Landfills leachates
The leachates used in this study were collected at Seropédica Landfill (active landfill with nine years of operation) and Gericinó Landfill (landfill operated for 27 years and currently closed) in Rio de Janeiro state, Brazil.
The Seropédica Sanitary Landfill has been in operation since 2011 and received an average of 10,000 tonnes per day of municipal solid waste from Seropédica, Itaguaí and Rio de Janeiro cities. The leachate production in this landfill is, on average, 1,000 m3/day (Ciclus Ambiental 2018; Almeida et al. 2020).
The Gericinó Controlled Landfill is located in Rio de Janeiro city that started operation in 1987 as an open dumpsite, but after being subjected to modifications, operated as a controlled landfill until its closure in 2014. The landfill received on average 2,000 tonnes per day of municipal solid waste, generating about 500 m3/day of leachate (Lima et al., 2017b).
In each landfill, leachate samples (n= 4) were collected, in a simple sampling, at the accumulation point, in different seasons from 2017 to 2019. The samples were maintained refrigerated (4 °C) for later physicochemical characterization (performed the day after collections) and preserved (-20 °C) for ecotoxicity assays (performed within a maximum period of 60 days after sample collection).
Evaluation of physicochemical parameters
The parameters pH, alkalinity, conductivity, chemical oxygen demand (COD), total organic carbon (TOC), total solids, total dissolved solids, total suspended solids, chloride, total ammonia nitrogen (TAN), turbidity, humic substances (HS) and absorbance at 254 nm, the latter related to the presence of aromatic organic compounds, such as humic substances (Zielińska et al., 2020) were evaluated for the physicochemical characterization.
The analyses were performed according to the methodology described in Standard Methods for the Examination of Water and Wastewater (APHA 2012), except for the humic substances parameter that was determined spectrophotometric/colorimetric method described by Lima et al. (2017a). All the tests were performed in triplicates, considering the standard deviation of the replicates.
Evaluation of leachates ecotoxicity
Aliivibrio fischeri (bioluminescence inhibition assay)
The marine bacterium Vibrio fischeri, currently designated as Aliivibrio fischeri (strain NRRL B- 11177), was used following the Brazilian Standard NBR 15411-3 (ABNT 2012) based on the international standard ISO 11348-3:2007, using the Microtox equipment (Modern Water and Model 500 Analyser). The bacterial suspensions used in the study were commercially obtained in lyophilized form (Umwelt Biotecnologia Ambiental, Blumenau, Brazil) and stored at a temperature between -18°C and -20 °C.
For this assay, the pH of the samples was adjusted to the range of 6.0-8.5 with HCl (1.0 mol/L) and NaOH (1.0 mol/L). Before analysis, the salinity adjustment of the samples was carried out with the osmotic solution (20% NaCl). The assay was performed at 15°C.
Reference assays with Zn2+ (ZnSO4.7H2O) and Cu2+ (CuSO4.5H2O) were used to check the sensitivity of each vial of bacteria. In addition, an additional control using 3,5-dichlorophenol and potassium dichromate were periodically evaluated for each bacterial batch used during the study, according to the methodology proposed by ABNT (2012).
Assay responses were expressed as reducing bioluminescence in EC50 (median effect concentration) after 30 minutes of exposure of the organism to the samples. All bioluminescence inhibition tests were performed in triplicate, considering the standard deviation of the replicates.
Activated sludge microorganisms (Respirometry assay)
Respirometry assay was carried out based on the Organisation for Economic Co-operation and Development methodology, method number 209 (OECD, 2010). Initially, the preparation of concentrated synthetic sewage was realized for feed for sludge during the test, as shown in Table 1.
Table 1 Composition of concentrated synthetic sewage (OECD, 2010)
Components
|
Concentration (g/L)
|
Peptone
|
16.0
|
Meat extract
|
11.0
|
Urea
|
3.0
|
NaCl
|
0.7
|
CaCl2. 2H2O
|
0.4
|
MgSO4. 7H2O
|
0.2
|
K2HPO4
|
2.8
|
The test was conducted under constant aeration, with the following composition: sample, biological sludge (concentration of 1.5 g/ L of suspended solids), and synthetic sewage (pH = 7.5 ± 0.5) in a beaker using a stirring plate and an air compressor connected to a silicone hose and a porous stone, for 180 min at a temperature of 20 ± 2 °C. The proportions of this mixture used in the test are shown in Table 2.
Table 2 Composition of Respirometry assays
Reagents
|
1
|
2
|
3
|
4
|
5
|
Blank
|
Abiotic
|
Sample (mL)
|
150
|
75
|
50
|
25
|
12.5
|
-
|
150
|
Synthetic sewage feed (mL)
|
16
|
16
|
16
|
16
|
16
|
16
|
16
|
Activated sludge suspension (mL)
|
134
|
134
|
134
|
134
|
134
|
134
|
-
|
Deionized Water (mL)
|
-
|
75
|
100
|
125
|
137.5
|
150
|
134
|
Total volume (mL)
|
300
|
300
|
300
|
300
|
300
|
300
|
300
|
|
|
|
|
|
|
|
|
|
Dissolved oxygen (DO) of the mixture (300 mL in a BOD bottle) monitoring reading started. DO monitoring (oximeter WTW OXI 7310 ) was performed until values lower than 2.0 mg DO/L.
The blank test (prepared with activated sludge and synthetic sewage, with no sample or reference substance), abiotic control (prepared without the addition of sludge biomass), and reference with Zn2+ (ZnSO4.7H2O) and Cu2+ (CuSO4.5H2O) were performed. In addition, all respiration inhibition tests were performed in triplicate and conducted at a temperature of 20 ± 2 °C, based on the required test condition (OECD, 2010).
The percentage of total inhibition (TI) of the sample was calculated using Equation 1, where: Rt = Total sample respiration rate; Rta = Total abiotic respiration rate, and Rtb = Total blank respiration rate.
Finally, the percentages of inhibition of oxygen consumption of the tested samples were plotted, and the EC50 values (%v/v) were obtained using the Trimmed Spearman-Karber statistical method (Hamilton et al. 1977), using the TSK program version 1.5 (USEPA, Cincinnati, Ohio).
Removal of leachate pollutants
Ammonia nitrogen and humic substances are found in high concentrations in landfill leachates and are identified as one of the main potential pollutants of this wastewater. In this context, the treatments of the raw leachate were carried out separately to remove ammonia by air stripping and humic substances by membrane filtration.
Ammonia Stripping
After pH adjustment (4 L), the raw leachate samples were submitted to air stripping at room temperature (25-27°C) to remove ammonia nitrogen. The pH was adjusted with NaOH 1.0 mol/L for pH = 11.0, and the tests were conducted in constant aeration and agitation, with the use of an air diffuser (rate of 360 L/ h) and agitation plate in order to maintain continuous contact between the air bubbles and the liquid phase.
The removal of total ammonia nitrogen (TAN) was performed for a long period, given the high ammonia nitrogen concentration in the leachate samples. Therefore, the ammonia concentration was monitored during the test period.
In order not to generate toxic effects for the studied organisms, the air stripping occurred until reaching a concentration lower than the no observed effect (NOEC) for A. fischeri (0.39 mg NH3/L) (Silva et al., 2018). In relation to the activated sludge microorganisms, ammonia concentration is not a concern; studies show that most species have a high tolerance to ammonia, except the ciliated protozoa, which exhibit a decrease in their diversity (Puigagut et al. 2005).
All samples were readjusted to pH initial after the treatment by adding 37% HCl or 1.0 mol/L NaOH and stored (4 °C) for subsequent physicochemical and ecotoxicological tests.
Membrane filtration
The filtration was conducted in a batch mode, using dead-end cell equipment (PAM Membranas Seletivas Ltda, BR) and the nitrogen gas for the pressure regulation in the filtration module.
Before membrane filtration, leachate samples were filtered in the vacuum system on a 0.45 µm glass fiber filter (Macherey-Nagel, Germany) to remove suspended solids. After this process, permeation was carried out using membranes of two different molecular weight cut-offs (10 kDa and 500 Da) to remove humic and fulvic fractions from landfill leachate samples.
In leachates from intermediate and mature landfills, humic acids (HA) constitute organic molecules with a molar mass greater than 10,000 Da, while fulvic acids (FA) constitute molecules with a molar mass between 500-1,000 Da (Chian, 1977; Trebouet et al., 2001 and Zolfaghari et al., 2018).
Ultrafiltration (UF) was performed using a 10 kDa polyethersulfone membrane (UP 010 - Microdyn-Nadir, USA) and transmembrane pressure of 5 bar. Nanofiltration (NF) was performed from the permeate of UF using a 500 Da polyethersulfone membrane (NP 030 - Microdyn-Nadir, USA) and transmembrane pressure of 20 bar. The filtration tests were performed at the sample's natural pH and room temperature (25-27°C). In this way, the UF permeate (with the humic portion removed) and UF+NF (with the humic and fulvic portion removed) were evaluated.
The use of UF and NF membranes can remove a part of ammonia nitrogen from the leachate. Therefore, to evaluate only the toxic potential of humic substances and fractions, the correction in the ammonia nitrogen concentration was performed with NH₄HCO₃ for the initial concentration of the raw leachate in UF and UF+NF permeates for further ecotoxicological evaluation. The ammonia nitrogen present in landfill leachate is associated with carbonates and bicarbonates (Campos et al., 2013).
The effluents from the ultrafiltration and nanofiltration permeate were stored (4 °C) for subsequent physicochemical and ecotoxicological tests.
Data analysis
Toxicity reduction
The toxic units (TU) of the samples were calculated using Equation 2.
The samples' acute ecotoxicity reduction was evaluated based on the TU of the raw leachates in TU of treated leachates, as calculated by Equation 3.
The Kolmogorov-Smirnov test assessed the normality of the data. The comparison among the means of the parameter's landfills leachate was performed using Fisher's test (Least Significant Difference- LSD) to correlate the studied landfills' characteristics. The bivariate relationship was assessed using the Pearson correlation, and the association between the physicochemical and ecotoxicological parameters was performed using the analysis of variance (ANOVA), principal component analysis (PCA) and linear regression. All statistical tests were performed with STATISTICA software version 7, licensed by STAT SOFT, except for the comparison test between the means of EC50 values considering the confidence intervals, in which it was performed based on the methodology proposed in USEPA (1985).