Genotoxicity evaluation of different types of leachate treated with Aspergillus flavus using Vicia faba micronucleus

Environmental Technology & Innovation 18 (2020) 100656

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Genotoxicity evaluation of different types of leachate treated with Aspergillus flavus using Vicia faba micronucleus Yassine Zegzouti a,b , Anas Aguelmous a,c,d , Ahmed Khadra a , Aziz Boutafda a , ∗ Loubna El Fels a,e , , Lalla Amina Idrissi Hassani f , Miloud El Hadek b , Mohamed Hafidi a,g a

Laboratory of Microbial Biotechnologies, Agrosciences and Environment, Faculty of Science Semlalia, Cadi Ayyad University, Marrakesh, 40 000, Morocco b Laboratory of Process Engineering Faculty of Sciences, Ibn Zohr University, Agadir, Morocco c Laboratory of Process Engineering and Environment, Faculty of Science and Technology, University Hassan II of Casablanca, Morocco d National High School of Chemistry, Univetsity Ibn Tofail Kenitra, Morocco e Higher Institute of Nursing Professions and Health Technics, Marrakech-Safi, Morocco f Laboratory of Plant Biotechnology, Department of Biology, Faculty of Sciences of Agadir, Ibn Zohr University, BP 8106, 80000 Agadir, Morocco g AgroBioSciences Program, Mohammed VI Polytechnic University (UM6P), Benguerir, Morocco

article

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Article history: Received 5 November 2019 Received in revised form 24 January 2020 Accepted 26 January 2020 Available online 30 January 2020 Keywords: Leachate Genotoxicity Bioremediation Aspergillus Micronucleus Vicia faba Risk assessment

a b s t r a c t In this study, the efficiency of Aspergillus flavus (A. flavus) was studied in the treatment of three landfill leachate types, namely young, intermediate and old ( YL, IL and OL ) in terms of removal of organic and nitrogen matter, as well as genotoxicity. The organic fraction of landfill leachate was measured by biological oxygen demand (BOD5 ) and chemical oxygen demand (COD), and the nitrogen fraction was measured by ammonium (NH+ 4 ). The genotoxicity was evaluated using the Vicia faba root-micronucleus test. The analysis of the three leachates revealed high values of COD ( 9517.4-19 523.33 mg/L), BOD5 ( 1756.8-11 600 mg/L), NH+ 4 ( 5931.25-3112.68 mg/L) and total chromium (Cr T: 0.06-3.78 mg/L). The three leachates also showed genotoxic effect on Vicia faba cells at a 1% leachate concentration. The treatment of leachates with A. flavus led to considerable reduction of pollutant load, and it was found to be more effective in reducing COD (30.12 %) and BOD5 (71.29%) in YL and NH+ 4 (98.21%) in IL after 30 days of treatment. A decrease in Micronucleus frequency (48.57-52.32 h) was observed in Vicia faba cells exposed to treated OL and IL compared to raw samples, while no such reduction was observed in the treated YL. The treatment of leachate with A. flavus showed more efficiency in genotoxicity removal for OL and IL. However, treated YL still possesses genotoxic character, even if it recorded the highest COD removal rate. Thus, the evaluation of leachate bioremediation on genotoxicity removal effectiveness should be included in landfill leachate risk assessment studies in order to ensure safe levels for its disposal in the environment. © 2020 Published by Elsevier B.V.

∗ Corresponding author at: Laboratory of Microbial Biotechnologies, Agrosciences and Environment, Faculty of Science Semlalia, Cadi Ayyad University, Marrakesh, 40 000, Morocco. E-mail address: [email protected] (L. El Fels). https://doi.org/10.1016/j.eti.2020.100656 2352-1864/© 2020 Published by Elsevier B.V.

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Y. Zegzouti, A. Aguelmous, A. Khadra et al. / Environmental Technology & Innovation 18 (2020) 100656

1. Introduction Environmental pollution by leachate percolating through sanitary landfills is a major concern throughout the world, particularly in developing countries. A wide variety of materials and organic compounds such as fatty acids, humic substances, heavy metals, and many other hazardous chemicals have been detected in landfill leachate (Hafidi et al., 2015; Zegzouti et al., 2019). A review of previous literature on the occurrence and concentration of these organic chemicals present in municipal solid waste (MSW) landfill leachates concluded that toxic and cancer-causing chemicals were detected in the leachates of all MSW landfills studied (Brown and Donnelly, 1988). These chemicals are difficult to treat by conventional wastewater treatment systems including activated sludge process due to high toxicity. Their migration away from the landfill and their release into water systems poses a serious threat to the living organisms inhabiting respective ecosystem and also tends to be bio-accumulated by long-term exposure in the food chain and in that manner poses a risk to human health as well (Sang and Li, 2004). The genotoxic effect of leachate emanating from landfill is well documented (Sang and Li, 2004; Gajski et al., 2012; Toufexi et al., 2013). Over the years, different physical and chemical processes including, natural evaporation, advanced oxidation processes, coagulation–flocculation, electrochemical treatment and membrane processes have been developed to enhance pollutants removal from leachate and attenuate their genotoxic effects (Sang and Li, 2004). Most of these processes removed the majority of the suspended colloidal organic materials; but, refractory and genotoxic compounds still remained in the effluent. To overcome these problems, bioremediation technologies have proven to be a very promising tool to convert toxic organic contaminants to innocuous and less-toxic constituents (Morris et al., 2018). Microbial bioremediation of landfill leachate is an environment eco-friendly, cost-effective and safe alternative to traditional chemical and physical methods. Only a few research experiments have been carried out on the bioremediation of landfill leachate using bacteria either fungi (Ellouze et al., 2008; Anastasi et al., 2013; Razarinah et al., 2015; Bardi et al., 2017; Morris et al., 2018; Smaoui et al., 2018; Spina et al., 2018; Zegzouti et al., 2020a,b). The majority of these researches have investigated extensively the bioremediation capacities of fungi in leachate, as most fungi are powerful and robust organisms and are generally more tolerant to a wide variety of toxic environmental compounds than bacteria (Magan et al., 2010). However, in all these studies the remediation goals were primarily based on removal of the target contaminants rather than on broader measures related to environmental health risks. Indeed, in some cases, contaminants may not be fully degraded by microorganisms, but transformed to an end products or metabolic intermediates, which may be equally or more toxic than the parent compound (Ganey and Boyd, 2004). Thus, the effectiveness of leachate bioremediation should not be based on the disappearance of the target pollutant alone but also on the reduction of toxicity, and thus, the minimization of risk. Indeed, Song et al. (2017) highlighted the importance to conducting health risk assessment, which in turn reflects the bioremediation effectiveness after treatment. Toward that end, several physico-chemical methods and genotoxicity bioassays have been used to assess the potential hazard and risk of wastewater before and after bioremediation (Giorgetti et al., 2011; Kumari et al., 2016). In many studies, genotoxicity bioassays have been recognized as being particularly efficient for evaluating the efficacy of bioremediation (Migid et al., 2007; Song et al., 2007; Hu et al., 2012; Chibwe et al., 2015). In most situations, it is not feasible to identify and quantify all contaminants using analytical method due to cost and available technology, especially in developing countries, which displays an array of problems including lack of knowledge and highly sophisticated analytical facilities. Therefore, the genotoxicity testing of landfill leachates might be more helpful and cost-effective in estimating the risks of several organic and inorganic contaminants present in this complex matrix than the chemical characteristics alone. The genetic effect of leachate has been shown in several higher plant systems, including biotests with plant roots which have provided cheaper, easier, sensitive, useful, reliable and valuable alternative methods for the determination of the adverse effects of environmental contaminants to the usual assays carried out on mammalian cell systems (Olorunfemi et al., 2011). Among them the Vicia faba micronucleus (MN) test which is recognized as a sensitive assay to evaluate genotoxicity effects and chromosomal aberrations from physical and chemical agents, as well as from complex mixtures. Many studies have been carried out using the Vicia faba micronucleus (MN) test to evaluate complex environmental mixtures, such as wastewater and industrial effluents (Sang and Li, 2004; El Hajjouji et al., 2007; Giorgetti et al., 2011), sludge from municipal treatment stations or compost (Khadra et al., 2012; El Fels et al., 2015; Khadra et al., 2019), soil samples (Hakkou et al., 2001) and sediments (Chen and White, 2004). On the other hand, the expected chemical quality of landfill leachate is highly site-specific and may vary mainly as result of landfill life cycle, also referred to as leachate age and generally categorized as young, intermediate and old (Alvarez-Vazquez et al., 2004). This implies that landfill leachate genotoxicity might also vary due to leachate aging and characteristics, which affect the leachate treatment process and therefore the technology selection. Thus, understanding the variation in leachate composition is essential to managing the leachate genotoxicity in the most efficient manner possible. The objective of this study was to investigate the effect of bioremediation on genotoxicity of three types of leachates: young, intermediate and old (YL, IL and OL) before and after biological treatment with A. flavus using the Vicia faba root-micronucleus test. 2. Material and method 2.1. Site description This study was conducted in Morocco in three different landfills located in Marrakesh city (Marrakesh Closed Landfill) (MCL), Al Mnabha village (Marrakesh New Landfill) (MNL) and Agadir city (Greater Agadir landfill) (GAL), (Table 1). These

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Table 1 General conditions of the landfill sites included in the study. Condition class

Marrakesh New Landfill (MNL)

Marrakesh Closed landfill (MCL)

Greater Agadir landfill (GAL)

Landfill type Period of landfilling Age of classification Daily average of waste disposed (tonnage) Waste type

Sanitary (operational) 2016-date Young 900 to 1000 Household

Sanitary (operational) 2010-date Intermediate 756 Household

Form of the leachate treatment Fate of generated landfill gas Coordinates at the landfill sites

Physical and Biological No facility 31◦ 54′ 47.6′′ N 8◦ 04′ 54.1′′ W

Non-sanitary (closed) 1980–2016 Old 703 Household, commercial and industrial Physical No facility 31◦ 42′ 09.8′′ N 8◦ 03′ 59.7′′ W

Physical and Biological No facility 30◦ 26′ 27.1′′ N 9◦ 30′ 47.2′′ W

locations were selected to be representative of landfills of different ages and types of waste. One of the three landfills (MCL) was a non-controlled municipal waste disposal site (i.e. without liners and leachate collection system), while the two others (MNL and GAL) were controlled (i.e. with liners and leachate collection systems). Among the three landfills included in the study, two were operational (MNL and GAL) and another one was closed (MCL) and it is currently under reclamation and re-planting operations. 2.2. Leachate sampling and physico-chemical characterization of landfill leachate The YL, IL and OL raw landfill leachate samples were collected from the three above-mentioned landfill sites as described by Zegzouti et al. (2019) The main physico-chemical characteristics of landfill leachate originating from MNL, MCL and GAL were previously analyzed as shown by Zegzouti et al. (2019). 2.3. Subculture and preparation of spore suspension The fungal culture used in this study was Aspergillus flavus (A. flavus-LC106118), which was isolated and molecularly identified in our previous study on screening and selection of autochthonous fungi from leachate contaminated-soil for bioremediation of different types of leachate (Zegzouti et al. in press). Generally, Aspergillus sp., was reported to be able to break-down long-chained pollutants, including textile dyes and crude oil contaminants (Mukherjee, 2016; Richardson and Rautemaa-Richardson, 2019). Spore suspensions of A. flavus were prepared following Aneja (2007). The fungal spores were initially harvested by aseptically and gently scraping the aerial mycelium from the medium surface of the plate with 200 ml saline solution containing 0.85% (w/v) with the aid of sterile loop. The spores suspension solution was then filtered through several layers of cheese-cloth to remove most of the fungal mycelium. Afterward, the concentration of spores in the suspension was measured by a cell-counting hemocytometer chamber (Neubauer Germany) after been germinated in an Erlenmeyer flask containing malt extract broth spiked with landfill, and giving an average count of 1 × 107 spores/mL. 2.4. Shake flask study for leachate treatment The raw landfill leachates were subjected to six treatments as detailed in Table 2. Each treatment was conducted in 250 ml Erlenmeyer flasks containing 150 mL of culture medium. The liquid leachate-based culture medium of each type of leachate samples was prepared at 50% concentration of landfill leachate (v/v) in distilled water). The choice of this concentration was not arbitrary but rather was based on the results stemmed from our previous study which has demonstrated that A. flavus was potentially tolerant against landfill leachate at a concentration below or equal to 50% (v/v) (Zegzouti et al. in press). Each flask was inoculated in triplicate with 10% (v/v) of the A. flavus sporal inoculum. A set of flasks of raw leachate was similarly prepared and were not inoculated to serve as control. All flasks of leachate medium were autoclaved before inoculating with spores suspension. Incubation was performed on an electric shaker (Sanyo-Gallemkemp, UK) with constant shaking at a 150 rpm/min at ambient temperature (28 ± 2 ◦ C) for a period of 30 days. Samples were collected and the content of BOD5 COD, NH4 + , NO3 − , pH before and after treatment was measured. 2.5. Micronucleus test The Vicia faba seeds bioassay was carried out according to Marcato-Romain et al. (2009). Dry Vicia faba seeds were soaked for 24 h in distilled water and allowed to germinate between two layers of moist cotton. After 3 days, when the primary emerged roots, were about 2–3 cm in length, they were used in the test and their tips were cut off in order to let the secondary roots grow. Five different concentrations of each raw and treated leachate samples were tested (0.1, 1, 5, 10 and 20% v/v). For each experiment three seeds were used per treatment. After the germination period, the direct contact method developed by Marcato-Romain et al. (2009) was used by placing germinated roots in cylindrical pots containing the LUFA standard soil for the assessment of the effect of their exposition to the three types of raw and treated leachates

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Y. Zegzouti, A. Aguelmous, A. Khadra et al. / Environmental Technology & Innovation 18 (2020) 100656 Table 2 Characteristics of raw leachate at Marrakesh (new and closed) and Greater Agadir landfills (MNL, MCL and GAL). Values represent means ± SD (n = 3) (Zegzouti et al., 2019). No

1 2 3 4 5 6 7 8 9 10 11 12

Parameter

pH COD (mg/L) BOD5 (mg/L) BOD5 /COD Total Nitrogen (TKN) (mg/L) Ammonia-N (mg/L NH4 + -N) Nitrate-N (mg/L NO3 − -N) Phenols (mg/L) Cr T (mg/L) Ni (mg/L) Zn (mg/L) Pb (mg/L)

Marrakesh landfills

Greater Agadir landfill

New Average

Closed Average

Intermediate age Average

6.8 ± 0.05 19 523.33 ± 674.78 11 600 ± 700.31 0.6 ± 0.05 6335.21 ± 390.84 5931.25 ± 353.01 35.03 ± 3.41 110 ± 9.53 0.06 ± 0 .01 0.12 ± 0.01 0 .17 ± 0.02 0,19 ± 0.02

9.1 ± 0.08 9517.4 ± 375.11 1756.8 ± 81.80 0.18 ± 0.02 4026.37 ± 194.51 3112.68 ± 203.47 12.22 ± 1.34 14 ± 1.35 3.78 ± 0.33 1.07 ± 0.15 1.95 ± 0.13 0.23 ± 0.01

8.1 ± 0.04 14 560.7 ± 481.11 5300.15 ± 253.99 0.36 ± 0.04 6149 ± 239.12 5226.13 ± 337.68 27.47 ± 2.25 35.23 ± 3.17 0.21 ± 0.02 0.23 ± 0.01 0.21 ± 0.02 0.08 ± 0.01

of different concentration. Moisture content was maintained at all times to 2/3 of water holding capacity by introducing water in the under cups. Distilled water was used as a negative control. Maleic Hydrazide 10−5 M solution was used as a positive control. Root tips were rinsed with distilled water, fixed in Carnoy’s solution (glacial acetic acid/ethanol 1:3, v/v) at 4 ◦ C overnight and transferred into 70% ethanol before storage. Then, they were hydrolyzed with 1N HCl for 5–7 min at 60 ◦ C. Three slides were prepared for each of the three seeds. After staining the root tips with 1% aceto-orcein for 3 min at 60 ◦ C, the interphase cells as described by Ma et al. (1995) were scored for micronucleus frequencies at 400 × magnification. MN frequencies were expressed in per 1000 cells while Mitotic index (MI) was expressed in %. The micronucleus test was performed only on root tips with a MI superior to 2% in order to avoid underestimation of the micronucleus frequency due to impaired cell proliferation rate. 3. Results and discussion 3.1. Physico-chemical analysis Basic physico-chemical properties and total heavy-metal contents in YL, IL and OL from the MNL, MCL and GAL, respectively, are depicted in Table 3. The YL leachate emanating from the MNL had typical properties of leachate in the transition phase (transition from aerobic to anaerobic conditions) (Kostova, 2006): acidic pH (6.8), high concentration of organic matter (COD-19 523.3) and typically high BOD5 /COD ratio > 0.3, showing high biodegradability potential. On the other hand, leachates originating from MCL and GAL was relatively less polluted, they contained relatively low concentration of ammonium nitrogen compared to leachate from MNL. The concentration of biodegradable organic matter (quantified as BOD5 ) was low (1756.8–5300.15 mg/L) and together with their high pH (8.1–9.1) signaled that the MCL and GAL were at the methanogenic phase. In addition to the pH variations, the BOD5 /COD (0.18–0.36) was very low indicated that the leachate from MCL and GAL contains a considerable amount of recalcitrant compounds such as humic and fulvic acid and thus presents a certain difficulty to be biologically biodegraded (Renoua et al., 2008). The most important heavy metals detected in the three landfill leachates in terms of concentrations were: Cadmium, copper, nickel, chromium and lead. The presence of metal ions in landfill leachate was reported to mediate the enhancement of genotoxic effect as well as cause inhibitory effect on the biological treatment of leachate (Salem et al., 2014). In general, concentration of heavy metals was low in the three leachates, except for Cr Total (Cr T), Ni and Zn in OL. In landfills, heavy metals are present in high concentration during the acidogenic phase when they are dissolved in leachate as a result of low pH caused by generation of organic acids (Kulikowska and Klimiuk, 2008). However, heavy metal concentrations in leachate were reported to be reduced during methanogenic phase, attributing this to the sorption of metals on waste material and precipitation of metal oxides due to the increased pH (Kalčíková et al., 2012). Indeed, organic matter is expected to be responsible for sorption of metals, and sulfides and carbonates are capable of forming precipitates with Cd, Ni, Zn, Cu and Pb. This explained the low concentration of metals obtained at the three landfills. The higher concentration of Cr T, Ni and Zn in OL samples can be due to the disposal of chrome tannery sludge and electronic devices like cell phones and computers at the MCL (uncontrolled landfill site). Another explanation is that there is no formation of any insoluble sulfide precipitate and thus the Cr T can leave the landfill with landfill leachate (Kjeldsen et al., 2002). According to the results of physico-chemical analysis and metal determination, the YL, IL and OL leachate is definitely not appropriate for discharge into the environment, because it exceeds many permissible legislative limits. On the other hand, the IL and OL leachate were less polluted than YL and it was evident as both landfills GAL and MCL has undergone methanogenic phase. In this phase concentrations of organic matter substantially decrease due to the strong decomposition of organic compounds to simpler molecules in the earlier acid phase of landfill stabilization (Renoua et al., 2008). In addition, the decrease of pollution of leachates in this phase is result of precipitation of insoluble metals as well as dilution which can

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Table 3 Leachate treatment applications used in the experiment. Treatments

Details of treatments

Y-50% RL Y-50%DRLI I-50% RL I-50%DRLI 0%–50% RL 0-50%DRLI

50% 50% 50% 50% 50% 50%

Raw Raw Raw Raw Raw Raw

Young Leachate + 50% distilled water (as control) Young Leachate with 10% inoculum of spores + 50% distilled water Intermediate Leachate + 50% distilled water (as control) Intermediate Leachate with 10% inoculum of spores + 50% distilled water Old Leachate + 50% distilled water (as control) Old Leachate with 10% inoculum of spores + 50% distilled water

occur during rainy seasons (Kalčíková et al., 2012). However, during methanogenic phase ammonium nitrogen (NH4 + ) content does not retain a similar signature as organic matter. The NH4 + become more concentrated with time as a result of the fermentation of organic matter containing proteins (Renoua et al., 2008). In general, NH4 + is one of the most harmful wastewater contaminants due to its toxicity to microorganisms and for human health. It is thus essential to evaluate the risks to public health and the environment from exposure to municipal waste landfill leachate. 3.2. Leachate organic and nitrogen matter after treatment by A. flavus The physico-chemical characteristics of landfill leachate after treatments with A. flavus showed a reduction of analyzed parameters (Table 4). The BOD5 , COD, NH4 + and NO3 − removal rates were much higher than those of the control experiments (Y- 50% RL, I- 50% RL and O- 50% RL) during the whole treatment process (data not shown). After treatment, the color of landfill leachate became light yellow. This was possibly achieved by degradation of organic substances present in landfill leachates. Treatment of leachates resulted in the reduction of BOD5 and COD by 55.76% and 20.95%, respectively in I-50% DRLI. This reduction in BOD5 and COD matter of landfill leachate might be attributed to the fungal degradation of organic contaminants to meet the nutritional and physiological requirements (Ghosh and Thakur, 2017). Under similar conditions, A. flavus reduced 71.29% and 30.12% of BOD5 and COD in Y-50% DRLI, respectively. Compared to IL and OL, A. flavus was more effective in the removal of organic matter from YL. This could probably because of the different characteristics and quality of each type of leachate as shown in Table 2. Indeed, Amaral et al. (2009) and Kalčíková et al. (2012) have also reported that YL contains a high concentration of easily hydrolyzed organic pollutants which would be more bioavailable. Our findings of the degradation capability of organic matter in landfill leachate by fungi are in agreement with earlier studies (Bardi et al., 2017; Morris et al., 2018; Smaoui et al., 2018; Spina et al., 2018). For instance, the level of COD and BOD5 reduction obtained in the present study after 30 days of treatment were slightly lower than those obtained by Razak et al. (2016) as they reported about 81.57% BOD5 and 44.66% COD reduction of landfill leachate after the same treatment duration with a ligninolytic fungi (Ganoderma Australe). This could highlight the ability of nonligninolytic fungi such as A. flavus used in the current study to bioremediate landfill leachate as the majority of studies carried out on landfill leachate bioremediation were performed using white rot fungi such as Phanerochaete chrysosporium, Ganoderma australe and Trametes trogii. On the other hand, the acceptance of bioremediation using A. flavus as a viable clean up strategy for the three studied leachates generated from MCL, MNL and GAL depends upon its capacity to compete with the other treatment strategies (e.g., physical, chemical and biological) in terms of efficiency as well as in terms of cost. In the case of the illustration, our results obtained in GAL site are consistent with a recent study carried at the same site which consist of treating leachate of the GAL by the infiltration–percolation technique using titaniferous sand (TFS) (Azougarh et al., 2019). Typically, ammonium nitrogen is one of the highly toxic nitrogen compounds and its microbial remediation offers an eco-friendly and cost-effective alternative to conventional treatment approaches. In this study, removal rates of NH4 + from Y-50% DRLI, I-50% DRLI and 0%–50% DRLI by A. flavus were 76.38%, 98.21% and 97.87%, respectively. These results proved the potential of A. flavus to metabolize the NH4 + . This was not surprising that the NH4 + removal rates were high, given that some studies have reported that fungi are heterotrophic nitrifiers with good efficiency (Lang and Jagnow, 1986; Odu and Adeoye, 1970). Aspergillus flavus has been reported to be one of the most interesting strains capable of performing heterotrophic nitrification because this is one of the few microorganisms able to convert ammonium to nitrate (Marshall and Alexander, 1962; Guest and Smith, 2002). However, with no knowledge of the end products amount (e.g., NO3 − ) of the heterotrophic nitrification by A. flavus, it is difficult to attribute the sharp decrease of NH4 + to the heterotrophic nitrification. Among the conditions in which nitrification occurs, pH is one of the majors (Guest and Smith, 2002). These authors reported that fungi cannot nitrify below pH 6–5. In our study the pH values recorded at the start up of the three above treatments perfectly meet the previous pH range requirements. The pH of leachate was increased from 6.42 to 9.43, 7.81 to 9.58 and 8.65 to 9.21 in Y-50% DRLI, I-50% DRLI and 0%–50% DRLI, respectively. This increase in pH, was not expected as the acidity was reported to be generated during heterotrophic nitrification (Stroo et al., 1986). This increase could be explained by the release of some alkaline substances during the organic matter degradation process which can in turn affect the pH at the end of the treatment. In addition, the accumulation of nitrate reached 485.15, 458.36 and 227.612 mg/L in Y-50% DRLI, I-50% DRLI and 0%–50% DRLI, respectively. This may indicate that the nitrification was occurring. However, these values feel well outside the normal expected for nitrification products, which raise questions to the other possible fates of NH4 + . One major possible explanation for the lack of nitrification end-products was that NH4 + might be assimilated into the cell mass for growth of new biomass (Guest

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Y. Zegzouti, A. Aguelmous, A. Khadra et al. / Environmental Technology & Innovation 18 (2020) 100656 Table 4 Physicochemical characteristics of 50% landfill leachates before and after treatment with A. flavus. Values represent means ± SD (n = 3). Treatments

Parameter

Raw leachate (mg L−1 )

Treated leachate (mg L−1 )

Removal efficiency (mg L−1 )

Y-50%DRLI

COD BOD5 NH4 + NO3 − pH

9761.66 ± 323.20 5800 ± 353.23 2965.625 ± 145.84 17.51 ± 2.19 6.42 ± 0.06

6821.45 ± 287.8 1665.18 ± 66.94 700.3 ± 42,15 485.15 ± 45.12 9.43 ± 0.07.

30.12% 71.29% 76.38% – –

I-50%DRLI

COD BOD5 NH4 + NO3 − pH

7280.35 ± 212.03 878.4 ± 120.56 2613.065 ± 174.63 13.73 ± 2.13 7.81 ± 0.07

5755.11 ± 172.65 388.6 ± 14.18 46.69 ± 2.5 458.36 ± 67.73 9.58 ± 0.08

20.95% 55.76% 98.21% – –

0-50%DRLI

COD BOD5 NH4 + NO3 − pH

4758.7 ± 186.12 2650.07 ± 37,34 1482.81 ± 101,39 6.11 ± 0.91 8.65 ± 0.07

2379.35 ± 52.82 1414.34 ± 31,39 17.51 ± 0,72 227.612 ± 23.61 9.21 ± 0.08

16.38% 46.6% 97.87% – –

and Smith, 2007). The high NH4 + removal rates obtained in this study were similar to those obtained by Ellouze et al. (2008). The findings from this study demonstrated that a stirred tank reactor (STR) operated by feeding an acclimatized consortia of nitrifying bacteria was able to remove 92% of NH4 + for an organic loading rate of 4.5 g/L per day from landfill leachate. 3.3. Micronuclei test 3.3.1. Raw landfill leachates genotoxicity The results of the Vicia faba root-micronuclei test of five concentrations of each leachate type samples showed that for leachate concentrations higher than 10%, root tips showed brown to black coloration, which in turn lead to the disruption of mitosis. Under these conditions the scoring of micronucleus was impossible. At the lowest concentration of leachate 0.1%, no significant increase in micronucleus frequency was recorded, compared with the negative control (data not shown). The results of the Vicia faba root-micronucleus test of the remaining concentrations ranging from 1% to 10% in which significant increases in micronucleus frequency were observed are presented in Table 5. Different capacities of leachate samples to significantly induce MN (3.62–16.7h) in Vicia faba root were obtained in comparison to the negative control (0.45h), which may suggest that the specific composition of each type of leachate had different genotoxic effect on Vicia faba cells. The first finding to be highlighted was that even in such a low concentration of raw the landfill leachate (1%), the three leachate samples showed the capacity to induce the formation of MN (3.62–7.07h) compared with the negative control. This ability of leachate to cause genotoxic effects at low leachate concentrations was previously observed in other studies using different bioassays. For instance, Srivastava et al. (2005) and Gamoń et al. (2019) demonstrated that leachate at 10% and 6.25% may cause mitotic and chromosomal abnormalities in Allium cepa root meristem cells and changes in their DNA. The strong genotoxic effect of raw leachates may result from chemical and physical characteristics of leachate samples such as the concentration of COD and NH4 + . Sang and Li (2004) has demonstrated the correlation between the genotoxicity on root tips of Vicia faba and the chemical measurement (COD) of leachate. Indeed, in our study the highest frequency of MN was detected in Y-50% RL (37–40 fold higher than negative control) at the concentration of 10% corresponding to the highest concentration of COD (19 523.33 mg/L) (Fig. 1). Thus, the genotoxicity of YL was higher than that attributed to OL and IL. This could be explained by the fact that YL contains a large number of compounds, including genotoxic compounds such as PHAs, PCB and heavy metals (Sang and Li, 2004; Gajski et al., 2012). It is noteworthy, that OL induced the formation of MN (3.62–7.83h); despite its classification as stable leachate with less toxic and biodegradable organic compounds. This was possibly due to the high concentration of chromium in OL (Table 2), which may enhance the micronucleus frequency in Vicia faba cell roots as it was demonstrated by El Fels et al. (2015). In general, organic compounds and heavy metals in the leachate may be involved in some processes that lead to genotoxicity such as formation of free radicals oxidative stress (Curtis et al., 1988; Sang and Li, 2004). It is important to emphasize that the MN frequency increased progressively with increasing concentration of each raw leachate. For example, the 10-fold increase of concentration (from 1% to 10% for Y-50% RL) was associated with about a 2-fold increase of number of micronuclei from 7.07 to 16.72h. The Mitosis index followed the same trend as that described for MN frequency, suggesting the presence of cytotoxic compounds which might also be affecting proteins synthesis and DNA and thus prevent the cell from entering mitosis (Kumari et al., 2016).

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Table 5 Micronucleus frequency and mitotic index values in Vicia faba roots exposed to different 50% raw landfill leachates concentrations. Values represent means ± SD (n = 3). Treatments

Leachate concentration (%) – –

Micronucleus (h)

Miotic index (%)

Positive control (MH) Negative control

18.12 ± 0.7 0.45 ± 0.4

5.4 ± 0.3 9.14 ± 0.7

Y-50%RL

1% 5% 10%

7.07 ± 0.86 8.06 ± 0.53 16.73 ± 1.1

3.64 ± 0.23 2.82 ± 0.30 2.60 ± 0.17

I-50%RL

1% 5% 10%

5.62 ± 0.33 7.14 ± 0.63 10.96 ± 0.95

4.49 ± 0.47 3.75 ± 0.21 2.44 ± 0.34

0%–50% RL

1% 5% 10%

3.62 ± 0.45 4.23 ± 0.21 7.83 ± 0.78

6.15 ± 0.67 5.21 ± 0.50 3.91 ± 0.22

Fig. 1. Micronuclei induced by 50% raw young landfill leachate at 10% in Vicia faba root cells. Micronuclei are marked with two arrows (400 × magnification).

3.3.2. Genotoxicity evaluation of landfill leachates after fungal treatment The results of the Vicia faba MN induction in roots exposed to different 50% treated landfill leachates concentrations (1, 5 and 10%) are exhibited in Table 6. The results showed that after Y-50% DRLI treatment leachate cause a similar degree of genotoxicity effect to raw leachate (Y-50% RL) in all leachates concentrations, with a MN frequency in the range of 8.24–15.27 h. This could be explained by the fact that during the Y-50% DRLI treatment, A. flavus attacked preferentially easily degradable organic compounds which are the most dominant in young leachate, and thus COD components that caused genotoxicity such as xenobiotics were still not metabolized. This observation of such inefficiency in genotoxicity elimination after treatment was in accordance with earlier studies by Gamoń et al. (2019) and Widziewicz et al. (2012). However, in another study, Bortolotto et al. (2009) assessed the effect of landfill leachate treatment in a system of anaerobic/facultative lagoons with a maximum COD removal efficiency of 90% and concluded that this did not affect the reduction of genotoxicity in the leachate. This raises the question of whether non change in genotoxicity after treatment may occur due to generation of by-products and microbial metabolites that are more toxic than the one from the raw samples (Claxton et al., 1995). Most types of microbial bioremediation treatment approaches, may produce additional compounds that add to the complexity of evaluating the potential genotoxic effect of a contaminated matrix during and after treatment (Claxton et al., 1995). As far as the I-50% DRLI and O-50% DRLI treatments are concerned, the results showed reduction in genotoxicity in all leachate concentrations (10%, 5%, and 1%), indicated by the decrease in the micronucleus frequencies and the improvement in mitotic index compared to the raw leachates. The result of our study showed also that the MN frequency increased progressively with the increasing concentration of treated leachates. The attenuation of genotoxicity in leachates was clearly illustrated by the reduction of the MN frequencies of 52.32% and 48.57% of the highest leachate concentration (10%) in vicia faba grown with treatments I-50% DRLI and O-50% DRLI, respectively. This strong decrease in MN frequencies proved the biodegradation activity of A. flavus and its ability to remove genotoxic compounds. Compared to Y-50% DRLI treatment, I-50% DRLI and O-50% DRLI treatments showed more efficiency to eliminate the genotoxicity, possibly because OL and IL contain predominantly biologically stable organic

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Y. Zegzouti, A. Aguelmous, A. Khadra et al. / Environmental Technology & Innovation 18 (2020) 100656 Table 6 Micronucleus frequency and mitotic index values in Vicia faba roots exposed to different 50% treated landfill leachates concentrations. Values represent means ± SD (n = 3). Treatments

Leachate concentration (%)

Positive control (MH) Negative control

Micronucleus (h)

Miotic index (%)

18.12 ± 0.7 0.45 ± 0.4

5.4 ± 0.3 9.14 ± 0.7

Y-50%DRLI

1% 5% 10%

8 24 ± 0.95 9,18 ± 0.88 15.27 ± 1.35

2.68 ± 0.22 2.45 ± 0.20 1.53 ± 0.12

I-50%DRLI

1% 5% 10%

1.71 ± 0.10 3.15 ± 0.17 5.74 ± 0.43

15.75 ± 1.13 9.24 ± 0.85 9.18 ± 0.71

0%–50% DRLI

1% 5% 10%

1.68 ± 0.11 2.14 ± 0.17 3.80 ± 0 .15

16.42 ± 1.34 11.99 ± 1.11 11.41 ± 0.97

component, including those possessing genotoxic character, which allow A. flavus to attack them as primarily target compounds. Specifically, fungi are capable of producing a wide variety of enzymes that can degrade recalcitrant organic compounds such as xenobiotics and mineralize other substances (Magan et al., 2010). 4. Conclusion The treatment of three leachates types YL, IL and OL with A. flavus was shown to be efficacious in decreasing the levels of BOD5 and COD in young leachate and NH4 + in intermediate leachate. In the analysis of the raw leachates genotoxicity by the Vicia faba micronucleus assay, the three leachates were shown to have a potential genotoxic effect. The genotoxicity of both treated IL and OL was markedly reduced. However, treated YL still shows genotoxic effect, raising the question of whether genotoxicity increase in YL leachate is due to the persistence of genotoxic substances or the formation of byproducts/intermediates more toxic than the starting parent contaminants during bioremediation. Thus, another important aspect for evaluating the effectiveness of bioremediation processes involves monitoring the biodegradation products and microbes and their metabolites for avoiding any risk of potential environmental health effects. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Yassine Zegzouti: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing. Anas Aguelmous: Software, Visualization, Investigation. Ahmed Khadra: Software, Visualization, Investigation. Aziz Boutafda: Software, Visualization, Investigation. Loubna El Fels: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing. Lalla Amina Idrissi Hassani: Writing - review & editing. Miloud El Hadek: Software, Validation. Mohamed Hafidi: Supervision. References Alvarez-Vazquez, H., Jefferson, B., Judd, S.J., 2004. Membrane bioreactors vs conventional biological treatment of landfill leachate: a brief review. J. Chem. Technol. Biotechnol. 79, 1043–1049. Amaral, M.C.S., Ferreira, C.F.A., Lange, L.C., Aquino, S.F., 2009. Characterization of landfill leachates by molecular size distribution, biodegradability, and inert chemical oxygen demand. Water Environ. Res. 81, 499–505. Anastasi, A., Tigini, V., Varese, G.C., 2013. The bioremediation potential of different ecophysiological groups of fungi. In: Fungi as Bioremediators. Springer, pp. 29–49. Aneja, K.R., 2007. Experiments in Microbiology, Plant Pathology and Biotechnology. New Age International. Azougarh, Y., Abbaz, M., Hafid, N., Benafqir, M., Ez-zahery, M., El Alem, N., 2019. Characterization and treatment of leachate of the great agadir discharge by infiltration–percolation onto titaniferous sand. Sci. Afr. 6, e00154. Bardi, A., Yuan, Q., Tigini, V., Spina, F., Varese, G.C., Spennati, F., Becarelli, S., Di Gregorio, S., Petroni, G., Munz, G., 2017. Recalcitrant compounds removal in raw leachate and synthetic effluents using the white-rot fungus Bjerkandera adusta. Water (Switzerland) 9, http://dx.doi.org/10.3390/w9110824. Bortolotto, T., Bertoldo, J.B., da Silveira, F.Z., Defaveri, T.M., Silvano, J., Pich, C.T., 2009. Evaluation of the toxic and genotoxic potential of landfill leachates using bioassays. Environ. Toxicol. Pharmacol. 28, 288–293. Brown, K.W., Donnelly, K.C., 1988. An estimation of the risk associated with the organic constituents of hazardous and municipal waste landfill leachates. Hazard. Waste Hazard. Mater. 5, 1–30. Chen, G., White, P.A., 2004. The mutagenic hazards of aquatic sediments: a review. Mutat. Res. Mutat. Res. 567, 151–225. Chibwe, L., Geier, M.C., Nakamura, J., Tanguay, R.L., Aitken, M.D., Simonich, S.L.M., 2015. Aerobic bioremediation of PAH contaminated soil results in increased genotoxicity and developmental toxicity. Environ. Sci. Technol. 49, 13889–13898.

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