Memoire Online - Evaluation des risques sanitaires et écotoxicologiques liés aux effluents hospitaliers

Les résultats des bioessais pratiqués sur les effluents hospitaliers, montrent ont l'écotoxicité d élevée de ces effluents sur les organismes aquatiques (GARTISER et ai, 1996; LEPRAT, 1998; JEHANNIN, 1999). Par ailleurs, la mise en oeuvre des tests de mutation génique AMES et HAMSTER prouve que les effluents des services cliniques et des laboratoires hospitaliers peuvent présenter un caractère de mutation génique (GARTISER et ai, 1996). L'une des hypothèse avancée sur la toxicité des rejets liquides hospitaliers est qu'elle est due aux désinfectants utilisés dans les services (DELOFFREBONNAMOUR, 1995; JouBois et al., 2002).

Parmi les différents désinfectants, les produits à base de glutaraldéhyde et les désinfectants à base de chlore (eau de Javel) sont largement utilisés dans presque tous les hôpitaux du monde. Il a été donc décidé, dans le cadre de cette thèse de réaliser une étude spécifique sur le devenir de l'hypochlorite de sodium et du glutaraldéhyde contenus dans les effluents hospitaliers. L'objectif de ce chapitre est de présenter les résultats de l'étude spécifique.

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

II. Toxicological effects of sodium hypochiorite disinfections on aquatic organisms and its contribution to AOX formation in hospital wastewater

Evens EMMANUEL¹r^2*, Yves PERRODIN¹, Gérard KECK³, Jean-Marie BLANCHARD², Paul VERMANDE²

¹ Laboratoire des Sciences de l'Environnement, École Nationale des Travaux Publics de l'État, Rue Maurice Audin, 69518 Vaulx-en-Velin, France

² Laboratoire d'Analyse Environnementale des Procédés et Systèmes Industriels, Institut National des Sciences Appliquées de Lyon, 20 avenue Albert Einstein, 69621 Villeurbanne Cédex, France

³ Unité d'Ecotoxicologie, Ecole Nationale Vétérinaire de Lyon, BP 83, 69280 Marcy l'Etoile, France

Keywords : Sodium hypoclorite, AOX, hospital effluents, toxicity, Daphnia.

* Corresponding author. Tel : +(33) 4 72 04 72 89; fax:+(33) 4 72 04 77 43 E-mail address : evemml eyahoo.fr

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Sodium hypochlorite (NaOCI) is often used for disinfecting hospital wastewater in order to prevent the spread of pathogenic microorganisms, causal agents of nosocomial infectious diseases. Chlorine disinfectants in wastewater, react with organic matters to give rise to organic chlorine compounds such as AOX (halogenated organic compounds adsorbable on activated carbon), which are toxic for aquatic organisms and persistent environmental contaminants. The aim of this study was to evaluate the toxicity on aquatic organisms of hospital wastewater from services using NaOCI in prechlorination. Wastewater samples from the infectious and tropical diseases department of a hospital of a big city of the southeast of France were collected. Three samples per day were done in the connecting well department at 9 A.M, 1 P.M. and 5 P.M. during eight days from 13 March to 22 March 2001, and a mixture was made at 6 P.M. with the three samples in order to obtain a representative sample for the day. For toxicity test, the 24-h EC₅₀ on Daphnia magna and a bioluminescence assay using Vibrio fischeri photobacteria were used. Fecal coliforms and physicochemical analysis such as: Total Organic Carbon (TOC), chloride, AOX, Total Suspended Solids (TSS) and Chemical Oxygen Demand (COD) were carried out. Wastewater samples highlighted an important acute toxicity on Daphnia magna and Vibrio fischeriphotobacteria. However, low most probable number (MPN) ranging from <3 to 2400 for 100 mL were detected for fecal coliforms. Statistical analysis, at a confidence interval of 95%, gave a strong linear regression assessed with r= 0,98 between AOX concentrations and EC₅₀ (TU) on daphnia. The identification of an ideal concentration of NaOCI in disinfecting hospital wastewater, i.e. its NOEC (Non Observed Effect Concentration) on algae and Daphnia magna, seems to be a research issue which could facilitate the control of AOX toxicity effects on aquatic organisms. Therefore, it would be necessary to follow-up at various dosages the biocide properties of NaOCI on fecal coliforms and its toxicity effects on aquatic organisms.

Sodium hypochlorite (NaOCI, CAS no. 7681-52-9 and EC no. 017-011-00-1), a solution containing between 12.5 to 25% active chlorine gas (Cl2), has a wide range of domestic, industrial, scientific and biomedical applications related to biocide properties (U.S. EPA, 1994a; Brondeau et al., 2000). When NaOCI is added to water and wastewater, the solution reacts readily with biological materials (including proteins and nucleotide bases) to produce a variety of organic chlorinated compounds (U.S. EPA, 1989a), which are mostly lipophilic, persistent, and toxic in aquatic environment (SalkinojaSalonen and Jokela, 1991).

The medical use of chlorine began in 1826 with chlorine water in delivery wards to prevent puerperal fever, and chloroform was first used for medical anesthesia in 1847 (Carey et al, 1998). Nowadays, the widespread biomedical use of NaOCI is particularly for the local surface and toilet disinfections, based on it very broad spectrum of biocide activity against bacteria, virus and fungi (DeloffreBonnamour, 1995).

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

In hospital, besides sodium hypochlorite, a variety of substances such as pharmaceuticals, radionuclides, solvents, are in use for medical purposes as diagnostics, disinfections and research (Erlandsson and Masson, 1978; Richardson and Bowron, 1985; Kümmerer et al, 1997). After application, many non-metabolized drugs are excreted by the patients and enter into wastewater (Halling-Sorensen et al, 1998). After their use disinfectants also reach the wastewater (Kümmerer, 2001). Due to laboratory and research activities or medicine excretion into wastewater, hospitals may represent an incontestable release source of many toxic substances in the aquatic environment (Jolibois et al, 2002).

Hospital effluents reveal the presence of organochlorine compounds in high concentrations (Leprat, 1998). AOX up to 10 mg/L were proved in the effluents of the hospitalization services of a university hospital center (Gartiser et al, 1996). Presence of AOX (adsorbable organic halogens) in hospital wastewater is usually associated with the presence of iodinated X-ray contrast media release by hospital radiography department. The assessment of AOX shows that those non conventional pollutants have a bad biodegradability and a bad behavior of adsorption (Sprehe et al, 1999).

Throughout the world, the assessment of wastewater discharges or effluents is focused on the precautionary principle, i.e., reduction of specific pollutants or substances in the framework of their emission policies (Kinnersley, 1990). In Directive 98/15/EEC, the European Commission (1998) proposes an emission limit of pollutants in wastewater for all member states. Based on a comparison of the measured values for chemical parameters in effluents and the proposed limited values, the directives on wastewater management in their application most often do not allow to evaluate the toxicity effects of hazardous substances, contained particularly in hospital wastewater, on organization levels of the aquatic food chains (Perrodin, 1988). However, a quick assessment of toxic effects of substances on aquatic organisms is possible by completing chemical characterization with toxicity assay (Perrodin, 1988, Schowanek et al, 2001, Sponza, 2003).

Cladocerans, a group of freshwater zooplankton, are broadly distributed in freshwater bodies, are present throughout a wide range of habitats, and are important links in many food chains (Abe et al., 2001). Daphnia sp. are extensively used to investigate the acute and chronic toxicity of industrial and agricultural chemicals in aquatic ecosystems (OECD, 1984; ASTM, 1994; Verschueren, 1996; Hanazato, 1998). Because they have a relatively short life cycle, require little space, are adaptable to laboratory conditions and are sensitive to a broad range of aquatic contaminants (Cooney, 1995). Application of Daphnia magna bioassay procedures on hospital wastewater had proved the high toxicity of these wastewater (Jehannin, 1999; Emmanuel et al, 2001). One of the assumptions on this toxicity is the presence of chlorinated disinfectants. The application of chlorine as a general oxidant in sewage treatment industries results in the creation of mixtures, AOX formed during chlorine disinfections of humic-rich raw water (Jokela et al., 1992), that are also emitted in the environment (Carey et al., 1998). The aim of this study was (i) to make a brief review of the chemistry and the

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

toxicology of chlorinated disinfectants in order (ii) to evaluate the toxicity on aquatic organism of hospital wastewater from services using NaOCI disinfectant.

11.3. The chemistry and toxicology of chlorinated disinfectants in water and wastewater 11.3.1. Chemical behavior of chlorinated disinfectants

In spite of the concern about the formation of toxic by-products usually associated with it use (Marhaba and Washington, 1998; Lopez et al, 2001), Chlorine (Cl₂) and its derive NaOCI are still the most widely used disinfectant (Baxter, 1994). When chlorine gas is dissolved in water, it hydrolyses rapidly according to the following equation to yield hypochlorous acid:

Hypochlorous acid is also formed when sodium hypochlorite (NaOCI) is used as the source of chlorine: NaOCI + H₂O --> HCIO + Na⁺ + OH^- (2)

Hypochlorous acid is a weak acid, and will undergo partial dissociation as follows:

The ratio [HCIO]/[ C10^-] is pH dependent (Lopez et al, 2001). In fact, in presence of pH between 6 and 9, both hypochlorous acid and hypochlorite ion will be present in waters; the proportion of each species is depending also on temperature (White, 1986). Hypochlorous acid is significantly more effective as a biocide than the hypochlorite ion. If bromide ions are present, chlorine will also oxidise bromide to form hypobromous acid (Von Guntent and Hoigné, 1995):

Hypobromous acid is an effective biocide, in this context the group [HBrO]/[Br0^-] are better oxidants than [HCIO]/[CI01, conversely, hypochlorous acid and hypochlorite ion are better halogenating substances (Lopez et al, 2001). When ammonia is also present, the competing reactions of chlorine with bromide and ammonia are likely to result in the rapid formation of both monochloramine and hypobromous acid. A number of other reactions can then occur:

HCIO and CIO^- can react with organic compounds by addition, substitution and oxidation (Boyce and Hornig, 1983). The reaction of chlorine with organic constituents in aqueous solution can be grouped into several types:

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Chlorine substitution can lead to the formation of halogenated compounds, such as chloroform (e.g. equation 11). Although such reactions are significant in terms of the resultant halogenated byproduct, it has been estimated that only a few percent of the applied chlorine ends up as halogenated organic products (White, 1986).

11.3.2. Environmental fate and toxicological effects of chlorinated disinfectants

In addition to the chlorinated water that is released into the environment from sewage treatment operations and other sources, chlorine is potentially released during its transport (NTP, 1992). Limited monitoring studies have detected chlorine in the ambient atmosphere at concentrations ranging from 1 to 3.7 mg/m³ or 0.344 to 1.27 ppm (U.S. EPA, 1994a). Chlorine may react with soit components to form chlorides; depending on their water solubility, theses chlorides are easily washed out from the soit (Seiler et ai, 1988). U.S. EPA (1989a) reports that chlorine hydrolyses very rapidly in water (rate constants range from 1.5x10-4 at 0°C to 4.0x10-4 at 25°C). However, there is no potential for the bioaccumulation or bioconcentration of chlorine (U.S. EPA, 1994a).

Chlorine gas (Cl2) or sodium hypochlorite (NaOCI) added to drinking water effectively inactivates bacteria in 20 minutes at concentrations of 0.03 to 0.06 mg/L at pH range of 7.0 to 8.5 and temperature range of 4°C to 22°C (NTP, 1992). Drinking water concentrations of higher than 90 ppm chlorine caused irritation of human membranes of throat and mouth (U.S. EPA, 1989a). In fact, chlorine is a primary irritant to the mucous membranes of the eyes, nose and throat and to the linings of the entire respiratory tract (Stokinger, 1982). Mice and rats exposed to chlorine at the RD50 concentration (9-11 ppm, 6h/day for 1, 3, or 5 days) developed degeneration of olfactory sensory cells in the olfactory mucosa, loss of cilia of the respiratory epitlelium, and cellular exfoliation primarily of the naso and maxilloturbinates (U.S. EPA, 1994a).

The major target organs for the subchronic/chronic toxicity of chlorine in humans are the respiratory tract and the blood. In animais, the major target organs are the immune system, the blood, the cardiovascular system and the respiratory tract (U.S. EPA, 1994a). EPA has derived an oral RfD (reference dose) of 0.1 mg/kg/day for chlorine, based on a no-obeserved-effect level of 14.4 mg/kg/day in a chronic drinking water study in rats (U.S. EPA, 1994b).

No conclusion on the carcinogenicity and on the developmental/reproductive toxicity of chlorine can be made on the limited information available from human and animal studies (NTP, 1992; U.S. EPA 1989 and 1994b). However, chlorine was mutagenic in Salmonella typhimurium strains, without metabolic activation, produced chromosome aberrations in human lymphocytes and other mammalian cells; interacted with DNA in E. coli polA (as sodium hypochlorite) and was negative for the induction of erythrocyte micronuclei or chromosome aberrations of bone narrow cells mice (up to 8 mg/kg/day of NaOCI) for up to 5 days (U.S. EPA, 1989a).

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Chlorine has high acute toxicity to aquatic organisms. Many toxicity values are less than or equal to 1 mg/L. Table 1 shows obtained results on toxicity studies of chlorine on some aquatic species (AQUIRE, 1994). Papillomas of the oral cavity in fish have been associated with exposure to chlorinated water supplies (NTP, 1992). Low level chlorination (0.05 to 0.15 mg/L) results in significant shifts in the species composition of marine phytoplankton communities (U.S. EPA, 1994a).

Chlorine is phytotoxic but is also essential to plant growth; crops need around 5 pounds or more of chlorine per acre (U.S. EPA, 1994a). Acute toxicity to plants is characterized by defoliation with no leaf symptoms and, in higher plants forms, by spotting of the leaves at 1.5 mg/m³, and marginal and interveinal injury at 150-300 mg/m³ (Seiler et al., 1988).

The wastewater from the infectious and tropical diseases department (ITDD), with a capacity of 144 beds, of a hospital of a big city of the southeast of France have been selected. Wastewater was collected before entering into the entire hospital sewer network, which discharges the total volume of effluents from the various departments into the urban wastewater network without pre-treatment. This choice was justified by the fact that the ITDD collector does not receive effluents containing iodized X-ray contrant media from radiography department, substances which could be contributed to AOX concentrations. Three samples per day were done in the connecting well department at 9 A.M, 1 P.M. and 5 P.M. during eight days from 13 March to 22 March 2001. A mixture has been made with the three samples at 6 P.M. in order to obtain a representative sample for the day. In the objective to avoid or decrease volatility process in samples, laboratory conditions controlling flow and turbulence have been considered during mixture preparations. Water sample was collected by means of a telescopic perch in a 1-L glass flask. pH was measured directly on site after sampling with a pH meter HANNA instrument HI 8417 (accuracy pH #177; 0.01pH, mV #177; 0.2 mV #177; 1, °C #177; 0.4°C) digit and standard electrode HI 1131 B (refillable glass combination pH electrode). All the water samples and the mixture were kept at 4°C until analysis.

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Total suspended solids (TSS) concentrations were determined in conformity with the European standard NF EN 872 (AFNOR, 1997a) after filtration through a 1.2 pm membrane and dewatering at 105 °C.

Chlorides were determined by following the European standard NF EN ISO 10304-1 (AFNOR, 1997b) on diluted and filtered samples at 0.45 pm by using DIONEX DX-100 ion chromatograph with suppressed conductivity detection from 0.0 to 1000 pS. Ionpac AS14 4x250 mm analytical column (P/N 046124) was used for chloride sample analysis. AOX were measured according to European standard EN 1485 (AFNOR, 1997a). COD was measured by potassium dichromate method using HACH spectrophotometer 2010 and test procedure provided by the supplier. Because hospital wastewater may record temporal physicochemical evolutions, measures of COD on the three samples and the mixture COD were determined for wastewater samples of March 20, 2001. This decision has been taken in order to analyze the variations of COD during the day.

Total Organic Carbon (TOC) was carried out on filtered samples at 0.45 pm and pre-treated with orthophosphoric acid (H3PO₄). French standard T90-102 (AFNOR, 1997a) was followed by using a carbon analyzer SPECTRA France, LABTOC model, with potassium per sulfate reagent (K2S208) and UV oxidation.

The determination of the inhibition of Daphnia magna mobility is a acute toxicity assay. Its objective is to identify the initial concentration of a pollutant in solution or an aqueous mixture which may immobilize in 24 or 48 hours 50% of exposed daphnia into polluted solutions. According to the European standard NF EN ISO 6341 (AFNOR, 1997c), the different assays were carried out on Daphnia sp. maintained in parthenogenetic culture in the laboratory (POLDEN of the National Institute of Applied Sciences of Lyon -- INSA de Lyon). The sensibility of the laboratory species was controlled by regular tests with potassium dichromate. Young female Daphnia, aged less 24 h were only used. The normal medium, without EDTA, was also used. The essays were realized at 20 #177; 2°C under darkness condition. All the assays were carried out in a limit of time from 6 to 48 h after sampling.

Because hospital wastewater is considered as toxic for aquatic environment, a volume of 250 mL unfiltered samples was taken for each assay. In order to understand the effects of color, turbidity and TSS present in hospital effluent samples on Daphnia sp., the toxicity of 250 mL filtered sample (0.45 pm pore size membrane) was studied by comparing the results with the unfiltered volume of the same sample. The three required conditions for the validity of assays were observed: (i) the concentration of dissolved oxygen (DO), in the control group, was 2 mg/L at the end of each assay; (ii) the

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

observed percentage of immobilization in the control group vessels was 10%; (iii) EC₅₀ in 24h of potassium dichromate was between 0.6 to 1.7 mg/L.

The bioassay on bacteria luminescence MICROTOX was carried out with a LUMIStox system (Dr Lange GmbH, Duesseldorf, Germany) following the standard procedure of the European standard NF EN ISO 11348-3 (AFNOR, 1999). Tests were performed using gram negative marine bioluminescent bacteria of the species Vibrio fischeri NRRL-B-11177 of the Vibrionaceae family. In order to prevent the interferences of TSS on the bacteria luminescence, samples were filtered using a 0.45pm pore size membrane. The samples were treated with NaCI solution of 20 g/L and brought to 50 mS/cm of conductivity before the analysis. Starting from the concentration of the sample, eight consecutive elutions were tested (dilution factor 1:2); the inhibition of bioluminescence was measured at a wavelength of 490 nm, with readings after 5 and 15 minutes of incubation at 15 °C. The EC₅₀ values were calculated as reported by Bulich (1979).

The immobilization percentage, of the initial total number of 20 daphnia used in the test, were determined for each sample concentration. EC₅₀ values for inhibition assays by Litchfield-Wilcoxon statistic method or probit analysis (Finney, 1971). Results of CE₅₀ 24h, with their confidence interval, are expressed in percentage of sample dilution in toxic unit TU (1 TU = 100 / CE₅₀)
·

Multiple regression analysis between y and x variables was performed using the statistical package Xlstat version 5.0 (b8.3) for Windows (Addinsoft, 2001). The multiple regression STEPWISE (Agresti, 1990) was used to determine the correlations between AOX (4 and EC₅₀ 24h on daphnia (y). The linear regression was assessed with r, r² and adjusted coefficient of determination. r, r² are respectively the coefficient of correlation and the coefficient of determination. Ail the statistical analysis were at a confidence interval of 95%.

In all samples (simple and mixture) pH was always in an alkaline range (7.9 -- 8.8) with a variation lower than 1 pH unit. The hospital wastewater composition from the infectious and tropical diseases department refers to the amounts of its physical, chemical constituents. These elements have been determined by measuring COD, TOC, TSS, pH, chlorides, AOX coupled with the bioassays of toxicity. The European Commission Directive 98/15/EC (1998) proposes medium discharge standards for all these parameters. The results of physicochemical characterization of hospital wastewater mixture samples are summarized in Table 2.

In the effluent samples, COD concentrations ranged from 362 to 1492 mg/L exceeded the discharge
standards. This could be attributed to the presence of toxic substances. The COD threshold values for

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

industrial wastewater which must be reached the sewer network is given by the Directive 98/15/EC and accounts 125 mg/L (European Commission, 1998). COD concentrations for punctual samples were compared with COD concentration of the mixture sample. The mixture sample value was different of mean value of the punctual samples. The variation of COD for this specific sample day is presented in figure 1.

Means	Minima		Maxima		SD		n
8.4	7.9		8.8		0.3		8
185	63.4		359.2		90.4		8
0.7	0.38		1.24		0.28		8
225	155		298		64		5
638	362		1492		435		6
218	160		350		78		6
3.43	2.01		4.26		0.92		6
1600
1400								1492

										1417
1200
1000

15)				1029
800
		792
0
0
0 600
400
200
0						m			a

Figure 1: Variations of COD concentrations between the three samples
and mixture of 6^th day

In conventional urban wastewater, TOC concentrations usually ranged from 80 to 290 mg/L (Metcalf and Eddy, 1991). In the hospital wastewater, TOC concentrations were from 160 to 350. The difference could be due to higher chloride concentrations in hospital wastewater. Seiss et al. (2001) reported that TOC degradation rate depends on the chloride concentration of the wastewater. The

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

different organochlorine compounds resulting from NaOCI disinfections of hospital wastewater could contribute to high TOC concentrations in these effluents.

TSS concentrations ranged from 155 to 298 mg/L and chloride values from 63.4 to 359.2 were detected. The linear regression analysis between TSS (y dependent variable) and chlorides (x independent variable) in the effluents revealed an acceptable linear correlation (r = 0.93, r² = 0.86, Durbin-Watson statistic DW = 1.23, P = 0.02). Chlorides and TSS levels were higher in the effluents samples where higher acute toxicity on Daphnia magna was observed. The same result was observed for AOX which concentrations ranged from 0.38 to 1.24 mg/L. The AOX permissible level 1 mg/L, given in French wastewater regulations (MATE, 1998), is exceeded in higher AOX concentration in the effluent mixture sample. However, AOX results generated by chlorine disinfections of hospital wastewater were lower than AOX concentration of 10 mg/L determined (Sprehe et al., 1999) in hospital wastewater containing iodized contrast media.

11.5.2. M icrobiological and toxicological characterizations of ITD D wastewater

Low concentrations of bacteria fora 2.4x10³/100mL were deducted for the hospital effluents (Table 3). Previous studies on microbiological characterization of hospital wastewater (Bernet and Fines, 2000) reported these effluents have bacteria concentrations lower than 10⁸/100mL generally present in the municipal sewage system (Metcalf and Eddy, 1991).

MICROTOX results in toxic unit (TU) explain the toxicity of hospital wastewater on V. fischery. Significant differences were observed between 5 and 15 min EC₅₀ values, showing that 5 min assay can be considered as no toxic because all the results are lower than 2 TU. The results of 15 min ranged from 2.47 to 4.15 TU greater than 2 TU had a toxicity similar to domestic wastewater (Table 3). However, all the results were lower than the means of 6.75 TU reported by the literature for the toxicity of hospital wastewater on V. fischerycompleted after 30 min of exposure (Jehannin, 1999).

The 24-h EC₅₀ values determined for each mixture sample toxicity on Daphnia magna, with the correspondent 95% confidence limits. The results are also presented in Table 3. In all the samples, the 24-h EC₅₀ values on Daphnia magna ranged from 9.8 to 116.8 TU greater than that 2 TU value proposed by French water agencies for industrial wastewater discharge. Potential toxicity is a suitable description of these effluents.

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Table 3. Microbiological and toxicological characterizations of the mixture samples

Parameters	Units	Means	Minima	Maxima	SD	n
Fecal coliforms	MPN		<3	2 400		4
EC₅₀ Vibrio fischeri	TU	1.42	1.1	1.54	0.25	4
(MICROTOX -- 5mn)
EC₅₀ Vibrio fischeri	TU	3.4	2.47	4.15	0.7	4
(MICROTOX -- 15mn)
EC₅₀ Daphnia	TU	44	9.8	116.8	34	8

Studies on hospital wastewater reported that these effluents are essentially domestic (i.e. sanitary wastewater from residential and commercial sources) and are characterized by pollutant concentrations of BOD₅, COD, TSS and TOC (EPA, 1989b; Mansotte and Justin, 2000). BOD₅ value was not analyzed in this study. In table 4 the results obtained for the hospital effluents were compared with the approximate ranges for domestic wastewater reported in the literature.

TOC and COD concentrations are greater than the values proposed by Metcalf and Eddy (1991) for domestic wastewater. COD and TOC were reacted to completion (Gray and Becker, 2002), and a good linear correlation was found between them in the results of this study (r = 0.95, r² = 0.9, DurbinWatson statistic DW = 1.96, P = 0.0039) the graph is presented in figure 2.

Parameters Units Approximate ranges of Approximate ranges of Results of the study

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

	400 350 300		y = 0.1707x + 85.10
2 a)	250		r= 0.95
E	200
c.) 0 1--	150		·		·
	100
	50
	0
		I	I	I		I	I	I	I	I

Information reported in the literature assume a COD/TOC of ratio 3 frequently found in many wastewater (Seiss et al., 2001). Gray and Becker (2002) reported a semi-empirical equation to determine the ratio between COD expressed in mg 0₂/L and TOC in mg C/L.

The variations of COD, TOC and the COD/TOC ratio in the samples of ITDD hospital wastewater, for the 6 first days of sampling, are depicted in figure 3. The COD/TOC ratio found in hospital wastewater range from 2.01 to 4.26. The difference could be an effect of the presence of inorganic substances in COD concentrations. The biodegradability of organic substances is a measure of the speed and completeness of its biodegradability by microorganisms (Sponza, 2003), and therefore the BOD₅/COD and COD/TOC ratios could be used to analyze the difficulty or not for organic substances to be degraded. In this study, the precise biochemistry knowledge of hospital wastewater, necessary to analyze the biodegradability by global parameters ratio, is not completed, because BOD₅ was not carried out. However, the data obtained from COD/TOC ratio, when compared with information reported in the literature on this issue, can be ranged in two groups: (i) COD/TOC from 2.01 to 3.00, and (ii) COD/TOC from 3.01 to 4.26. In the first group the organic substances would be degraded by microorganisms with out difficulties, but in the second group the substances would be difficult to degrade. In the future, it will be necessary to verify this analysis by comparing the COD/TOC ratio with the BOD₅/COD ratio.

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Figure 3: Variations of COD, TOC and COD/TOC ratio in hospital effluents of ITDD

11.6.2. Influence of chloride on AOX formation and toxicity on aquatic organisms

Organic halogen compounds are formed by reaction of chlorine with organic wastewater compounds. The hypothesis that sodium hypochlorite, used as elementary chlorine in disinfections of hospital wastewater of ITDD, is responsible for the disinfecting effect of AOX on aquatic organisms seems to be verified. In fact, the major mass carriers for the AOX in hospital effluents are most likely iodized Xray contrast media, solvents, disinfectants, cleaners and drugs containing chlorine. In general, the maximum contribution of drugs to the AOX is not above 11% (Kümmerer et al., 1998). Beyond that it is also known that the AOX concentration in the urine of patients not treated with drugs is very low. It is normally between 0.001 to 0.2 mg/L (Schulz and Hahn, 1997). Due to the dilution effect, no substantial contribution from this source is consequently expected (Kümmerer, 2001).

Previous studies on AOX presence in wastewater explained the formation of this "non-conventional" pollutants by the presence of organochlorine compounds (Seiss et al., 2001; Lôpez, 2003), and by the oxidation of bromide by hydroxyl radicals (von Gunten and Oliveras, 1998) or by the oxidation of iodized X-ray contrast media (Sprehe et al., 2001). Brominated organic compounds are negligible for the AOX in the hospital effluents (Kümmerer, 2001). The conditions of sampling taken for this study, i.e. choice of a wastewater collector which does not receive iodized X-ray contrast media effluents

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

form radiography department, lead to the conclusion that the AOX formation, in hospital wastewater from the infectious and tropical diseases department, is mainly due to the influence of sodium hypochlorite (chlorine). The lower chloride concentrations correspond to lower concentration of reduction of AOX formation. The effluent samples showing the higher chloride concentration were the same generating the higher AOX results and the most important acute toxicity on Daphnia magna.

11.6.3. Acute toxicity of physicochemical parameters of the ITDD effluents on the aquatic organisms

Fecal coliform populations of ITDD wastewater were affected because chloride values of hospital wastewater are greater than values generally uncounted in domestic wastewater. Although, hospital use and discharge (into sewer network) large amounts of water (U.S. EPA, 1989b; Leprat, 1998; Emmanuel et al., 2002) which allows the dilution of higher pollutant concentrations to lower values, it seems necessary to follow-up the behavior of microbial populations of urban wastewater treatment plants which receive these hospital effluents containing higher chloride and AOX concentrations.

Each of the two species responded differently to hospital wastewater samples. Acute toxicity on marine organism was performed using the MICROTOX test to study the inhibition of the bacteria bioluminescence, and acute toxicity on freshwater zooplankton was studied using Daphnia magna bioassay.

The ITDD effluents had an inhibition on the bacteria bioluminescence. It is difficult to attribute this toxicity to one or all the determined physicochemical parameters. The variation of toxicity on Vibrio fischery in all the samples did not have good correlations with chloride and AOX concentrations. Because the assay was realized only under 5 and 15 min of exposure, it seems that the toxic effects, of organochlorine compounds containing in hospital wastewater samples, were not completed. The sodium hypochlorite needs a contact time of 20 minutes to inactivate bacteria. Although in all cases the pH values in the samples were ranged from 7.9 to 8.8, the exposure time lower than 20 minutes is probably the cause of absence of a good correlation among the toxicity and the physicochemical concentration. Beside the pH conditions, it seems that 30 minutes of exposure time is the ideal contact time to evaluate toxicity of organochlorine containing in hospital wastewater using MICROTOX test.

Although the results of AOX concentrations in some cases were lower than the discharge regulation, the results of toxicity test of hospital wastewater on Daphnia magna indicated potential toxicity. Ail the samples demonstrated the presence of an acute toxicity of hospital effluents on aquatic organisms. A good linear regression was found between EC₅₀ results of toxicity on Daphnia magna and AOX assessed with r = 0.98, r² = 0,95, DW = 1.13, P = 0,0001 (figure 4). No significant difference was observed between the EC₅₀ Daphnia -- AOX correlation and the EC₅₀ Daphnia -- chlorides correlation (r = 0.98, r² = 0,95, DW = 1.75, P = 0,0001). The variations of EC₅₀ (UT)

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Daphnia, pH, chlorides and AOX concentrations of ITDD hospital wastewater are highlighted in figure 5.

The weight of TSS on the most toxic sample (EC₅₀ of 116.8 TU) was studied by passing sample through Giving a 0.45 pm pore size membrane and performing the Daphnia magna bioassay. The 24h EC₅₀ of filtered sample was 29.7 TU. It seems that TSS contributed in increasing toxicity of hospital wastewater on Daphnia magna approximately at 74.57%. Because, the application of NaOCI as elementary chlorine was made before the sedimentation and filtration processes of effluents, it allowed the formation of organochlorine compounds such as AOX resulting from chemical reaction between the organic matters in wastewater an chlorine.

Figure 4: Linear regression between EC₅₀ (UT) Daphnia and AOX concentrations

The results of this study on toxicological effects of NaOCI and its contribution to AOX formation in hospital wastewater allows to conclude that chloride could be used as indicator of the toxicity of hospital wastewater on aquatic organisms when NaOCI was used in elementary chlorine of wastewater collector which does not receive effluents from hospital radiography department. It seems necessary to verify in other studies, these first observations on toxicity of hospital effluents.

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Figure 5: Variations of EC₅₀ (UT) Daphnia, pH, chlorides and AOX concentrations
of ITDD hospital wastewater

The results of this study showed that the application of NaOCI in wastewater can reduce bacteria water pollution and give rise to toxicity effects on aquatic organisms. Low MPN ranging from <3 to 2400 for 100 mL were detected for fecal coliforms. Toxicity test using MICROTOX showed the toxicity of hospital wastewater on Vibrio fischeri, however bad correlations were observed between this toxicity and the concentrations of chloride and AOX. It seems that 30 minutes of exposure time is the ideal contact time to evaluate toxicity of organochlorine containing in hospital wastewater using MICROTOX test. The results of 24-h EC₅₀ on Daphnia magna demonstrated the high toxicity effect of hospital wastewater on these species. Some good correlation were observed between these toxicity results and the concentrations of chloride and AOX.

The identification of an ideal concentration of NaOCI in disinfecting hospital wastewater, i.e. its NOEC (Non Observed Effect Concentration) on algae and Daphnia magna, seems to be a research issue which could facilitate the control of AOX toxicity effects on aquatic organisms. Therefore, it would be necessary to follow-up at various dosages the biocide properties of NaOCI on fecal coliforms and its toxicity effects on aquatic organisms.

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Abe T., Saito H., Niikura Y., Shigeoka T., Nakano Y. Embryonic development assay with Daphnia magna application to toxicity of analine derivatives. CHEMOSPHERE, 45:487-495, 2001.

Addinsoft. Aide XlstatPro, XLStat version 5.0 (b8.3). Copyright 2000-2001, Addinsoft, Paris; 2001. AFNOR (Association Française de Normalisation). Qualité de l'eau, tome 2 : méthodes d'analyse 1, analyse organoleptique, mesures physico-chimiques, paramètres globaux, composés organiques. Recueil de normes françaises 1997, 2^e édition, Paris; 1997a.

AFNOR (Association Française de Normalisation). Qualité de l'eau, tome 3 : méthodes d'analyse 2, éléments majeurs, autres éléments et composés minéraux. Recueil de normes françaises 1997, 2^e édition, Paris; 1997b.

AFNOR (Association Française de Normalisation). Qualité de l'eau, tome 4 : méthodes d'analyse 3, analyse biochimique et biologique, analyse microbiologique, textes réglementaires. Recueil de normes françaises 1997, 2^e édition, Paris; 1997c.

AQUIRE (Aquatic Information Retrieval). EPA ERL-Duluth's Aquatic Ecotoxicology Data Systems. U.S. EPA (United States Environmental Protection Agency), Duluth, MN; 1994.

ASTM (American Society for Testing and Materials). Standard guide for conducting renewal life-cycle toxicity tests with Daphnia magna. In.: Annual Book of ASTM Standards, E 1193-94. ASTM, Philadelphia, 1994.

Banerjee S., Howard P.H., Rosenberg A.M., Dombrowski A.E., Sikka H., Tulis D.L. Development of a general kinetic model for biodegradation and its application to chlorophenols and related compounds. Envron Sci Technol, 18:416-422; 1984.

Baxter G. Chlorine disinfection -- the industry standard. In.: International Speciazed Conference: Disinfection of Potable Water. Kruger National Park, South Africa, 1998.

Bernet S., Fines M. Effluents du CHU de CAEN : Etude qualitative et quantitative de la flore microbienne et recherche de bactéries multirésistantes. Poster. Quatrième journée du Réseau Régional d'Hygiène de Basse-Normandie, Caen, 2000.

Boyce S.D., Hornig J.F. Reaction pathways of trihalomethane formation from the halogenation of dihydroxyaromatic model compounds for humic acid. Environ. Sci. Technol., 17:202-211, 1983. Brondeau M.T., Falcy M., Jargot S., Miraval S., Protois J.C., Reynier M., Scheneider O. Eaux et extraits de Javel. Fiche toxicologique N° 157 de l'INRS. Cahiers de notes documentaires -- Hygiène et sécurité du travail, 178:11-15, 2000.

Bulich A.A. Use of luminescent bacteria for determining toxicity. In.: ASTM STP 667 Aquatic Toxicity, Philadelphia, 1979.

Carey J., Cook P., Giesy J., Hodson P., Muir D., Owens W., Solomon K. Ecotoxicological risk assessment of the chlorinated organic chemical. In.: Carey J., Cook P., Giesy J., Hodson P., Muir D., Owens W., Solomon K., (ed) Workshop on Environmental Risk Assessment for Organochlorine Compounds. SETAC (Society of Environmental Toxicology and Chemistry), Pensacola-Florida, 1998.

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

CCLIN (Comité de Coordination de Luttes contre les Infections Nosocomiales). Élimination des effluents liquides des établissements hospitaliers -- Recommandations. Institut Biomédical des Cordeliers, Paris, 1999.

Cooney J.D. Freshwater tests. In.: Rand G.M. (Ed.). Fundamentals of aquatic toxicology: Effects, environmental fates and risks assessment. Second ed., Taylor & Francis, Washington DC, 1995. Deloffre-Bonnamour N. Les rejets des établissements de santé : des effluents liquides aux déchets solides. Mémoire de Maîtrise, Université Claude Bernard-Lyon1, Institut Universitaire Professionnalisé, Génie de l'Environnement--Ecodéveloppement, Lyon; 1995.

Emmanuel E., Blanchard J.-M., Keck G., Perrodin Y. Caractérisation chimique, biologique et écotoxicologique des effluents hospitaliers. Déchets Sciences et Techniques, revue francophone d'écologie industrielle, 22:31-33; 2001.

Emmanuel E., Perrodin Y., Keck G., Blanchard J-M., Vermande P. Effects of hospital wastewater on aquatic ecosystem. Proceedings of the XXVIII Congreso Interamericano de Ingenieria Sanitaria y Ambiental. Cancun, México, 27-31 de octubre, 2002. CDROM.

Erlandsson B., Matsson S. Medically used radionucides in sewage sludge. Water, Air, and Soil Pollution, 2:199-206; 1978.

European Commission. Directive 98/15/EEC amending Council Directive 91/271/EEC: Urban wastewater treatment. Brussels; 1998.

Finney D.J. Probit analysis. 3r^d ed. Cambridge Univ. Press, Cambridge, 1971.

Gartisser St., Brinkler L., Erbe T., Kümmerer K., Willmund R. Contamination of hospital wastewater with hazardous compounds as defined by § 7a WHG. Acta hydrochim. Hydrobio 24, 2:90-97; 1996. Gray S.R., Becker N.S.C. Contaminant flows in urban residential water systems. Urban water, 4:331346; 2002.

Halling-Sorensen B. Occurrence, fate and effects of pharmaceutical substances in the environnement -- A review. CHEMOSPHERE, 36:357-393; 1998.

Hanazato T. Growth analysis of Daphnia early juvenile stages as an alternative method to test the chronic effect of chemicals. Chemosphere, 36:1903-1909; 1998.

Jehannin P. Caractérisation et gestion des rejets liquides hospitaliers -- Etude particulière de la situation du C.H. de Hyères (Var). Mémoire de fin d'études, Ecole Nationale de la Santé Publique, Rennes; 1999.

Jokela J.K., Eloma E., Salinoja-Salonen M.S. Adsorbable organic halogens (AOX) in drinking water and aquatic environment in Finland. J Water Supply Res. Technol, 41:4-11, 1992.

Jolibois B., Guerber M., Vassal S. Glutaraldehyde in hospital wastewater. Arch. Environ. Contam. Toxicol. 42:137-144; 2002.

Kinnersley D. Discharge consent and compliance policy: a blueprint for the future. NRA (National Rivers Authority) Water Quality Series, No. 1, Bristol; 1990.

Kümmerer K. Drugs in the environment : emission of drugs, diagnostic aids and disinfectants into wastewater by hospitals in relation to other sources -- a review, Chemosphere 45:957-969; 2001.

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Kümmerer K., Meyer M., Steger-Hartmann T. Biodegradability of the anti-tumour agent Ifosfamide and its occurrence in hospital effluents and communal sewage. Wat. Res., 11:2705-2710, 1997. Kümmerer K., Gartiser St., Erbe T., Brinkler L. AOX-emissions from hospital into municipal wastewater. Chemosphere, 36:2437-2445, 1998.

Leprat P. Les rejets liquides hospitaliers, quels agents et quelles solutions techniques ? Troisième journée du Réseau Régional d'Hygiène de Basse-Normandie, Caen, 1998.

Lopez A., Mascolo G. Ciannarella, Tiravanti G. Formation of volatile halogenated by-products during chlorination of isoproturon aqueous solutions. Chemosphere, 45:269-274; 2001.

Lôpez M.C.C. Determination of potentially bioaccumulating complex mixtures of organochlorine compounds in wastewater: a review. Environment Int. 28:751-759; 2003.

Mansotte F., Jestin E. Les rejets liquides des établissements de santé : Caractérisation à la source et impact sur l'environnement marin côtier. Direction Départementale des Affaires Sanitaires et Sociales de la Seine Maritime, Agence de l'Eau de la Seine Normandie, Nanterre, 2000.

Marhaba T.F., Washington M.B. Drinking water disinfection and by-products: history and current practice. Adv. Environ. Res. 2:103-115; 1998.

MATE (Ministère de l'aménagement du territoire et de l'environnement). Arrêté du 2 février 1998 relatif aux prélèvements et à la consommation d'eau ainsi qu'aux émissions de toute nature des installations classées pour la protection de l'environnement soumises à autorisation. 3.0 (Journal Officiel) n° 52 du 3 mars 1998 page 3247, Paris.

Metcalf & Eddy, Inc. Wastewater Engineering: Treatment, disposai, and reause. 3^1t1 ed. Revised by Tchobanoglous G., Burton F.L. Irwin/McGraw-Hill, New York, 1991.

NTP (National Toxicology Program). Toxicology and carcinogenesis studies of chlorinated water (CAS NOS 7782-50-5 and 7681-52-9) and chloraminated water (CAS No. 10599-90-3) (Deionized and charcoal-filtrered) in F344/N rats and B6C3F1 mice (drinking water studies). U.S. Dept. of Health and Human Services, National Institutes of Health, Research Triangle Park, NC, 1992; NTP TR 392.

OECD (Organization for Economic Cooperation and Development). Daphnia sp. Acute immobilization test and reproduction test. In. : OECD Guidelines for testing of chemicals, 211. OECD, Paris; 1984. Perrodin Y. Proposition méthodologique pour l'évaluation de l'écotoxicité des eflluents aqueux : Mise au point d'un Multi-Test Macroinvertébrés (M.T.M.) -- Application aux lixiviats de décharges et à leurs composants caractéristiques. Thèse de doctorat. Institut National des Sciences Appliquées de Lyon; 1988.

Richardson M. L., Bowron J. M. The fate of pharmaceutical chemicals in the aquatic environment. J. Pharm. Pharmacol. 37:1-12; 1985

Salinoja-Salonen M.S., Jokela J.K. Measurement of organic halogen compounds in urine as an indicator of exposure. Scand J Workplace Environ Health, 17 :75-78; 1991.

Schowanek D., Fox K., Holt M., Schroeder F., Koch V. GREAT-ER: a new tool for management and risk assessment of chemicals in river basin. Water Sci. Technol. 43:179-185, 2001.

Seiler H.G., Sigel H., Sigel A. Handbook on toxicity of inorganic compounds. Marcel Dekker, Inc., New York, 1988.

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Schulz S., Hahn H.H. Der kanal als reactor-undersuchugen zur AOX-Bildung durch wirkstoffe in reinigunsmittenln. gwf Wasser Abwasser, 138:109-120; 1997.

Seiss M., Gahr A., Niessner R. Improved AOX degradation in UV oxidative wastewater treatment by dialysis with nanofiltration membrabe. Wat. Res., 13:3242-3248; 2001.

Sponza D.T. Application of toxicity tests into discharges of the pulp-paper industry in Turkey. Ecotoxicology and Environmental Safety, 54:74-86; 2003.

Sprehe M., GeiBen S.-U., Vogelpohl A. Traitement of AOX-containing wastewater from hospitals -- Degradation of iodized X-ray contrast medium. Korrespondenz Abwasser, 46, 4:548-558; 1999.

Sprehe M., GeiBen S.-U., Vogelpohl A. Photochemical oxidation of iodized X-ray contrast media (XRC) in hospital wastewater. Water Science and Technology, 5:317-323; 2001.

Stokinger H.E. The halogens and the nonmetals boron and silicon. In: Clayton G.D., Clayton F.E., Eds. Patty's industrial hygiene and toxicology, 3`^l ed., Vol 2B., John Wiley & Sons, New York; 1981.

U.S. EPA (United States Environmental Protection Agency) Drinking water criteria document for chlorine, hypochlorous acid and hypochlorite ion. Environmental Criteria and Assessment Office, U.S. EPA, Cincinnati, OH; 1989a.

U.S. EPA (United States Environmental Protection Agency) Chemical summary for chlorine. Office of pollution prevention and toxics, U.S. EPA, Cincinnati, OH; 1994a. EPA 749-F-94-010a.

U.S. EPA (United States Environmental Protection Agency) Reportable quantity document for chlorine, draft. Environmental Criteria and Assessment Office, U.S. EPA, Cincinnati, OH; 1994b.

U.S. EPA (United States Environmental Protection Agency) Preliminary data summary for the hospitals point source category. Office of water regulations and standards, Office of water, U.S. EPA, Washington, D.C., 1989b. EPA 440/1-89/060-n.

Verschueren K. Handbook of environmental data on organic chemicals. Third ed., Van Nostrand Reinhold Company Inc., New York, 1996.

Von Gunten U., Hoigné J. Bromate formation through ozone and hydroxyl radicals. In.: Minear R.A., Amy G.L. (Eds.) Desinfection by-products in water treatment. Lewis Publisher, Boca Raton, 1995.

Von Gunten U., Oliveras Y. Advanced oxidation of bromide containing waters: bromate formation mechanism. Environ. Sci. Technol. 332:63-70, 1998.

White G.C. Handbook of chlorination. 2nd edition, Van Nostrand Reinhold Company Inc., New York;

Chapitre VI Etude spécifique sur le devenir de deux désinfectants largement utilisés dans les hôpitaux : l'hypochlorite de sodium et le glutaraldehyde

Evaluation des risques sanitaires et écotoxicologiques liés aux effluents hospitaliers

CHAPITRE VI ETUDE SPECIFIQUE SUR LE DEVENIR DE DEUX DESINFECTANTS LARGEMENT UTILISES DANS LES HOPITAUX : L'HYPOCHLORITE DE SODIUM ET LE GLUTARALDEHYDE

I. Introduction

II. Toxicological effects of sodium hypochiorite disinfections on aquatic organisms and its contribution to AOX formation in hospital wastewater