Assessment of the genotoxicity of antibiotics and chromium in primary sludge and compost using Vicia faba micronucleus test

Ecotoxicology and Environmental Safety 185 (2019) 109693

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Assessment of the genotoxicity of antibiotics and chromium in primary sludge and compost using Vicia faba micronucleus test

T

Ahmed Khadraa, Eric Pinellib, Ezzariai Aminec, Oubane Mohameda, Georges Merlinab, Karim Lyamloulid, Lamfeddal Kouisnid, Mohamed Hafidia,d,∗ a

Laboratoire Ecologie et Environnement (Unité Associée Au CNRST, URAC 32), Faculté des Sciences Semlalia, Université Cadi Ayyad, BP 2390, Marrakech, Morocco EcoLab, Laboratoire Ecologie Fonctionnelle et Environnement, Université de Toulouse, CNRS, INPT, UPS, Avenue de l’Agrobiopôle, F-31326, Castanet-Tolosan, France c LBE, Université de Montpellier, INRA Narbonne, France d University Mohammed VI Polytechnic (UM6P), Agrobiosciences Program, Benguerir, Morocco b

ARTICLE INFO

ABSTRACT

Keywords: Dewatered primary sludge Co-composting Antibiotics Chromium Genotoxicity Agriculture

The objective of this study was to investigate chemical, biological and eco-toxicological parameters of a compost produced through the co-composting of dewatered primary sludge (DPS) and date palm waste to evaluate in which extent it can exploited as a bio-fertilizer. DPS and date palm waste were co-composted in aerobic conditions for 210 days. Physico-chemical parameters were evaluated during composting (total organic carbon, total nitrogen, pH, available forms of phosphorus). Furthermore, heavy metals (Cd, Cu, Cr, Pb, Ni, Zn) and antibiotics (fluoroquinolones, macrolides and tetracyclines) content were analyzed in the DPS. To evaluate the genotoxicity of substrates, Vicia faba micronucleus test was carried out. Single and combined toxicities of a mixture of antibiotics (ciprofloxacin, enroflxacin, nalidixic acid, roxithromycin and sulfapyridin) and chromium (Cr2 (SO4)3 and K2Cr2O7) were examined. Although the final compost product showed a significant decrease of the genotoxicity, almost 50% of the micronucleus frequency still remained, which could be explained by the persistence of several recalcitrant compounds such as chromium and some antibiotics. Overall, the presence of antibiotics and chromium showed that some specific combination of contaminants represent an ecological risk for soil health and ecosystems even at environmentally negligible concentrations.

1. Introduction In Morocco, the volume of sewage sludge produced annually is estimated at 18,000 tons of dry matter. The sewage sludge, which is a byproduct of treated wastewater, is considered as a source of organic matter and mineral nutrients that could be used to improve the soil fertility (Albiach et al., 2001; Garcia-Gil et al., 2004). However, the sewage sludge is also contaminated by organic and inorganic contaminants such as polycyclic aromatic hydrocarbons, phthalic acid, pharmaceutical compounds, and heavy metal (Golet et al., 2002; Amir et al., 2005a; Clarke and Smith, 2011; Alvarenga et al., 2015; Ezzariai et al., 2017). In the specific case of Marrakesh, the wastewater treatment plant (WWTP) receives both domestic wastewater as well as industrial wastewater generated by the tanning sector, which is the most important source of the industrial wastewater. Chromium sulphate (trivalent chromium Cr (III)) is the most widely used substance in the tanning sector (EL Fels et al., 2015; Shahid et al., 2017). All the used

salts in the tanning industry, especially the chromium salts, are evacuated with the wastewater stream toward the WWTP and they end up in the sludge process (Wionczyk et al., 2006). The WWTP of Marrakesh produces 140 ton/day (50.4 ton/day of primary sludge and 89.6 ton/ day of secondary sludge) (Ezzariai et al., 2017). All the produced sludge is evacuated directly toward a controlled landfill without any preliminary treatment. In addition, there is no valorization sector of the sludge by using a biological/physicochemical process and several industrial experiments still in progress. The presence of certain minerals, such as MnO2, could enhance the oxidation of Cr (III) to hexavalent chromium Cr (VI). At alkaline pH, Cr (VI) bioavailability, mobility and solubility in water is significantly increased, thus furthering its deleterious effect on living organisms, and more particularly at the level of cell membranes (Marchese et al., 2008). In the same manner, WWTP convenes also a significant portion of pharmaceutical compounds. For instance, antibiotics concentration in sewage sludge can fluctuate from few nanograms to up to 100 mg/kg (Ezzariai et al., 2018a). Antibiotics

∗ Corresponding author. Laboratoire Ecologie et Environnement (Unité associée au CNRST, URAC 32), Faculté des Sciences Semlalia, Université Cadi Ayyad, BP 2390, Marrakech, Morocco. E-mail address: [email protected] (M. Hafidi).

https://doi.org/10.1016/j.ecoenv.2019.109693 Received 21 May 2019; Received in revised form 14 September 2019; Accepted 17 September 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 185 (2019) 109693

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are inherently persistent as they are consistently released into the environment, which is mainly due to the development of antibiotic resistance properties by the microbial community, including pathogenic bactria (Daughton and Ternes, 1999; Hernando et al., 2006; Selvam et al., 2012; Kang et al., 2011). In the light of what has been stated, untreated sewage sludge, poses assuredly serious environmental hazards, with potential negative impact at the sanitary level. Thus, there is a need to provide potent solutions to overcome such threat. Composting could constitute an efficient waste management strategy. Indeed, sludge treatment through composting is used to alleviate organic compounds (Aguelmous et al., 2018), heavy metals concentrations (Amir et al., 2005) as well as pathogens (Jiménez, 2007). Even if several studies addressed the fate of these organic or inorganic pollutants after the sludge composting (Amir et al., 2005a; Hafidi et al., 2008; Ezzariai et al., 2018a, 2018b), the genotoxic effect of the final compost product has been scarcely investigated. However, the assessment of the genotoxic potential using genetic short-term bioassays, especially toxicity bioassays, has gained special attention during the last decades (Shahid et al., 2011; Khadra et al., 2012). Such assays are able to predict the genotoxic potential of pollutants by measuring damage caused to chromosomes and DNA. Plant assays are highly sensitive, quite easy to conduct, inexpensive, and good predictors of carcinogenicity (Panda and Panda, 2002). More specifically, the Vicia faba micronucleus test is very sensitive for the detection of both clastogenic and aneugenic effects for organic and inorganic pollutants (Iqbal, 2016). For instance, through using the Vica faba micronucleus test, EL Fels et al. (2015) observed that the toxicity of sewage sludge contaminated with Cr (VI) decreased after the composting. On another note, few studies were focused on the simultaneous influence of organic and inorganic pollutants in mixtures, which remain highly important as, toxico-dynamic properties of chemical compounds can interchange because of antagonistic or synergistic reactions between pollutants (Mochida et al., 2006; Oleszczuk, 2006; Khadra et al., 2012; Cedergreen et al., 2017). Bearing this in mind, the objectives of this study were (i) to assess the effects of DPS – palm waste co-composting on the genotoxicit, and (ii) to evaluate the genotoxicity associated to chromium and antibiotics using the Vicia faba micronucleus test.

aqueous extract was filtered through 45 μm filter (Millipore). The heavy metals were determined by the inductively coupled plasma optical emission spectroscopy (ICP OES Thermo IRIS Intrepid II XDL Duo). Antibiotics extraction and analysis were conducted according to Golet et al. (2002) with minor modifications (Khadra et al., 2019). Briefly, a Dionex ASE 200 with extracting solvent (50 mM aqueous phosphoric acid (pH 2.0) and acetonitrile (1:1, v/v)) was used. Analysis was carried out by 1200 Agilent LC system using SB-Zorbax C18 column coupled to tandem 6410A Agilent mass spectrometer. 2.4. Phytotoxicity assay Phytotoxicity test was conducted based on the seeds germination of cress (Lepidium sativum) and turnip (Brassica rapa) species. Twenty seeds were germinated in water-soluble extracts of composts (10 g/ 100 ml distilled water) in the dark at ambient temperature (25 °C) for 72 h. Three replicates were made for each sample. The phytotoxicity was assessed using the germination index (GI), which measures both the germination and the root growth (Cunha-Queda et al., 2007) based on the formula below:

%GI = (NGext×LRext)/(NGwater×LRwater)×100 NGext and NGwater refer the number of the germinated seeds in the aqueous extract and the distilled water respectively; LRext and LRwater refer to root length in the extract and in water treatments respectively. 2.5. Vicia faba micronucleus assay (MN assay) The MN assay was carried out according to Marcato-Romain et al. (2009) and Cotelle et al. (2015). Vicia faba seeds were soaked for 4 h in water. The seed coats were removed and seeds were germinated between two layers of moist cotton. After 4 days, the primary roots, having reached 2–3 cm in length, were selected for the MN assay. Before transplanting the Vicia faba seedlings to soil hydroponic conditions, the primary root tip was cut off (2 mm) to stimulate the emergence of secondary roots. Four days were necessary to obtain secondary roots of suitable length (1–2 cm) for the test. Exposure time was three days. The LUFA soil wetted with distilled water represent the negative control (NC), or the maleic hydrazide at a concentration of 10−5 M (1.12 mg L−1) after dilution in distilled water was used as positive control (PC). The moisture content was maintained at 2/3 of the water holding capacity by introducing water in the under cups. After treatment, root tips were fixed during one night in a carnoy solution, also called solution of farmer (glacial acetic acid/ethanol 1:3 v/v), which allows to fix the cells in metaphase, then rinsed with deonised water for 10 min and transferred into ethanol before the storage. Afterward, root tips were hydrolysed with 1 N HCl for 6 min at 60 °C and stained with 1% aceto-orcein for 3 min at 60 °C. Five slides were prepared, for each seed (one slide per root tip), and at least 1500 cells were counted per seedling. The MN frequency was obtained from at least six thousand cells per treatment. The interphase cells as defined by Ma et al. (1995) were scored for MN frequencies at 1000× magnification. The slides were observed under microscope to count the total number of cells, the number of cells in division (mitosis) and cells with micronucleus (Fig. 1). This counting makes it possible to calculate: mitotic index (MI) and micronucleus (MN) frequency as below

2. Materials and methods 2.1. Co-composting DPS produced from the WWTP of Marrakesh was co-composted with palm date waste in a pile (3.5 m3; v/v) for 210 days. The mixture was turned over every 15 days to maintain aerobic conditions. Sampling was carried out at various time of the composting process, namely at T0 (Before composting), T15 (after 15 days of composting), T30 (after 30 days of composting), T60 (after 60 days of composting), T90 (after 90 days of composting) and T120 (after 120 days of composting). 2.2. 2. Physico-chemical analysis The temperature was measured every day at different composting time with sensors equipped with data memory (PH 0700115 model 1.20, Ector-Traçability software, ECTOR France). The pH was measured in an aqueous extract of the compost at room temperature (1 g/10 ml of distilled water). The total organic carbon (TOC) and ash contents were determined after calcination in a muffle furnace at 600 °C for 6 h. The total Kjeldahl nitrogen (TKN) was evaluated in 0.5 g samples by using the classical Kjeldahl procedure.

MI(%) = Number of cells in mitosis× 100/total number of cells observed MI(%) = Number of micronuclei ×1000/total number of cells observed

2.3. Trace metals content and antibiotics analysis

In order to avoid underestimation of the MN frequency due to impaired cell proliferation rate, the MN test was performed only concerning roots tips with a mitotic index greater than 2% (Ma et al., 1995; Cotelle et al., 2015). For the statistical analysis, the Mann–Whitney U test was used to

The metal extraction from the DPS samples was conducted by using an aqua regia digestion (AFNOR (1995)). The DPS sample (1 g) was digested with a mixture of nitric acid and perchloric acid (1:1; v/v). The 2

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Fig. 1. Microscopic observation of tip root of Vicia faba of negative control (a) and positive control (b).

determine the significance level against the negative control in each experimental (Béraud et al., 2007; Cotelle et al., 2015). The direct contact method was applied for three concentrations (100, 50 and 30%) of the DPS and three stage of the sludge composting (0, 120 and 210 days). Five replicates were processed for each concentration. Antibiotics (ciprofloxacin, enroflxacin, nalidixic acid, roxithromycin and sulfapyridin) and chromium (Cr2(SO4)3 and K2Cr2O7) were used. Table 1 shows the physico-chemical parameters of the used antibiotics. Three different concentrations of antibiotics were tested, 0.04, 0.4 and 4 mg/kg respectively. Also, three different concentrations of Cr2 (SO4)3 (10, 100 and 200 mg/kg) and K2Cr2O7 (1, 10 and 100 mg/ kg) were evaluated. The concentrations of antibiotics and chromium were predetermined according to previous literature studies (Golet et al., 2002; Shahid et al., 2017; Ezzariai et al., 2018a). The nominal concentrations of antibiotics and chromium are shown in Table 2. For all the experiments, the mitotic index was greater than 2%, and no significant difference, with the control treatment was observed. The final concentration of methanol was less than 0.05%, without any genotoxic effect (data not shown).

Table 2 Nominal concentration of mixture of MA combined with two type of chromium.

Quinolones Fluorquinolones Macrolides Sulfonamide

Nalidixic acid Ciprofloxacin Enrofloxacin Roxythromycin Sulfapyridine

232 331 359 836 249

5.95 6.38–5.9 6.27 8.8 8.4

0.47 0.4 1.1 2.75 0.35

Cr2(SO4)3 10

MA 0.2

K2Cr2O7 1

MA 0.2

Cr2(SO4)3 10

K2Cr2O7 1

3.3. Phytotoxicity test

Table 1 Physical-chemical characteristics of antibiotics used in experiment. Log KOW

MA 0.2

M3

Fig. 2 shows the total content of heavy metals in DPS. Compared to the French standard (AFNOR, 1996), the heavy metal content in the compost are very low except for chromium (a concentration of 1000 mg/kg DW was observed). Total chromium concentration is for instance higher than the one obtained in composted sludge (EL Fels et al., 2015) and tannery composting (Shukla et al., 2009). According to the previous results of EL Fels et al. (2015), the primary sludge coming from the WWTP of Marrakesh was windrowed with palm waste to conduct a composting experiment with the same ratio and in the same composting platform. Results shown that no significant removal of total chromium was observed after the composting process. In a previous study, Khadra et al. (2019) analyzed the presence of antibiotics in the DPS of the WWTP of Marrakesh and their fate during co-composting. Antibiotics were detected in the DPS with concentrations of 2.9–4.2 μg/Kg DM for fluoroquinolones, 0.20 for ampicillin, 1–2 μg/kg DM for the macrolides and 0.2 for tetracyclines. After composting, a significant removal of 46%, 40% and 35% was observed for ampicillin, macrolides and tetracyclines respectively. On the other hand, fluoroquinolones (ciprofloxacin and ofloxacin) were not removed significantly. Generally, the evacuated antibiotics toward the WWTP are coming from some pharmaceutical laboratories or after its using in the human medicine. Nevertheless, these concentrations are very low compared to those reported in other studies (Golet et al., 2002; McClellan and Halden, 2010).

The evolution of the physico–chemical parameters during the cocomposting is shown in Table 3. The thermophilic stage is characterized by a rise in the temperature, which peaked at 67 °C. The temperature increase is due to the microbial activity resulting from the organic matter degradation (Barje et al., 2012). The pH increased slightly and stabilized after 210 days of maturation at 7.9 until the end of the cocomposting. The pH increase is mainly due to the degradation of the organic acids and the release of ammonia, while its decrease is due to the nitrifying process or the synthesis of the phenolic compounds (Liu et al., 2012). After 210 days of composting, the TOC value decreased from 34.4% to 26.7% which is related to the organic matter degradation. At the end of the co-composting process, the TN slightly increased (2.3%), which is attributed to carbonaceous degradation (Bernal et al., 1998). Ultimately, upon co-composting achievement, decomposition rate of the organic matter reached 55.3%, and C/N ratio drawn close to 11. These

pKa

Compounds Concentrations (mg/Kg)

M2

3.2. Concentration of heavy metals and antibiotics in DPS and during composting

3.1. Evolution of the physico-chemical parameters during the co-composting

MW

M1

results are in agreement with the literature and confirmed the maturity of the final compost product (Jouraiphy et al., 2008; Barje et al., 2012; El Fels et al., 2014; Ezzariai et al., 2017).

3. Results and discussion

Antibiotic

Mixtures

The phytotoxicity test is a common assay used routinely to evaluate the compost maturity and its phytotoxicity (Miaomiao et al., 2009). On the other hand, the germination index is a sensitive parameter indicating both low toxicity affecting root growth and high toxicity affecting germination (Zucconi et al., 1985). For both cress and turnip a low germination index (GI) of 8.1% and 16.4%, respectively was recorded for DPS (Table 4). In this specific case, DPS toxicity could be explained by the high content of inorganic 3

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Table 3 Physical–chemical properties of compost. Parameters

Co-composting

Moisture

pH

TOC

TNK

Primary Sludge Waste palm date Mixture

– – 0d 120 d 210 d

64.7 24.6 66.5 58.3 54.4

7.8 6.6 7.5 7.9 7.9

31.8 ± 3 57.8 ± 0.3 34.4 ± 0.4 27.1 ± 0.5 26 ± 0.3

3.1 1.5 2.1 2.2 2.3

a

a

± ± ± ± ±

0.7 0.1 0.3 0.4 0.5

C/N

%DECa

10.1 35.2 16.3 11.5 11.4

– – – 52.4 55.3

Results expressed per unit of dry matter weight, TOC = Total Organic Carbon; TKN = Total Kjeldahl Nitrogen; DEC = Decomposition rate.

and composted sludge. For the higher concentrations (50% and 100%), roots appeared necrotic and blackened at the end of the exposure, revealing an acute toxicity. In addition, due to their rigidity, it was almost impossible to mount the microscope slide, therefore those roots have not been analyzed, and micronuclei could not be counted in these conditions. At a concentration of 30%, data showed that the primary sludge resulted in a significant induction of MN in Vicia faba root tips (12.56 ± 1.1) compared to the negative control (1.4 ± 0.3). Indeed, the MN frequencies were not significantly different between the primary sludge and the initial co-composting stage with the same concentration (30%). After 4 months of the sludge composting, the genotoxicity was significantly reduced by almost 53%. Moreover, no significant difference was observed between the compost at 4 month and the final compost product. Sludge is well known for containing high levels of inorganics and organic compounds with gentoxic and mutagenic properties (Corrêa Martins et al., 2016). In Marrakesh city, Tannery effluents are one of the most important source of chromium pollution. Despite the thermodynamic stability of Cr (III), the presence of certain naturally occurring minerals, especially MnO2, could enhance oxidation of Cr (III), which lead to the formation of the toxic form Cr (VI) (Avudainayagam et al., 2003). More importantly, compared to Cr (III), Cr (VI) is highly mobile in soil, extremely toxic to living organisms with mutagenic, carcinogenic and teratogenic effects, and constitutes a serious health risk (Wise et al., 2010; Prado et al., 2016). Our data confirm the role of co-composting in reducing sludge toxicity. They also showed the potential efficiency in root cell mitosis of the soil amended by final compost. EL Fels et al. (2015) reported that after co-composting of raw sludge with bulking agent, the MN rate in cells of Vicia faba roots decreased significantly by 77.2% in organic matter aqueous extract produced during the compost with decreasing of Cr (VI) concentration to 58%. The genotoxic activities of two forms of chromium and five antibiotics alone and in mixture were evaluated by the Vicia faba MN assay (Table 6). At the lowest concentrations, Cr (III) and the mixture of five antibiotics had no significant effect on the MN frequency compared to the negative control. However, a toxic effect was observed at 2 mg/kg of Cr (VI). More precisely a fifteen-fold increase was registered for Cr (VI) alone, with regards to micronucleus frequency compared to the negative control. When Vicia faba seedlings were exposed to Cr (III) and Cr (VI) alone and in mixture, the micronucleus frequency increased significantly. At the two highest concentrations of the antibiotics mixture, MN frequencies were increased. The most marked effect was observed on Vicia faba roots exposed to the highest concentration (4 mg/ kg), reaching 33% of the effect obtained in the positive control. The presence of antibiotics and chromium in mixtures revealed a significant MN induction. Regardless of the evaluated mixture, the highest genotoxic effect was observed when antibiotics, Cr (III) and Cr (VI) were applied at 0.2, 1 and 10 mg/kg, respectively, which represented 88% of the positive control. Interestingly, the range of maximal effects obtained with each molecule alone at 5–10 mg/kg (30–49%) was obtained with the mixture at 0.2, 1 and 10 mg/kg respectively of antibiotics, Cr (III) and Cr (VI). In these experimentations, no sign of toxicity was observed on the root tips, except for the high concentration of Cr (VI). In the same way, the number of mitosis

Fig. 2. Content of total metals (Pb, Cu, Ni, Zn, Cd and Cr) in DPS. Table 4 Evolution of the germination index in compost water extract primary sludgedate palm waste. Composting stages

T0d T210d

Germination index GI cress

Turnip

8.1 ± 0.2 81.3 ± 5.2

16.4 ± 1.3 88.7 ± 2.5

and organic contaminants, such as total chromium (440 mg/kg DM in DPS) organic acids, ammonia, and heavy metals. Phytotoxicity of such substances has been reported by several studies (Piotrowska et al., 2006; Nóvoa-Muñoz et al., 2008; El Fels et al., 2014). At the final stage of composting, the GI increased and reached 81.3% and 88.7% for cress and turnip respectively. The high value of the GI from the maturation phase could be explained by the significant reduction of phytotoxic substances such as lipids, ammonia, organic acids and lipids (Ait Baddi et al., 2004; Jouraiphy et al., 2008). 3.4. The effect of co-composting on the primary sludge genotoxicity as a function of chromium and antibiotics concentration Table 5 presents the evolution of Vicia faba MN induction of DPS Table 5 Results of the Vicia faba micronucleus test by direct contact in Lufa 2.2 soil. Samples

Mitotic index%

Micronucleus (‰)

% Effect

Negative control Positive control (MH) Primary sewage sludge T0d T120d T210d

7.4 ± 0.3 6.2 ± 1 6 ± 0.6 6.6 ± 0.6 7.4 ± 0.6 6. ± 0.4

1.4 ± 0.3 23 ± 1.5 12.6 * ± 1.1 12.1* ± 1.3 6.4* ± 0.7 6.7* ± 0.3

0 100 54.61 52.8 28.4 26.3

Each value represents mean ± SD of 5 independent experiments * and ** represent statistically significant value at p0.05 and p0.01, respectively. 4

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sludge, or other incurred samples, and to assess the fate of these genotoxic effects during a sludge treatment like composting. The presence of chromium at significant concentrations in the compost hinders its potential exploitation for agricultural applications. Moreover, the persistence of antibiotics and the genotoxic effects of the mixture, even at low concentrations, highly question the use of both sludge and compost in agriculture, as in view of the risk of biological effects, bacteria resistance induction, and antibiotic transfers in the trophic chain, it becomes mandatory to legislate on the limit values of antibiotics in sludge.

Table 6 Evaluation of genotoxic effect of chromium and mixture of antibiotics using the micronucleus test. Vicia faba roots were exposed by direct contact to soil contaminated by Cr (III), Cr (VI) and the mixture of antibiotics.

Negative control Positive control (MH) K2Cr2O7 (mg/Kg) 2 20 200 Cr2(SO4)3 (mg/Kg) 20 200 400 Mixture of chromium (mg/Kg) Cr2(SO4)3 K2Cr2O7 100 10 200 100 Mixture of antibiotics (mg/Kg) 0.04 0.4 4 M1 M2 M3

Mitotic Index (%)

Micronucleus (‰)

% Effect

8.05 ± 1.3 7.1 ± 1.2

0,4 ± 0,7 24.6 ± 2.5

0 100

6.9 ± 1.6 6.1 Not shown

6.1* ± 1 11* Not shown

24.4 44.7

7.4 ± 1.3 7.3 ± 0.3 6.3 ± 0.8

3.4 * ± 1 9* ± 0.7 15.1* ± 3.2

13.8 36.6 61.4

7.8 ± 0.7 6.3 ± 0.5

5.6* ± 0.6 8.6 * ± 0.7

22.8 35

9.1 7.1 7.4 8.3 6.9 7.9

2±1 6.2 * ± 0.5 9.0* ± 0.7 8.8* ± 1.2 13.9* ± 3.2 21.7* ± 2.9

8 25 36.6 35.8 56.5 88.2

± ± ± ± ± ±

2.3 0.9 0.5 2.6 3.4 1.2

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Efficiency of co-composting process to remove genotoxicity from sewage sludge contaminated with hexavalent chromium. Ecol. Eng. 82, 355–360. Ezzariai, A., Barret, M., Merlina, G., Pinelli, E., Hafidi, M., 2017. Evaluation of the antibiotics effects on the physical and chemical parameters during the co-composting of

Each value represents mean ± SD of 5 independent experiments * and ** represent statistically significant value at p0.05 and p0.01, respectively.

remained constant irrespectively the concentration of quinolones and fluoroquinolones. Robinson et al. (2005) assessed the toxic effects and environmental risk of fluoroquinolones to aquatic organisms such as cyanobacterium (Microcystis aeruginosa), duckweed (Lemna minor) and the green algae (Pseudokirchneriella subcapitata) by using a multi-organism approach. Those authors showed that the greatest toxicity observed was with prokaryotic organisms. Furthermore, Ding et al. (2017) reported toxicity of fluoroquinolones and tetracyclines to a zebra fish based on biochemical biomarkers and histological observation. The phytotoxicity of enrofloxacin, which is a precursor compound for ciprofloxacin, reduced growth of primary root, hypocotyl, cotyledons and growth/number of leaves for Cucumissativus, Lactucasativa, Phaseolus vulgaris and Raphanussativus at 5 mg.L−1 in growth medium (Migliore et al., 2003). In the Mediterranean area, from 15 to 45 ton per hectare of compost is applied to the agriculture soils (Morra et al., 2010; Montiel-Rozas et al., 2018). In Morocco, 6.25 million tons of organic matter are needed annually (Rihani et al., 2010), which could reach 15 ton per hectare in the long-term. To reach at least 3.5% of organic carbon for fertilization (26% of organic carbon was observed in the final compost), concentrations of 2.6–3.8, 0.1, 0.6–1 and 1.2 mg/ha for fluoroquinolones, ampicillin, macrolides and tetracyclines respectively could be observed after each compost application. In addition, the most detected heavy metal (chromium) could be observed at 501.6 mg/ha. Several effects of antibiotics (Khadra et al., 2012; Ezzariai et al., 2018a, 2018b) and heavy metals (EL Fels et al., 2015) were observed even at low concentrations. However, these concentrations must be used to evaluate the potential effect on fauna and flora, and thus to choice suitable frequency and rate of compost before its soil application. 4. Conclusions Overall, data regarding the effect of pollutants mixtures in the environment are very scarce, which is mainly due to the high diversity of chemicals involved, hence inducing complex interactions in fields, which can limit or exacerbate the effect of the chemical compounds on organisms. Vicia faba micronucleus assay could be used successfully to assess the genotoxic effects, in relation to the presence of pollutants in 5

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