Annals of Occupational Hygiene Advance Access originally published online on September 5, 2005
Annals of Occupational Hygiene 2006 50(2):189-196; doi:10.1093/annhyg/mei051
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© 2005 British Occupational Hygiene Society Published by Oxford University Press
Original Article |
Hospital Wastewater Genotoxicity
1 Laboratoire de Toxicologie, Faculté de Médecine et de Pharmacie, 22 Blvd Gambetta, F-76183 Rouen cedex, France; 2 Service Hygiène Hospitalière, Centre Hospitalier de Compiègne, 8 avenue Henri Adnot, F-60321 Compiègne Cedex, France
* Author to whom correspondence should be addressed. Tel: +33 3 44 23 66 28; fax: +33 3 44 23 66 27; e-mail: bjolibois001{at}ch-compiegne.rss.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Hospitals represent an incontestable release source of many chemicals compounds in their wastewaters, and which may have an impact on the environment and human health. Indeed, 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. To study the toxicity and the risk associated with these releases biological tests, such as genotoxicity tests, can be used. An evaluation of the genotoxic potential of the wastewaters from a university hospital was performed with the SOS chromotest and the Salmonella fluctuation test. The samples were taken for six 1-week periods between May 2001 and April 2003. Out of a total of 38 samples tested, 31 were positive in at least one assay (82%). Distribution, proportion and intensity of the genotoxic response were different among the six sampling periods. The two genotoxicity tests had different sensitivities. It must be emphasized that whatever the sampling period, Monday samples were always genotoxic in at least one assay. This work shows that this hospital wastewaters samples are very often genotoxic, the response intensity being inflected by rain levels. Efforts must be undertaken by hospitals to integrate the knowledge and the control of their wastewaters in infection and environmental control programs.
Keywords: hospital wastewater • genotoxicity • mutagenicity tests • chemical water pollution
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Hospitals represent an incontestable release source of many chemicals compounds in the aquatic environment due to laboratory activity or medicine excretion into wastewater (Kümmerer, 2001
). However the knowledge about the hospital wastewater toxicity is scarce and must be studied. Indeed hospital wastewater may have an impact on the environment and human health. Different reviews of the literature have reported the fate of some of pharmaceutical compounds as well as their occurrence and effects in the aquatic environment (Richardson and Bowron, 1985; Halling-Sorensen et al., 1998). 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 investigations have shown a link between genotoxic drinking water intake and a rise in cancers (Koivusalo et al., 1994, 1995, 1997). 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 the population exposure. 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 pollutants because they usually occur in concentrations too low to allow analytical determination, and putative mutagens, with few exceptions, have never even been identified. Moreover, the composite effects of mixtures cannot be readily assessed via analytical methods. Thus toxicity is often evaluated by means of biological tests, as well as by bacterial genotoxicity tests which do not require a priori knowledge of toxicant identity and/or physical–chemical properties.
There are only few studies dealing with the hospital wastewater genotoxicity (Giuliani et al., 1996; Steger-Hartman et al., 1997; Hartman et al., 1999; Jolibois et al., 2003). Even if no standard followed protocols for sample collection, sample processing, or selection of tests exist, and all the studies show that the hospital wastewater could have a genotoxic potential (Table 1). In the present study the hospital wastewater genotoxicity from a university hospital (2600 beds, 1500–2000 m3 wastewater daily) was investigated. The samples were taken for six 1-week periods between May 2001 and April 2003. The genotoxicity was studied by combination of two tests: the SOS chromotest and the Salmonella fluctuation test on strains TA 98, TA 100 and TA 102. The Salmonella fluctuation test on Salmonella typhimurium is a version in liquid medium 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 (TA 100 and TA 102 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 Salmonella test (Monarca et al., 1985). Moreover this test allows to incorporate a greater sample volume in the assay and thus to detect genotoxic compounds at lower concentration without requiring a concentration method. As no extraction method exists that recovers all relevant substances from the sample in equal fractions (Stahl, 1991), the Salmonella fluctuation test has the advantages of an extraction method without the drawbacks. The second genotoxicity test used was the SOS chromotest which allows the detection of primary DNA damaging agents on Escherichia coli. These two tests are not equivalent but complement each other (Rosenkranz et al., 1999) and have been jointly selected in order to broaden detection capacity and to evaluate the overall genotoxic risk present in the hospital wastewater.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Sampling of hospital wastewater
The samples were taken for six 1-week periods between 2001 and 2003: 28 May and 3 June 2001, 10 and 15 September 2001, 22 and 28 November 2001, 29 April and 4 May 2002, 13 and 19 January 2003, 31 March and 06 April 2003.
For the 2001 and 2002 samples, two-liter wastewater samples were collected proportional to the time from the main sewer of the hospital by an autosampler (ORI Mic B type, Fischer Bioblock Scientific, France). The samples were taken during the maximal hospital activity period (8:00 a.m. to 6:00 p.m.). For the 2003 samples, ten-liter wastewater samples were taken proportionally over time by an autosampler during a 24-h period (Isco 3700). The samples were partitioned into aliquots in polyethylene bottles and stored up to 7 days at –25°C until tested. Before genotoxicity assay the samples were filtered through cellulose acetate filters (0.45 µm pore size; Sartorius Minisart, Germany), although this action can result in substantial losses of genotoxic activity (White et al., 1996).
SOS Chromotest
The SOS chromotest allows the detection of primary DNA damaging agents on a genetically engineered bacterium E. coli PQ37. This tester strain was kindly provided by M. Hofnung (Institut Pasteur, Paris, France). The SOS chromotest was performed without metabolic activation as described by Quillardet and Hofnung (1985) with modifications provided by Mersch-Sundermann et al. (1991) and Kevekordes et al. (1999). ß-Galactosidase (ß-gal) and phosphatase alkaline activity (PAL) were determined at 405 nm using a reference solution with no bacteria. The samples (20 µl) were tested as neat samples and half diluted (2001 and 2002 samples) or 10-fold concentrated by means of a Speedvac® concentrator (2003 samples). The sample concentrations are expressed as the percentage of sample volume contained in the medium (1.6 and 3.3% for the 2001 and 2002 sampling periods, 3.3 and 33% for the 2003 sampling period).
The genotoxic activity for a concentration c of the sample is expressed in the ratio Rc = ß-gal/PAL. 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 ultrapure water). The criterion to consider a sample as positive in the SOS chromotest differs between authors (Dayan et al., 1987; Mersch-Sundermann et al., 1992; Ruiz and Marzin, 1997). We choose to consider a sample as an SOS repair system inducer if the IF is >1.5, the ß-gal activity significantly increases compared to the solvent control and the result is reproducible. All results are expressed as the mean of three experiments (±SD). Sterile ultrapure water is used as negative control and 4-nitroquinoline-1-oxide as positive control (2.5 µg ml–1).
Salmonella fluctuation test
The tester strains TA 98, TA 100 and TA 102 were a gift from V. André (Laboratoire de Cancérologie Expérimentale, Centre François Baclesse, Caen, France). The Salmonella fluctuation test is a version in liquid medium of the Salmonella mutagenicity test usually performed in agar plate (Maron and Ames, 1983). The fluctuation test was performed as described in Legault et al. (1994). The assay was conducted without metabolic activation. Three samples volumes were tested (0.2, 2 and 4 ml), which correspond to a sample concentration of, respectively, 1, 10 and 20% in the medium. The plates were sealed in plastic bags and incubated at 37°C for 3–5 days. Mitomycin C (1 ng ml–1 in fluctuation medium), sodium azide (5 ng ml–1 in fluctuation medium) and 2-nitrofluorene (50 ng ml–1 in fluctuation medium) were used as positive controls for TA 102, TA 100 and TA 98 strains, respectively and sterile ultrapure water as negative control.
All yellow, partially yellow or turbid wells are considered positive, and all purple wells negative. Chi-square analysis was used for statistical evaluation of the treated plates versus the control plates (Gilbert, 1980). 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 (±SD).
Expression of the genotoxicity results
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 categories are: slightly, moderately and strongly genotoxic (Table 2).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The results of the genotoxicity tests (SOS chromotest and Salmonella fluctuation test) on the hospital wastewaters are presented in Tables 3 and 4, and summarized in Table 5.
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Out of a total of 38 samples tested, 31 were positive in at least one assay (82%). Distribution of the genotoxic response was different among the six sampling periods. All the samples in September 2001, November 2001, January 2003 and April 2003 was positive. In April–May 2002, 50% of the samples were positive in at least one test and 43% in May–June 2001.
The proportion and intensity of the genotoxic responses were different among the sampling periods (Fig. 1). Some periods are characterized by an overall high proportion and intensity of positive responses (September 2001, November 2001, April 2003), other periods by a weak proportion and an overall high intensity of positive responses (May–June 2001, April–May 2002), and another period by a high proportion and an overall weak intensity of positive response (January 2003).
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The two genotoxicity tests had different sensitivities. Indeed, the Salmonella fluctuation test allowed the detection of 68% of the samples as genotoxic (26 out of 38) and the SOS chromotest 45% (17 out of 38).
It must be emphasized that whatever the sampling period, Monday samples were always genotoxic in at least one assay.
Table 6 presents the proportion and intensity of the genotoxic responses according to rainfall. The proportion of the genotoxic responses corresponds to the number of positive responses obtained in relation to the number of samples tested. The intensity of the genotoxic response for a period corresponds to the median of the daily genotoxic response intensities. A response intensity decrease is observed between periods of high and low rainfall on the two Salmonella strains (strongly to moderately genotoxic), but not for the SOS chromotest.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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An evaluation of the genotoxicity of the hospital wastewaters was performed with the SOS chromotest and the Salmonella fluctuation test over six 1-week periods between May 2001 and April 2003 (38 samples). These tests show that this hospital's wastewaters present a genotoxic effect. Indeed, out of a total of 38 samples tested, 31 were positive in at least one assay (82%). It is difficult to compare these results with other studies because many parameters can influence the genotoxicity test response (composition of the sampling, hospital size and its degree of activity, nature of the medicines used in treatments, nature of the genotoxicity tests, etc.), and there are few hospital wastewater studies in the literature. Even if samples are of a different origin, studies of Steger-Hartmann et al. (1997)
on the umuC test, and Hartmann et al. (1999) on the umuC test, Salmonella test and V79 chromosomal aberration assay exhibited, respectively, 50 and 56% genotoxic samples.
Twenty-six samples (68%) were positive on the Salmonella fluctuation test. In the study of Hartmann et al. (1999) only 2 samples out of 25 were positive (8%) on the strains TA 98 and TA 100. Even if samples were of a different origin, the use in our study of greater sample volumes leading to detection of genotoxic compounds at lower concentration could explain the difference observed with Hartmann et al. (1999). This difference could also be explained by the use of strain TA 102. Indeed, this strain was the most sensitive and detected 22 samples as genotoxic out of 31 (71%). Strain TA 102 is very sensitive because the histidine mutation has been introduced into a multicopy plasmid (pAQ1) of which 
30 copies are found by strain, and not on the bacterial chromosome like other strains (Levin et al., 1982). Strain TA 102 detects a variety of oxidative mutagens (like fluoroquinolone), and many anticancer drugs (mitomycin, adriamycin, bleomycin, daunomycin, etc.) which are not or rarely detected by the other strains.
The SOS chromotest allows the detection of 45% of the samples as genotoxic (17 out of 38). Similar results have been found in the studies of Steger-Hartmann et al. (1997) or Hartmann et al. (1999) on the umuC test (similar to the SOS chromotest) with, respectively, 50 and 40% of positive response. If we consider only the samples tested without a concentration method (2001 and 2002 samples), the SOS chromotest allows the detection of 17% of the samples as genotoxic (4 out of 24) which is in agreement with the results of Giuliani et al. (1996) who found 13% of positive response. On the 2003 samples, the use of a concentration method (10-fold) allowed to increase the detection of genotoxic samples. Indeed, 13 samples out of 14 were found genotoxic (93%).
It must be emphasized that whatever the sampling period, Monday samples were always genotoxic in at least one assay. No real explanation can be given for this phenomenon. However a higher level of hospital activity after the week-end, and thus a more important rejection, could be a hypothesis. A study on release of glutaraldehyde in the same hospital (Jolibois et al., 2002) has shown a concentration peak on Monday morning which can be explained by the weekly disinfecting solutions renewal in hospital departments. It is possible that the higher hospital activity at the beginning of the week leads to liquid rejections in larger quantity, and thus a higher wastewater genotoxicity than on the other days of the week.
The six sampling periods are characterized by different proportions and intensities of the genotoxic response (Fig. 1). Even if a variation in the hospital activity could partially explain this phenomenon, the amount of rain could also interfere. In order to study the influence of rainfall, a comparison was carried out on samples with a comparable mode of sampling and for which data of high and low rainfall are available (Table 6). A response intensity decrease is observed between periods of high and low rainfall on the two Salmonella strains (strongly to moderately genotoxic), but not for the SOS chromotest. On the other hand, in terms of proportion of positive responses, no conclusion can be drawn. Indeed high rainfall involves either a reduction (SOS chromotest), or an increase (TA 100), or no change (TA 98) of the proportion of genotoxic responses.
The dilution of hospital wastewater which occurs in the periods of high rainfall must be the origin of a decreased intensity of the genotoxic response obtained on the Salmonella test.
Moreover, periods of dryness could lead to an important genotoxic response. A similar observation was carried out by De Lima-Moraes and Jordao (2001) on their study of river water cytotoxicity and genotoxicity. On the other hand, in our case, rainfall does not seem to influence the proportion of genotoxic samples, which is certainly more dependent on hospital activity than of a possible dilution of the liquid discharges by rainwater.
The simultaneous use of the SOS chromotest and the Salmonella fluctuation test allowed us to carry out a large screening of the complex mediums like hospital wastewaters. These two genotoxicity tests are sensitive to different genotoxic effects (substitution mutation, frameshift mutation or primary DNA damage) and the variability of the obtained results tends to show a large variety of the compounds responsible. However the correlation between a particular compound and the observed genotoxicity is a much more difficult task. Very few data are available today, such as Hartmann et al. (1998, 1999) who have correlated primary DNA damage but no mutagenicity with ciprofloxacin concentrations in German hospital wastewaters. Among the numerous pharmaceutical compounds used in this university hospital, different classes can be considered as possible causative agents: the anticancer drugs (ifosfamide, cisplatin, doxorubicin, ...), the antimicrobial agents (ciprofloxacin, ...), as well as other compounds able to be formed in situ.
This work shows that this hospital's wastewater samples are very often genotoxic in bacteria, the response intensity being inflected by rain levels. We have broadened this study on the wastewaters of the city in which the hospital lies. A genotoxic activity was detected on the wastewater network, and the hospital was identified as a source of this contamination (Jolibois and Guerbet, 2005a). Another study conducted on the wastewater treatment plant which processes, notably, hospital wastewaters showed that the process used was effective in removing the genotoxicity (Jolibois and Guerbet, 2005b). However, for other hospitals and other wastewater treatment plants, it is not obvious that the genotoxic contamination disappears after treatment in the wastewater treatment plant, and thus this contamination could be found in drinking water (Waters et al., 1989; Doerger et al., 1992; Filipic and Toman, 1996). Since hospitals are one of the sources of rejections of genotoxic compounds in wastewater, efforts must be undertaken by hospitals in order to integrate the knowledge and the control of their wastewaters, and thus the environment management, in the infection and environmental control programs.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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This work is dedicated to Serge Vassal, responsible for the hospital's hygiene laboratory, who died in October 2001. The authors are grateful to E. Jolibois for her advice in editing the manuscript. This study was carried out thanks to the technical and financial support of the ‘Agence de l'Eau de Seine-Normandie’ (R. Goujon and L. Guezennec), the ‘Agglomération de Rouen Haute-Normandie—Direction de l'assainissement’ (I. Maillet) and the ‘Service de Navigation de la Seine’ (S. Durel).
Received January 29, 2005; in final form August 8, 2005
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