Monitoring hospital wastewaters for their probable genotoxicity

In contemporary lifestyle, pharmaceutical products are increasingly and inevitably consumed. However, considerable amounts of these products are released in the environment and are responsible for aquatic pollution, including pollution of river environments, drinking water sources, and lakes. The rapid and the considerable spread of pharmaceutical-based pollution has become a real threat and is receiving significant interest worldwide.

There is growing concern regarding the toxic effects of pharmaceutical products on human health, because they are designed to elicit specific physiological functions in targeted regions of the human body at relatively low concentrations. Hospitals constitute the principal place where pharmaceuticals are consistently employed to treat diseases (Azuma 2018). Previous studies (Sdiri-Loulizi et al. 2010; Nasri et al. 2017a, 2017b; Souza et al. 2018) have shown that hospital wastewaters contain large amounts of pathogenic organisms, dangerous and persistent substances such as pharmaceuticals, radionuclides, solvents, disinfectants, and metals. More recently, the discharge of pharmaceutical residues, which may eventually be discharged into the aquatic environment, raises the question of their impact on human and environmental health. In fact, hospital effluent may release a significant amount of pollutants with teratogenic, carcinogenic, and mutagenic hazards (Jobling et al. 2002; Phillips & Foster 2008; Pérez-Alvarez et al. 2018). On the other hand, many studies have detected the presence of endocrine disrupting compounds and antibiotics causing genotoxic and cytotoxic effects and inducing much damage in the tissue of marine species (Zouiten et al. 2016; Nasri et al. 2017b; Tahrani et al. 2017, 2018). Data according to health hazards associated with long-term exposure to hospital wastewaters are scant. In fact, hospital effluents are generally discharged directly into the public sanitation network. Currently, there are no rules or limits on hospital wastewater treatment before discharging (Yilmaz et al. 2017).

Therefore, our study focused on the characterization of three different hospital effluents in Tunisia and the evaluation of their genotoxic effects. Genotoxicity and cytotoxicity assays were performed using Salmonella typhimurium TA104 and the hepatic tissue of an animal model periodically treated with wastewater.

Wastewater samples were collected from three Tunisian hospitals: Hedi Chaker University Hospital in Sfax, Tahar Sfar University Hospital in Mahdia, and the Regional Hospital of Gafsa, during the same period (March 2017).

The sampling points chosen received the highest amount of wastewater and collected discharges from different services including: general medicine, general surgery, intensive care units, maternity, gynecology, oncology, psychiatry, rheumatology, hematology, hepatic-gastroenterology, several radiology units, and laboratories. Wastewater samples collected were transported to the laboratory prior to determining the different physicochemical parameters and/or immediately stored at −20 °C until further analysis.

The vitotox test was carried out on genetically modified Salmonella typhimurium TA104 as previously described by Verschaeve et al. (1999). Briefly, an overnight pre-culture containing 100 μL of each strain, 625 μL of tetracycline (0.8 mg mL⁻¹), and 312.5 mL of ampicillin (8 mg mL⁻¹) were placed in two Erlenmeyer's flasks in a water bath (300 rpm, 36 °C) for 16 h. Next, the two Erlenmeyer's flasks were placed on ice for 15 min and 20 mL of mineral medium, 160 μL of previously cultured bacteria were added and stirred during 1 h in an appropriate water bath (300 rpm, 36 °C). These genox and cytox work-cultures were then ready for testing. A mixture of 2.125 mL of mineral medium, 350 μL of bacterial suspension, and 1 mL of the post-mitochondrial supernatant fraction S9 was prepared and transferred to a black 96-well microplate with 100 μL from each sample (diluted at ½ and ¼ in distillate water).

In the absence of S9, 1 mL of mineral medium was added. Positive controls 4-NQO (0.4 mg mL⁻¹) and B(a)P (0.8 mg mL⁻¹) were used in the presence and absence of S9, respectively. A microplate illuminometer was used to measure genotoxicity and cytotoxicity; the light was analyzed every 5 min over a 4-h time span. All the measurements were automatically performed between 60 and 240 min of incubation. The samples were added to the bacteria in the presence and the absence of the S9 mix (fraction obtained from the livers of aroclor-treated rats). The signal to noise ratio (S/N) was measured using the formula of Verschaeve et al. (1999): S/N = the light production of exposed cells divided by the light production of nonexposed cells for each measurement and for each strain separately. It was previously demonstrated that genotoxicity always takes place when the maximum S/N (genox)/maximum S/N (cytox) is higher than 1.5. Cytotoxicity is assumed when S/N in genox/cytox is lower than 0.8 (Verschaeve et al. 1999, 2012; Verschaeve 2002). This test shows DNA damage by emitting a readily detectable signal of bioluminescence after exposure to mutagenic factors.

Sixty adult male mice (weighing 180–200 g) were purchased from SIPHAT, Tunisia. The standard pellet diet was purchased from the Industrial Society of Rodents' Diet (SNA, Sfax, Tunisia). The animals were maintained under constant temperature (22 ± 1 °C) and humidity (50%), with diurnal lighting (12 h light/12 h dark). Mice were fed a standard laboratory diet and given free access to tap water. All animals were treated according to the local Institute Ethical Committee Guidelines for the Care and Use.

Animals were left for 2 weeks for adaptation, and then were randomly separated into ten groups of six mice in each group (Table 1) and effluents were administrated orally to all the groups by gavage during 28 consecutive days.

After 28 days of the administration of effluents, the mice were sacrificed by cervical decapitation. Liver tissues were excised immediately from the animals and stored in a liquid nitrogen container to avoid protein degradation and were used for molecular studies.

Qualitative damage to genomic DNA was estimated by agarose gel electrophoresis, as previously described by Kanno et al. (2004). DNA was extracted in duplicate from the liver with equal volumes of phenol-chloroform-isoamyl alcohol (25:24:1), and precipitated with twice the volume of ethanol. DNA samples (3 μg of DNA/lane) were electrophoresed on 1.4% agarose gel containing ethidium bromide (final concentration, 0.16 μg/ml), and the fragmentation was visualized under UV light.

The monitoring of water contamination for potentially genotoxic compounds represents a major concern for human health. This study provides important data to consider such a concern using samples from three hospital wastewaters and different toxicity bioassays.

All the wastewater samples showed a direct genotoxic effect (genox/cytox ratio >1.6) in a dose-dependent manner when tested in the absence of S9 mix (Figure 1). However, no genotoxic effect was observed when hospital wastewaters were tested in the presence of S9 mix. In fact, the genox/cytox ratio does not exceed 1.5 (Figure 2).

Liver DNA electrophoresis showed the hallmark of apoptosis, characterized by a mixed smearing and laddering of the DNA extracted from mice treated with 100% concentrations of the pharmaceutical effluent (lanes 4 and 8). Treated animals with 25% and 50% concentrations of the pharmaceutical effluent collected from Gafsa and Mahdia (lanes 2, 3, 6, and 7) and control group (lanes 1, 5, and 9) revealed an intact band (Figure 3).

Effluents collected from the hospital in Sfax caused DNA fragmentation in the livers of treated mice (lanes 10, 11, and 12) independently from the sample concentration.

Many studies have reported the important occurrence of micropollutants and toxic substances in hospital wastewaters (Emmanuel et al. 2009; Verlicchi et al. 2010). Nowadays, the detection and the quantification of these residues in hospital effluents require particular attention. These substances are potentially genotoxic and may cause harmful environmental effects even at very low concentrations (Kummerer et al. 2009; Toolaram et al. 2014). In a previous study, our team detected heavy metals and antibiotics in hospital wastewaters collected from three different regions in Tunisia (Nasri et al. 2017a).

It is evident now that prescription trends and patients' consumption are closely reflected by the pharmaceutical residues found in hospital effluents (Dorival-Garcia et al. 2013). The presence of high concentrated pharmaceutical products such as enrofloxacin, marbofloxacin, oxytetracycline, pipemidic, acid sulfamethoxazole, sulfonamides, acetaminophen, mefenamic acid, atenolol, carbamazepine, and caffeine was revealed.

Personal care products such as benaophenone-3 and benzotrizole were the most frequently detected in the three hospitals' effluents (results not shown). Effluents collected from the hospital in Sfax revealed the presence of ciprofloxacin, which may explain its highest genotoxicity (results not shown).

These observations confirm the fact that hospital effluents are the main source of heavy metals (e.g., Cr, Co, Ni, Cu, Zn, Cd, Pb, and Hg) release into the environment (Nasri et al. 2017a).

It is well known that hospital effluents, industrial effluents, and domestic wastewaters can contain pharmaceutical compounds, drugs, chemicals, and pesticides; detergents and disinfectants could also be detected in the wastewaters (Kummerer 2001).

The genotoxic activity of such mixtures has already been demonstrated in many previous studies (Hamer et al. 2000; Dizer et al. 2002; Durusoy & Kambur 2003; Zegura et al. 2009).

The genotoxic activity was generated by all the effluents tested, suggesting the presence of different genotoxic compounds' formation.

The hospital wastewaters; genotoxic response was not affected by the addition of S9 mix addition which is in agreement with other studies which showed that the putative genotoxins in surface waters and municipal wastewaters are primarily direct-acting, i.e., S9 addition does not advance the response and the municipal wastewaters having a low genotoxic response. However, we were in disagreement with Kittinger et al. (2015), where a toxic signal could only be found after addition of S9 mix.

Indeed, data provided by the conventional water quality indices and chemical analysis are not sufficient to confirm possible aftermaths and the numerous formed substances that can threaten human health (Dizer et al. 2002; Wang et al. 2011). Therefore, to detect changes in cellular organics, further histological assays should be performed, such as for the presence of micronuclei after exposure to different concentrations of wastewaters. Thus, chemical agents can induce micronuclei through spindle disturbance or chromosome breaks (Ventura-Camargo et al. 2011).

The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical process, which plays an important role in the development and maintenance of homeostasis in most of the multicellular organisms (Smaili et al. 2003). To detect the genotoxic and mutagenic assessment of xenobiotics, DNA fragmentation test is the most widely used because of its technical simplicity and rapidity (Krishna et al. 2000). Our study revealed that sub-acute exposure to different concentrations of the pharmaceutical effluent collected from Gafsa, Sfax, and Mahdia increased the DNA fragmentation in mice livers, a hallmark of apoptosis which joins previous observations in suggesting a link between genotoxicity and pharmaceutical effluent poisoning (Houk 1992; Snyder & Green 2001; Babatunde & Bakare 2006; Siddique et al. 2008; Adeoye et al. 2015).

This cellular process can be triggered by the activation of pro-inflammatory cytokines that ultimately culminates in the activation of the caspase family of proteases (Yano et al. 2007), or by activation of free radicals, either through pharmacodynamics, auto-oxidation, or enzyme-catalyzed oxidation of electrophilic molecules of the pharmaceutical effluent (Bakare et al. 2009). Indeed, reactive oxygen species, endogenously generated on exposure to the effluent, could induce lipid peroxidation in the tissues by reacting with the lipid content of the cell membrane, thereby causing breakage of the DNA chain by oxidating the base component of the membrane. In addition, the free radicals generated by effluent could also have reacted with those protein-enzymes involved in the DNA repair mechanism, the alteration of repair enzyme activity resulting in increased frequency in DNA damage (Marnett & Burcham 1993).

In conclusion, our study focused on the detection of heavy metals, antibiotics, and plasticizers in hospital wastewaters from three hospitals in Tunisia. The results obtained assessed the toxicological risk and pointed to cytotoxic and genotoxic hazards caused by hospital effluents. Although there was a low concentration, harmful damage was observed; therefore, wastewater treatment requires special attention. It is mandatory to acquire additional information about the quality of hospital effluents and environmental control programs must be designed for the pre-treatment of wastewater before its release into municipal sewage systems. Controlling wastewaters should start by reducing wastewater generation and by using natural biological processes to treat them. Bioremediation by microorganisms, algae, and aquatic organisms should be promoted for wastewater treatment.