Horticulturae 2792868 Peer Review v1

Article 1
Assessment of Phytotoxicity in Untreated and Electrochemi- 2
cally Treated Leachates Through the Analysis of Early Seed 3
Growth and Inductively Coupled Plasma–Optical Emission 4
Spectroscopy Characterization 5
Alfredo Martínez-Cruz and *María Neftalí Rojas-Valencia 6
Institute of Engineering, National Autonomous University of México, External Circuit, University City, 7
Coyoacan Delegation, Mexico City 04510, México; amartinez@iingen.unam.mx 8
* Correspondence: Correspondence: mrojasv@iingen.unam.mx; Tel.:+52-(55)-5633600 (ext. 8663) 9
Abstract: The treatment of stabilized leachates, characterized by a biodegradability index (IB) of 10

0.094 and dissolved organic carbon (COD) of 3.4 gL-1, was accomplished through the application of 11
electro-coagulation (EC) and electro-oxidation (EO) technologies. As a result, an effluent with an IB 12
of 0.46 and COD of 0.6 gL-1 was obtained, achieving removal rates of 82%, 86%, 99%, and 81% for 13
COD, color, and nitrogen, respectively. The biodegradable COD increased from 26% in the crude 14
leachate to 39% in the EC process and to 58% in the EO process effluent. Particulate COD in the 15
crude leachate decreased from 48% to 23% in the EC effluent and 33% in the EO effluent. The EC 16
process removed 69% and 63% of humic and fulvic acids, respectively, whereas the electro-oxidation 17
process removed 39% and 55% of humic and fulvic acids, respectively. The proposed treatment sys- 18
tem successfully broke the aromatic structures of the humic substances present in the raw leachate, 19
thereby increasing the biodegradable material content. The phytotoxicity tests showed that the pro- 20
posed treatment system reduced the phytotoxicity of the generated effluents. The parameters that 21
contributed the most to toxicity in the treated leachates were aluminum, copper, iron, zinc, and 22
calcium. 23
Keywords: Electro-coagulation; electro-oxidation; mature leachates; organic matter; phytotoxicity 24

25
Assessment of Phytotoxicity in Untreated and Electrochemically Treated Leachates Through the 26

Analysis of Early Seed Growth and inductively coupled plasma–optical emission spectroscopy 27
Characterization 28
1. Introduction 29
The overall generation of municipal solid waste (MSW) has steadily increased be- 30
cause of urbanization, increased commerce, population growth, and economic develop- 31
ment (Anand and Palani, 2022). By the end of the next three decades, it is projected that 32
global MSW generation will reach 3.4 billion tonnes, up from 2.01 billion tonnes generated 33
in 2016 (Kaza et al., 2018). The prevalent approach for MSW disposal in developing and 34
underdeveloped countries is landfilling (Kamaruddin et al., 2017). Given the moisture 35
content and seasonal fluctuations in MSW, the production of hazardous liquid, known as 36
landfill leachate (Anand and Palani, 2022), is a natural by–product of landfills (hereinafter 37
referred to as "leachates" in this document). 38
From the viewpoint of environmental pollution, the crucial components of leachates 39
include organic pollutants, such as volatile fatty acids (VFA) and humic substances (HS), 40
and inorganic pollutants, including heavy metals, ammoniacal nitrogen (NH 3–N), 41
Horticulturae 2023, 9, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/horticulturae

Horticulturae 2023, 9, x FOR PEER REVIEW 2 of 23
sulfates, sodium, magnesium, calcium, and other xenobiotic compounds such as aromatic 42
hydrocarbons and halogenated organic compounds(Ghosh et al., 2017). 43
Untreated leachates have the potential to inflict severe and irreversible harm on both 44
aquatic and terrestrial ecosystems 2016 (Anand and Palani, 2022). The categorization of 45
leachates is based on their age, which can be classified as young (less than 5 years), inter- 46
mediate (5–10 years), or stabilized (> 10 years), as reported by (Kow et al. (2016). For bio- 47
logical treatment of leachates, the ideal BOD/COD ratio is >0.5 (Dhanke and Wagh, 2020), 48
but previous investigations conducted by our group have determined that the biodegra- 49
dability index (BOD/COD ratio) of the leachates used in this project (Bordo Poniente land- 50
fill, Mexico City, México) was 0.094±0.03 (Martínez-Cruz et al., 2021; Martínez-Cruz and 51
Rojas-Valencia, 2023). 52
Leachates are among the most complex forms of wastewater, and their treatments 53
can be classified into three categories: biological (aerobic/anaerobic), physical and chemi- 54
cal (sedimentation/flotation, coagulation/flocculation, adsorption, and chemical oxida- 55
tion), and combined or hybrid treatment methods (Nidheesh et al., 2023; Teng et al., 2021). 56
It is worth noting that, as the age of the leachate increased, the concentration of recalcitrant 57
organic matter increased significantly. Conventional treatment methods have proven in- 58
effective for treating recalcitrant organic matter, leading to the growing popularity of ad- 59
vanced oxidation processes (AOPs) for leachate treatment (Teng et al., 2021). It is of utmost 60
importance to thoroughly understand the characteristics of leachates to evaluate their po- 61
tential to cause harm to the environment and to determine the most effective treatment 62
methods. Although these physicochemical treatment methods are effective, they are not 63
cost–efficient and produce a significant amount of sludge that must be processed further, 64
as reported by Ghosh et al. (2017) and Kamaruddin et al. (2017). 65
In contrast, electrochemical technologies have the potential to become dominant so- 66
lutions for the removal of biorefractory substances (Martínez-Huitle et al., 2015). Among 67
the electrochemical technologies, electro–oxidation (EO) has been demonstrated to be ef- 68
fective in significantly reducing recalcitrant organic compounds (Nidheesh et al., 2018; 69
Sirés et al., 2014; Vasudevan and Oturan, 2014). The treatment of stabilized leachates via 70
EO can be a challenging endeavor because of the presence of colloidal and hydrophobic 71
humic substances, which can hinder the efficiency of the process and increase the energy 72
costs. However, by implementing electro–coagulation (EC) process as a preliminary treat- 73
ment, it is possible to separate these humic substances. This results in a more efficient 74
treatment process and elimination of interference from these colloidal species (Baiju et al., 75
2018; Mahmud et al., 2012; Martínez-Cruz and Rojas-Valencia, 2023; Moradi and Ghan- 76
bari, 2014). The EC process has been demonstrated to be effective in treating various com- 77
plex wastewaters, including leachate, as reported in several studies (Gandhimathi et al., 78
2015; Ghanbari et al., 2020; Martínez-Cruz and Rojas-Valencia, 2023). 79
Assessment of the toxic effects of leachates can be conducted through various toxicity 80
tests that utilize diverse bioassays, such as microorganisms (Ames mutagenicity test, lu- 81
minescent bacteria test, etc.), plants (seed germination study, micronucleus test, comet 82
assay, etc.), and animals (e.g., mortality and contact tests). These tests were performed 83
using standardized protocols to evaluate the potential toxicity of leachates to living or- 84
ganisms (Anand and Palani, 2022; Luo et al., 2020). Plant bioassays, such as seed germina- 85
tion studies, are commonly employed to evaluate the toxicity of hazardous waste, includ- 86
ing leachates. These tests are particularly sensitive to the toxic effects of substances such 87
as low–molecular–weight acids, NH3–N, heavy metals, and salts present in leachates. To 88
quantify the toxicity of leachates, both the germination index (GI) and root inhibition of 89
the seeds can be measured. Furthermore, it should be noted that seed germination studies 90
are not only rapid but also economical compared to other toxicity assessment methods 91
(Anand and Palani, 2022). 92
Although numerous studies have explored leachate treatment (Abbas et al., 2009; Ál- 93
varez-Vázquez et al., 2004; Alver and Altaş, 2017; Chaudhari and Murthy, 2010; Gomes et 94
al., 2019; Kulikowska, 2016; Kurniawan et al., 2006; Luo et al., 2020; Martínez-Cruz et al., 95
2021; Nájera-Aguilar et al., 2019; Oulego et al., 2015; Ozturk et al., 2003; Pastore et al., 2018; 96
Renou et al., 2008; Seibert et al., 2019; Torretta et al., 2017; Verma and Naresh Kumar, 2016; 97
Vlyssides et al., 2003), most studies have focused on changes in parameters, such as chem- 98
ical oxygen demand (COD) and biochemical oxygen demand (BOD5), without considering 99
the impact of treatment on the toxicity levels of the resulting effluents. Based on the lim- 100
ited data available, it is difficult to establish a correlation between leachate characteristics, 101
effluent leachate results subsequent to the chosen treatment, and potential modification 102
in toxicity levels in both leachates and effluents. Despite the dearth of reports on the tox- 103
icity of leachates to seeds using germination and root inhibition assays, the root growth 104
assay continues to be a highly efficacious method for toxicity evaluation. 105
The assessment and quantification of toxic chemical elements, particularly those of 106
utmost concern, are crucial components of chemical analysis. Spectroscopic inductively 107
coupled plasma–optical emission spectroscopy (ICP–OES) has emerged as the preferred 108
method for elemental analysis owing to its ability to simultaneously measure multiple 109
elements. This method employs inductively coupled plasma to produce excited atoms and 110
ions that emit the characteristic electromagnetic radiation (Bulska and Wagner, 2016). 111
ICP–OES is known for its exceptional accuracy, precision, and low detection rate (Douvris, 112
2023). There has been limited prior research establishing a correlation between phytotox- 113
icity and comprehensive analysis by ICP–OES in both the influents and effluents of a treat- 114
ment system, such as that proposed in this project. 115
This study aimed to investigate the phytotoxicity of early seed growth in raw leacha- 116
tes and raw leachates treated with an EO–EC system. Previously, the optimal conditions 117
for the EC–EO treatment system were identified to remove organic matter, as measured 118
by COD. Additionally, an investigation was conducted on the raw leachates and gener- 119
ated effluents, which included an examination of any potential connections between the 120
phytotoxicity results and the ICP-OES characterization. 121
2. Materials and Methods 122

2.1. Landfill Leachate Collection and Characterization of Leachates and Eﬄuents 123
Leachate samples were collected from the Bordo Poniente Landfill Stage III. This 124
landfill is located southwest of the former Lake Texcoco five kilometers from Mexico City’s 125
international airport (longitude 99°00 14.51 and 99°02 36.21 west; latitude 19°26 09.36 and 126
19°29 09.22 north), has an area of 670 ha, and was operated from 1985 to 2011. This landfill 127
is the largest in Latin America and contains 76 million tons of urban solid waste (Martínez- 128
Cruz and Rojas-Valencia 2023). Leachate samples (80 L) were collected and transferred to 129
plastic containers, which were subsequently stored in a refrigerator at 4 °C until analysis. 130
American Standard Test Methods were used to determine the dissolved organic car- 131
bon (DOC) and color (ASTM, 2009, 2019). Standardized APHA methods were used to de- 132
termine pH, electrical conductivity, COD, BOD5, chlorides, and NH3–N (APHA, 2022). 133
Fractions of COD –determination of biodegradable COD by aeration and soluble COD 134
by coagulation with zinc sulfate (Abu Amr et al., 2014; Bashir et al., 2013), and humic 135
substances (separation of humic acids by acidification and fulvic acids by adsorption) 136
were determined (Dia et al., 2018). 137
2.2. Leachate Treatment System 138

In this study, a two–stage treatment system was developed and implemented. The 139
initial stage involved the application of a process known as electro–coagulation to the raw 140
leachate. This was followed by the electro–oxidation process, which was optimized using 141
response surface methodology and a fractional orthogonal design to achieve the maxi- 142
mum removal of organic matter. The goal of this second stage was to produce the final 143
effluent with the highest possible degree of organic matter removal. The details of this 144
procedure have been documented in a previous study conducted by the same research 145
team (Martínez-Cruz and Rojas-Valencia, 2023). 146
Two distinct rectangular plexiglass reactors were employed for the EC and EO pro- 147
cesses. The reactor used for the EC process had dimensions of 15 cm × 10 cm × 15 cm, with 148
a volume of 1.5 L, and comprised an Fe anode and a stainless–steel cathode, each measur- 149
ing 10 cm × 10 cm x 0.4 cm, with an effective area of 160 cm² and a distance between elec- 150
trodes of 1 cm. The EO reactor, on the other hand, had dimensions of 12 cm × 10 cm × 12 151
cm, with a volume of 1.5 L, and comprised a boron–doped diamond (BDD) anode and a 152
stainless-steel cathode, each measuring 7 cm × 5 cm x 0.1 cm, with an effective area of 60 153
cm². The EO process employed a filter–press arrangement in an upper frame that held the 154
electrodes, with the option of varying the distance between the electrodes at 0.5, 1.0, and 155
1.5 cm, and a continuous flow of input and output using a recirculation vessel at a rate of 156
1.5 L min−1. Both EC and EO electrodes were connected in monopolar mode in parallel to 157
a digital DC power supply (Steren PRL258, Steren®, Mexico City, Mexico) in batch oper- 158
ation mode. 159
2.3. Evaluation of Phytotoxicity 160

Lactuca. sativa seeds are widely recognized as standardized seeds for phytotoxicity 161
testing and have been endorsed by the Organization for Economic Cooperation and De- 162
velopment (OECD, 2006), United States Environmental Protection Agency (USEPA, 1996), 163
and American Society for Testing and Materials (ASTM, 1990). Inclusion of Cucumis seeds 164
sativus and Phaseolus. vulgaris in the phytotoxicity tests was intended to provide data on 165
the potential effects of contaminants on plant communities in and around functioning and 166
closed landfills. These seeds were selected because of their sensitivity to environmental 167
stress, ease of handling, relatively short life cycle, and the ability to provide consistent and 168
reproducible results. Additionally, they are commonly cultivated in areas near landfills in 169
Mexico, making them particularly relevant for horticultural considerations. 170
The phytotoxicity testing method utilized the International Organization for Stand- 171
ardization ISO 11269–2, Organization for Economic Cooperation and Development 172
OECD–Test Guideline 208, and the United States Environmental Protection Agency Test 173
Guidelines OPPTS 850.4200 (ISO, 2017; OECD, 2006; USEPA, 1996) to evaluate the effects 174
of different concentrations of leachates and effluents on the growth of lettuce (Lactuca sa- 175
tiva), cucumber (Cucumis sativus), and bean (Phaseolus vulgaris) in a garden soil substrate. 176
Gardening land was purchased from a local specialty store in Mexico City, Mexico. The 177
seeds were purchased from 8 g packs in Rancho Los Molinos ® (Mexico City, Mexico). 178
The dilutions were prepared by mixing raw leachate and treated leachates with tap 179
water under conditions that maximized COD removal by EC and EO. The seeds were 180
subsequently placed within the treated soil and assessed for growth and development 181
seven days after the emergence of 50% of the seedlings in the control group. Measure- 182
ments and observations were compared to those of the untreated control. 183
Following the test, all plants, including their roots, stems, and leaves, were carefully 184
removed from the pots. The thin and thick roots were thoroughly washed with tap water 185
to remove any soil residue. All the samples were subsequently dried in an oven at 75°C, 186
after which the dry matter content was determined. 187
The seeds were subjected to a formal tone and soaked in tap water for two hours 188
prior to the experiment. The seeds were then evenly distributed in germination containers, 189
which consisted of 10 seeds of L. sativa and five seeds each of C. sativus and P. vulgaris, in 190
a container filled with commercial garden soil that had been soaked with 5 mL of the 191
evaluated effluent. The germination vessels, made of polypropylene, had 12 cells with di- 192
mensions of 0.3 x 0.3 m, and the research was conducted with 12 replicates (Figure 1). The 193
germination vessels were kept in a greenhouse under a translucent plastic film cover that 194
allowed the passage of light but not rain. 195
196
Figure 1. Germination tray used in the phytotoxicity experiments. 197
The germination rates (Ec. 1), radicle length (Ec. 2); growth inhibition: biomass (Ec. 198
3) and germination index (Ec. 4), was subsequently determined as part of the phytotoxicity 199
test. 200
Number of seeds germinates

% Germination rate = 100 … (1) 201
Total of seeds
(Raditle lenght control − radicle lenght dilution)

% Growth inhibition = 100 … (2) 202
Raditle lenght control
(average weight in control − average weight in treatment)

% Growth inhibition = 100 … (3) 203
average weight in control
radicle lenght dilucion germinated seed dilution

Germination index = … (4) 204
radicle lenght control germinated seed control
2.3.1. Experiment Design 205

The experimental design was a factorial design with 12 replicates. The independent 206
variables were the seed type (L. sativa, C. sativus, and P. vulgaris), the dilution of the leach- 207
ate and treated leachates (0, 2.5, 10, 30, 60, and 90%), the pH (unadjusted pH, and neutral 208
pH of 7.0 using citric acid or NaOH 0.1 N), and the leachate (row leachate, effluent electro– 209
coagulation, and effluent electro–oxidation). To avoid the effects of oxidation on the or- 210
ganic matter in the samples, citric acid (20 wt.), following the recommendations of agroe- 211
cological companies (Control Bio 2020; Romero 2007). 212
The response variables were the germination rate, growth inhibition (radicle length), 213
growth inhibition (biomass), and germination index. The half maximal effective concen- 214
tration (EC50) was calculated from the dose–response relationship between the germina- 215
tion rate and the dilution of the leachates or effluent using probit analysis with confidence 216
limits of 95%. 217
MINITAB Release 18 statistical software was used to conduct a thorough analysis 218
supported by the statistical methods. The results are presented as the mean±standard de- 219
viation, and the statistically significant difference between the control and treated groups 220
was determined using one–way and two–way ANOVA. 221
2.4. Analysis Using Inductive Coupling Plasma with Optical Emission Spectrophotometer 222
(ICP–OES) 223
Undeniably, the significance of resource and time optimization is paramount. Con- 224
sequently, the roots obtained from the effluent of the complete treatment system (electro– 225
coagulation electro–oxidation) underwent ICP–OES analysis. Additionally, the analysis 226

was extended to include the garden land used in the tests. The methodology is described 227
as follows: 228
2.4.1. Chemical Reagents and Materials and Preparation of Samples 229

High–purity (99.99%) argon was used as the plasma, auxiliary, and nebulizer gas. 230
Nitric acid and hydrogen peroxide grade AR were obtained from Merck ® (Darmstadt, 231
Germany) for use in this experiment. All glassware used in the experiment was of "A" 232
grade quality and was calibrated. A micropipette with a calibrated range of 100–1000 μl 233
was used in the experiment. 234
The quantification of roots and soil was conducted, with approximately 0.4 g of each 235
being measured. Subsequently, 5 mL of leachate samples were collected. Nitric acid and 236
hydrogen peroxide were added to the digestion tube at a volume ratio of 5 mL. An assisted 237
microwave digestion technique, as outlined by Stadler and Michaelis (2021), was used to 238
heat the organic matter in the samples to 120 °C for 30 min. After digestion, the samples 239
were filtered through Whatman No. 41 paper filter in volumetric flasks with a capacity of 240
25 mL. The volume was adjusted using 5% nitric acid solution to achieve the desired quan- 241
tity. 242
2.4.2. ICP–OES Equipment 243

The equipment used was the Agilent–Technologies 5100 ICP–OES (Santa Clara, CA, 244
USA), with a concentric nebulizer, a gas flow rate of 12 L/min, and an auxiliary gas flow 245
rate of 0.7 L min–1 (argon). The radiofrequency power used was 1.2 kW, and the pump 246
speed was 12 rpm. A charge–coupled device (CCD) detector and 21 CFR 11 version 4.1.0 247
software for the data acquisition and processing. 248
For the calibration curve preparation of the instrument, a standard multi–element 249
solution of 1000 μg mL–1 K and 100 μg mL–1 for the remaining elements were used (Quality 250
Control Standard 26, hps ®, Charleston, South Carolina, USA). During ICP–OES studies, 251
the vertical height of the plasma was maintained at a constant level of 7 mm. The sample 252
uptake time was set at 30.0 seconds, followed by a 5–second delay and a 10–second rinse 253
time. The initial stabilization period was 10 s, and the time between replicate analyses was 254
fixed at 5 s. These parameters were maintained throughout the study period. 255
3. Results and Discussion 256

3.1. Treatment System and Characterization of Leachates and Eﬄuents 257
The optimal conditions for achieving significant removal of organic matter, as meas- 258
ured by COD, during electro–coagulation were a current density of 23.3 mA cm–2, stirring 259
at 100 rpm, and a pH of 7. These conditions resulted in COD, DOC, color, and NH 3–N 260
removal rates of 63, 69, 94, and 50%, respectively. The most effective conditions for elec- 261
tro–oxidation were an NaCl concentration of 1 g L–1, electrode distance of 0.75 cm, current 262
density of 33.3 mAcm–2, and pH of 7. Under these conditions, COD, DOC, color, and NH3– 263
N were removed at rates of 82, 86, 99, and 81%, respectively (compared to raw leachate). 264
The attributes of the raw leachate, electrocoagulation, and electro–oxidation effluents 265
are listed in Table 1. The pH of the raw leachates was found to be 8.4±0.1, which aligns 266
with the results reported in other studies on mature leachates. Poblete and Pérez (2020) 267
reported a pH value of 8.9 in mature leachates. Typically, mature leachates have a pH 268
greater than 7 because of the microbiological reduction of volatile fatty acids and a de- 269
crease in the leaching force during the methanogenic phase (Hussein et al., 2019; Zakaria 270
and Aziz, 2018). These high pH values are consistent with the stages that MSW undergoes 271
in landfills, including acetogenic and methanogenic stages (Wijekoon et al., 2022). During 272
electro–coagulation, the pH of the effluent increases because of the presence of hydroxyl 273
groups (Yazici et al., 2019). The decrease in pH during electro–oxidation was attributed to 274
the generation of H+ ions (Ghanbari et al., 2020). 275
Table 1. Characterization of raw leachates and effluents generated. 276
Electro– Electro–
Raw
Parameter coagulation oxidation
leachate
effluent effluent
Ph 8.4±0.1 9.5±0.1 5.6±0.1
Biodegradability
0.094 0.26 0.48
index
Electrical
8.5±1 2.2±0.5 1.7±0.4
conductivity
Chlorides 6.7±0.1 3.6±0.1 1.9±0.4
BOD5 0.32±0.01 0.338±0.01 0.288±0.01
Color 3200±90 200±10 20±1
DOC 1.2±0.2 0.36±0.01 0.16±0.01
NH3–N 0.66±0.03 0.33±0.01 0.12±0.01
Total COD 3.4±0.1 1.3±0.5 0.6±0.01
Soluble COD 1.77±0.1 0.97±0.2 0.4±0.1
Biodegradable COD 0.87±0.04 0.49±0.1 0.35±0.01
Non–biodegradable
0.89±0.04 0.48±0.02 0.052±0.02
soluble COD
Humic acid 1.94±0.04 0.61±0.03 0.37±0.02
Fulvic acid 0.77±0.03 0.29±0.01 0.13±0.01
Hydrophilic fraction 0.87±0.04 0.34±0.01 0.1±0.01
Units in g L–1, except for the color (Pt–Co U) and electrical conductivity (mScm–1). The pH and bio- 277
degradability indices are dimensionless. Results are expressed as mean±standard deviation. 278
The biodegradability index value obtained for the raw leachates was 0.094, which is 279
less than 0.1. This indicates that the leachate sampled at landfill Bordo Poniente was of the 280
mature type, as confirmed by Naveen et al. (2016).The results obtained were consistent 281
with the closure of Stage III of the landfill, which has been in place since 1994 and has an 282
age well above 10 years. According to Abunama et al. (2021), a biodegradability index of 283
less than 0.1 signifies a substantial quantity of non–biodegradable organic matter. This 284
finding is consistent with the non–biodegradable COD level of 74% identified in the crude 285
leachate in this study. The treatment system resulted in changes in the biodegradability 286
index. For electro–coagulation, a value of 0.26 was obtained, and for the electro–oxidation 287
process, it reached 0.48. In the case of electro–coagulation, the destabilization of colloidal 288
material is achieved by coagulating organic matter (mainly humic substances). Regarding 289
the electro–oxidation process, the increase in the biodegradability index is justified by the 290
removal of organic matter caused by the action of •OH radicals formed at the BDD anode. 291
The raw leachate exhibited a color value of 3200±90 U Pt–Co. The color of leachates 292
is commonly associated with the presence of organic substances, particularly during the 293
final phase of degradation, which is primarily composed of humic substances, such as 294
humic and fulvic acids (Ibrahim and Yaser, 2019; Tripathy and Kumar, 2019). The discol- 295
oration observed in the electro–coagulation process (95%) is primarily attributable to the 296
removal of humic substances, which constitute the majority of the organic matter present 297
in mature leachates. Therefore, the reduction in color is indicative of a concomitant de- 298
crease in the aforementioned humic substances caused by electro–coagulation (Dia et al., 299
2018). The electro–oxidation process effectively reduced the remaining 90% of the color, 300
resulting in a final reading of 23 Pt–Co units. This significant decrease can be attributed to 301
the removal of humic substances, particularly fulvic acids, which are more challenging to 302
remove through electro–coagulation. 303
The concentration of NH3–N in the raw leachates collected was 658 mg L–1. This result 304
can be ascribed to the biological degradation of amino acids and other nitrogenous organic 305
substances present in municipal solid waste (MSW); a process previously documented by 306
Abunama et al.(2021). Electro–coagulation removed 50% of NH3–N. According to Singh 307
and Dash (2023), the use of electro–coagulation results in the removal of NH3–N, which 308
can be attributed to the increase in temperature and pH levels. These changes disrupted 309
the equilibrium of nitrogen present in the NH3–NH4+ leachates, promoting the release of 310
NH3–N as a gas into the atmosphere. According to Fudala–Ksiazek et al. (2018), the de- 311
crease in NH3–N during electro–oxidation is attributed to its indirect oxidation resulting 312
from the formation of reactive chloride oxidants, including Cl2 and HOCl, which accounts 313
for 81% reduction. 314
In the electro–coagulation process, the destabilization of colloidal material caused by 315
the presence of iron hydroxides is responsible for the removal of organic matter, as meas- 316
ured by the chemical demand for oxygen and dissolved organic carbon. Similarly, in the 317
electro–oxidation process, complete oxidation of a portion of the organic material to CO 2 318
is achieved through the action of •OH radicals formed at the anode, resulting in the re- 319
moval of organic matter (De Battisti y Martínez–Huitle, 2018). 320
In the raw leachates examined, the fractions of humic substances, as determined by 321
COD, were as follows: humic acids in 1.94±0.04 g L –1, comprising 54% of the total, and 322
fulvic acids in 0.77±0.03 mg L–1, comprising 22% of the total. Previous studies reported 323
comparable results for mature leachates. Specifically, Dia et al. (2018) reported 27% humic 324
acid and 42% fulvic acid, collectively accounting for 76% of the total COD. The presence 325
of humic substances in the leachates confirmed their maturity. 326
In the electro–coagulation process, it was observed that the biodegradable fraction of 327
COD increased from 26% to 39% owing to the elimination of hydrophobic substances from 328
humic acids (Dia et al., 2018). Conversely, the particulate fraction of COD decreased from 329
48 to 23%, which could be attributed to the removal of colloids (Hakizimana et al., 2017). 330
The electro–oxidation process resulted in a noticeable increase in biodegradable COD 331
from 39% to 58%. This increase can be attributed to indirect electro–oxidation mediated 332
by • OH radicals, which oxidize nonbiodegradable organic matter. Consequently, the sol- 333
uble COD fraction decreased from 77% to 67%, primarily because of oxidation of the hy- 334
drophilic fraction during the process. Additionally, the biodegradable soluble COD frac- 335
tion significantly increased from 51% to 87% because of the action of • OH radicals that 336
oxidize recalcitrant organic matter, such as humic and fulvic acids (Ding et al., 2018; Man- 337
dal et al., 2017). 338
3.2. Evaluation of Phytotoxicity 339

3.2.1. Research on Seed Germination 340
By analyzing the data from the early germination of seeds, it was possible to gain 341
insight into the phytotoxicity of leachates and effluents. If the phytotoxicity was high, seed 342
germination was inhibited; however, if the phytotoxicity was low, seed germination oc- 343
curred, and root growth could be measured. In this study, control experiments were con- 344
ducted, and the seeds of C. sativus germinated on the second day, while the seeds of L. 345
sativa and P. vulgaris germinated on the third day. The germination rates are shown in 346
Table 2. At the control dilution, the germination rates of all three seeds were higher than 347
90% in all experiments. The average germination values were higher in leachates with 348
neutral pH than those without adjustment (p<0.05), and no statistically significant differ- 349
ences were observed between the three seeds evaluated (p>0.05). 350
Table 2. Germination rate in phytotoxicity tests. 351
Germination rate (%)

Raw leachate (dilutions in percent)
Seed Neutral pH Unadjusted pH (8.4)
Control 2.5 10 30 60% 90% Control 2.5% 10 30 60 90

L. sativa 97±4 97%±4 97±3 52±2 24±1 2±0.1 92±4 94±3 96±4 40±2 12±0.5 0.8±0.01
C. sativus 97%±4 97%±3 97±4 62%±3 23±1 2±0.1 93±3 93±2 95±4 54±2 15±0.5 0.0
P. vulgaris 98%±4 97%±4 97±4 58%±3 33±1 0 93±4 95±2 95±4 48±2 27±0.5 0.0
Electro–coagulation effluent (dilutions in percent)

Control 2.5 10 30 60 90 Control 2.5 10 30 60 90
L. Sativa 97±4 97±4 97±4 55±0.1 26±0.1 7±0.3 95±4 96±3 94±4 51±2 20±1 5±0.2
C. sativus 98±4 98±4 97±4 67±0.3 28±0.1 8±0.4 95±3 95±3 93±4 60±3 20±1 5±0.2
P. vulgaris 98±3 97±4 98±3 63±0.3 42±0.2 8±0.4 95±3 93±3 95±3 60±2 38±1 5±0.1
Electro–oxidation effluent (dilutions in percent)
Control 2.5 10 30 60. 90. Control 2.5 10 30 60 90

L. sativa 99±5 99±4 97±4 61±2 32±1 12.5±0.5 98±5 98±4 97±4 57±1 29±1 10±0.5
C. sativus 100±4 100±4 98±4 72±3 40±2 15.0±0.5 98±4 98±3 95±4 67±3 33±1 11±0.5
P. vulgaris 100±4 98±4 100±4 72±3 53±2 21.7±1 98±4 95±4 97±5 65±3 47±2 18±1
Results are expressed as mean±standard deviation. 352
The germination rate decreased as the leachate content increased, with the exception 353
of the 2.5% and 10% dilutions. At these two concentrations, the germination rates did not 354
show a statistically significant difference compared with the control tests (p > 0.05). From 355
30% to 90% dilutions, there was a statistically significant decrease in germination rate val- 356
ues in all cases (p < 0.05). At 90% dilution of raw leachates with unadjusted pH in the seeds 357
of P. vulgaris and C. sativus, germination rates of 0% were obtained, and with pH adjust- 358
ment to neutral, only the seeds of P. vulgaris had a germination rate of 0%. It is worth 359
noting that in the case of the treated leachates, there were no germination rates with a 360
value of zero, even at the highest leachate content of 90%. This suggests that the treatment 361
system proposed for leachates was successful in reducing the phytotoxicity. 362
pH adjustment was found to be an important variable for the germination rate. At a 363
dilution of 30%, the pH adjusted for neutrality generated higher germination rates when 364
compared with the corresponding values of pH without adjustment, obtaining statisti- 365
cally significant values (p<0.05). When comparing the germination rates of the various 366
liquids evaluated, it was discovered that there were statistically significant differences 367
(p<0.05) starting from the 30% dilution, and the order was as follows: raw leachate < elec- 368
tro–coagulation effluent < electro–oxidation effluent for all three seeds and at both pH 369
levels evaluated. This suggests that the decrease in organic matter achieved during the 370
treatment process, in conjunction with any structural changes that may have occurred, 371
played a role in the observed differences in germination rates. The correlation between the 372
enhanced biodegradability index of the treated effluents in the proposed system and the 373
reduction in phytotoxicity appears to be significant. 374
3.2.2. Effects of Radicle Length and Acquired Biomass on Growth Inhibition. 375
Figures 1 and 2 depict the outcomes obtained for the inhibition of growth based on 376
the length of the radicle and biomass acquired. In the two lowest dilutions (2.5% and 10%), 377
the growth inhibition values were negative in nearly all experiments, suggesting that plant 378
growth was beneficial to some extent in these two dilutions. When comparing the growth 379
inhibition between the dilutions of 2.5 and 10%, there were statistically significant 380
variations in the three seeds evaluated (p<0.05); the dilution of 10% demonstrated im- 381
proved growth. This was evident, as the most negative growth inhibition values were ob- 382
served at that particular dilution. 383
384
Figure 1. Growth inhibition through radicle length in toxicity tests. 385
386
Figure 2. Growth Inhibition through biomass gained in toxicity tests. 387
When comparing the outcomes of growth inhibition across the three types of leacha- 388
tes, statistically significant differences were observed (p<0.05). The order of these differ- 389
ences in values was inverse to that of the germination rate: crude leachate > electro–coag- 390
ulation effluent > electro–oxidation effluent. Furthermore, the growth inhibition value was 391
negatively correlated with germination, the higher the germination rate, the lower the 392
growth inhibition. These findings demonstrate that the treatment system successfully re- 393
duced the phytotoxicity of leachate effluents. 394
The impact of pH was investigated, and it was discovered that there were statistically 395
significant differences in the growth inhibition values among the three seeds and the three 396
leachates assessed (p<0.05). The unadjusted pH value yielded the highest growth inhibi- 397
tion values, indicating that neutral pH promotes root growth. It is worth mentioning that 398
citric acid was used to adjust pH, which has been demonstrated to have positive effects 399
on plant growth. Yang and Zhang (1998) documented an increase in seedling height, fresh 400
mass, and dry root mass of P. vulgaris in response to citric acid applications. 401
3.2.3. Results of Germination Index 402

The information presented in Figure 3 depicts the germination index values obtained 403
from a series of experiments. The germination rate serves as a measure of the relative ger- 404
mination of seeds, which is determined by the growth of the radicle. This rate indicates 405
the effects of numerous factors on the promotion or inhibition of germination. Notably, 406
the lowest dilutions in all cases, specifically 2.5% and 10%, were statistically different 407
(p<0.05) from the remaining concentrated dilutions. This observation can be attributed to 408
the positive influence of these dilutions on the radicle growth, as previously discussed. 409
410
Figure 3. Germination index in toxicity tests. 411
The findings revealed that an increase in leachate content negatively affected the ger- 412
mination rate across all instances, with statistical significance (p<0.05). Conversely, the 413
treatment system had a positive influence on the germination rate of all three seeds eval- 414
uated, with statistical significance (p<0.05). Notably, the electro–oxidation effluent exhib- 415
ited the highest germination index value, which was statistically significant (p<0.05). 416
The lowest values for the germination index were recorded for the three seeds in the 417
dilution with the highest concentration of raw leachates (90%) at both pH levels of neutral 418
and pH 8.4. This suggested the presence of phytotoxicity in the leachates, as previously 419
noted by several researchers (Bicelli et al., 2023; Storck et al., 2023; Yang et al., 2023). 420
One aspect that must be considered when considering the low germination index 421
values observed in raw leachates is the presence of NH 3–N. According to Fuentes (2004), 422
elevated concentrations of ammonia nitrogen can have detrimental effects on seed germi- 423
nation in various plant species. At the highest concentration of raw leachates, a zero-ger- 424
mination rate may be observed because non–biodegradable organic matter interferes with 425
enzyme activities, which in turn would significantly impact plant growth, as noted by 426
Arunbabu et al.(2017). 427
For the germination index, no values were recorded, even when the leachate concen- 428
tration reached 90%. As the concentration of leachate increased, a decrease in the germi- 429
nation rate was observed at a dilution of 30%, indicating an increase in phytotoxicity. The 430
pH values of the samples were also analyzed in relation to germination rates, and it was 431
observed that the pH adjusted to neutrality was higher than that without adjustment in 432
all cases (p<0.05). The electro–oxidation effluent had the highest germination rates at con- 433
centrations of 2.5% and 10% of the treated leachates, which were higher than 100%. Ob- 434
taining germination rates greater than 100% in phytotoxicity tests with treated leachates 435
has been reported in previous research using Fenton treatment in mature leachates: Pob- 436
lete et al. (2019) using L. sativa, and Li et al. (2017) with Z mays. 437
3.2.4. Half Maximal Effective Concentration 438

The half-maximal effective concentration is a statistical calculation that represents the 439
expected concentration of a substance in a medium that produces a specific effect on 50% 440
of a given population of organisms under defined conditions (Jiang et al., 2023). The av- 441
erage effective concentration values obtained from the germination rates in the tests are 442
presented in Table 3. 443
Table 3. Results of median effective concentrations in evaluated seeds. 444
Half maximal effective concentration (%)

Raw leachate
Seed
pH 7 pH 8.4
L. sativa 2.1±0.1 1.7±0.1
C. sativus 2.3±0.1 2.0.±0.1
P. vulgaris 2.9±0.1 1.6±0.1
Electro–coagulation effluent
Seed
pH 7 pH 9.5
L. sativa 18.9±1 16..6±1
C. sativa 20.5±1 18.1±2
P. vulgaris 22.8±2 21..1±1
Electro–oxidation effluent
Seed
pH 7 pH 5.6
L. Sativa 49.8±2 47.0±2
C. sativus 55.7±2 50.3±2
P. vulgaris 62.0±3 56.2±2
Results are expressed as mean±standard deviation. 445
Upon comparing the mean effective concentration values obtained for various 446
leachates evaluated under identical conditions, it was evident that statistically significant 447
differences existed (p<0.05) in the following descending order: raw leachate < electrocoag- 448
ulation effluent < electro–oxidation effluent. 449
An increase in the mean effective concentration is typically accompanied by a de- 450
crease in toxicity, as demonstrated by Ward et al.(2005). Notably, studies on leachate treat- 451
ment systems utilizing the Fenton process have revealed an increase in mean effective 452
concentration values (Bolobajev et al., 2014; Poblete et al., 2019). These findings suggest 453
that the proposed treatment system effectively reduces the toxicity of leachates. 454
The lowest mean effective concentration was noted in the raw leachate with no ad- 455
justment of pH in L. sativa at 2.1 ± 0.1%. Conversely, the highest mean effective concentra- 456
tion value was observed in the electro–oxidation effluent with a neutral pH in P. vulgaris 457
at 62.0 ± 2.8%. 458
The enhancement of the mean effective concentration serves as evidence that the 459
treatment system proposed in this project effectively diminishes the toxicity of the gener- 460
ated effluents. This outcome can be attributed to a substantial reduction of 83% in organic 461
matter, as measured by COD, through the treatment system. Moreover, the biodegrada- 462
bility index demonstrated a significant improvement, increasing from 0.094 in the crude 463
leachate to 0.46 in the electro–oxidation effluent. This increase in biodegradable content, 464
caused by the oxidation of non–biodegradable material in the electro–oxidation process, 465
positively affects the average effective concentration. 466
Upon evaluating the average effective concentrations per seed under identical condi- 467
tions, it was determined that P. vulgaris exhibited the highest values, followed by C. sativa 468
and L. sativa, the latter of which had the lowest values (p<0.05). In the electro–oxidation 469
effluent, significant differences were observed in the values obtained per seed, with the 470
highest values recorded as 49.82%, 55.72%, and 62.03% for L. sativa, C. sativus, and P. 471
vulgaris, respectively (p<0.05). Additionally, P. vulgaris seeds were the least affected by 472
leachates at the mean effective concentration (p<0.05). 473
pH was found to be a crucial variable in all experiments. Under the same seed and 474
dilution conditions, the mean effective concentration was consistently higher at neutral 475
pH than at an unadjusted pH (p<0.05). The raw leachate obtained using P. vulgaris had a 476
neutral pH of 2.9±0.1%, whereas the unadjusted pH resulted in a concentration of 477
1.6±0.1%. When applying the electro–oxidation effluent with P. vulgaris, the values ob- 478
tained at neutral and unadjusted pH levels were 62.0±3 and 56.2±2, respectively. A neutral 479
pH promotes germination and growth upon dilution (Kaur et al., 2017) Additionally, the 480
phytotoxicity results of this study should consider the high salinity levels present in the 481
leachates from the Bordo Poniente landfill. Kalčíková et al. (2012) identified high salinity 482
as a contributing factor to leachate toxicity. 483
3.3. Analysis Using Inductive Coupling Plasma with Optical Emission Spectrophotometer (ICP– 484
OES) 485
Metals and metalloids in the soil are prevalent in both urban and rural settings and 486
pose a persistent threat to the environment. Unlike organic molecules, these substances 487
do not break down and, therefore, require careful attention. Given that many sanitary 488
landfills are situated near crop areas, it is imperative to conduct phytotoxicity tests on the 489
raw and treated leachates. In the RSBP, Stage IV employs a 1 mm HDPE geomembrane as 490
a waterproofing solution (Alcantar, 2015; Parra and Ovando, 2011). Table 4 presents the 491
findings of the ICP–OES analysis of the garden land employed in this study, along with a 492
parallel examination of the recommended values put forth by the New York State Depart- 493
ment of Environmental Conservation (NYSDEC, 2006). 494
Table 4. Results of ICP–OES analysis of garden soil used in phytotoxicity tests and comparison with 495
recommended values. 496
Concentration Maximum recommended value (mg Concentration Maximum recommended value (mg
Parameter Parameter
(mg kg–1) kg–1) * (mg kg–1) kg–1) *
Ag <0.6 K 35600±10
Al 3201±110 Mg 10615±550
As <6 16 Mn 486±10
Ba 98±4 350 Ni 140
Be <0.6 Pb 7±0.20 400
Ca 2268±110 Se <6
Cd 2±0.05 2.5 Ti 1184±50
Co 13±0.06 Tl <6
Cu 23±1 270 V 50±2
Fe 24125±1120 Zn 97±4 2200

*Recommended maximum values for agricultural soil as recommended by NYSDEC (2006). 497
According to the suggested maximum limits for metal content in cultivation soil stip- 498
ulated by the New York State Department of Environmental Conservation (NYSDEC, 499
2006), the levels of metals present in the garden soil utilized in this research project were 500
below the aforementioned maximum limits. This observation validated the suitability of 501
the soil used in the phytotoxicity assays and confirmed that it did not have any adverse 502
impact on the test outcomes. The findings obtained through ICP–OES analysis of the roots 503
during phytotoxicity testing proved to be highly enlightening, shedding light on the in- 504
terplay between the elements absorbed by the roots and their potential effects on growth 505
inhibition and germination rates. As shown in Table 5, root characterization was facilitated 506
by ICP–OES. 507
Table 5. ICP–OES analysis of roots collected in phytotoxicity tests. 508
Parameter(mg kg–1)
Seed Dilution (%)
Al Ba Ca Cu Fe K Mg Mn Ni Zn
0 2599±130 43±2 3013±150 15±0.5 3222±160 30399±1500 3739±140 115±5 15±0.6 76±3
2.5 2788±150 45±2 3093±140 15±0.5 3391±140 31999±1400 3936±160 121±6 15±0.4 80±2
10 2987±180 47±2 3082±160 16±0.5 3570±120 33683±1600 4143±210 127±5 16±0.6 84±3
L. sativa
30 3025±150 51±1 3037±140 20±0.5 3609±120 35452±110 3681±140 104±4 17±0.5 90±3
60 3176±140 53±1 3189±120 21±0.5 3739±110 36724±120 3865±150 109±5 17±0.4 93±4
90 3192±150 53±1 3247±150 21±1 3753±160 36851±110 3848±160 111±5 19±5 94±4
0 2442±120 61±3 7067±380 18±0.6 1457±1.1 20805±80 4094±210 89±4 7±4 65±3
2.5 2919±80 62±3 7018±270 20±0.8 1591±0.6 23552±110 4287±180 90±4 7±3 66±3
10 3442±150 76±6 7108±480 20±0.2 2417±110 25532±120 4413±120 71±3 7±3 67±2
C– sativus
30 3631±140 93±4 7035±300 21±0.3 2626±90 30868±150 3595±140 77±4 10±4 69±2
60 3994±120 102±4 7113±320 21±0.4 2889±160 33955±140 3754±170 75±4 11±5 73±3
90 4393±200 113±5 7139±410 21±1 3178±180 37350±160 3950±200 105±5 12±5 80±3
0 654±40 44±2 2058±140 9±0.3 422±300 14557±700 1753±120 29±1 7±3 54±2
2.5 682±30 50±2 2117±160 9±0.8 557±40 15518±100 1753±180 31±1 7±3 63±2
10 736±30 56±2 2264±100 14±0.4 578±30 18838±110 1910±80 36±1 7±2 68.3±2
P. vulgaris
30 868±40 56±2 2225±110 14±0.6 670±20 18627±80 1447±110 46±2 9±2 69±3
60 955±40 58±2 2358±80 16±0.4 737±30 20489±1000 1692±120 50±2 9±4 75±3
90 1050±50 65±2 2413±120 17±0.6 811±40 22538±1000 1961±140 55±2 10±0.5 83±4
Values expressed as mean±standard deviation. 509
A direct relationship was observed between the evaluated metals (Al, Ba, Cu, Fe, Ni, 510
and Zn) and the leachate content for all metals. Specifically, the higher the concentration 511
of leachates, the higher the metal content in all cases, which was statistically significant 512
(p<0.05) for all dilutions. It was also observed that P. vulgaris has a lower retention capacity 513
for the evaluated metals, as it obtained the lowest values for aluminum, copper, iron, 514
nickel, and zinc. In contrast, C. sativus had the highest retention capacities for Al, Ba, Cu, 515
and Zn. 516
Therefore, the effects of Al on plant growth are crucial. Al is recognized as a limiting 517
factor for crop productivity (Yan et al. 2023). Al toxicity was first observed in the root sys- 518
tems. Roots are more susceptible to the toxicity of this element, resulting in poor growth 519
by inhibiting elongation and cell division (Pokharel et al., 2024). Chen et al. (2020) sug- 520
gested that L. sativa adapts to oxidative stress caused by aluminum through polyphenolic 521
biosynthesis, which could have been observed in the phytotoxicity tests of this project, 522
particularly in the case of electro–oxidation effluent, where the lowest values of growth 523
inhibition were obtained compared to crude leachate and electro–coagulation effluent. 524
The highest Al content in the roots was observed in C. sativus, at 4393±200 mg kg–1 with 525
90% raw leachate, and the lowest was 654±40 mg kg –1 with P. vulgaris in the control sam- 526
ple. Aluminum may have been a negative contributor to plant development during the 527
phytotoxicity tests. 528
Barium can be considered to have a slightly detrimental effect on plant growth as it 529
competes with calcium, which is necessary for plant growth. However, this effect would 530
only occur if the Ba levels in the soil exceeded the recommended maximum value, which 531
was not the case in this study. The highest level of Ba recorded in this study was in the 532
roots of C. sativus, which had a raw leachate content of 90% and a level of 113±8 mg kg –1. 533
This level did not exceed the recommended maximum value of 350 mg kg –1 (NYSDEC, 534
2006), and it can be concluded that Ba did not have a negative impact on the phytotoxicity 535
tests conducted. 536
Copper is a micronutrient that is essential for plants in small amounts. It plays a role 537
in the formation of lignin in the cell walls and enhances resistance to oxidative stress. 538
However, excessive Cu in the growing medium can have a negative impact on the devel- 539
opment of the root system by burning its tips, leading to excessive lateral growth, reduced 540
branching, and ultimately plant deterioration (Gemin et al., 2023). In the current study, 541
the highest Cu content was recorded at 21±3 mg kg–1 in C. sativus with 90% crude leachate, 542
which exceeded the recommended maximum of 10 mg kg–1 (Gemin et al., 2023).This find- 543
ing implies that Cu is another element impeding the growth of the analyzed seeds. It is 544
worth noting that the Cu content in the seeds, with the exception of the roots of C. sativus 545
with 2.5% raw leachate and the control sample at 9 mg kg–1, was above the recommended 546
maximum limit. 547
Iron (Fe) plays a fundamental role in plant growth and is involved in various bio- 548
chemical processes, including respiration, chlorophyll synthesis, pathogen defense, the 549
generation and elimination of reactive oxygen species, nitrogen assimilation, and photo- 550
synthesis. Both iron deficiency and excess result in harmful effects on plant development 551
such as chlorosis (Wang et al., 2023). In these tests, the maximum iron value was observed 552
in C. sativus at 90% of the raw leachate at 3753±160 mg kg–1, which was much higher than 553
the recommended maximum of 200 mg kg–1 (Wang et al., 2023). The iron content in all 554
evaluated roots was above the maximum recommended value. Although it is true that 555
phytotoxicity tests carried out in this project did not involve the evaluation of chronic 556
effects on plant growth, In the case of chlorosis, it can be considered that this excess iron 557
provided by the leachates negatively influenced the growth of the evaluated seeds. 558
Nickel, a micronutrient essential for plant growth, is a component of various com- 559
pounds that plays a crucial role in enzymatic catalysis. Despite being less toxic than other 560
heavy metals, such as copper, excessive nickel can negatively impact plant growth by im- 561
pairing enzyme function. The highest concentration of Ni was observed in L. sativa, at 19±5 562
mg kg–1, and the recommended maximum value for this element in the roots was 22 mg 563
kg–1 (Rodríguez–Jiménez et al., 2016). Interestingly, no negative effects on phytotoxicity 564
were observed when the Ni content was below the recommended maximum for all the 565
evaluated roots. 566
Zn is commonly present in an insoluble form in the soil and is an essential micronu- 567
trient for plants. It plays a critical role in the synthesis of carbohydrates during photosyn- 568
thesis and in the metabolism of hormones by regulating the levels of auxins (a plant hor- 569
mone that promotes plant growth and development). However, excessive Zn levels can 570
have a detrimental effect on plant growth (Gupta et al., 2023). Suitable values for plant 571
development ranged from 15 to 20 mg kg–1 zinc. The highest concentration of zinc in the 572
analyses was presented in L. sativa at 94±4 mg kg–1 with 90% raw leachate, four times the 573
appropriate value. Therefore, Zn could be considered to have contributed negatively to 574

the phytotoxicity test results, as it was higher than those recommended for all the evalu- 575
ated roots. 576
No direct correlation was observed between leachate dilution and root content for 577
Ca, K, Mg, and Mn. For instance, the roots of C. sativus in the control sample had a calcium 578
concentration of 7067±380 mg kg–1, whereas in the sample with 90% crude leachate, the 579
concentration was 7139±410 mg kg–1, with both values not statistically significant (p > 580
0.05). Calcium is a structural element in plants that includes the cell wall and membrane, 581
and plays a fundamental role in cell division and elongation (Sharma and Singh, 2023). 582
Ca deficiency symptoms are commonly observed in growing organs, including apical me- 583
ristems, which promote growth when plants germinate (Sheeba et al., 2023). Given the 584
importance of calcium in plant growth, it was concluded that calcium influenced the phy- 585
totoxicity of the evaluated plants. 586
Potassium (K), a crucial plant nutrient, plays an indispensable role in plant health 587
and development. As a significant macronutrient, it comprises the majority of inorganic 588
cations in plants and accounts for 10% of plant dry weight. This essential nutrient is pri- 589
marily sourced from the soil (He et al. 2023). According to the ICP–OES analysis of the 590
roots, potassium levels exceeded the rest, with L. sativa at 90% leachate registering 36±1 g 591
kg–1 and P. vulgaris control sample showing 14±0.7 g kg–1. Upon examining the potassium 592
content of the seeds, it was observed that P. vulgaris roots had the lowest potassium con- 593
tent (p<0.05). Given that plant potassium sufficiency levels range from 10 to 50 g of potas- 594
sium per kg of dry matter (Sardans and Peñuelas, 2021), it can be inferred that potassium 595
does not adversely affect leachate phytotoxicity. 596
Magnesium (Mg) plays a critical role in plant metabolism and its mobility within 597
plants is highly beneficial for growth. As a fundamental component of chlorophyll, Mg 598
enables plants to effectively perform photosynthesis, making it essential for crop health 599
and productivity (Tang et al., 2023). Analysis of the Mg values of the evaluated seeds re- 600
vealed that the roots of P. vulgaris had the lowest values (p<0.05). According to the litera- 601
ture, Mg sufficiency values in plants range from 2 to 10 g per kg of dry mass (Ishfaq et al., 602
2022; UME, 2023). As the Mg values were below the maximum range, it can be inferred 603
that Mg did not adversely affect the phytotoxicity of the leachates. 604
Manganese is an essential micronutrient crucial for the proper functioning of various 605
plant processes, including root cell elongation. Plants can actively absorb Mn in the form 606
of Mn 2+, but excessive levels of Mn can have detrimental effects on plant growth and de- 607
velopment by replacing Mg in enzymatic reactions (Nong et al., 2023). When comparing 608
the Mg values by seed, it was observed that the root of P. vulgaris had the lowest value 609
(p<0.05). According to the literature, Mg sufficiency values in plants range from 50 to 150 610
mg per kg of dry mass (Integri, 2023; Li et al., 2019). All Mn values in the roots were below 611
the maximum value, indicating that Mn did not influence leachate toxicity. 612
4. Conclusions 613
• The characterization of the leachates employed in this research project indicates that 614
they possess a biodegradability index of 0.094, a chemical oxygen demand of 3.4±0.1 615
g L–1, a dissolved organic carbon of 1.2±0.05 gL–1, a color of 3200±100 Pt–Co U, and an 616
ammonia nitrogen content of 0.66±0.03 gL–1. Consequently, it can be asserted that the 617
leachates in question are mature. 618
• The parameters for the enhanced elimination of organic matter, as measured by COD, 619
were established for both electro–coagulation and electro–oxidation processes. For 620
electro–coagulation, the optimal current density was found to be 23.3 mA cm –2, with 621
a stirring rate of 120 revolutions per minute and a pH of 7. For electro–oxidation, the 622
conditions were determined to be a NaCl concentration of 1.0 g L -1, an electrode dis- 623
tance of 0.75 cm, a current density of 33.3 mA cm–2, and a pH of 7. 624
• Under conditions of greater removal of organic matter, measured as COD, removal 625
values were reached in the chemical demand of oxygen, dissolved organic carbon, 626
color, and ammonia nitrogen in the electro–coagulation process of 63%, 69%, 94%, 627
and 50%, respectively. For the electro–oxidation process, these values were 82, 86, 99, 628
and 81%, respectively. 629
• The proposed treatment system resulted in a significant enhancement of biodegrada- 630
ble organic matter. The concentration of biodegradable COD increased from 26% in 631
the raw leachate to 39% following the electro–coagulation process and further in- 632
creased to 58% in the effluent of the electro–oxidation process. Additionally, the bio- 633
degradability index, which was initially 0.094 in the crude leachate, improved to 0.26 634
with the electro–coagulation process and attained a value of 0.46, following the elec- 635
tro–oxidation process. 636
• The concentration of particulate COD in the electro–coagulation effluent decreased 637
from 48% to 23%. The electro–coagulation process effectively removed colloidal spe- 638
cies that could have impeded the subsequent electro–oxidation process, demonstrat- 639
ing its suitability as an initial treatment stage. 640
• The conversion of a portion of the recalcitrant organic matter present in raw leachates 641
into biodegradable materials and CO2 was achieved through both electro–coagula- 642
tion and electro–oxidation processes. These processes resulted in a significant altera- 643
tion in the chemical structure of the recalcitrant organic matter. The biodegradable 644
material content was enhanced owing to the breakdown of the aromatic structures 645
present in the humic substances included in the raw leachate. 646
• By analyzing the organic matter content in an electron–coagulation and electro–oxi- 647
dation system used to treat mature leachates, the structural changes that enhance the 648
biodegradability of the resulting wastewater were uncovered. 649
• Based on the data collected in this study, it can be concluded that the parameters that 650
significantly contributed to the toxicity in the leachates examined were aluminum, 651
copper, iron, zinc, and calcium. 652
• The findings of the phytotoxicity assessments indicated that the proposed treatment 653
approach led to a diminution of the phytotoxicity of the effluents produced. This out- 654
come can be ascribed to alterations in the molecular composition of the organic mat- 655
ter, mainly a decline in the degree of aromaticity. 656
Recommendations 657
• In this investigation, it was found that garden soil was a consistent factor in all of the 658
experiments conducted. Therefore, future phytotoxicity trials should assess the ef- 659
fects of different soil types, such as sandy and clayey soil, to better understand the 660
relationship between plants and the chemical composition of leachates in the soil. 661
• A valuable area of inquiry is to evaluate the influence of emerging pollutants on phy- 662
totoxicity tests. Although these pollutants have received considerable attention in re- 663
cent times, the cessation of operations at the Bordo landfill in 2012 restricts the ap- 664
plicability of this variable. Therefore, it is proposed that phytotoxicity tests incorpo- 665
rate leachates from landfills with nearer closure dates, or even those that continue to 666
function. 667
Author Contributions statement: Alfredo Martínez-Cruz: Investigation; conceptualization; meth- 668

odology; performed the experiments; analyzed and interpreted the data; writing—original draft 669
preparation. María Neftalí Rojas Valencia: Validation; formal analysis; resources; contributed rea- 670
gents; conceived and designed the experiments; analysis tools or data, writing—review and editing; 671
supervision; project administration; funding acquisition. 672
Declaration of interests statement: The authors declare no conflict of interest. 673
674
675
676
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Article 1

Assessment of Phytotoxicity in Untreated and Electrochemi- 2

cally Treated Leachates Through the Analysis of Early Seed 3

Growth and Inductively Coupled Plasma–Optical Emission 4

Alfredo Martínez-Cruz and *María Neftalí Rojas-Valencia 6

Abstract: The treatment of stabilized leachates, characterized by a biodegradability index (IB) of 10

Keywords: Electro-coagulation; electro-oxidation; mature leachates; organic matter; phytotoxicity 24

Assessment of Phytotoxicity in Untreated and Electrochemically Treated Leachates Through the 26

Horticulturae 2023, 9, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/horticulturae

2. Materials and Methods 122

2.2. Leachate Treatment System 138

2.3. Evaluation of Phytotoxicity 160

Number of seeds germinates

(Raditle lenght control − radicle lenght dilution)

(average weight in control − average weight in treatment)

radicle lenght dilucion germinated seed dilution

2.3.1. Experiment Design 205

coagulation electro–oxidation) underwent ICP–OES analysis. Additionally, the analysis 226

2.4.1. Chemical Reagents and Materials and Preparation of Samples 229

2.4.2. ICP–OES Equipment 243

3. Results and Discussion 256

Table 1. Characterization of raw leachates and effluents generated. 276

3.2. Evaluation of Phytotoxicity 339

Table 2. Germination rate in phytotoxicity tests. 351

Germination rate (%)

Raw leachate (dilutions in percent)

Seed Neutral pH Unadjusted pH (8.4)

Control 2.5 10 30 60% 90% Control 2.5% 10 30 60 90

Electro–coagulation effluent (dilutions in percent)

Seed Neutral pH Unadjusted pH (9.5)

Electro–oxidation effluent (dilutions in percent)

Seed Neutral pH Unadjusted pH (5.6)

Control 2.5 10 30 60. 90. Control 2.5 10 30 60 90

Results are expressed as mean±standard deviation. 352

3.2.3. Results of Germination Index 402

3.2.4. Half Maximal Effective Concentration 438

Table 3. Results of median effective concentrations in evaluated seeds. 444

Half maximal effective concentration (%)

Ba 98±4 350 Ni 140

Be <0.6 Pb 7±0.20 400

Cd 2±0.05 2.5 Ti 1184±50

Cu 23±1 270 V 50±2

Fe 24125±1120 Zn 97±4 2200

Table 5. ICP–OES analysis of roots collected in phytotoxicity tests. 508

Values expressed as mean±standard deviation. 509

appropriate value. Therefore, Zn could be considered to have contributed negatively to 574

Author Contributions statement: Alfredo Martínez-Cruz: Investigation; conceptualization; meth- 668

Declaration of interests statement: The authors declare no conflict of interest. 673

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