only with the Salmonella test, and solely in the two influents (sites 3 and 5). The process used in the wastewater treatment plants has been effective in removing the genotoxicty detected in the wastewater treatment plants influents [44]. The results of this study show that the different wastewaters tested present a genotoxic risk, and that the wastewater treatment plants were able to remove this genotoxicity. Complementary studies should be undertaken in the analytical field in order to try to identify and quantify the compounds responsible for the genotoxicity. This difficult task will be necessary to identify the sources of toxic contaminants and thus to take preventive and/or curative measures in order to limit the toxicity of the wastewater.

Acknowledgements The authors are grateful to E. Jolibois for her advice in editing the manuscript. This study was carried

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

out thanks to the technical and financial support of the “Agence de l’Eau de Seine-Normandie” (R. Goujon and L. Guezennec), the “Agglom´eration de Rouen Haute-Normandie—Direction de l’assainissement” (I. Maillet) and the “Service de Navigation de la Seine” (S. Durel).

[14]

References

[16]

[1] P.A. White, J.B. Rasmussen, The genotoxic hazards of domestic wastes in surface waters, Mutat. Res. 410 (1998) 223–236. [2] M.L. Richardson, J.M. Bowron, The fate of pharmaceutical chemicals in the aquatic environment, J. Pharm. Pharmacol. 37 (1985) 1–12. [3] S. Halling, N. Nors, P.F. Lanzky, F. Ingerslev, L. Holten, S.E. Jorgensen, Occurrence, fate and effects of pharmaceutical substances in the environment—a review, Chemosphere 36 (1998) 357–393. [4] M. Koivusalo, J.J. Jaakkola, T. Vartiainen, T. Hakulinen, S. Karjalainen, E. Pukkala, J. Tuomisto, Drinking water mutagenicity and gastrointestinal and urinary tract cancers: an ecological study in Finland, Am. J. Public Health 84 (1994) 1223–1228. [5] M. Koivusalo, T. Vartiainen, T. Hakulinen, E. Pukkala, J.J. Jaakkola, Drinking water mutagenicity and leukemia, lymphomas, and cancers of the liver, pancreas, and soft tissue, Arch. Environ. Health 50 (1995) 269–276. [6] M. Koivusalo, E. Pukkala, T. Vartiainen, J.J. Jaakkola, T. Hakulinen, Drinking water chlorination and cancer—a historical cohort study in Finland, Cancer Causes Control 8 (1997) 192–200. [7] B. Jolibois, M. Guerbet, S. Vassal, Detection of hospital wastewater genotoxicity with the SOS chromotest and Ames fluctuation test, Chemosphere 51 (2003) 539–543. [8] L. Shen, J.Y. Wu, G.F. Lin, J.H. Shen, J. Westendorf, H. Huehnerfuss, The mutagenic potentials of tap water samples in Shanghai, Chemosphere 52 (2003) 1641–1646. [9] T. Morisawa, T. Mizuno, T. Ohe, T. Watanabe, T. Hirayama, H. Nukaya, T. Shiozawa, Y. Terao, H. Sawanishi, K. Wakabayashi, Levels and behavior of 2-phenylbenzotoriazole-type mutagens in the effluent of a sewage treatment plant, Mutat. Res. 534 (2003) 123–132. [10] S. Monarca, D. Feretti, C. Collivignarelli, L. Guzzella, I. Zerbini, G. Bertanza, R. Pedrazzani, The influence of different disinfectants on mutagenicity and toxicity of urban wastewater, Water Res. 34 (2000) 4261–4269. [11] A. Hartmann, E.M. Golet, S. Gartiser, A.C. Alder, T. Koller, R.M. Widmer, Primary DNA damage but not mutagenicity correlates with ciprofloxacin concentrations in German hospital wastewaters, Arch. Environ. Contam. Toxicol. 36 (1999) 115–119. [12] L. Shen, G.F. Lin, J.W. Tan, J.H. Shen, Genotoxicity of surface water samples from Meiliang Bay, Taihu Lake, Eastern China, Chemosphere 41 (2000) 129–132. [13] T. Haider, R. Sommer, S. Knasmuller, P. Eckl, W. Pribil, A. Cabaj, M. Kundi, Genotoxic response of Austrian groundwater

[15]

[17]

[18]

[19]

[20]

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28] [29]

161

samples treated under standardized UV (254 nm)-disinfection conditions in a combination of three different bioassays, Water Res. 36 (2002) 25–32. T. Ohe, P.A. White, D.M. DeMarini, Mutagenic characteristics of river waters flowing through large metropolitan areas in North America, Mutat. Res. 534 (2003) 101–112. G.d.A. Umbuzeiro, D.A. Roubicek, C.M. Rech, M.I.Z. Sato, L.D. Claxton, Investigating the sources of the mutagenic activity found in a river using the Salmonella assay and different water extraction procedures, Chemosphere 54 (2004) 1589–1597. S. Monarca, R. Pasquini, G.S. Sforzolini, Mutagenicity assessment of different drinking water supplies before and after treatments, Bull. Environ. Contam. Toxicol. 34 (1985) 815–823. R.G. Stahl, The genetic toxicology of organic compounds in natural waters and wastewaters, Ecotoxicol. Environ. Saf. 22 (1991) 94–125. R. Langevin, J.B. Rasmussen, H. Sloterduk, C. Blaise, Genotoxicity in water and sediment extracts from the St. Lawrence river system, using the SOS chromotest, Water Res. 26 (1992) 419–429. P.A. White, C. Blaise, J.B. Rasmussen, Detection of genotoxic substances in bivalve molluscs from the Saguenay Fjord (Canada), using the SOS chromotest, Mutat. Res. 392 (1997) 277–300. H.S. Rosenkranz, S. Mersch, G. Klopman, SOS chromotest and mutagenicity in Salmonella: evidence for mechanistic differences, Mutat. Res. 431 (1999) 31–38. D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113 (1983) 173–215. Y. Sayato, K. Nakamuro, H. Ueno, R. Goto, Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water, Mutat. Res. 242 (1990) 313–317. A.J. Hendriks, J.L. Maas-Diepeveen, A. Noordsij, M.A. Van der Gaag, Monitoring response of XAD-concentrated water in the Rhine delta: a major part of the toxic compounds remains unidentified, Water Res. 28 (1994) 581–598. L.C. Waters, R.L. Schenley, B.A. Owen, P.J. Walsh, A.W. Hsie, R.L. Jolley, M.V. Buchanan, L.W. Condie, Biotesting of wastewater: a comparative study using the Salmonella and CHO assay systems, Environ. Mol. Mutagen. 14 (1989) 254–263. M.H. Green, W.J. Muriel, B.A. Bridges, Use of a simplified fluctuation test to detect low levels of mutagens, Mutat. Res. 38 (1976) 33–42. D. Gatehouse, Detection of mutagenic derivatives of cyclophosphamide and a variety of other mutagens in a microtitre fluctuation test, without microsomal activation, Mutat. Res. 53 (1978) 289–296. R. Legault, C. Blaise, D. Rokosh, R. Chong-Kit, Comparative assessment of the SOS chromotest kit and the Mutatox test with the Salmonella plate incorporation (Ames test) and fluctuation tests for screening genotoxic agents, Environ. Toxicol. Water 9 (1994) 45–57. R.I. Gilbert, The analysis of fluctuation tests, Mutat. Res. 74 (1980) 283–289. P. Quillardet, M. Hofnung, The SOS Chromotest, a colorimetric bacterial assay for genotoxins: procedures, Mutat. Res. 147 (1985) 65–78.

162

B. Jolibois, M. Guerbet / Mutation Research 565 (2005) 151–162

[30] S. Mersch-Sundermann, S. Kevekordes, S. Mochayedi, Sources of variability of the Escherichia coli PQ37 genotoxicity assay (SOS chromotest), Mutat. Res. 252 (1991) 51–60. [31] S. Kevekordes, S. Mersch, C.M. Burghaus, J. Spielberger, H.H. Schmeiser, V.M. Arlt, H. Dunkelberg, SOS induction of selected naturally occurring substances in Escherichia coli (SOS chromotest), Mutat. Res. 445 (1999) 81–91. [32] J. Dayan, S. Deguingand, C. Truzman, M. Chevron, Application of the SOS chromotest to 10 pharmaceutical agents, Mutat. Res. 187 (1987) 55–66. [33] S. Mersch-Sundermann, S. Mochayedi, S. Kevekordes, Genotoxicity of polycyclic aromatic hydrocarbons in Escherichia coli PQ37, Mutat. Res. 278 (1992) 1–9. [34] M.J. Ruiz, D. Marzin, Genotoxicity of six pesticides by Salmonella mutagenicity test and SOS chromotest, Mutat. Res. 390 (1997) 245–255. [35] H. Steger-Hartmann, K. Kummerer, A. Hartmann, Biological degradation of cyclophosphamide and its occurrence in sewage water, Ecotoxicol. Environ. Saf. 36 (1997) 174– 179. [36] D.E. Levin, M. Hollstein, M.F. Christman, E.A. Schwiers, B.N. Ames, A new Salmonella tester strain (TA102) with A × T base pairs at the site of mutation detects oxidative mutagens, Proc. Natl. Acad. Sci. U.S.A. 79 (1982) 7445–7449.

[37] F. Giuliani, T. Koller, F.E. Wurgler, R.M. Widmer, Detection of genotoxic activity in native hospital waste water by the umuC test, Mutat. Res. 368 (1996) 49–57. [38] A. Hartmann, A.C. Alder, T. Koller, R.M. Widmer, Identification of fluoroquinolones antibiotics as the main source of umuC genotoxicity in native hospital wastewater, Environ. Toxicol. Chem. 17 (1998) 377–382. [39] S.M. Rappaport, M.G. Richard, M.C. Hollstein, R.E. Talcott, Mutagenic activity in organic wastewater concentrates, Environ. Sci. Technol. 13 (1979) 957–961. [40] J.R. Meier, D.F. Bishop, Evaluation of conventional treatment processes for removal of mutagenic activity from municipal wastewaters, J. Water Pollut. Control Fed. 57 (1985) 999–1005. [41] J.R. Meier, W.F. Blazak, E.S. Riccio, B.E. Stewart, D.F. Bishop, L.W. Condie, Genotoxic properties of municipal wastewaters in Ohio, Arch. Environ. Contam. Toxicol. 16 (1987) 671–680. [42] J.G. Babish, B.E. Johnson, D.J. Lisk, Mutagenicity of municipal sewage sludges of American cities, Environ. Sci. Technol. 17 (1983) 272–277. [43] V.S. Houk, The genotoxicity of industrial wastes and effluents, Mutat. Res. 277 (1992) 91–138. [44] B. Jolibois, M. Guerbet, Efficiency of two wastewater treatment plant in removing the genotoxicity, Arch. Environ. Contam. Toxicol., in press.