New insights on abatement of organic matter and reduction of toxicity from landfill leachate treated by the electrocoagulation process

Accepted Manuscript Title: New insights on abatement of organic matter and reduction of toxicity from landfill leachate treated by the electrocoagulation process Authors: Aline Roberta de Pauli, Fernando Rodolfo Espinoza-Qui˜nones, Isabella Cristina Dall’Oglio, Daniela Estelita Goes Trigueros, Aparecido Nivaldo M´odenes, Caroline Ribeiro, Fernando Henrique Borba, Alexander Dimitrov Kroumov PII: DOI: Reference:

S2213-3437(17)30517-1 https://doi.org/10.1016/j.jece.2017.10.017 JECE 1927

To appear in: Received date: Revised date: Accepted date:

21-8-2017 19-9-2017 8-10-2017

Please cite this article as: Aline Roberta de Pauli, Fernando Rodolfo Espinoza-Qui˜nones, Isabella Cristina Dall’Oglio, Daniela Estelita Goes Trigueros, Aparecido Nivaldo M´odenes, Caroline Ribeiro, Fernando Henrique Borba, Alexander Dimitrov Kroumov, New insights on abatement of organic matter and reduction of toxicity from landfill leachate treated by the electrocoagulation process, Journal of Environmental Chemical Engineering https://doi.org/10.1016/j.jece.2017.10.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

New insights on abatement of organic matter and reduction of toxicity from landfill leachate treated by the electrocoagulation process Aline Roberta de Pauli1, Fernando Rodolfo Espinoza-Quiñones1, , Isabella Cristina Dall’Oglio1, Daniela Estelita Goes Trigueros1, Aparecido Nivaldo Módenes1, Caroline Ribeiro1, Fernando Henrique Borba2, Alexander Dimitrov Kroumov3 1

Postgraduate Program of Chemical Engineering, West Paraná State University, Rua da Faculdade 645, Jd. Santa Maria, 85903-000,

Toledo, PR, Brazil 2

Postgraduate Program of Environment and Sustainable Technologies, Federal University of Fronteira Sul, Rua Major Antônio

Cardoso 1580, 97900-00, Cerro Largo, RS, Brazil The “Stephan Angeloff” Institute of Microbiology - Bulgarian Academy of Sciences, Acad. G. Bonchev str., Bl. 26, Sofia 1113,

3

Bulgaria

Corresponding

author: Tel.: +55 45 3379 7092, fax: +55 45 3379 7002 E-mail: [email protected]

Graphical abstract

Highlights 

EC method has evidenced good inorganic matter removals from landfill leachate



EC-treated landfill leachates were assessed by their toxicity response



The highest toxicity was persistent in electrolysis times below 40 min



Treatment times above 90 min showed best results on median lethal concentration



Remaining toxicity on EC-treated leachates was attributed to ammoniacal nitrogen

1

ABSTRACT In this work, new insights on organic matter (OM) removal performance along with the assessment of the toxicity of landfill leachate (LL) treated by Electrocoagulation (EC) method were investigated. In the context of response surface methodology (RSM), the optimal EC experimental conditions were sought by applying a 33 complete factorial design, regarding pH, current density and electrolysis time as operational parameters. The sum of dissolved organic and inorganic carbon, namely as dissolved total carbon (DTC), was chosen as a global response parameter. Other EC experiments were performed, keeping fixed pH and current density at local optimal values and varying the electrolysis time. Determination of the median lethal concentration (LC50) from bioassays based on Artemia salina and Lactuca sativa indicators was performed. A second-order polynomial function was statistically validated with good predictions of the DTC data, indicating best removals by setting values of pH, current density, and electrolysis time at 5, 128.57 Am-2 and 120 min, respectively. Additionally, removals above 90% were achieved for color, turbidity, iron concentration and dissolved inorganic carbon, whereas reductions on related-to-organic matter parameter values were around 60%. Although the EC treatment reduced the LL effluent toxicity, as verified by toxicity bioassays, 90 min treatment times showed best results on LC 50, but higher toxicity was persistent in electrolysis times below 40 min. Thus, a second stage of treatment based on a biological process could be suitably included in order to abate recalcitrant OM and decrease remaining toxicity in a more efficient integrated treatment system.

Keywords: LL leachate effluent; EC process; RSM; toxicity assessment.

1

INTRODUCTION

Nowadays, there are many environmental problems in our technological societies being, in great part, caused by their strong interferences and contributions with discharging of any type of wastes in the environment. In this context, one of the main environmental issues that resulted from urbanization was urban solid waste disposal [1]. To reduce the anthropic impact on environmental compartments, landfills have been used to minimize the contamination of water and soils by any type of organic or inorganic pollutant, being a common practice to dispose of solid waste in large and medium-sized cities [2]. Although the correct disposal of solid wastes was apparently solved, another environmental problem has originated from the natural degradation of solid wastes along with rainwater percolation, which allows for the formation of landfill leachate (LL) effluents in large amounts. In addition, LL effluents are mainly characterized by containing a great variety of pollutants based on recalcitrant substances and other inorganic and organic pollutants, such as aromatic hydrocarbons, acids, esters, alcohols, amides, ammonia nitrogen, and heavy metals [3, 4] – all of which produce an usually strong dark color and an unpleasant

2

odor. Due to their complex compositions and their high potential for contamination, if these pollutants were directly discharged into groundwater and surface waters, then LL wastewaters would be a serious environmental concern. The simplest way to reduce their impact is to keep LL effluents in aerobic lakes. Likewise, other more sophisticated treatment methods could be applied to solve this environmental problem, such as trying to drive the removal of high pollutant charges so that they reach the recommended environmental requirements before being discharged into bodies of water as proposed by the authors [5, 6]. To remove the pollutants from LL effluents, many unconventional technologies have been proposed; examples include the advanced oxidation processes, the membrane filtration process, a biologic process, and coagulation-flocculation methods [7]. Among these methods or processes, few have exhibited good performance in treating LL effluents by reducing their environmental impacts, but unfortunately, their costs were higher than other methods due to the complexity and different natures of the LL pollutants. Hence, new approaches were introduced for increasing the treatment method’s performance and reducing costs – among them, the electrocoagulation (EC) method [8, 9] is highlighted. In the framework of the EC method, organic and inorganic compounds undergo electrochemical reactions, which are supported by the addition of coagulants via oxidation of the electrodes along with the water hydrolysis, to form long chains of metallic hydroxides, where the electrically modified pollutants are confined so that they are quickly separated from the aqueous medium in a simple way [10]. Although the EC technique had a high performance and reliability as an LL treatment alternative [11], experimental results showed that some recalcitrant pollutants remained after the EC treatment, which demonstrated the requirement for further purification activities. As an environmentally responsible way to elucidate the possible impact of treated effluents on living organisms found in freshwater bodies, toxicity assessments are usually demanded. In this context, the toxicity assessments for LL effluents are traditionally based on physico-chemical analyses as reported by Klauck et al. [12]. These analyses reveal low sensitivity and are unable to represent the real toxic effects of the remaining pollutants on biota and fauna. Hence, another approach, which uses bioassays for the toxicity assessments [13], should be performed to evaluate the real environmental risks, such as the method reported by Sobrero and Ronco [14]. In the framework of bioassay testing, it was possible to evaluate the biological effects and other factors, such as the bioavailability and interactions with toxicants in an integrated way [15]. As an

3

evaluation criterion, the median lethal concentration (LC50) is usually employed, which provides an integrated response in time to all the toxicological effects [13]. Two special living organisms, Lactuca sativa and Artemia salina, have usually been recommend and employed for assessing the toxicological effects of wastewaters [16-18, 12]. This is because the Artemia-salina-based toxicity tests show precision and reliability in assessing the toxicological effects of EC treated LL effluents [19]. In this work, the minimum environmental impact of LL effluents treated using the EC method was investigated. This study was performed by applying a two-stage strategy. First, an evaluation of the best operational conditions for the EC process was investigated based on the response surface methodology, which involved applying a 3³ experimental design. Second, the removal performance of organic and inorganic pollutants was evaluated by examining the main components of the LL effluents for the dissolved total carbon (DTC), the chemical oxygen demand (COD), the biochemical oxygen demand (BOD), the ammonia nitrogen concentration (N-NH3), the turbidity, and the color, as well as, for the concentration of a series of chemical elements. The toxicity index for raw and treated LL effluents was statistically inferred from the LC50 determination.

2 2.1

MATERIALS AND METHODS Collection, monitoring, and preservation of LL samples

In the Cascavel municipality, which is located in the western region of Brazil’s Parana state, almost all the solid waste collected from urban residences are often disposed of in a modern municipal sanitary landfill. Since this practice began, this landfill has generated a huge amount of gases that were generated by the degradation of organic matter, which enables the production of enough electricity to be used as a source of power generation. Additionally, a large amount of landfill leachate was produced and stored in aerobic lakes for pretreatment. For the purposes of employing and studying the EC method as a part of the LL treatment system in this landfill, about 100 liters of leachate effluent were collected in plastic cylindrical containers and then preserved using refrigeration at 4ºC in a laboratory, which is as the methodology described in the Standard Methods [20]. A few physico-chemical parameters, such as temperature, pH, the dissolved oxygen, and the electrical conductivity, were measured in situ using a multi-parameter meter (Hanna, HI 9828 model), whereas other parameters were measured under lab conditions. Throughout one year, the main physico-chemical and pluviometric parameters were

4

monitored monthly; the pluviometric data was provided for research purposes by the Meteorological System in the Brazilian state of Paraná.

2.2

Chemicals and stock solutions

All stock solutions and dilutions were prepared by using chemicals of analytical grade and ultra-pure water obtained from a reverse osmosis filtration system (Millipore® Direct-Q-Model). According to the Standard Methods [20], chemical solutions were prepared and stored for analysis of the chemical oxygen demand (COD), the biochemical oxygen demand (BOD), the ammoniacal nitrogen, and the color. Additionally, a certified standard solution of Gallium, with a 1.0 gL-1 concentration in a 5% nitric acid medium, and ICP grade (Sigma-Aldrich®, CAS 16639) were used to analyze the total reflection X-ray fluorescence (TXRF) by applying the internal standard method to determine the concentrations of chemical elements in aqueous samples. According to the protocol proposed by Meyer et al. [16], a nutrient solution consisting of 23.0 g of NaCl, 11.0 g of MgCl2·6H2O, 4.0 g of Na2SO4, 1.3 g of CaCl2.2H2O, and 0.7 g of KCl were placed in 1.0 L of Milli-Q water, which was prepared for all the toxicity tests using A. salina. The pH of the nutrient solution was adjusted to 9.0 by adding aliquots of Na 2CO3 solution. The nutrient solution, with pH = 9.0, was used as a diluent and as a negative control for the bioassays with A. salina. Likewise, as proposed by Sobrero and Ronco [14], reconstituted hard water, which consists of 1.01 g of MgSO4·7H2O, 0.72 g of NaHCO3, 0.30 g of KCl, and 0.48 g of CaSO4 in 4.0 L of Milli-Q water, was prepared for the Lactucasativa-based toxicity tests. The hard water was used as a diluent and as a negative control in the bioassays with Lactuca sativa.

2.3

Characterization of the LL effluents

To follow the performance of the EC process and its improvement, a series of physico-chemical indicators, which were mainly associated with the content of organic and inorganic matter, were used. Reductions in the COD, the BOD, the color, the turbidity, the ammoniacal nitrogen, the dissolved organic carbon (DOC), and the dissolved inorganic carbon (DIC) were related to the raw effluent used as a response parameter for the evaluation of the EC process performance, whereas concentrations of iron and other chemical elements were associated with inorganic origins. The amounts of organic and inorganic carbon in the raw LL effluent were estimated to have a concentration range above 100 mgL-1, and different sized particulates were noticed in the raw LL effluent,

5

which probably introduced undesirable interference that prevented a reliable analysis of the DOC and the DIC concentrations. Therefore, diluted and filtered samples of raw LL effluent were prepared and analyzed. The concentration of the DTC (DOC+DIC), the DOC, and the DIC were measured using a TOC analyzer (Shimadzu, model TOC-L), which was equipped with an OCT-L sampler. Following the analysis described by the Standard Methods [20], the COD was determined using the closed reflux method. The BOD was measured using the respirometric method. The ammoniacal nitrogen analysis was performed using the phenate method, and the phosphate was analyzed with the ascorbic acid method. The turbidity was determined using a turbidimeter (Tecnal, model TB1000), while the color analysis was performed using the Pt-Co method based on a calibration curve obtained from a UV-vis spectrophotometer. The totals solids (TS) in the raw LL samples were measured using the evaporation process in an oven at a temperature of 105°C until a constant weight was achieved, where the dried weight is considered to be the total solids. A portable benchtop TXRF spectrometer (Bruker, model S2 PICOFOX), controlled by the Spectra 7 TM software, was used to identify and quantify the chemical elements in an aqueous solution. A series of characteristic X-rays, such as the Kα and Kβ spectral lines for low atomic numbers and L spectral lines for intermediate and high atomic numbers respectively, were used to identify each chemical element; the area of the main X-ray spectral line was used to determine the concentrations of chemical elements in the LL samples. A set of fourteen chemical elements (S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn and Br) with atomic number ranging from 13 to 36 was identified by their K-X ray spectral lines (X-ray energies below 17 keV), whereas only two chemical elements (Ba and Pb) were identified by their L-Xray spectral lines within the range of 1 to 17 keV. Other chemical elements, such as Na and Mg, with atomic number below 13 were impossible to identify and quantify because they exhibited lowest elements sensitivities in TXRF analysis than those ones of transitional chemical elements. All the TXRF-based concentration measurements were performed in triplicate, where the internal standard method was applied in this simple and reliable procedure. As an internal standard, a certified solution of Ga (1.0 gL-1) was used. In a cleaned, 2-mL conical tube with a cover, a volume of 50 μL of Ga solution was added to 950 μL of either a raw or an EC-treated LL sample that was homogenized and stored. On a siliconized quartz reflector disc (30 mm diameter, 3 mm thickness), an aliquot of 5 μL of a Ga-doped LL sample was pipetted at its center, and dried in an environment free of contaminants for 24 h, which formed a small, very fine spot of

6

particulates that were stored in a sample holder for posterior TXRF measurements. A set of three discs, which contained a reference material (Kraft XIII) and were provided by Bruker, were included for a recovery test of several chemical elements at the same certified concentration of 1.0 mgL -1. All the discs were mechanically positioned in the total reflection condition for irradiation by a monochromatic Mn-Kα X-ray beam and XRF detection by a Silicon drift detector Xflash® that covers a solid angle of almost 2π. In the Spectra 7TM software, a method analysis was built for each type of sample, including a list of identified chemical elements and a time setting of 1000 s for every TXRF analysis. To identify the infrared vibrational bands for a series of organic functional groups and to follow their changes in intensity due to pollutant removal using the EC process, Fourier Transform Infrared (FTIR) spectroscopy was applied. In this regard, a mechanically pressed-built pellet, containing 100 mg of KBr and 1 mg of a dried LL sample, was prepared for FTIR analysis. An FTIR-NIR spectrometer (PerkimElmer, model Frontier), with a spectral resolution of 4 cm-1, was used to scan KBr pellets made with raw and treated samples in the range of 4000 to 400 cm-1 and to store the infrared vibrational spectra.

2.4

An aluminum-based EC reactor

Due to the fact that both Fe and Al electrodes are cheaper than other materials and have shown higher efficiency during the removals of organic and inorganic matter by the electrocoagulation (EC) process [21], they were usually the most used and most often proposed for treatment systems based on EC. Our previous experience has shown that relatively better performances on organic matter removal were obtained when Al electrodes were used instead of Fe ones in treatment system of wastewaters [7, 22-24]. It must be noted that, a high Fe concentration was found in landfill leachate, which required its removal as one of the main goals. Based on these facts, we concluded that the best choice for such scenario was to apply Al electrodes. A benchtop electrocoagulation reactor was built, as shown in Fig. 1, which consists of walls made of 0.6cm nylon plates and a system of six electrodes made of aluminum (10 cm x 15 cm x 2 mm), positioned upright, and possessing a separation gap of 2 cm parallel between plates. For this reactor dimension and electrode distribution, an effective volume of 1 liter, and an effective area of 350 cm2 between the Al plates were established. The set of aluminum (Al) plates were electrically connected, in parallel, to form five electrochemical cells. All the electrochemical cells were kept under the same electric potential

7

difference (EPD), which was controlled by a Direct Current (DC) power supply (Instrutemp, model FA 1030) with a maximum EPD of 30 V and a maximum DC of 10 A. Because the electrical conductivity of the raw LL effluent was found to be relatively high, lower EPD values of around 1-2 V were required to set the electric current density values in the tested interval of 42 to 128 Am-2. As the EC process usually tended to cause the formation of an undesirable dielectric layer on the aluminum electrodes over time, which provoked a performance loss in the treatment, the polarity of the Al electrodes was reversed after every 15 min of electrolysis time. At the end of each EC treatment, all the Al plates underwent a cleaning procedure. Based on an EC process with Al electrodes, a typical chemical reaction, as Al oxidation given by Eq. 1 [25], is expected to occur at the anode, allowing forming aluminum hydroxide at alkaline and acidic condition, as given by Eqs. 2 and 3, respectively. Further, H2 bubble production is expected to proceed at the cathode along with OH-, resulting in an increase in pH values of the solution [26], as given by Eq 4. In the presence of Al(OH)3 floccules and aggregates could be formed, as described by Eq. 5 [27], providing thus a way for removing of pollutants from landfill leachates. 𝐴𝑙 − 3𝑒 → 𝐴𝑙 3+

(1)

𝐴𝑙 3+ + 3𝑂𝐻 − → 𝐴𝑙(𝑂𝐻)3

(2)

𝐴𝑙 3+ + 3𝐻2 𝑂 → 𝐴𝑙(𝑂𝐻)3 + 3𝐻 +

(3)

2𝑒 − + 2𝐻2 𝑂 → 𝐻2 + 𝑂𝐻 −

(4)

𝐴𝑙 + 3𝐻2 𝑂 → 𝐴𝑙(𝑂𝐻)3 (𝑠) + 32𝐻2 (𝑔)

(5)

2.5

A complete factorial design for the EC experiments

In the RSM framework [28], preliminary EC experiments were performed following a 3 3 CFD. Three EC parameters – the initial pH (q1), the electric current density (q2), and the electrolysis time (q3) – were used as variables in the CFD experiments, which covered 27 different EC conditions, according to three levels (lower, central, and higher values) in intervals of 5 to 9, 42 to 128 Am-2, and of 30 to 120 min respectively. The TDC removal was proposed as the main parameter response to be monitored, assessed, and improved by seeking the best experimental conditions along with the lowest toxic effects on the EC treatment of raw effluent. In the context of the RSM, a second-order polynomial response function, as given by Eq. 6, was used to model the set of experimental data obtained from the 3 3 CFD. The data was fitted using the software

8

Statistica™. The analysis of variance (ANOVA) and the F-test were applied to validate the mathematical representation of the data. All coefficient values of linear (β), quadratic (γ), binary (δ) and ternary interactions () from the validated response function were estimated using a 95% confidence level. 3

3

3

3

2

2

2 𝑗

𝑅 = 𝛼0 + ∑ 𝛽𝑖 𝑞𝑖 + ∑ 𝛾𝑖 𝑞𝑖2 + ∑ [∑ 𝛿𝑖𝑗 𝑞𝑖 𝑞𝑗 ] + ∑ [∑ (∑ 𝜎𝑖𝑗𝑘 𝑞1𝑖 𝑞2 𝑞3𝑘 )] + 𝜀 𝑖=1

𝑖=1

𝑖=1 𝑗≠1

𝑖=1 𝑗=1

𝑘=1

(6) A better value for the electric current density (128 Am-2) corresponds to an improved pollutant removal performance. Another EC experiment was performed, where the pH value of the raw LL effluent was kept fixed at its natural value (7.8), while the electrolysis time was varied from 5 to 120 min to evaluate the real toxicity of the EC-treated effluents.

2.6

Toxicity tests on the EC-treated LL effluents

Although the treatment of the LL samples using the EC method could exhibit good performance in removing pollutants, there were a few substances that remained within the treated LL samples that could be highly toxic to living organisms. These toxic substances could have originated from the effects of EC on destabilizing organic matter or through a similar explanation. Therefore, any effluent that has undergone a treatment process must be evaluated in relation to its real toxicity before it is discharged into bodies of water. If there were very low amounts of organic and inorganic pollutants with an unknown toxicity level in any of the EC-treated LL effluents, then bioassays for the direct evaluation of their toxicities should be and were performed using two sensible, living organisms: micro-crustaceans of the Artemia salina species and lettuce seeds of the Lactuca sativa species.

2.6.1 Bioassays using A. salina The use of A. salina cysts as a toxicity indicator was previously reported by Svensson et al. [19], and has the advantages, in respect to indirect methods, of low cost, great sensitivity to toxic substances, as well as a quick response that is used to directly estimate the real toxicity levels. Based on the methodology described by Meyer et al. [16], A. salina cysts were hatched in a nutrient solution after an interval of 48 h. To achieve a partitioned reduction of the aqueous sample’s toxicity, a dilution procedure was applied to it using different v/v fractions of 20, 40, 60, 80, and 100%. A sample of each EC-treated effluent, with an

9

undiluted toxicity, was also included in the bioassays to define a set of diluted toxic samples. Five mL of the toxic samples, which were either diluted or undiluted, were pipetted and stored in 10-mL tubes for bioassay testing. Ten nauplii of A. salina specie were put into each of the 5-mL toxic samples that were either diluted or undiluted. The bioassays were carried out in triplicate, where all the samples were kept under the presence of light in a laminar flow cabinet for 24 hours. At the end of each bioassay test, the number of living nauplii of A. salina specie was recorded to determine the median lethal concentration (LC50) using software functions based on the Trimmed Spearman-Karber Method [29].

2.6.2 Bioassays using L. sativa Based on its rapid growth and germination index, lettuce seeds of the L. sativa species were previously used to directly reveal the toxicity of effluents, soils, or sediments [30, 13]. This species was recognized as a good bio-indicator because it allowed the evaluation of the inhibition of its germination process and root elongation after it was exposed to the presence of low concentrations of toxic compounds. Based on the methodology described by Sobrero and Ronco [14], the bioassays were carried out using lettuce seeds of the L. sativa species with a germination potential of 95%. For our purposes, a partitioned reduction of the aqueous sample’s toxicity was carried out in the L.-sativabased bioassays by applying a dilution procedure using different v/v fractions of 1, 3, 10, 30 and 100%. Each diluted toxic sample was obtained by adding the proper volume of nutrient solution to an EC-treated LL effluent. As a control sample for the L.-sativa-based bioassay, pure, reconstituted hard water, or a 100% diluted toxic sample, was considered. When carrying out all the L.-sativa-based bioassays, a set of Petri plates was used, where qualitative filter paper was placed on their bottoms to serve as seedbeds. About 4 mL of a diluted toxic sample was allowed to soak into each seedbed. After that, 20 lettuce seeds were uniformly distributed on the seedbed containing a diluted toxic sample. Each toxicity test was performed in triplicate. The set of closed Petri plates was placed in an incubator (Solab, model SL 200) at a controlled temperature of 22 ± 2°C, where the germination and the growth of lettuce seeds should occur over a period of 5 days. After the incubation period, response parameters for the toxicity tests, such as the number of seeds, the lengths of the root and the radicle in sample sets containing both diluted toxic and electrolysis time conditions, were manually measured, which allowed the determination of their average values. Based on these response parameters, other parameters, which are related to the germination, growth, and the

10

inhibition of seeds, were deduced. To follow the changes in the germination indicators for toxic aqueous samples, the absolute germination (𝐺𝑒𝑟𝑚𝑎𝑏𝑠 ) and the germination exposed to the control (𝐺𝑒𝑟𝑚𝑐𝑜𝑛𝑡 ) values were estimated. As given by Eq. 7, 𝐺𝑒𝑟𝑚𝑎𝑏𝑠 was obtained by comparing the average number of the germinated seeds exposed to diluted toxic samples (𝐴𝑁𝑒𝑑𝑡,𝐺𝑠 ) to the total number of the initial seeds exposed to diluted toxic samples (𝑇𝑁𝑒𝑑𝑡,𝑖𝑠 ). Likewise, 𝐺𝑒𝑟𝑚𝑐𝑜𝑛𝑡 was obtained from the ratio of the average number of germinated seeds exposed to diluted toxic samples (𝐴𝑁𝑒𝑑𝑡,𝐺𝑠 ) to the average number of germinated seeds exposed to diluted toxic samples (𝐴𝑁𝑒𝑐,𝐺𝑠 ) as given by Eq. 8. 𝐺𝑒𝑟𝑚𝑎𝑏𝑠 =

AN𝑒𝑑𝑡,𝐺𝑠

𝐺𝑒𝑟𝑚𝑐𝑜𝑛𝑡 =

TN𝑒𝑑𝑡,𝑖𝑠 AN𝑒𝑑𝑡,𝐺𝑠 AN𝑒𝑐,𝐺𝑠

(7) (8)

By considering the ratio of morphologic parameters, e.g., the average root length in the germinated seeds exposed to diluted toxic samples (𝐴𝑅𝐿𝑒𝑑𝑡,𝑔𝑠 ) and in the germinated seeds exposed to the control (𝐴𝑅𝐿𝑒𝑐,𝑔𝑠 ), the root length index related to the control (𝑅𝑜𝑜𝑡_𝐿𝑐𝑜𝑛𝑡 ) was estimated using Eq. 9. Likewise, the ratio of the average radicle growth in the germinated seeds exposed to diluted toxic samples (𝐴𝑅𝐺𝑒𝑑𝑡,𝐺𝑠 ) and in the germinated seeds exposed to the control (𝐴𝑅𝐺𝑒𝑐,𝐺𝑠 ) were used to define the radicle growth index related to the control (𝑅𝑎𝑑_𝐺𝑐𝑜𝑛𝑡 ) as given by Eq. 10. 𝑅𝑜𝑜𝑡_𝐿𝑐𝑜𝑛𝑡 = 𝑅𝑎𝑑_𝐺𝑐𝑜𝑛𝑡 =

𝐴𝑅𝐿𝑒𝑑𝑡,𝑔𝑠 𝐴𝑅𝐿𝑒𝑐,𝑔𝑠 𝐴𝑅𝐺𝑒𝑑𝑡,𝐺𝑠 𝐴𝑅𝐺𝑒𝑐,𝐺𝑠

(9) (10)

By including the germination related to the control, the germination index (𝐺𝑒𝑟𝑚𝑖𝑛𝑑𝑒𝑥 ) and the growth index (𝐺𝑟𝑜𝑤𝑡ℎ𝑖𝑛𝑑𝑒𝑥 ) were also estimated using Eqs. 11 and 12 respectively. 𝐺𝑒𝑟𝑚𝑖𝑛𝑑𝑒𝑥 = 𝐺𝑒𝑟𝑚𝑐𝑜𝑛𝑡 × 𝑅𝑜𝑜𝑡_𝐿𝑐𝑜𝑛𝑡

(11)

𝐺𝑟𝑜𝑤𝑡ℎ𝑖𝑛𝑑𝑒𝑥 = 𝐺𝑒𝑟𝑚𝑐𝑜𝑛𝑡 × 𝑅𝑎𝑑_𝐺𝑐𝑜𝑛𝑡

(12)

Based on these parameters (𝑅𝑜𝑜𝑡_𝐿𝑐𝑜𝑛𝑡 and 𝑅𝑎𝑑_𝐺𝑐𝑜𝑛𝑡 ), the inhibition index related to the control for the root and radicle growth, when exposed to diluted toxic samples, were determined by Eqs. 13 and 14 respectively. 𝐼𝑛ℎ𝑖𝑏_𝑅𝑜𝑜𝑡𝑖𝑛𝑑𝑒𝑥 = 1 − 𝑅𝑜𝑜𝑡_𝐿𝑐𝑜𝑛𝑡

(13)

𝐼𝑛ℎ𝑖𝑏_𝑅𝑎𝑑𝑖𝑛𝑑𝑒𝑥 = 1 − 𝑅𝑎𝑑_𝐺𝑐𝑜𝑛𝑡

(14)

11

Regarding the absolute germination (𝐺𝑒𝑟𝑚𝑎𝑏𝑠 ) and germination index (𝐺𝑒𝑟𝑚𝑖𝑛𝑑𝑒𝑥 ) data obtained from the L.-sativa-based bioassays, the LC50 value corresponding to each EC-treated LL sample was determined by applying the Trimmed Spearman-Karber Method [29].

3 3.1

RESULTS AND DISCUSSION Characteristics of the raw LL effluent

A huge amount of organic matter was found in the raw LL effluent, as revealed by high concentrations of the COD (6.5 gL-1O2) and the BOD (3.0 gL-1O2) with a precision of about 10%. Additionally, the concentration values for the N-NH3 and the DIC were found to be 1.2 and 1.1 gL-1 respectively, while the alkalinity value was 6.4 gL-1 as CaCO3. It was remarkable that the concentration of the N-NH3 in the raw LL effluent was 50-fold above the allowed maximum value (20 mgL-1) recommended by the Brazilian Council of Environment (BCE) in its 430/2011 Act for the disposal of industrial effluents in fresh water bodies [31]. This resulted in significant concern for the real environment impact of the effluents. According to the TXRF technique, a high Fe concentration of about 50 mgL-1 was found, which was three-fold above its allowed maximum value (15 mgL-1) as recommended by the 430/2011 BCE Act. Conversely, the raw LL effluent exhibited DO, electrical conductivity, and pH values of 0.32 mgL-1, 14.17 mS cm-1, and 7.3 respectively. Although the performance of the EC process in removing organic pollutants was previously reported as being strongly dependent on the initial pH of the effluent [32], another work reported that an improvement in the EC performance could be achieved by setting the initial pH to around 7 [9]. Likewise, the electrical conductivity of the effluents undergoing the EC process was reported to be an important physical parameter for quickly starting a treatment that had a lower energy power consumption, and a reduction in the effective treatment time [33]. Because suitable physicochemical characteristics were used, the raw LL effluent could be treated without initial changes to its natural pH. The set of main parameters that characterized the raw LL effluent was monitored over one year (2015/2016), and they exhibited great variation in their physical-chemical parameters, except the natural pH value, which remained at a nearly unchanged value near 7. These parameters are summarized in Table 1. Because of the variability of the monthly-cumulative pluviometric precipitation (MCPP) during the collecting year along with different compositions of the garbage, the characteristics of the raw LL effluent suffered changes over time.

12

The amount of raw LL samples, which were collected and used in both the EC treatments and in the toxicity evaluation, exhibited physico-chemical parameter values that were in good agreement with the annual representative characteristics obtained during the 2015/2016 monitoring program, but their variations could be justified by the behavior of the MCPP as shown in Table 1. A Pearson correlation among the values for the physicochemical parameters and the MCPP was performed. Some physicochemical parameters showed poor correlations among them, but a few of them (e.g., the pH, the COD, the TS, and the Fe content) had negative correlations of around -0.5 when the MCPP was set to a confidence level of 95% as shown in Table 2. A negative Pearson correlation, upon increasing the MCPP, is an indicator of diluted values for the pH, the COD, the TS, and the Fe content.

3.2

RSM-based improvement of the EC processes’ performance

Although a set of several inorganic and organic variables were investigated as possible response parameters in order to seek the optimal experimental conditions for the EC process, the main goal was to reduce the organic matter in a significant way. By applying the CFD to the LL effluent treatment using the EC method, a set of 27 responses to the DTC removal was obtained, in triplicate, as summarized in Table 3. From all the EC experiments, it was verified that the DTC removals varied from 11.5 to 62.7%, which shows that it is possible to achieve a maximum DTC removal of around 62%. From the statistical analysis using the RSM framework, the EC reactor was operated at a pH of 5, with a current density of 128 Am-2, and an electrolysis time of 120 min, which were found to provide the maximum DTC removal. In the context of the ANOVA and using 80 degrees of freedom, the mathematical model, as given by the Eq. 15, represents the DTC removal data that was actually validated, as shown in Table 4, and the F-test was passed when Fcal was 450-fold greater than Ftab, as well as, when it exhibited an extremely small p-value. According to this model, a good correlation coefficient value (r2=0.96) between the observed and predicted values for DTC removal were achieved as shown in Fig. 2. 𝑅𝐷𝑇𝐶 = −150 + 1.44𝑞3 + 6.3𝑞2 + 85𝑞1 − 0.040𝑞22 − 8.5𝑞12 − 2.6𝑞1 𝑞2 − 1.3𝑞1 𝑞3 − 5 × 10−5 𝑞2 𝑞32 + 9.6 × 10−6 𝑞3 𝑞22 + 1.7 × 10−2 𝑞1 𝑞22 + 5.3 × 10−3 𝑞1 𝑞32 + 0.16𝑞3 𝑞12 + 0.24𝑞2 𝑞12 − 8𝐸 −4 𝑞12 𝑞32 − 1.4 × 10−3 𝑞12 𝑞22 + 2.6 × 10−2 𝑞1 𝑞2 𝑞3 − 1.2 × 10−7 𝑞12 𝑞22 𝑞32 − 1.5 × 10−4 𝑞1 𝑞2 𝑞32 − 1.5 × 10−4 𝑞1 𝑞3 𝑞22 − 3.6 × 10−3 𝑞2 𝑞3 𝑞12 + 8.9 × 10−7 𝑞1 𝑞22 𝑞32 − 2.2 × 10−5 𝑞2 𝑞12 𝑞32 + 1.9 × 10−5 𝑞3 𝑞12 𝑞22

(15)

Besides this, the validated mathematical function (Eq. 15) that represents the DTC removal data was used to visualize their graphical representations in the space of the EC-reactor’s operational parameters. Keeping the current density and the electrolysis time fixed at their best values (128.57 Am-2 and 120 min

13

respectively), three-dimensional surface responses were built for removing TDC as shown in Fig. 3. Maximum TDC removals of around 60, 55 and 50 % were achieved using pH values of 5, 6 and 7 respectively, which showed a slight drop of about 10-15% in the EC performance as the initial pH of the raw LL effluent was changed from 5 to 7. To set the pH values to 5, it was necessary to add a chemical to the raw LL effluent, which would increase the EC treatment costs. Hence, other EC experiments were performed using a current density of 128.57 Am-2 at the natural pH value (near to 7) of the LL effluent, while the electrolysis times were varied.

3.3

Effect of the electrolysis time on the removal of pollutants

As can be observed in Eq. 15, removals of DTC were strongly linear dependent on pH, current density and reaction time. Simulations with the equation showed that the reaction time in electrocoagulation process was an important parameter that has drove the treatment towards better removal results when an increase on time was regarded. Increased reaction time most probably reflected to an increase of Al ions, resulting subsequently to an increasing on amounts of hydroxides flocs along with a high rate of bubble generation. When using the best electrical current density (128.57 Am-2) at the natural pH (7.3) of the raw LL effluent, the performance for the pollutant removal using the EC treatment process was quick and significantly increased for short electrolysis times below 40 min, but the performance was almost unchanged when electrolysis times above 90 min were used (see Fig. 4). Additionally, an electrolysis time of 90 min was consistent with the optimal condition obtained from the RSM analysis, which suggested operating the EC reactor for up to 90 min to significantly enhance organic pollutant removals. Concentrations of the Fe content and the DIC were very efficiently reduced, to almost 100%, during the first 30 min of electrolysis time. The removal efficiency was slightly reduced to achieve color and turbidity values of about 90%. For the physico-chemical parameters linked to organic matter (the COD, the BOD, and the DOC), a removal performance of about 40% was obtained. This fact could be explained by the destabilizing agents originating from the EC process, which would not be enough to attack or remove organic pollutants and suggests the need to add more oxidizing agent to degrade the organic matter. Residual values of around 60% were found for the COD, the initial BOD, and the DOC in ECtreated LL effluent, which were not in agreement with the 470/2011 BCE Act, and indicates that it is necessary to evaluate the remaining toxicity before the effluent is discharged into bodies of water.

14

3.4

Toxicity bioassays

Regarding the set of five diluted toxic samples, where the effluent was treated using EC, the LC50 values at a 95% confidence interval were estimated based on the mortality ratio related to the initial population of nauplii of the A. salina species as shown in Table 5. Among all the monitored physico-chemical parameters in the EC-treated LL effluents, the amount of N-NH3 was found to be strongly correlated with the behavior of the LC50 values. The presence of ammonia and other toxic components were of serious concern because, in a high concentration, they could reduce the LL treatability through conventional processes [34, 35]. In this regard, Dave and Nilsson [36] reported that the acute toxicity in raw LL samples may be attributed to high concentrations of ammoniacal nitrogen. Further study by Manenti et al. [37] suggested that the intermediate compounds formed during the electrocoagulation process are most likely responsible for the negative results. Among these compounds, EC can produce ammonia during treatment because nitrates can be electrochemically reduced to nitrites and then they can be transformed in ammonia at the cathode, as given by Eqs. 16 and 17 [38]. 𝑁𝑂3− + 𝐻2 𝑂 + 2𝑒 − ↔ 𝑁𝑂2− + 2𝑂𝐻 −

(16)

𝑁𝑂2− + 5𝐻2 𝑂 + 6𝑒 − ↔ 𝑁𝐻3 + 7𝑂𝐻 −

(17)

After the first 30 min. of EC treatment, a high mortality ratio of above 50% of the nauplii of the A. salina species suggested that the presence of the remaining pollutants, which have undesired toxicity, are probably from an organic source. However, this toxic effect was systematically reduced by increasing the electrolysis time, which also improved the LC50 to values of about 30% when the treatment time was set above 60 min. In other words, there was a persistent toxicity from the remaining pollutants that should correspond to 70% of probably organic matter, which induces lethal effects in living organisms that are sensitive to toxicity. Therefore, other treatment processes have to be integrated to decrease the toxicity by removing the mainly organic matter, which includes the high amount of residual ammonia. The toxicity tests based on root germination and growth was proposed by US government agencies to partially evaluate the potential contamination from disposing of effluents in the environment [17]. Among the possible environment contaminants, ammonia, volatile organic acids, heavy metals, and saline content may cause harmful effects during plant growth, such as the inhibition of seed germination or root growth [39].

15

Conversely, toxicity responses could be a little different in another bioindicator, such as in the seeds of the L. sativa species. In fact, both A.-salina and L.-sativa bioindicators were different in terms of their metabolism and physiology, which could probably be expected for different responses to the same toxic substance. Additionally, the EC treatment showed a greater performance in reducing inorganic matter (nearly to 95-98%) rather than organic matter (near to 40-50%). In this context, it could be expected that one of them would be more sensitive to inorganic matter and the other to organic matter, but in a mixture of organic and inorganic matter, their sensitivities could also be the same. For this reason, L.-sativa-based toxicity tests were performed using a set of five diluted toxic samples for the series of treatment times. To determine the LC50 values at the 95% confidence interval, germination parameters related to the initial seed population or to the control solution were employed and are summarized in Table 6. When treatment times were below 30 min, all the EC-treated LL effluents with diluted toxicities were observed to be highly toxic. Besides this, the EC-treated LL effluents became less toxic as the treatment time was increased from 30 to 90 min, where the lethal and sub-lethal effects on the germination of lettuce seeds were reduced in samples with a diluted toxicity of 30%. It was obvious that the most diluted samples had a lower lethality index due to the lower concentration of the toxic agent. When no dilution procedure was applied to the EC-treated LL effluent (100%), there was no germination for any studied treatment time, which agrees with similar results reported by Zamora and Garcia [40]. During the last stages of electrolysis, an increase in the sub-lethal effects were observed when the germination index was lower than that value found for the raw effluent. A possible cause for this effect could be the formation of intermediate compounds that caused an increase in the toxic effects of the medium. Among these compounds, it should be mentioned that the organochlorines, such as the chloramines, for instance, produced a considerable chlorine concentration in the effluent, which were on the order of 1 gL-1 as measured by the TXRF technique.

3.5

Toxic effects on the germination and inhibition index of seeds

The mechanism that produces organochlorine compounds was reported by Anglada et al. [41] and by Bashir et al. [42]. They discussed the reaction between chlorine radicals and a high concentration of dissolved organic matter and ammonia that increased the effluent’s toxicity. Landfill leachates generally have a high concentration of chlorine. During electrocoagulation, pollutants can be removed due to indirect oxidation using chlorine or hypochlorite formed by the anodic oxidation of the chlorine present in

16

the slurry, resulting in an electrochemical oxidation [43]. According to Deng and Englehardt [44], the most probable mechanism and involved chemical reactions in the electro-oxidation of chlorine can be presented by Eqs. 18-22. At the end of these processes hypochlorite (OCl-) was generated. It is well known that hypochlorite is a strong oxidant for organic compounds. It must be noted, these processes could drive towards to the formation of organochlorine compounds that are often more dangerous than the own organic matter. 2𝐶𝑙 − → 𝐶𝑙2 + 2𝑒 −

(18)

6𝐻𝑂𝐶𝑙 + 3𝐻2 𝑂 → 2𝐶𝑙𝑂3− + 4𝐶𝑙 − + 12𝐻 + + 32𝑂2 + 6𝑒 −

(19)

2𝐻2 𝑂 → 𝑂2 + 4𝐻 + + 4𝑒 −

(20)

𝐶𝑙2 + 𝐻2 𝑂 → 𝐻𝑂𝐶𝑙 + 𝐻 + + 𝐶𝑙 −

(21)

𝐻𝑂𝐶𝑙 → 𝐻 + + 𝑂𝐶𝑙 −

(22)

The features of chloramines and their volatile nature were responsible for the toxicity of the leachate. A way to follow the real toxicity of the EC-treated LL effluent was indirectly performed by contrasting the response-to-toxicity parameters, such as those related to germination, growth, and the inhibition of seeds of the L. sativa species, to the electrolysis time used for five EC-treated LL effluents with diluted toxicities as shown in Fig. 5. Although germination has exhibited a lethal point and used to assess toxicity, it was not actually the most sensitive indicator. Instead of this, the root length has exhibited a sub lethal endpoint, proving to be an actual more sensitive parameter, but its determination would be more difficult to perform in comparison with that for germination [45]. Indeed (see Fig. 5 a, b, and c), the average number of the germinated seeds exposed to diluted toxic samples (𝐺𝑒𝑟𝑚𝑐𝑜𝑛𝑡 ) (see Fig. 4a) was less sensitive to the toxicity than the inhibition index related to the control for root (𝐼𝑛ℎ𝑖𝑏_𝑅𝑜𝑜𝑡𝑖𝑛𝑑𝑒𝑥 ) and radicle (𝐼𝑛ℎ𝑖𝑏_𝑅𝑎𝑑𝑖𝑛𝑑𝑒𝑥 ) growth, as shown respectively in Figs. 5b and 5c, when exposed to diluted toxic samples. No negative effects were observed on the germinated seeds in toxic samples diluted to 1-10%. At 1% and 3% dilutions, the roots and rootlets sometimes showed higher growth than the own control, as indicated by the negative inhibition values. At 1% and 3% dilutions, the roots and rootlets showed sometimes higher growth than the own control, as indicated by the negative inhibition values. According to Arunbabu et al. [46], the dilution of toxic pollutants would be expected to occur in low concentrations, as well as, the best use of nutrients from the landfill leachate that could justify thus this higher growth. However, seed germination was seriously compromised in toxic samples diluted to 30% after an

17

electrolysis time lower than 90 min was used, and it completely stopped after being exposed to pure ECtreated effluent regardless of the treatment time that was used. There was almost no inhibition of the length of radicles in the seeds in toxic samples diluted to 1-3% regardless of the electrolysis time. However, there was a low inhibition index for the length of roots in seeds in the toxic samples diluted to 1-3% and for electrolysis times up to 90 min, but a nearly 100% inhibition index was observed for electrolysis times larger than 120 min. In the toxic samples diluted to 1-3%, there was a larger concentration of electrolytes than in the toxic sample diluted to 10-30%, which came from the hard water used to prepare the samples. It could be the cause of germination and the easier growth of the lettuce seeds.

3.6

Organic pollutants in EC-treated LL samples

Some types of organic matter, which are expected to be present in the effluents, could be identified by their series of molecular vibrational modes that have characteristic absorptions in the IR region. From the FTIR analysis, the raw LL effluent showed a characteristic IR spectrum with some broad bands in the region of 400 to 1750 cm-1 (see Fig. 6). Functional groups, which are characterized by showing different vibrational modes, were expected to exhibit their respective signatures in the IR spectra if they were present in the organic pollutants. When EC treatments were applied to the raw LL samples using different electrolysis times (of 90 and 120 min), IR spectral characteristics of the respective functional groups were perturbed as shown by changes in the respective band intensity. The changes in the IR spectra could be explained by the removal of organic matter from the raw LL sample. The characteristic band at 1560 cm-1 represents the in-plane N-H vibrational mode, and has been commonly identified as part of the molecular signature for N-NH4+ in effluent, such as by Lenz et al. [47]. Bands around 3400-3300 cm-1 are attributed to the stretching vibrational mode for O-H and N-H, which were also identified and reported, for instance, by Tahiri et al. [48]. Long-chains of aluminum hydroxides are expected to be formed in an EC process, and are confirmed by the increasing intensity of the stretching vibration mode of the OH functional group. Another band, which was identified at 1418 cm-1, could be attributed to the presence of carboxylic groups [47]. These compounds were still identified after the leachate treatment, and they are most probably responsible for the remaining toxicity.

18

Despite observing a toxicity reduction, when applied as a single process, the electrocoagulation method was not completely efficient at removing all the leachate pollutants. It was because there was not a complete reduction of organic pollutants and toxicity. Marian et al. [49] showed the strategy to treat the leachate, which included different treatment methods and their combinations. In this way, by employing the EC method in combination with a biological treatment method, it was possible to obtain an effective decrease in toxicity, in the quantity of pollutants, and in the LL samples.

4

CONCLUSION

The EC process has shown an improvement on performance in removing pollutants from raw LL effluent when optimal conditions are applied: an electric current density of 128.57 Am-2, an initial pH of 5, and an electrolysis time of 120 min. Regarding the natural pH (7.3) of the raw LL effluent, similar pollutant removal results were achieved without adding chemicals to correct the pH. Additionally, kinetic experiments have shown that the EC process was an effective treatment technique for satisfactorily removing inorganic matter, but this process also showed low removal performance for organic matter. Ammoniacal nitrogen was identified as the main pollutant with lowest removal rate, and it is responsible for the remaining toxicity after the EC treatment of the LL effluents. Although the EC-treatment process reduced the toxicity of the LL effluents, as verified by both the A.-salina and L.-sativa bioindicators, treatment times longer than 90 min showed significant reductions in the toxicity, but there was also a persistent lethal contribution to the toxicity when shorter electrolysis times were used. This may have been due to the presence of recalcitrant compounds and to the conversion of some organic compounds to their more toxic forms. Thus, a second stage of treatment based on a biological process could be suitably included in order to abate recalcitrant OM and decrease remaining toxicity in a more efficient integrated treatment system.

Acknowledgment F.R. Espinoza-Quiñones thanks the Brazilian Ministry of Science, Technology, Innovations and Communications (CNPq) for financial support under project #455.954/2014-3. A.R. de Pauli thanks the National Council for the Improvement of Higher Education (CAPES) for the awarded doctoral scholarship.

19

5

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[42] M.J.K Bashir, H.A. Aziz, S.Q. Aziz, S.S. Abu Amr. An overview of electrooxidation processes performance in stabilized landfill leachate treatment, Desalination Water Treat. 51 (2013) 2170-2184. [43] A. Fernandes, M.J. Pacheco, L. Ciríaco, A. Lopes, Review on the electrochemical processes for the treatment of sanitary landfill leachates: Present and futures, Applied Catalysis B: Environmental 176 (2015) 183-200. [44] Y. Deng, J.D. Englehardt, Electrochemical oxidation for landfill leachate treatment, Waste Manage. 27 (2007) 380-388. [45] A. Priac, P.M. Badot, G. Crini, Treated wastewater phytotoxicity assessment using Lactuca sativa: Focus on germination and root elongation test parameters, C. R. Biologies 340 (2017) 188-194. [46] V. Arunbabu, K.S. Indu, E.V. Ramasamy, Leachate pollution index as na effective tool in determining the phytotoxicity of municipal solid waste leachate, Waste Manage. 68 (2017) 329-336. [47] S. Lenz, K. Bohm, R. Ottner, M. Huber-Humer, Determination of leachate compounds relevant for landfill aftercare using FT-IR spectroscopy, Waste Manage. 55 (2016) 321-329. [48] A. Tahiri, A. Richel, J. Destain, P. Druart, P. Thonart, M. Ongena, Comprehensive comparison of the chemical and structural characterization of landfill leachate and leonardite humic fractions, Anal. Bioanal. Chem. 408 (2016) 1917-1928. [49] T.L. Marian, D. Nghiem, Landfill leachate treatment using hybrid coagulation-nanofiltration processes, Desalination 250 (2010) 677–681.

23

FIGURE CAPTIONS

Figure 1. Sketch of the EC reactor used to treat landfill leachates.

Figure 2. Predicted values of the DTC removal, which are in good agreement with the observed values according to the good correlation coefficient of R2=0.96.

24

Figure 3. The 3-dimensional response surface for the DTC removal, which was achieved by setting the initial pH value to (a) 5, (b) 6, and to (c) 7 as a function of the other two EC parameters.

25

Figure 4. Removal profiles for the COD, the BOD, the color, the turbidity, iron concentration, the DOC, and the DIC after applying the electrocoagulation technique and using electrolysis times that ranged from 0 to 180 min.

26

Figure 5. Behavior for (a) germination relative to the negative control, (b) the inhibition of radicle growth relative to the negative control, and (c) the inhibition of root growth relative to the negative control and as a function of the electrolysis time.

27

Figure 6. FTIR spectra for raw and EC-treated LL samples that correspond to EC-process times of 5, 90, and 120 min.

28

TABLES

Table 1. Variations of the values for the main physico-chemical parameters pertaining to raw LL and MCPP in a 2015/2016 monitoring program.

Collects month/year 05/2015 06/2015 07/2015 08/2015 09/2015 10/2015 11/2015 12/2015 01/2016 02/2016 03/2016 04/2016 Annual average value 95% confidence interval

Values of main parameters pH PO4-3 Alcalinity (±0.1) (mg L-1) (mgL-1 CaCO3) 7.0 7.8 7.0 7.3 7.2 7.3 7.4 7.3 7.5 7.3 7.5 7.9

5.6 ± 0.2 10.7 ± 1.4 7.4 ± 0.8 5.4 ± 0.5 6.4 ± 0.2 6.2 ± 0.2 4.6 ± 0.2 5.9 ± 0.3 6.3 ± 0.2 6.0 ± 0.2 4.1 ± 0.2 5.2 ± 0.3

4860 ± 102 6416 ± 137 5822 ± 85 6048 ± 56 5083 ± 9 5133 ± 40 5319 ± 117 7436 ± 102 5224 ± 71 5116 ± 68 5048 ± 83 6055 ± 110

Turbidit y (NTU) 115 ± 2 100 ± 3 88 ± 2 97 ± 3 42 ± 2 43 ± 1 41 ± 3 86 ± 4 97 ± 6 83 ± 5 52 ± 2 86 ± 3

N-NH3 (mgL-1)

COD (mgL-1)

TS (mgL-1)

Fe (mg L-1)

MCPP

847 ± 54 999 ± 59 880 ± 55 935 ± 48 734 ± 21 792 ± 29 751 ± 20 769 ± 13 877 ± 16 903 ± 22 443 ± 19 657 ± 25

4185 ± 183 6556 ± 664 4251 ± 142 5428 ± 154 4238 ± 179 4439 ± 146 4258 ± 165 4020 ± 190 6023 ± 155 6316 ± 130 4654 ± 166 6047 ± 150

6631 ± 196 9891 ± 117 4858 ± 83 6587 ± 120 5747 ± 96 5645 ± 155 5063 ± 28 4558 ± 98 5588 ± 115 5690 ± 58 5477 ± 108 8726 ± 96

33 ± 1 56 ± 1 33 ± 1 43 ± 2 11 ± 1 20 ± 2 7.2 ± 0.5 14 ± 1 11 ± 1.2 1.4 ± 0.2 23 ± 1 38 ± 2

261 91 385 55 135 81 270 318 206 250 149 40

7.3

6.1

5630

78

799

5034

6205

24

187

7.2 – 7.5

5.2 – 7.1

5202-6057

63-92

715-882

4489-5580

5305-7104

15-34

124-250

(mm)

29

Table 2. Values of the Pearson correlation among physicochemical parameters characterizing the raw LL effluent and the MCPP at a 95% confidence level in the 2015/2016 monitoring program. pH

PO4-3

Alcalinity

Turbidity

N-NH3

COD

TS

Fe

pH

1.00

PO4-3

0.23

1.00

Alcalinity

0.33

0.34

1.00

Turbidity

0.15

0.36

0.32

1.00

N-NH3

-0.16

0.67

0.21

0.57

1.00

COD

0.73

0.39

0.07

0.42

0.37

1.00

TS

0.74

0.53

0.18

0.40

0.25

0.67

1.00

Fe

0.40

0.50

0.35

0.51

0.27

0.29

0.75

1.00

MCPP

-0.61

-0.04

0.07

0.13

0.12

-0.46

-0.64

-0.42

MCPP

1.00

30

Table 3. Operational parameters for the EC reactor (pH, current density, and the electrolysis time) and parameter responses (DTC removal) within the 33 CFD in the RSM framework. EC reactor operational parameters

Removals of DTC

5

Current density (q2) (Am-2) 42.85

Time (q3) (min.) 30

7

42.85

30

9

42.85

5

pH (q1)

% DTC1

% DTC2

% DTC3

42.3

44.0

44.0

25.9

25.8

26.1

30

11.5

13.1

13.1

85.71

30

44.9

46.5

45.1

7

85.71

30

34.0

33.4

33.3

9

85.71

30

20.6

22.2

21.6

5

128.57

30

56.1

55.7

56.2

7

128.57

30

47.1

47.8

47.9

9

128.57

30

32.6

31.9

32.7

5

42.85

75

47.3

47.9

46.9

7

42.85

75

30.2

30.5

30.4

9

42.85

75

24.9

27.7

26.6

5

85.71

75

62.6

59.2

61.1

7

85.71

75

31.2

30.2

31.7

9

85.71

75

20.8

21.5

22.3

5

128.57

75

59.2

59.0

59.7

7

128.57

75

46.0

46.3

46.3

9

128.57

75

24.5

24.7

25.6

5

42.85

120

44.1

45.7

48.2

7

42.85

120

27.2

26.9

27.1

9

42.85

120

22.8

26.0

25.6

5

85.71

120

55.7

57.8

57.7

7

85.71

120

32.5

32.9

34.6

9

85.71

120

33.3

33.3

33.4

5

128.57

120

61.3

61.6

62.7

7

128.57

120

50.6

50.3

50.5

9

128.57

120

36.5

36.5

36.7

31

Table 4. Results of the ANOVA for validation of the mathematical model given by Eq. 15, which represents the DTC removal data. Freedom

Means

squares

degrees

squares

q1

10081

2

5041

6438

<10-64

q2

2912

2

1456

1860

<10-49

q3

452

1

452

577

<10-29

q1:q2

417

4

104

133

<10-26

q1:q3

300

4

75

96

<10-23

q2:q3

255

2

127

163

<10-22

q1:q2:q3

356

8

44

57

Regression

14773

23

642

770

1.72

<10-62

residual

47.5

57

0.834

Lack of fit

5.24

3

1.75

2.23

2.77

0.10

Parameters

Sum

of

of

Fcal.

Ftab.

p-value

32

Table 5. Evaluation of the A.-salina-based toxicity in EC-treated LL effluent performed using electrolysis time intervals of 0-120 min.

Time (min)

N-NH3 (mg L-1)

Mortality ratio in diluted toxicity in ECtreated effluents 20%

40%

60%

80%

100%

LC50 value (%)

95% confidence Interval

0

1221

30/30

30/30

30/30

30/30

30/30

NA

NA

5

1182

20/30

30/30

30/30

30/30

30/30

NA

NA

10

1275

17/30

30/30

30/30

30/30

30/30

NA

NA

15

962

13/30

30/30

30/30

30/30

30/30

21.7

NA

20

1042

10/30

30/30

30/30

30/30

30/30

23.8

NA

30

1073

8/30

28/30

30/30

30/30

30/30

25.5

22-29

45

937

4/30

30/30

30/30

30/30

30/30

26.8

NA

60

735

6/30

21/30

30/30

30/30

30/30

30.3

26-35

75

938

3/30

29/30

30/30

30/30

30/30

27.5

26-29

90

643

4/30

20/30

30/30

30/30

30/30

32.1

28-36

120

721

3/30

21/30

30/30

30/30

30/30

31.9

28-36

180

731

1/30

25/30

30/30

30/30

30/30

30.4

28-33

*NA: Not assessed.

33

Table 6. Toxicity behavior of the EC-treated LL effluents, which were exhibited in the diluted toxic samples by employing Lactuca sativa. Germination in diluted toxicity in EC-treated effluents Time

3

Confidence Interval (95%)

10

30

100

Germabs Germabs

Germabs

Germabs

Germabs

0

100.0

100.0

100.0

0.0

0.0

17.3 NA

5

100.0

100.0

100.0

0.0

0.0

17.3 NA

10

100.0

100.0

100.0

0.0

0.0

17.3 NA

15

100.0

100.0

100.0

0.0

0.0

17.3 NA

20

100.0

100.0

100.0

0.0

0.0

17.3 NA

30

100.0

100.0

100.0

15.0

0.0

20.6 17-25

45

100.0

100.0

100.0

15.0

0.0

20.6 17-25

60

100.0

100.0

100.0

35.0

0.0

25.9 20-33

75

100.0

100.0

100.0

45.0

0.0

29.1 23-38

90

100.0

100.0

100.0

50.0

0.0

30.8 24-40

120

100.0

100.0

100.0

65.0

0.0

36.6 29-47

180

100.0

100.0

100.0

65.0

0.0

36.6 29-47

(min)

1

LC50

*NA: Not assessed.

34