OJEpi Open Journal of Epidemiology 2165-7459 Scientific Research Publishing 10.4236/ojepi.2021.114033 OJEpi-112999 Articles Medicine&Healthcare Characterization of Four University Hospitals Wastewater in Cotonou, Benin Ghislain Emmanuel Sopoh 1 Cyriaque Degbey 2 Judicaël Todedji 1 Fidèle Suanon 3 Arouna Yessoufou 3 Ferdinand Goudjo 4 Jean Wilfried Hounfodji 5 Daouda Mama 3 Justin Somadje 3 Regional Institute of Public Health, Department of Environmental Health, University of Abomey-Calavi, Ouidah, Benin Laboratory of Applied Hydrology, University of Abomey-Calavi, Cotonou, Benin Laboratory of Theoretical Chemistry and Molecular Spectroscopy, Faculty of Science and Technology, University of Abomey-Calavi, Cotonou, Benin Laboratory of Inorganic Chemistry and Environment, Faculty of Science and Technology, University of Abomey-Calavi, Cotonou, Benin University Clinic of Hospital Hygiene, National University Hospital Center Hubert Koutoukou Maga, Cotonou, Benin 11 10 2021 11 04 402 419 23, August 2021 5, November 2021 8, November 2021 © Copyright 2014 by authors and Scientific Research Publishing Inc. 2014 This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Introduction: Because of the specificity of their care activities, hospital wastewater contains various contaminants such as germs, disinfectants, metals, pharmaceuticals and chemical reagents, potentially infectious or toxic, which can be harmful to living organisms, or create ecological damage. This study aimed to analyze the quality of wastewaters produced by university hospitals of Cotonou in Benin. Methods: We conduct an analytical cross-sectional study including 30 wastewater samples from selected hospitals. An analysis of variance was performed at the significance level of 0.05. Results: Microbiological analysis showed the presence of germs such as total coliform types, faecal coliforms, fecal streptococcus, staphylococcus, yeasts and Clostridium perfringens in hospital wastewater samples. There was a significant difference (p < 0.05) in the microbial loads of various germs, except staphylococcus, between hospitals. With regard to the physicochemical parameters, only the conductivity complied with the standards for the discharge of wastewater into the environment. A significant difference was noted between hospitals, for the electrical conductivity of wastewater (p < 0.05). The average levels of metals detected in the wastewaters were in line with Benin’s standards. There was no difference between the average concentrations of metals except for copper (p < 0.05). Conclusion: The wastewater produced by the university hospitals of Cotonou is of poor quality and therefore requires adequate prior treatment before discharge into the environment.

Germs Physicochemical Contaminants Hospital Wastewater Risks Benin 1. Introduction

Water is essential for the hygiene and functioning of hospitals [1]. The wastewater discharged by hospitals presents health and environmental risks because of the nature and the importance of the substances they contain [2]. Thus, hospital managers must keep great interest in a better treatment and management of wastewater. The management of hospital wastewater is a real problem in developing countries, due to the inexistence of wastewater treatment and purification stations [3]. This hospital wastewater is continuously discharged into natural receptacles such as the ocean and lake, despite the ecotoxicity of 5 to 15 times greater than urban’s [4]. Despite the ignorance of the quality of the wastewater produced by hospitals in Benin, they are continually discharged into Lake Nokoué and sea. It is of utmost importance to know the current quality of the wastewater discharged into environments [5].

Wastewaters from hospitals contain potentially pathogenic micropollutants. The presence of substances in the environment is considered an emerging subject due to uncertainties related to the risk that they represent for terrestrial and aquatic ecosystems [6] [7]. Despite the health and environmental risks of hospital wastewater, there are almost no regulations on their treatment before discharge into the environment. The World Health Organization (WHO) document “safe management of healthcare waste” [8] is the only existing directive concerning hospital wastewater. In Benin, there is no specific directive concerning the management of hospital wastewater, but decree n˚ 2001-109 of April 4, 2001 defines the standards quality for wastewater applications in the country and the procedures for discharging wastewater [9].

The rejection of hospital wastewater in the environment without prior treatment has become a subject of global concern in the fields of the environment and public health and arouses the interest of scientists and public authorities. In recent decades, the scientific community has focused on the biological, physical and chemical characteristics of hospital wastewater in order to assess the potential risks associated with their discharges into aquatic environments. Pollutants such as total and fecal coliforms, chemical residues (detergents), pathogens (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella and Vibrio) and potentially toxic metals (cadmium, copper, cyanide, iron, gadolinium, nickel, lead, platinum, zinc, phenol, etc.) have been quantified in hospital wastewater. The chronic exposure of aquatic organisms to these substances explains the emergence of various phenomena such as hormonal imbalances, resistance to antibiotics and all other harmful impacts on the environment [8] [10].

The discharge of untreated hospital wastewater into the surface waters in Benin poses ecotoxicological risks and threatens public health since the pollutants from these wastewaters can enter the food chain through consumed fish products [5]. It, therefore, becomes important to know the quality of the wastewater produced in university hospitals which are the greatest producers of hospital wastewater. Hence, this study aimed to assess the microbiological, physicochemical and toxicological quality of the wastewater from university hospitals in Cotonou.

2. Material and Methods 2.1. Study Sites

The study was conducted in four of the six university hospitals of the city of Cotonou, in the Littoral department of Benin: the National University Hospital “Hubert Koutoukou MAGA” (CNHU-HKM), the University Hospital “de la Mere et de l’enfant” (CHU-MEL), the Army Instruction University Hospital of Cotonou (HIA-CHU); and the District University Hospital of Suru Léré (CHUZSL). The university hospitals are national referral centers in the health system, and training setting for health professionals. These four centres were chosen because of the diversity of disciplines, and the medical and technical services available as well as because of the variety of pathologies treated there, at contrary to the other two centres which, rather, are specialized.

The CNHU-HKM is the most important health establishment in Benin. It offers referral care in several surgical and medical specialties, and various diagnostic exploration specialties. It has a competent medical team. The CNHU-HKM has an activated sludge wastewater treatment station with a capacity of 6000 m3, with an initial design flow of 350 m3 day, which unfortunately is no longer functional.

The CHU-MEL is a referral hospital in the fields of pediatrics and gynecology-obstetrics. It includes neonatal, pediatric, pediatric surgery, gynecology and obstetrics, as well as diagnostic exploration services (biology, radiology). The CHU-MEL has a wastewater treatment station and uses the septic tank system. The treatment station receives part of the wastewater produced by the various services.

The CHUZ-SL’s mission is to meet the health needs of the populations of its coverage area and elsewhere. It includes emergency departments, pediatrics, general medicine, surgery, maternity, ophthalmology, physiotherapy and diagnostic exploration services.

The HIA-CHU of Cotonou is under the supervision of the Directorate of Army Health Services (DSSA) attached to the Ministry of Defense. It is also a referral hospital in Cotonou.

The CHUZ-SL and the HIA-CHU do not have a treatment station. Wastewater and sewage are drained to septic tanks, designed to collect, settle and digest, by anaerobic fermentation, organic matter contained therein. Septic tanks, generally made up of three compartments, are made of concrete, inserted into the ground.

 2.2. Study Type

This is an analytical cross-sectional study involving 30 samples from hospital wastewater treatment station and septic tanks.

 2.3. Sampling Method and Technique

The health services whose septic tanks were sampled were selected using a reasoned and non-probabilistic technique. The wastewater collected were that of health services at high risk of infection. The 30 hospital wastewater samples were collected from the four hospitals included in the study. The number of samples in each hospital was proportional to the number of septic tanks receiving wastewater from the health services.

 2.4. Collection and Transport of Samples

The samples were collected in 500 mL pre-sterilized glass bottles for microbiological analysis and in 1.5 L plastic bottles for physico-chemical and toxicological analyses. Before being filled, on-site, the bottles were washed three times with the wastewater to be collected. They were then filled to the brim and then closed with a screw cap to avoid any gas exchange with the atmosphere. They were transported in coolers fitted with frozen accumulators at a temperature between 2˚C and 8˚C to keep the samples cool, preserve them from light, and thus, prevent them from undergoing biological, physical or even chemical transformations. The analyses were carried out within 48 hours after the sampling.

 2.5. Data Processing and Analysis

The laboratory analyzes were carried out at the Public Health Research and Expertise Laboratory of Hospital Hygiene service of the CNHU-HKM.

2.5.1. Microbiological Analysis

The microbiological analysis aimed to identify and enumerate the total coliforms, fecal coliforms, fecal streptococcus, staphylococcus, yeasts and clostridium. The analyses were proceeded by the membrane filtration method. These microorganisms were identified and counted by filtering 100 mL of the samples on a 0.45 μm in diameter pores membrane. The membranes were then placed on selective media for a defined temperature and time (37˚C and 44˚C for 24 hours to 48 hours in a thermostated controlled oven immediately after filtration) depending on the type of germs sought. The specific agar media used for the enumeration of coliforms are Eosin Methylene Blue (EMB), bile esculin azide agar (BEA) for fecal streptococcus, chapman medium for staphylococcus; Tryptone Sulfite Neomycin (TSN) medium for Clostridium perfringens. Mushrooms and yeasts were searched by culture on sabouraud + chloramphenicol. The dishes were incubated at 25˚C or at room temperature for 3 to 7 days.

Additional tests were done to determine the type of bacteria. The API 20E gallery was used for coliforms and gram-negative bacilli isolated on EMB while the DNase tests, catalase, coagulase were carried out for cocci isolated on Chapman medium.

 2.5.2. Physico-Chemical Analysis

The global pollution parameters were used to determine the physico-chemical quality of hospital wastewater: electrical conductivity, turbidity, biochemical oxygen demand after 5 days (BOD5), chemical oxygen demand (COD), suspended matter (SM), dissolved oxygen, total ammonium and phosphorus. They were analyzed according to the protocols recommended by Rodier et al. (2009) [11]. Electrical conductivity, dissolved oxygen and turbidity were determined in situ using a Multi 3320 WTW system. BOD5 was determined by the dilution and seeding method with the addition of allyl-thiourea according to standard NF EN 1899-1. The COD was determined according to the protocol described by standard NF T90-101. The assay was carried out by the potassium dichromate method. The suspended matter (SM) was determined by filtration through a fiberglass filter. The total ammonium and phosphorus were determined by the ascorbic acid method (Spectrophotometric method).

2.5.3. Toxicological Analysis: Analysis of Metals

The analysis of heavy metals consisted in finding in the wastewater the following elements: Lead (Pb), Cadmium (Cd), Iron (Fe), Copper (Cu), Zinc (Zn), Manganese (Mn), Arsenic (Ar), Total Chromium (Cr), Hexavalent Chromium (CHV), Nickel (Ni) and Colbat (CO). Heavy metals were analysed using a flame atomic absorption spectrophotometer, by Dithizone spectrometric method, after mineralization of the samples.

 2.6. Data Analysis

The results of the laboratory analyze were processed using a combination of statistical methods. A first statistical approach was based on a simplified analysis of the results of studied parameters (calculation of the average concentrations) in comparison with the standards of the WHO (2014) [12] and those of Benin [9]. A second approach was based on the analysis of variance with a factor (ANOVA) for each parameter. The mean comparison graphs with their 95% confidence interval (95% CI) were produced. This test was followed by the Bonferroni test to determine the significant differences between pairs of University Hospitals. The significance level used is α = 0.05. The various statistical analyzes were carried out with SPSS version 21 software.

3. Results 3.1. Microbiological Characterization

The results of microbiological analyzes of University Hospital’s wastewater in Cotonou are shown in Table 1 and Figure 1.

Average microbial loads (CFU/100mL) of university hospital’s wastewater in Cotonou, compared to the WHO standards
Variable NE Min Max Average Standard deviation WHO standards
TC * (105) 30 0.8 17.30 4.32 4.04 LS
FC* (105) 30 0.11 3.81 0.59 0.84 2 × 103
FS* (105) 30 0 28.00 4.32 5.61 LS
Sta (105) 30 0 6.24 1.31 1.74 LS
Clos* (105) 30 0 0.20 0.05 0.058 LS
yeasts * (105) 30 0 61.00 5.63 14.03 LS

*Significant difference; NE: Number of sample; Min: Minimum value; Max: Maximum value; TC: Total coliforms; FC: Fecal coliforms; FS: Fecal streptococcus; Sta: Staphylococcus; Clos: Clostridium; LS: Lack of standard; CFU: Colony forming unit.

Total and fecal coliforms were highlighted in all the samples taken. Total coliform concentrations range from 0.8 × 105 CFU/100mL to 17.3 × 105 CFU/100mL with an average of 4.32 × 105 CFU/100mL. The concentrations of fecal streptococcus vary from 0 CFU/100mL to 28 × 105 CFU/100mL with an average of 4.32 × 105 CFU/100mL. Fecal coliforms are less abundant in the wastewater analyzed. Their concentrations varied from 0.11 × 105 to 3.81 × 105 CFU/100mL with an average of 0.59 × 105 CFU/100mL. Microbial loads varied significantly between hospitals (p < 0.05) except for staphylococcus, which was found to be insignificant (p > 0.05).

Microbiological analyses showed the presence of germs of total coliform types, fecal coliforms, fecal streptococcus, staphylococcus, yeasts and Clostridium perfringens in hospital wastewater.

The average concentrations of staphylococcus obtained at CNHU-HKM were higher than those obtained in the other three centers (CHU-MEL, HIA-CHU, CHUZ-SL). The hospital wastewater of the CHU-MEL had a higher average microbial load of fecal streptococcus than those of the other CHU with a significant difference between the microbial loads of the CHU-MEL and HIA-CHU on the one hand and between those of the CHU-MEL and the CHUZ-SL on the other hand (p < 0.05). The microbial loads of fecal coliforms varied significantly between the CNHU-HKM and the three other hospitals (p < 0.05).

 3.2. Physico-Chemical Characterization

The average concentrations of the physicochemical parameters of the four CHUs are presented in Table 2.

The results of analyzes were compared to the WHO standard and those adopted in Benin. Apart from the conductivity and the dissolved oxygen, all the other parameters values were higher than the standards defined for wastewater to be discharge in the environment. The average of EC is significantly lower than the standards, when the DO average is above the standards, even not significantly different from the standards.

Results of physico-chemical analyzes of wastewater (mg∙L<sup>−1</sup>) of university hospital wastewater in Cotonou, compared to the WHO and Benin standards
Variable NE Min Max Average Standard deviation WHO and Bénin standard
EC* 30 295.0 3210 1310.33 918.60 <2000
Turbidity 30 4.90 198.1 74.14 57.20 <5
SM 30 9.00 264 98.83 73.0 35a
DO 30 0.24 4.20 1.43 1.30 >5
BDO5 30 1.97 470.00 98.14 132.31 <25*
COD 30 5.33 874.20 181.14 251.12 <125*
Am 30 0.10 494.52 66.63 123.22 <0.2
TP 30 5.00 300.0 42.32 62.16 <10

*Significant difference; aBenin standard only; NE: Number of samples, Min: Minimum value; Max: Maximum value; EC: Electrical conductivity; SM: Suspended matter; BOD5: Biochemical oxygen demand; COD: Chemical oxygen demand; DO: Dissolved oxygen; Am: Ammonium; TP: Total phosphorous.

A significant difference was noted between the hospitals, for the electrical conductivity (p < 0.05). No significant difference (p > 0.05) was observed between the other global pollution parameters (Figure 2).

The results showed that hospital wastewater from CHUs were moderately mineralized and had very high organic pollution. The EC of the wastewater varies between 295 μS/cm and 3210 μS/cm, with an average of 1310.33 ± 918.60 μS/cm. The wastewater had high levels of nutritive salts, including nitrogenous compounds, with contents of 0.102 mg/L to 494.52 mg/L and an average of 66.63 mg/L for Am (Figure 2).

The average EC (2361.63 μS/cm) of the CHUZ-SL wastewater was higher than that of the three other Hospitals with a significant difference (p < 0.05) between the values obtained at the CHU-MEL and those of the HIA-CHU. The EC values of CNHU-HKM, CHU-MEL and HIA-CHU were 974.50 μS/cm, 743.63 μS/cm and 1018.17 μS/cm, respectively. The wastewater of the CNHU-HKM presented higher values of turbidity, BOD5, and COD followed by HIA (BOD5 =135.31; COD =256.33) which indicates an important organic pollution compared to the other centers, but with no significant difference (p > 0.05).

The wastewater of HIA-CHU and CHUZ-SL had a slightly high MES, Am and TP values compared to the other two CHUs but with no significant difference

(p > 0.05).

 3.3. Toxicological Characterization

The average heavy metal concentrations of the four CHUs are presented in Table 3.

The average concentrations of the heavy metals observed in the wastewater were in conformity with the Benin standards for the discharge of wastewater into the environment.

The wastewater of the CHUs had globally a high concentration of copper. However, the one of the CNHU-HKM (2.76 mg/L) seems higher than that of the three other centers (1.89 mg/L at the CHU-MEL; 2.10 mg/L at the HIA-CHU, 1, 62 mg/L at CHUZ-SL), but without a significant difference (p > 0.05). The wastewater sampled at CHUZ-SL had a higher lead content (0.24 mg/L) than that of CNHU-HKM (0.17 mg/L), HIA-CHU (0.18 mg/L) and CHU-MEL (0.22 mg/L) but without significant difference (p > 0.05).

The average manganese concentrations in the CNHU-HKM wastewater were higher than those of the other CHUs and varied between 0.10 mg/L and 0.18 mg/L with an average of 0.14 mg/L. Those of CHU-MEL and HIA-CHU wastewater were approximately equal but not significant (p > 0.05). On the one hand, the manganese concentrations vary significantly between CNHU-HKM and CHUMEL (p < 0.05) and on the other hand between CNHU-HKM and HIA (p < 0.05).

There is a significant difference between the average concentration of Zinc at CNHU-HKM and those obtained at CHU-MEL, HIA-CHU and CHUZ-SL with a higher value obtained at CNHU-HKM. The mean concentration of cobalt at

Average value of the concentrations in mg/L of heavy metals in university hospitals wastewater compared to the WHO and Benin standards
Parameters NE Min Max Average Standard deviation Benin’s standard
Lead 30 0.006 0.49 0.21 0.12 1
Cadmium 30 0.001 0.28 0.08 0.08 1
Zinc 30 0.050 0.61 0.38 0.15 5
Iron 30 8.32 23.88 13.89 4.53 -
Cupper* 30 1.02 2.93 1.96 0.54 2.5
Manganese 30 0.050 0.19 0.12 0.04 -
Chromium 30 0.038 0.10 0.06 0.01 2.5
Nickel 30 0.001 0.62 0.02 0.11 2.5
Cobalt 30 0.002 0.53 0.07 0.10 -
CHV 30 0.020 0.38 0.04 0.06 0.1

DL = detection limit, *significant, CHV: Chromium hexa valent. There was no significant difference (p > 0.05) between the average levels in each hospital, except for copper (p < 0.05). Moreover, no trace of Arsenic was found in hospital wastewater studied.

CNHU-HKM (0.14 mg/L) and CHU-MEL (0.08 mg/L) were significantly higher than those obtained at HIA-CHU (0.07 mg/L), CHUZ-SL (0.04 mg/L) (Figure 3).

3.4. Correlations between Variables

Table 4 presents the correlation analysis between all the variables taken in pairs to determine the potential links between the variables and the possible sources of contamination.

Bivariate analysis of linear correlations provides information on the strength of associations between the parameters studied. Pearson correlation coefficients higher than 0.5 and significant (p < 0.05) are shown in bold at the table level.

3.4.1. Correlation between Microbiological Parameters

There were significant and positive correlations between total coliforms and fecal coliforms (r = 0.854), total coliforms and fecal streptococcus (r = 0.816), and between total coliforms and staphylococcus (r = 0.566). A significant and positive correlation was noted between fecal coliforms and fecal streptococcus (r = 0.814).

 3.4.2. Correlations between Physico-Chemical Variables

There were significant and positive correlations between turbidity and suspended solids (r = 0.806), with BOD5 (r = 0.789) and with COD (r = 0.795). A significant

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