In vivo toxicities of the hospital effluent in Mahdia Tunisia

Hospital effluent (HE) is one of the most important sources of pharmaceuticals released into the environment. This kind of pollution is a recognized problem for both human health and aquatic life. Consequently, in the present study, we assessed the effects of untreated hospital effluent on mice via biochemical and histopathological determinations. Female mice were given free access to water bottles containing untreated HE at different dilutions for 21 days. Then clinical biochemistry and histopathology evaluation were conducted. Serum biochemistry analysis showed the presence of significant increase in cholesterol, triglycerides, glycaemia and total bilirubin. However, phosphatase alkaline and urea activities have been significantly decreased compared to the control group. No significant variation was observed for the rest of the studied parameters (high-density lipoproteins; low-density lipoproteins and uric acid). Additionally, multiple alterations, including cellular necrosis, leucocyte infiltration and congestion, were observed in different tissues of mice exposed to the tested HE.


INTRODUCTION
Pharmaceuticals are a group of chemically active compounds used for diagnosis, treatment and prevention. They play an important role in the population's well-being.
Pharmaceutically active compounds (PhACs) are continuously released into the aquatic environment (Seifrtová et al. ) due to the increase of these drugs' consumption in human populations especially in industrialized countries (Mendoza et al. ). One of the most important sources of this type of pollution is hospital effluents (HEs), which are generated in high quantities (between 200 and 1,200 L bed À1 day À1 ) (Oliveira et al. ). Nevertheless, in most countries, these effluents are usually considered as domestic wastewaters and are, therefore, collected with municipal effluents without any pre-treatment and discharged into the received environment after conventional treatment in wastewater treatment plants (WWTPs) (Carraro et al. ). However, previous studies showed a low effectiveness of WWTPs, which are not designed to remove complex chemicals, namely pharmaceuticals (Verlicchi et al. b; Lindholm-Lehto et al. ). As a result, pharmaceutical compounds are detected ubiquitously in the environment (e.g., mussels, fish, vegetables and drinking water). Due to their pseudo-persistent character and depending on their physio-chemical properties, these compounds and/or their interaction products may pose a long-term toxic effect on the aquatic organisms (Santos et al. ) and consequently, the human body is the final target of these pollutants via the food chain. In Tunisia, there are no quality references or HE management methods imposed by the competent authorities, and thus, HEs are directly released into the sewerage system, and thereafter discharged into the waterbody after conventional treatment in WWTP. Recently, our research team has observed that wastewater coming from the university hospital of Mahdia (centre of Tunisia) contains high levels of pharmaceuticals ranging from a few nanograms to hundreds of micrograms as well as heavy metals (Nasri et al. ; Afsa et al. ). These compounds were also detected in the seawater, even after treatment in a municipal WWTP, which may affect negatively the aquatic environment and human health (Afsa et al. 

Effluent collection and physico-chemical analysis
Hospital untreated wastewater samples were collected from the Tahar Sfar university hospital located in Mahdia city into a sterile glass bottle and stored at 4 C during the study period. The studied characteristics of effluent were as follows: pH, BOD (biochemical oxygen demand), COD (chemical oxygen demand), TOC (total organic carbon), TSSs (total suspended solids) NO 3 À (nitrate) and AOX (absorbable organically bound halogens).

Histopathology and clinical biochemistry analyses
To investigate the histopathological changes in the tissues of HE-treated mice, slices of heart, livers and kidneys were fixed in 10% neutral buffered formalin. In brief, the tissues were dehydrated in the ascending order of ethyl alcoholwater concentrations, cleared in xylene and embedded in paraffin. Sections of 3-μm thickness were obtained using a rotary microtome. Prepared slides were stained with haematoxylin and eosin (H&E) and observed at 400× magnification under a light microscope by a trained pathologist.
For biochemical analysis, one millilitre of venous blood of each mouse was collected in plastic tubes. Blood sera were obtained by cooling centrifuge 3,000 rpm for 15 min at þ4 C and stored at À80 C prior to biochemical analysis.

Physico-chemical parameters of untreated HE
Physico-chemical parameters of HE are presented in Table 1. All the measurements were below the national allowable limits (NT 106.0021989).

Effect of untreated HE on body and organ relative weight
The total body weight has not significantly increased in the treated groups during the exposure period compared to the control group (Table 2). Organ relative weights have also been evaluated, and the obtained results have shown a significant decrease in the relative weight of heart in the group treated with HE diluted to 50% (Table 3). A mortality percentage of 17% was observed in treated groups with dilutions of 25 and 50%. There was no mortality found in the group treated with the dilution of 10% and the control group.

Effect of untreated HE on biochemical parameters
Compared to the control group, the exposure of mice to HE at different dilutions has induced a significant variation in some serum biochemical parameters, mainly in the 50% treated group. The obtained results have shown an increase in CHOL, TG, GLY and TB, whereas the values of URA and ALP have decreased (Table 4).

Histopathological assessment
Sections from the negative control showed normal tissues.
Histological changes were observed in the different treated Concerning heart sections, enlargement and disruption of connective tissue have been observed as well as leucocyte infiltration and necrosis ( Figure 3). Additionally, in the present study, we have detected cancerous cells in skin tissue of only one mouse exposed to HE diluted to 50% (Figure 4).

DISCUSSION
In this study, physico-chemical parameters were within the permissible limits of the Tunisian authorities (NT 106.0021989). Nevertheless, based on our previous results, HEs represent a major source of the release of pharmaceuticals and other contaminants into the environment (Nasri    The increase of glycaemia observed in our study could be related to the disruption of the glucose uptake at the cellular level, and it could also suggest a high-energy demand of organism metabolic pathways to cope with stressors. The elevated level of cortisol is one of the physiologically conserved responses to stress in vertebrates, which maintains an increased plasma glucose level to fuel the necessary energy and re-establish body homoeostasis. Previously, it has been reported that municipal wastewater perturbs cortisol response in fish (rainbow trout) (Ings et al. a, b).
Other authors have linked the disruption of these parameters by the impairment of the expression of liver metabolism key genes in exposed mice, which probably induce the alteration of glucose and lipid homoeostasis be related to cellular and tissue alterations. Thus, the histopathological study was conducted to better understand the effects of untreated HE in several organs of exposed mice.
As can be seen in Figures 1-3  These inhibitory effects of metals were associated with oxidative injuries in the liver and kidneys of exposed animals. The hepatotoxic effects of paracetamol may be related to its highly toxic P450 metabolite N-acetyl-p-benzoquinone imine (NAPQI), which may lead to hepatic necrosis at toxic doses (Kavitha et al. ). Histochemical analysis has shown that the β-blocker propranolol, at environmental con- those organs, although, in this study, we have also evaluated the impact of the HE on the heart tissue of mice. Under stress conditions, the heart increases blood flow as an adaptive response to enhance the body's capacities to get rid of invasion (Ings et al. a). Thus, the heart plays an important role in protecting the body; however, its function could also be affected and unbalanced by different environmental toxicants. Therefore, this organ could be a useful indicator, giving an insight into the state of toxicity. Interestingly, the obtained results showed leucocyte infiltration, necrosis, congestion, disruption and destruction of connective tissue. These results suggest that HE could influence heart tissue and its function. It is well known that increased levels of cholesterol could be considered as a risk factor of cardiovascular diseases (Zhang et al. ), and thus, the observed damage in heart tissue probably is linked to the disruption of lipid metabolism.
Additionally, histological abnormalities, such as cellular infiltration and necrosis, were observed in heart tissues of fish In the present study, we have detected sporadic cancerous cells in skin tissue, which may translate to the high genotoxicity of untreated HE. It is not surprising to obtain these results, since the same tested effluent has been recently reported to be highly cytotoxic and genotoxic in exposed rat

CONCLUSION
Based on our findings, the toxicity of HE cannot be ruled out even after a short (sub-chronic) exposure term. Consequently, more attention should be given to HE management by the concerned authorities. Given the high variability of HEs, future analyses should be undertaken to consolidate our results and to better understand the mechanism by which complex mixtures could negatively affect mammals (e.g, chemical analysis of HE, chronic exposure of mice, use of biomarkers of oxidative stress defence system and molecular biomarkers of DNA damage).

ETHICAL APPROVAL
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