Ecotoxicological study of land fi ll leachate treated in the ANAMMOX process

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 Cd2þ, Cu2þ, Zn2þ, 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


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. ).As rainwater percolates through layers of the decomposed waste, landfill leachate is formed (Christensen et al. ).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 1.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. ; Zheng-Yong et al. ).In order to remove nitrogen from ammonium rich wastewater without the need of organic carbon, the ANAMMOX process can be used 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

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. () 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. ).
Regular measurements of ammonium, nitrite, and nitrate nitrogen were conducted using fast photometric

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 (NH 4 þ -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, NaNO 2 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  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 Concentration of heavy metals in untreated leachate is presented in Table 3.

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.
High phytotoxicity of the untreated leachate and the influent might be caused by the presence of heavy metals.2), resulting from the lower nitrogen removal in the sample II by the ANAMMOX process, NH 3 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  The genetic similarity degree also shows that H 2 O 2 caused more DNA damage than EMS, which is shown in The dendrograms generated for the OPA04 primer from its RAPD profiles are presented in Figure 3(a)-3(i).
In Figure 3 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 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  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.

(
Liang & Liu ; Zheng-Yong et al. ; Miao et al. ).The ANAMMOX process, which consists of an autotrophic nitrogen removal performed by ANAMMOX bacteria, uses ammonium nitrogen (NH 4 þ -N) as an electron donor and nitrite (NO 2 --N) as an electron acceptor, to convert nitrogen into dinitrogen gas (van de Graaf et al. ; Strous et al. ).This process does not use oxygen nor an organic carbon source, it produces less sludge, and it emits less CO 2 (Strous et al. ; Reginatto et al. ).The most commonly used stoichiometry of the ANAMMOX process is shown in Equation (1) (Strous et al. ): 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 H 2 O and positive controls EMS (ethyl methanesulfonate), H 2 O 2 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 0 -AATCGGGCTG-3 0 ) was used (Williams et al. ).The PCR procedure optimized by Szulc et al. () 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 MgCl 2 (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 ¼ 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.
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 NH 4 þ -N and COD, but a low concentration of NO 2 --N and NO 3 --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.
Figure 1(a)-1(c).Lower phytotoxicity and growth stimulation may be caused by a high NH 4 þ removal and an increased NO 3 -concentration, because Lemna minor requires nitrogen for proper development (Cheng & Stomp ).However, NO 3 -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. ().Bortolotto et al. () used anaerobic/facultative lagoons for wastewater treatment.Bortolotto et al. () 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 Zn 2þ , may cause the growth stimulation.Ociepka-Kubicka & Ociepka () demonstrated that Zn 2þ 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 ).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. () 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 NH 4 þ (Table2), resulting from the lower nitrogen removal in the

Figure 1 |
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 2
Figure 2(a)-2(c).These results stand in contrast to research described by Ziembin ´ska-Buczyn ´ska et al. () 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 H 2 O 2 and EMS, may induce different genotoxicity.Rank et al. () obtained different genotoxicity in two different samples of Allium cepa induced by di(2-ethylhexyl) (a), groups consisting of positive controls and the samples incubated on the highest leachate dilution were observed.Additionally, similar groups were detected in Figure3(c), 3(g) and 3(i).As can be seen in Figure3(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 (H 2 O 2 ) and negative control (H 2 O), 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 NaCl addition) causes chromosomal aberrations in Allium cepa genome.It was stated by Arbašic ´et al. (), 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.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.
al. ; Ganigue et al. ; Zheng-Yong et al. ).Ranges of chosen physicochemical parameters of landfill leachate are shown in Table

Table 1 |
Typical physicochemical parameters of landfill leachate

Table 2
-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%.

Table 2 |
Physicochemical parameters of untreated wastewater and the leachate before and after treatment in SBR reactor with ANAMMOX process a mmol L -1 , b no unit.