Abstract

The exacerbated production of solid residues represents a major problem in the management and handling of urban wastes. The by-product of stored municipal and industrial solid waste production is landfill leachate. Leachate is characterized by a high concentration of organic compounds, ammonia, and the presence of heavy metals. Because of its composition, this kind of wastewater can cause serious environmental pollution and should be treated to reduce its toxic effects. Increasingly, the interest is directed to the application of the ANAMMOX (anaerobic ammonium oxidation) process for the landfill leachate treatment. In this study, for the first time, the effect of treatment with the ANAMMOX process on the toxicity of leachate was investigated. Based on the research performed in this study, it could be stated that the untreated landfill leachate from the municipal landfill and the influent of the ANAMMOX reactor present phytotoxicity to Lemna minor, due to a correlation of high concentrations of organic compounds, heavy metals, such as Cd2+, Cu2+, Zn2+, and the presence of an unionized form of ammonia (NH3). The results of the Allium cepa test demonstrated that the treatment was not efficient in eliminating the genotoxic substances that are responsible for the mutagenic potential in the effluent.

This article has been made Open Access thanks to the kind support of CAWQ/ACQE (https://www.cawq.ca).

INTRODUCTION

Increasingly, affluent lifestyles and industrial development in many countries worldwide have been accompanied by a rapid increase in both industrial and municipal solid waste production, which are stored in landfills (Renou et al. 2008). As rainwater percolates through layers of the decomposed waste, landfill leachate is formed (Christensen et al. 2001). Landfill leachate is characterized by high concentrations of organic compounds, ammonium and inorganic salts, including in some cases heavy metals such as Cd, Cr, Cu, Pb, Ni, Zn (Horan et al. 1997; Ganigue et al. 2007; Zheng-Yong et al. 2010). Ranges of chosen physicochemical parameters of landfill leachate are shown in Table 1.

Table 1

Typical physicochemical parameters of landfill leachate

ParameterRange (mg L–1)
pHa 4.5–9.0 
Total solid 2,000–60,000 
Biological oxygen demand (BOD) 20–57,000 
Chemical oxygen demand (COD) 140–152,000 
BOD/COD (ratio) 0.02–0.80 
Total organic carbon (TOC) 30–29,000 
Organic nitrogen 14–2,500 
Ammonium-N 100–5,500 
ParameterRange (mg L–1)
pHa 4.5–9.0 
Total solid 2,000–60,000 
Biological oxygen demand (BOD) 20–57,000 
Chemical oxygen demand (COD) 140–152,000 
BOD/COD (ratio) 0.02–0.80 
Total organic carbon (TOC) 30–29,000 
Organic nitrogen 14–2,500 
Ammonium-N 100–5,500 

The ranges are based on Christensen et al. (2001) and Renou et al. (2008).

aNo unit.

This kind of wastewater has to be removed by means of complicated processes, which generate high costs of treatment. Because of the high concentration of ammonia and low biodegradable organic matter content, nitrogen from landfill leachate is difficult to remove by means of conventional biological methods. Treating leachate using autotrophic nitrification and heterotrophic denitrification would be expensive, due to high costs of aeration and of the addition of an external carbon source (Hellinga et al. 1998; Zheng-Yong et al. 2010). In order to remove nitrogen from ammonium rich wastewater without the need of organic carbon, the ANAMMOX process can be used (Liang & Liu 2008; Zheng-Yong et al. 2010; Miao et al. 2015).

The ANAMMOX process, which consists of an autotrophic nitrogen removal performed by ANAMMOX bacteria, uses ammonium nitrogen (NH4+-N) as an electron donor and nitrite (NO2-N) as an electron acceptor, to convert nitrogen into dinitrogen gas (van de Graaf et al. 1996; Strous et al. 1998). This process does not use oxygen nor an organic carbon source, it produces less sludge, and it emits less CO2 (Strous et al. 1998; Reginatto et al. 2005). The most commonly used stoichiometry of the ANAMMOX process is shown in Equation (1) (Strous et al. 1998):  
formula
(1)
Typical indicators of the treated wastewater quality include: chemical oxygen demand (COD), biological oxygen demand (BOD), nitrogen and phosphorus compounds content, and total suspended solids (TSS). However, several reports have demonstrated that not only these indicators should be considered during industrial wastewater treatment, but also ecotoxicological ones (Wiszniowski et al. 2009; Wei et al. 2012; Zhu et al. 2013). Phytotoxicity may be investigated using Lemna minor (duckweeds (Mohan & Hosetti 1997; Singh & Singh 2006)). Lemna minor is a floating, vascular, fast growing plant, small in size, easy in culturing, reproducing vegetatively, cost effective, and commonly encountered in fresh water bodies. Lemna is sensitive to heavy metal pollution and high values of COD. Moreover, Lemna minor also has the ability to accumulate heavy metals and to remove ammonia nitrogen from wastewater efficiently, which makes it a good indicator in the toxicity tests of landfill leachates (Mohan & Hosetti 1997; Naumann et al. 2007; Ge et al. 2012), especially for short-term phytotoxicity tests.

Toxic substances may change the appearance, behavior, or cause DNA damage of indicators used in the toxicity tests. In order to investigate the effect of a toxicant at the genome level changes, the PCR-RAPD (polymerase chain reaction-random amplified polymorphic DNA) method can be applied. This technique, based on the PCR, allows the detection of genetic polymorphism, using arbitrary primers (Williams et al. 1990). Plants are known to be useful for environmental mutagenesis studies. Some plants, such as Allium cepa (onion), have been widely used in the research of chromosomal aberration, caused by chemical mutagens. Moreover, Allium cepa tests are inexpensive and the results can be easily analyzed (Wilkie et al. 1997; Ziembińska-Buczyńska et al. 2016). These advantages make this species useful for laboratory toxicity testing, and are more beneficial than other plants.

The aim of this study was to determine how efficiently the ANAMMOX process reduces the phytotoxicity and genotoxicity of landfill leachate. In order to test the wastewater phytotoxicity, Lemna minor was used in the growth inhibition tests, and in order to test genotoxicity Allium cepa was used in the RAPD assay.

MATERIALS AND METHODS

ANAMMOX reactor characteristics and physiochemical analysis

A 20 L sequencing batch reactor (SBR) was operated at a temperature of 33 ± 1 °C (standard deviation, SD), pH 7.8 ± 0.2 (SD), dissolved oxygen (DO) below 0.1 mg L–1, 1.9 ± 0.3 g L–1 (SD) of volatile suspended solids (VSS) and hydraulic retention time (HRT) equal to 1 day. Before the experiment, the reactor was fed with a mineral medium adapted from van de Graaf et al. (1996) for 830 days. After this time, the mineral medium was replaced with landfill leachate and treated for 120 days. The performance of the reactor during the treatment of synthetic and real landfill leachate was described in a previous paper (Tomaszewski et al. 2018).

Regular measurements of ammonium, nitrite, and nitrate nitrogen were conducted using fast photometric tests (MERCK Millipore) with a photometer (MERCK Spectroquant® NOVA60). Temperature and pH were monitored by JUMO tecLine HD – a pH combination electrode. DO concentration was measured by ELMETRON Conductivity/Oxygen Meter CCO-505 with the ELMETRON COG-1 oxygen sensor. The concentration of VSS was measured according to Standard Methods (APHA 2005).

Landfill leachate preparation and sampling

The real landfill leachate used for this experiment was taken from a municipal landfill in southern Poland. The feeding strategy was based on an increase of the nitrogen loading rate (NLR) when the nitrogen removal efficiency was stable. The ammonium (NH4+-N) concentration was regulated by diluting the wastewater with tap water. In order to achieve an adequate ammonium:nitrites ratio (1:1.32) required for the ANAMMOX process, NaNO2 was added, which simulated the wastewater after a partial nitrification process.

Samples for ecotoxicological assays were collected three times: at the 35th (sample I), at the 70th (sample II), and at the 98th (sample III) day of the landfill leachate treatment. Samples were taken before dilution (untreated wastewater) and from the influent and the effluent of the ANAMMOX reactor.

Phytotoxicity test

The growth inhibition tests on Lemna minor were performed in triplicate. The concentrations of each sample of wastewater were 6.25, 12.5, 25, 50 and 100%. As a negative control (NC), a standard growth medium for macrophytes was used, which was prepared according to the OECD Test No. 221 (2006). L. minor plants, with a total of at least 12 leaves, were placed in plastic Petri dishes containing 20 mL of the test solution and incubated in a thermostatic cabinet, at 24 ± 1 °C under 16 hours photoperiod (16 hours of light:8 hours of darkness). After 14 days of incubation, the number of dead and live plants was counted and the percentage of the growth inhibition was determined.

Genotoxicity test: DNA isolation, PCR-RAPD procedure, and the results analysis

Germinated seeds of Allium cepa were placed in triplicate in 1.5 ml test-tubes containing wastewater solution in distilled water in concentrations of 6.25, 12.5, 25, 50 and 100%. As a negative control H2O and positive controls EMS (ethyl methanesulfonate), H2O2 were used. After 72 hours incubation at room temperature DNA was isolated. Genomic DNA of Allium cepa was isolated using the Genomic Mini AX Plant Spin (A&A Biotechnology, Poland). For PCR-RAPD the primer OPA04 (5′-AATCGGGCTG-3′) was used (Williams et al. 1990). The PCR procedure optimized by Szulc et al. (2012) was used. Amplification of DNA was performed in a 30 μL reaction mixture, consisting of 15.5 uL MiliQ water, 6 μL 5 × Green GoTaq Flexi Buffer (Promega), 2.4 μL MgCl2 (25 mM, Promega), 1.3 μL dNTPs Mix (1 mM, Promega), 0.5 μL of the primer, and 0.3 μL GoTaq DNA Polymerase (5 u/μL), Promega). DNA was added in a volume of 4 μL. Amplification was performed in C-100 Thermocycler (BioRad) programmed as follows: a preliminary 12 min denaturation at 95 °C; 30 cycles of 1 min denaturation at 95 °C, 1 min annealing at 37 °C, 1.5 min extension at 72 °C; and 5 min final extension at 72 °C. PCR products were separated with gel electrophoresis on 1% agarose gel (EURx) in 1 × TBE buffer (100 mM Tris pH = 8.3, 90 mM boric acid, 1 mM EDTA) with ethidium bromide (10 μL/ml, Promega). The electrophoresis was performed at 100 V for 90 min. A 1 kb DNA ladder (Promega) was used for the DNA bands size assessment. The gel was visualized under UV light and photographed. The fingerprints were analyzed with Quantity One 1D Software (BioRad). Dendrograms of the genetic distance and diagrams presenting genetic similarities were constructed. Dice coefficient was applied for constructing dendrograms using the neighbor-joining algorithm.

RESULTS AND DISCUSSION

The samples of untreated landfill leachate, influent, and effluent of the ANAMMOX reactor were collected at three sampling times: at the 35th (sample I), at the 70th (sample II) and at the 98th (sample III) days of the landfill leachate treatment. Physicochemical parameters of untreated leachate, influent, and effluent of SBR with the ANAMMOX process are presented in Table 2. Untreated leachate was characterized by a high concentration of NH4+-N and COD, but a low concentration of NO2-N and NO3-N. pH of untreated leachate was close to 8. Concentrations of heavy metals in the untreated landfill leachate were provided by the municipal landfill in southern Poland. Concentration of heavy metals in untreated leachate is presented in Table 3.

Table 2

Physicochemical parameters of untreated wastewater and the leachate before and after treatment in SBR reactor with ANAMMOX process

 Untreated (mg L−1)
Influent (mg L−1)
Effluent (mg L−1)
 IIIIIIIIIIIIIIIIII
NH4+-N 820–1,070 470 320 440 0.0 27.4 6.2 
NO2-N 2.21–2.15 580 530 630 0.6 10.0 5.0 
NO3-N 8.1–12.0 12.4 8.6 9.5 100 103 88 
TN 830.31–1,084.15 1,140 900 1,090 118 190 110 
NH3 22.8–55.6 11,7 7.4 10.0 0.0 0.9 1.0 
COD 2,440–2,450 1,816 1,670 2,070 938 880 1,050 
11.7 3.6 4.6 4.8 5.0 4.0 4.6 
Cl 2,200 1,400 1,040 1,490 2,210 1,580 2,290 
Alkalinitya 50.5 14.88 34.60 34.20 12.01 24.9 26.3 
pHb 7.71–7.99 7.66 7.63 7.62 7.72 7.84 7.75 
 Untreated (mg L−1)
Influent (mg L−1)
Effluent (mg L−1)
 IIIIIIIIIIIIIIIIII
NH4+-N 820–1,070 470 320 440 0.0 27.4 6.2 
NO2-N 2.21–2.15 580 530 630 0.6 10.0 5.0 
NO3-N 8.1–12.0 12.4 8.6 9.5 100 103 88 
TN 830.31–1,084.15 1,140 900 1,090 118 190 110 
NH3 22.8–55.6 11,7 7.4 10.0 0.0 0.9 1.0 
COD 2,440–2,450 1,816 1,670 2,070 938 880 1,050 
11.7 3.6 4.6 4.8 5.0 4.0 4.6 
Cl 2,200 1,400 1,040 1,490 2,210 1,580 2,290 
Alkalinitya 50.5 14.88 34.60 34.20 12.01 24.9 26.3 
pHb 7.71–7.99 7.66 7.63 7.62 7.72 7.84 7.75 

ammol L–1, bno unit.

Table 3

Concentration of heavy metals in untreated leachate provided by the municipal landfill in southern Poland

Heavy metalConcentrationUnit
Cu 26.2–26.6 μg L–1 
Zna 0.47–2.97 mg L–1 
Pb <5 μg L–1 
Cd <0.08 μg L–1 
Hg <0.0001 μg L–1 
Cr(VI)a <0.01 mg L–1 
Heavy metalConcentrationUnit
Cu 26.2–26.6 μg L–1 
Zna 0.47–2.97 mg L–1 
Pb <5 μg L–1 
Cd <0.08 μg L–1 
Hg <0.0001 μg L–1 
Cr(VI)a <0.01 mg L–1 

amg L–1.

As shown in Table 2, in each sample (I–III) almost the whole content of N-NH4+ and N-NO2 was removed from the influent, indicating a good ANAMMOX activity. In the effluent of samples I and III the concentrations of Cl were 2,210 and 2,290 mg L–1, respectively. In sample II the concentration decreased to 1,580 mg L–1. Furthermore, an increase of the pH value in sample II (7.84) was observed, compared to samples I and III (7.72 and 7.75). The removal of COD, despite the different concentration in the influent, was stable and remained at an efficiency of 42 ± 2%.

Lemna minor bioassay

The results of the Lemna minor test for the three samples are reported in Figure 1(a)1(c). The test shows that duckweed incubated in increasing concentrations of the untreated leachate and the influent was close to the growth inhibition of 100%, except for the highest dilution of the influent in the sample II where a 55% growth inhibition was observed. Treatment results in a decrease of the growth inhibition in almost all samples, except for the non-diluted effluent in sample II, where a 100% inhibition was observed. Furthermore, in samples II and III in dilutions between 25–6.25% and 50–6.25%, respectively, growth stimulation was observed.

Figure 1

Growth inhibition (%) towards Lemna minor of concentration between 6.25% and 100% of untreated leachate, influent and treated leachate analyzed by bioassay. (a) Sample collected at the 35th, (b) sample collected at the 70th, (c) sample collected at the 98th day of the landfill leachate treatment. *Statistically significant difference (t-Student test, p < 0.05).

Figure 1

Growth inhibition (%) towards Lemna minor of concentration between 6.25% and 100% of untreated leachate, influent and treated leachate analyzed by bioassay. (a) Sample collected at the 35th, (b) sample collected at the 70th, (c) sample collected at the 98th day of the landfill leachate treatment. *Statistically significant difference (t-Student test, p < 0.05).

High phytotoxicity of the untreated leachate and the influent might be caused by the presence of heavy metals. Naumann et al. (2007) and Mohan & Hosetti (1997) reported a toxic effect of some heavy metals on Lemna minor. Moreover, Naumann et al. (2007) indicated that Cd2+, Cu2+, Ni2+ were the most toxic for duckweed. The presence of Cd2+ and Cu2+ in the tested untreated leachate was detected in the tested samples. Additionally, the highest concentration in the untreated leachate was detected for Zn2+ (0.496–2.92 mg L–1). Despite that, a low concentration of Zn2+ may be assimilated by Lemna minor, a high level of Zn2+ contamination causes undesirable effects (Radića et al. 2010). Thus, the occurrence of Cd2+, Cu2+ and Zn2+ could have contributed to the increased phytotoxicity of the untreated leachate and influent.

Some studies have demonstrated that organic compounds are important constituents of landfill leachate, influencing its toxicity (Bortolotto et al. 2009). The concentration of COD in the untreated landfill leachate and the influent were 2,445 and 1,852 mg L–1, respectively. Bortolotto et al. (2009) detected that for non-diluted untreated leachate, with the COD concentration of 832.5 mg L–1, the inhibition of the root growth in Allium cepa was calculated to be 87.76%. It is worth mentioning that COD was not the only factor influencing phytotoxicity. However, a concentration level of COD lower than in the untreated leachate and in the influent may suggest that COD is a significant compound of leachate influencing its phytotoxicity, because of the 100% growth inhibition in almost all dilutions in the untreated leachate and the influent.

Another factor which may influence the phytotoxicity of leachate are nitrogen compounds, especially ammonia (Cheng & Stomp 2009). The importance of ammonia in the leachate toxicity has been demonstrated by Clément & Merlin (1995), especially with a high pH value. At the pH level of ∼8, ammonia was changed into free ammonia (NH3), which influenced the toxicity of Lemna minor cells (Cheng & Stomp 2009). For the wastewater studied in this work, the concentration of FA, based on Anthonisen et al. (1976), was calculated. FA concentrations in untreated, influent and effluent were 22.8–55.6, 10.0–11.6 and 0.8–1.0 mg L–1, respectively. Körner et al. (2001) investigated the effects of NH3 concentrations on the growth of Lemna gibba at pH 6.8–8.7 using domestic wastewater and reported that, within the examined range of pH, NH3 contributed to the toxicity at NH3-N concentrations below 1 mg L–1 (Cheng & Stomp 2009). It could be suspected that free ammonia may be the reason for high toxicity of the untreated leachate, due to its pH value of 7.71–7.99 and NH4+ concentration.

The ANAMMOX process led to the reduction of the growth inhibition, observed for the effluent in Figure 1(a)1(c). Lower phytotoxicity and growth stimulation may be caused by a high NH4+ removal and an increased NO3 concentration, because Lemna minor requires nitrogen for proper development (Cheng & Stomp 2009). However, NO3 is the only absorbable form of nitrogen. In addition, the reduction of the COD concentration resulted in a decrease in phytotoxicity, which was also reported by Bortolotto et al. (2009). Bortolotto et al. (2009) used anaerobic/facultative lagoons for wastewater treatment. Bortolotto et al. (2009) reported that the concentration of COD decreased with a 90% efficacy. Furthermore, a reduction of heavy metals was demonstrated, which allowed the assumption that the reduction of COD and heavy metals concentrations could decrease the phytotoxic effects of landfill leachate. In some cases a low concentration of some heavy metals, such as Zn2+, may cause the growth stimulation. Ociepka-Kubicka & Ociepka (2012) demonstrated that Zn2+ can stimulate plants growth, caused by the proper metabolic function. Both the deficiency and excess of this element limits the growth and development of plants (Ociepka-Kubicka & Ociepka 2012). It is possible that the ANAMMOX process caused a reduction of zinc concentration, thus its toxic properties changed into stimulating ones, which could be observed in the effluent of samples II and III. Lotti et al. (2012) claimed that a minute concentration of zinc and copper adsorbed on the ANAMMOX biomass supports the validity of our results that in the ANAMMOX process the concentration of heavy metals may be decreased.

High toxicity of the non-diluted effluent in the sample II (Figure 1(b)) may result from the presence of free ammonia. Due to the pH value 7.84 and a high concentration of NH4+ (Table 2), resulting from the lower nitrogen removal in the sample II by the ANAMMOX process, NH3 may be formed, as described above.

Induction of genotoxicity in Allium cepa

PCR-RAPD was applied to show changes in DNA as the genotoxic effect of the untreated landfill leachate, the influent, and the effluent of SBR with the ANAMMOX process. For amplification the OPA04 primer, specific for Allium cepa, was used (Szulc et al. 2012). According to Szulc et al. (2012) the OPA04 primer had the lowest genetic similarity to the negative control (H2O) in RAPD profiles, so it is the most efficient in the detection of DNA changes in Allium cepa roots. On the basis of the RAPD profile obtained with the primers OPA04, the genetic similarity to the negative control (H2O) was calculated, which is shown in Figure 2(a)2(c). The results of the RAPD similarity profiles to the negative control suggest that all the concentrations of landfill leachate used in the experiment caused changes in the DNA of Allium cepa cells. It allows us to state that the tested leachate was mutagenic.

Figure 2

The comparison of RAPD genetic similarity to negative control (H2O) obtained for untreated leachate, influent and effluent of SBR reactor with ANAMMOX process in concentration of 6.25–100% and positive controls. (a) Sample collected at the 35th, (b) at the 70th, (c) at the 98th day of the landfill leachate treatment. *No detected fingerprints.

Figure 2

The comparison of RAPD genetic similarity to negative control (H2O) obtained for untreated leachate, influent and effluent of SBR reactor with ANAMMOX process in concentration of 6.25–100% and positive controls. (a) Sample collected at the 35th, (b) at the 70th, (c) at the 98th day of the landfill leachate treatment. *No detected fingerprints.

Different RAPD similarity profiles in each sample were obtained for the untreated landfill leachate used in the experiment, which may suggest that due to the composition leachate in each phase had different genotoxic effects on Allium cepa cells. It is worth mentioning that the RAPD similarity profiles obtained by the negative control are lower or comparable to the RAPD similarity profiles obtained by the positive controls (H2O2, EMS). This allows us to assume that even in such a low concentration of the untreated landfill leachate, the influent and the effluent may generate higher changes in the indicator's DNA than the positive controls. Srivastavia et al. (2005) demonstrated that even a low concentration of the untreated landfill leachate (10%) may cause mitotic and chromosomal abnormalities in Allium cepa root meristem cells. Additionally, these abnormalities were probably the outcome of the impact of leachate on the spindle apparatus (Srivastavia et al. 2005).

The degree of similarity in all the samples is not correlated with the landfill leachate concentration (Figure 2). It could be expected that the highest similarity to the negative control should be obtained for the lowest leachate concentration (6.25%) and should decrease as the leachate concentration increases. Similar results were obtained for hospital wastewater by Ziembińska-Buczyńska et al. (2016). Such a situation may occur because the DNA changes caused by landfill leachate appear randomly. Moreover, for non-diluted leachate (100%) in Figure 2(a)2(c), the value of the similarity profiles was higher than of the similarity profiles of the lowest dilution of leachate (6.25%), compared to the negative control. Optionally, a high concentration of leachate may damage the DNA of Allium cepa cells, leading to the intensification of DNA repairing processes; therefore, less DNA damage may be detected through PCR-RAPD. This may support the hypothesis that after passing the critical level of DNA damage, the efficient cellular repairing processes are being turned on (Ziembińska-Buczyńska et al. 2016).

The genetic similarity degree also shows that H2O2 caused more DNA damage than EMS, which is shown in Figure 2(a)2(c). These results stand in contrast to research described by Ziembińska-Buczyńska et al. (2016) in which toxicity of hospital wastewater was investigated. It is possible that a different variety of Allium cepa plant was used for these tests, thus the same toxic factors, such as H2O2 and EMS, may induce different genotoxicity. Rank et al. (2002) obtained different genotoxicity in two different samples of Allium cepa induced by di(2-ethylhexyl)phthalate (DEHP). For one sample, DEHP was not found to be genotoxic, in contrast to the second sample, in which a high level of aberrant cells was detected. These results confirm the earlier hypothesis that different groups of Allium cepa may exhibit different genotoxicity pathways for the same positive controls.

The dendrograms generated for the OPA04 primer from its RAPD profiles are presented in Figure 3(a)3(i). In Figure 3(a), groups consisting of positive controls and the samples incubated on the highest leachate dilution were observed. Additionally, similar groups were detected in Figure 3(c), 3(g) and 3(i). As can be seen in Figure 3(f), positive and negative controls create one cluster of the dendrogram, another group is created by samples incubated on the leachate dilution. Figure 3(e) contains two groups: one consisting of the positive (H2O2) and negative control (H2O), and the other group with the positive control (EMS) and the sample incubated on the lowest (6.25%) leachate dilution. Clusters constructed of the negative control and the sample incubated on the lowest (12.5 and 6.25%) leachate dilutions were detected in Figure 3(c) and 3(i). For samples I and III incubated on the untreated leachate (Figure 3(a) and 3(g)) and the effluent of SBR (Figure 3(c) and 3(i)) similar clusters were observed. This situation suggests similar genotoxic effects of untreated and treated wastewater. Potentially, it resulted from chemical parameters of both samples, such as the concentration of Cl and COD. However, the Cl concentrations in the untreated leachate and the effluent of samples I and III were similar (Table 1), but the concentration of COD was lower in both samples. It is possible that during the treatment the components of COD that caused genotoxicity were not biodegradable; therefore, the effluents included similar toxic fractions of COD as the untreated leachate. These results may also suggest that the concentration of ammonia did not influence genotoxicity, because of different concentrations of the total nitrogen in both groups. However, there is some evidence that salinity (as NaCl addition) causes chromosomal aberrations in Allium cepa genome. It was stated by Arbašić et al. (1995), who observed that macroscopic changes in terms of root growth slow-down is caused by NaCl, representing phenotype expression of the changes in genetic material of root tip cells.

Figure 3

Dendrograms constructed with neighbor-joining algorithm for RAPD profiles with primer OPA04 for A. cepa root meristem cell; samples incubated with increasing concentration of landfill leachate range between 6.25% and 100%; and EMS and H2O2 – positive control, H2O – negative control. Sample I from untreated (a), influent (b), effluent (c), sample II from untreated (d), influent (e), effluent (f), sample III from untreated (g), influent (h), effluent (i).

Figure 3

Dendrograms constructed with neighbor-joining algorithm for RAPD profiles with primer OPA04 for A. cepa root meristem cell; samples incubated with increasing concentration of landfill leachate range between 6.25% and 100%; and EMS and H2O2 – positive control, H2O – negative control. Sample I from untreated (a), influent (b), effluent (c), sample II from untreated (d), influent (e), effluent (f), sample III from untreated (g), influent (h), effluent (i).

The dendrograms generated from the RAPD profiles presented a long genetic distance between each dilution of landfill leachate and the positive controls with a proven mutagenic effect. Additionally, the genetic distances presented at the dendrograms are not correlated with the leachate concentrations used in this experiment, indicating randomness of genotoxic mechanisms.

Analyzing the genotoxicity test in Allium cepa in which plants were exposed to different concentrations of the untreated leachate, the influent, and the effluent of SBR obtained for three samples, the correlation between the concentration of components of landfill leachate, such as heavy metals or COD on the ANAMMOX process, was not found. It can be expected that the untreated leachate and the influent could have the highest toxicity, because of the presence of heavy metals and a higher concentration of COD. Additionally, the treatment process had to decrease the concentration of COD and heavy metals as described above, which may result in the reduction of the effluent genotoxicity. However, the results show that the treated leachate caused a similar degree of the DNA damage to the untreated leachate, as described above. It may be caused by low efficiency of the COD removal (47 ± 2%). However, in the study concerning the landfill leachate treatment, Bortolotto et al. (2009) used anaerobic/facultative lagoons with a high COD removal efficiency (90%) and pointed out that this did not affect the decrease of genotoxicity in the leachate. It allows us to assume that even high organic compound removal did not cause a decrease of genotoxic effects of landfill leachate. Furthermore, it confirms the above hypothesis, that toxic fractions of COD may not be biodegradable during the treatment process. Leachate may contain large concentrations of organic compounds, which can be more or less easily biodegradable. Xenobiotics are resistant to biodegradation compounds which are considered as toxic substances (Christensen et al. 2001). Tiushin et al. (2013) proved that xenobiotics such as salicylic acid caused chromosomal aberrations in Allium cepa genome. This allows the assumption that the presence of xenobiotics in landfill leachate may be responsible for DNA damage.

It is possible that some DNA damage was caused during the DNA isolation procedure, due to some substances contained inside the cell. Allium cepa has the ability to store various substances inside its cells, such as flavonoids, phenols, and sterols included in leachate, which can be released from its vacuoles during the isolation of the genetic material and could interfere with DNA (Abu-Romman 2011; Ziembińska-Buczyńska et al. 2016).

Both genotoxicity and phytotoxicity could result from the combination of heavy metals or other organic compounds present in the landfill leachate or other components of the leachate which were not studied (Kjeldsen et al. 2002; Srivastavia et al. 2005). Additionally, in favorable conditions of pH close to 8, phytotoxic effects may be caused by ammonia. The differences between the results of genotoxic and phytotoxic tests suggest that the ANAMMOX process caused a decrease of phytotoxicity in Lemna minor, but did not influence the reduction of genotoxicity in Allium cepa. The reduction of phytotoxicity could be affected by the decrease in the COD concentration and the increase in NO3 in the effluent, due to the treatment process. It is not possible to determine the influence of the ANAMMOX process on the reduction of the concentration of heavy metals, as described by Lotti et al. (2012); furthermore, decreased phytotoxicity in the effluent suggests a lower concentration of heavy metals. Similar genotoxicity in the untreated leachate and in the effluent suggests that the ANAMMOX process did not cause the reduction of components influencing the DNA damage; moreover, these components were not tested, so it is not possible to distinguish what caused genotoxicity.

Further studies will be needed in order to evaluate the effects of the treatment on other organisms of the trophic chain. Furthermore, it is necessary to detect exactly which components of the landfill leachate influence the toxicity of wastewater.

CONCLUSIONS

Due to its composition, landfill leachate is one of the major environmental problems. Treatment of this kind of wastewater is important for the purposes of reducing its toxic effect on the environment, which was the subject matter of previous studies. In this experiment we used a phytotoxic test on Lemna minor and PCR-RAPD for genotoxicity of Allium cepa roots performed in a dilution between 6.25 and 100% of the untreated leachate, the influent, and effluent of SBR with the ANAMMOX process. Based on the research performed in this study, it could be stated that the untreated landfill leachate from the municipal landfill in southern Poland, and the influent of the SBR reactor, demonstrate phytotoxicity for Lemna minor due to the correlation of a high concentration of organic compounds, heavy metals, such as Cd2+, Cu2+, Zn2+, and the presence of a unionized form of ammonia (NH3). The present study allows us to state that ANAMMOX could be an important tool to reduce the phytotoxicity of landfill leachate as observed in the Lemna minor test. The results of the Allium cepa test demonstrated that the treatment was not efficient in eliminating the genotoxic substances responsible for the mutagenic potential of the effluent. Since genotoxic potential is observed, even after the treatment of the leachate, this does not appear to be associated with the effects of heavy metals, such as the formation of free radicals or the presence of organic compounds. Genotoxicity may be caused by other substances in the leachate not tested in the study.

ACKNOWLEDGEMENTS

The study was financed by the Polish National Science Centre: UMO-2013/09/D/NZ9/02438. The authors would like to thank Tomasz Lewek for laboratory assistance.

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