Article 1

Application of humic and fulvic acids as an alternative method 2

of cleaning water from plant protection products residues 3

Eirini A. Makrigianni 1, Eirini S. Papadaki 2, Theodoros Chatzimitakos 2, Vassilis Athanasiadis 2, Eleni Bozinou 2, 4
and Stavros I. Lalas 2,* 5

1 Department of Chemical Engineering, University of Western Macedonia, GR-50100, Kozani, Greece; 6

eirhnhmakrygiannh@gmail.com 7
2 Department of Food Science and Nutrition, University of Thessaly, GR-43100 Karditsa, Greece; 8
eirinipap@uth.gr (E.S.P.); tchatzimitakos@uth.gr (T.C.); vaathanasiadis@uth.gr (V.A.); empozinou@uth.gr 9
(E.B.) 10
* Correspondence: slalas@uth.gr; Tel.: +30-24410-64783 11

Abstract: Humic acids (HA) and Fulvic acids (FA) are naturally occurring compounds that influence 12
the fate and transportation of various compounds in the soil. Although HA and FA have multiple 13
uses, the reports about their sorbent potential for environmental pollutants are scanty and sparse. 14
In this study HA and FA, isolated from lignite samples from two mines in Greece, were studied as 15
sorbent materials of three active compounds of plant protection products, namely glyphosate (herb- 16
icide), cypermethrin (pyrethroid insecticide), and azoxystrobin (fungicide). According to the results, 17
both HA and FA are promising sorbent materials of these active compounds, with HA achieving 18
better sorption for cypermethrin and azoxystrobin, while FA was found to be more efficient for 19
glyphosate. Moreover, their performance was not compromised by other components, commonly 20
found in commercially available herbicides/insecticides/fungicides. Also, no significant leaching of 21
the sorbed compounds was recorded. Finally, the two materials achieved similar extraction effi- 22
ciency of the compounds from lake water. 23

Keywords: Humic acids; fulvic acids; lignite; binding ability; sorption; herbicides; pesticides; insec- 24
ticides; fungicides 25
26
Citation: Lastname, F.; Lastname, F.;
Lastname, F. Title. Separations 2022,
9, x. https://doi.org/10.3390/xxxxx
1. Introduction 27
Academic Editor: Firstname Last- Water is vital for the sustenance of life. It has multiple uses, including drinking and 28
name
cooking, farming, industry, agriculture, etc. Among the various uses, around 70% of wa- 29
Received: date ter is used for irrigation purposes [1]. In order to sustain the increasing population of earth 30
Accepted: date and cover the need for food, various compounds, such as fertilizers, herbicides, fungi- 31
Published: date cides, and insecticides are extensively being used in agriculture to either increase the yield 32

Publisher’s Note: MDPI stays neu-

of the crops, or to fight weeds, diseases, and pests, and as such, protect plants. Around 33

tral with regard to jurisdictional

200,000 metric tons of herbicides are being used annually, to assist the production of 34

claims in published maps and institu- crops, followed by the ~100,000 tons of insecticides and ~20,000 tons of fungicides [2]. 35
tional affiliations. Despite their benefits, the abovementioned compounds are like a double-edged sword. 36
An amount of these compounds can be washed off during irrigation or rain and be trans- 37
ferred into the underground water bodies, where they can have detrimental conse- 38

Copyright: © 2022 by the authors.

quences. Studies have found that exposure to such compounds is linked with high toxicity 39

Submitted for possible open access

publication under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
ativecommons.org/licenses/by/4.0/).
and mortality rates, obesity, endocrine disruption, reproductive disorders, and various 40
types of cancer [3,4]. Owing to this, leaching of the compounds in the environment is a 41
renowned problem, discussed by many scientists for decades [5,6]. 42
To address the issue, much effort has been placed to remove the compounds from 43
the environment, including physical methods (e.g., membrane technology and sorption), 44

Separations 2022, 9, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/separations

Separations 2022, 9, x FOR PEER REVIEW 2 of 9

chemical methods (e.g., electrochemical remediation and advanced oxidation technolo- 45

gies), and biological methods [7]. Between the physical methods, sorption is much simpler 46
and does not have the limitations accompanied by the use of membranes (e.g., fouling, 47
size of membranes, etc.). To date, many sorbent materials have been developed, to be used 48
in remediation processes. Activated carbon, is one such material, with very good sorption 49
properties. However, its high manufacturing costs are making it difficult for commercial 50
production [7,8]. Nanomaterials are also widely exploited, including carbon-based nano- 51
materials, metallic nanomaterials, etc. [9–12]. However, issues also arise, concerning their 52
toxicity [13]. As a result, natural materials, such as lignin, hemicellulose and cellulose, 53
clays, and zeolites are also exploited [7]. One parameter that affects the efficiency of the 54
sorbents, is the presence of organic matter in the samples. Organic matter consists mainly 55
of HA and FA. These compounds can interfere with the sorption of herbicides/insecti- 56
cides/fungicides (H/I/F) and promote their desorption from the sorbent material [14,15]. 57
Due to their ubiquitous presence in the environment, HA and FA inevitably interact 58
with H/I/F, affecting the efficiency of the latter and their transportation in the environment 59
[16,17]. Owing to their multiple surface groups and the interspaces between them, HA 60
and FA have great potential to be used in sorption processes. For instance, in a previous 61
study, it was showcased that HA and FA can bind benzo(a)pyrene and pentachlorophe- 62
nol, with HA being able to adsorb more hydrophobic compounds, compared to FA [18]. 63
Similarly, HA was also found to be able to adsorb tylosin and sulfamethazine [19]. Re- 64
cently, HA and FA were successfully used to reduce formulated herbicides (imazamox 65
and a mixture of thifensulfuron methyl-tribenuron methyl) contaminating drinking water 66
[20]. 67
In this study, we examined the potential of HA and FA for the removal of H/I/F from 68
water. With this aim, one representative compound of herbicide, one of insecticide and 69
fungicide classes, (namely glyphosate, cypermethrin, and azoxystrobin, respectively) 70
(Figure 1) were used as water contaminants. Their sorption by HA and FA was studied. 71
To resemble the real-world conditions, instead of using pure chemical compounds, com- 72
mercially available plant protection products were used. This decision was made in order 73
to obtain a better overview of the performance of HA and FA when other ingredients 74
(deriving from the commercial products) were present. 75

76
Figure 1. Chemical structures of azoxystrobin, cypermethrin, and glyphosate. 77

2. Materials and Methods 78

2.1. Chemicals and reagents 79

Sodium hydroxide and hydrochloric acid (37% w/w) were purchased from Panreac 80
(Barcelona, Spain). Acetonitrile and acetic acid were purchased from Merck (Darmstadt, 81
Separations 2022, 9, x FOR PEER REVIEW 3 of 9

Germany). Three plant protection products were chosen, namely, Fuego 36 SL (herbicide), 82
Fasthrin 10 EC (insecticide), and Epathlon 25 SC (fungicide), all products of Agrotechnica 83
general industrial partnership (Thessaloniki, Greece). Concerning the active compounds, 84
Fuego 36 SL contained glyphosate (36% w/v), Fasthrin 10 EC contained alpha-cyperme- 85
thrin (10% w/v), and Epathlon 25 SC contained azoxystrobin (25% w/v). Solutions were 86
prepared according to the label instructions. Lignite used in the current study was ob- 87
tained from two lignite mines in the Kozani area (Western Macedonia, Greece), South 88
Field (SF), and Mavropigi (MP). Lake water was collected from Plastiras lake near 89
Karditsa City, Greece. The water was filtered with a paper filter and used directly. 90

2.2. Instrumentation 91
Chromatographic separations were carried out at a Shimadzu CBM-20A liquid chro- 92
matograph (Shimadzu Europa GmbH, Duisburg, Germany), equipped with a SIL-20AC 93
autosampler and coupled to a diode array detector (Shimadzu SPD-M20A). A Phenom- 94
enex Luna C18 column (5 µ m, 4.6 × 250 mm) (Phenomenex Inc., Torrance, CA, USA) was 95
used in this study. Separations were carried out at a constant temperature (40 °C). The 96
mobile phase consisted of (A) water and (B) acetonitrile, both containing 0.5% v/v formic 97
acid. Gradient elution was employed by changing the concentration of B from 5% to 95% 98
in 40 min. Spectra were recorded between 220 and 360 nm. Identification of the com- 99
pounds (glyphosate, alpha-cypermethrin, and azoxystrobin) was carried out by compar- 100
ing the retention time and the UV spectra with that of standard compounds. 101

2.3. HA and FA isolation 102

Isolation of HA and FA was carried out as described in our previous study [20]. In a 103
beaker, lignite (150 g) was mixed with double distilled water (850 g) and sodium hydrox- 104
ide (0.5 M) was added so that the pH of the mixture was adjusted to 9. The mixture was 105
left to stand for 24 h and then it was centrifuged (4,000 rpm for 10 min). Next, the pH of 106
the solution was adjusted to 1, using concentrated hydrochloric acid and the mixture was 107
centrifuged again. The precipitate (HA) and supernatant (FA) were collected, freeze- 108
dried, and stored in amber glass vials. 109

2.4. Removal of organic compounds 110

In order to study the removal of H/I/F, aqueous solutions containing 50 ppm of each 111
of the active substance was prepared (the concentration of the active compounds was val- 112
idated with HPLC). For the removal of the organic compounds, HA or FA (600, 1000, or 113
2000 ppm) were added to 50 mL of deionized water containing one of the H/I/F. The mix- 114
tures were vortexed for 1 min and then placed on an orbital shaker (100 rpm) for 30 min. 115
Next, samples were centrifuged (10,000 rpm for 5 min), filtered with 0.45 μm syringe fil- 116
ters and injected into the HPLC system. All experiments were repeated five times and 117
extraction efficiency is expressed as the average of five replicate analyses. The amount of 118
each compound, sorbed by HA or FA was calculated from the difference between the area 119
of the peak in the initial solution and the area of the peak in the final solution. 120

2.5. Statistical analysis 121

The normality of the data was examined using the Shapiro-Wilk test. Since data were 122
not found to be normally distributed, statistically significant differences (for p < 0.05) were 123
examined with Kruskal-Wallis test. All statistical tests were examined using IBM SPSS 124
Statistics (ver. 26) (SPSS Inc., Chicago, IL, USA). 125

3. Results and discussion 126

To evaluate the sorption performance of HA and FA, extracted from lignite, originat- 127
ing from two different sites, three representative plant protection compounds were cho- 128
sen. These active compounds are present in many commercially available products 129
Separations 2022, 9, x FOR PEER REVIEW 4 of 9

(herbicides, insecticides, fungicides), and as such are commonly employed in agriculture. 130
Parameters that are often examined in sorption studies, such as sample pH and ionic 131
strength, were not examined, since it would be unrealistic for the scale up of the proce- 132
dure. In addition, commercially available products were used, instead of pure chemicals, 133
so as to have a matrix that consists of all the compounds present in the commercially 134
available products. In all cases, the equilibrium time was examined (data not shown) and 135
it was found that 30 min were adequate to achieve results with less than 5% variation. 136
Longer extraction times did not result in better extraction, while lower extraction times 137
were inadequate to achieve maximum efficiency. As such 30 min were used as the opti- 138
mum extraction time to compromise between extraction efficiency and short duration of 139
the process. 140

3.1. Glyphosate sorption 141

Undoubtedly, herbicides have promoted the development of agriculture. They can 142
interact in multiple ways with organisms, such as incorporation in the DNA, affecting cell 143
division, etc. [21]. Among others, glyphosate is one of the most efficient herbicides. Some 144
claim that exposure to glyphosate below regulatory permissible levels is safe, while others 145
oppose [22]. Although more studies are probably needed to conclude, chronic exposure 146
will certainly lead to unwanted health issues. Thus, its removal from the environment is 147
necessary. 148
The sorption efficiency of glyphosate from HA or FA is presented in Figure 2. As can 149
be seen, a dose-dependent increase in extraction efficiency was recorded for all examined 150
sorbents. Also, no differences were recorded between the two different HA and FA exam- 151
ined. Between HA and FA, the latter was more efficient, achieving almost 35% sorption 152
efficiency (using 2000 ppm), compared to about 15% of HA. This difference can be at- 153
tributed to the different chemical structures of HA and FA. For instance, in a previous 154
study, it was reported that the alkyl and hydroxyalkyl groups of FA have a looser confor- 155
mation compared to HA [23]. Also, it was found that a stronger interaction between FA 156
and glyphosate is developed, compared to HA-glyphosate, suggesting the formation of 157
more hydrogen bonds between the carboxyl and phosphonate groups of glyphosate and 158
the oxygen moieties of FA [23]. 159

160
Figure 2. % Extraction efficiency of glyphosate using humic acid (HA) or fulvic acid (FA) extracted 161
from different concentrations of lignite from South Field (SF) or Mavropigi (MP) mines; Bars repre- 162
sent the average extraction efficiency of five replicate analyses; Statistically significant differences 163
(p < 0.05) are depicted with small and capital letters for the same concentration is sorbent used. 164

3.2. Cypermethrin sorption 165

Separations 2022, 9, x FOR PEER REVIEW 5 of 9

Cypermethrin is one of the most hazardous synthetic pyrethroids. It has catastrophic 166
effects on aquatic organisms and due to the mechanisms via which toxicity is induced, no 167
known antidote exists up to date [24]. Although cypermethrin levels in soil and surface 168
waters are relatively low, the fact that it is not easily transported to deeper levels may 169
increase in time its concentration in surface waters, resulting in increased toxicity [25]. 170
As can be seen in Figure 3, both HA and FA can achieve high sorption of cyperme- 171
thrin. More specifically, at the highest tested concentration, HA can achieve 100% sorp- 172
tion, while FA can achieve nearly 75%. Sorption of cypermethrin is achieved by three po- 173
tential interactions: (1) π-π interactions, (2) hydrophobic interactions, and (3) hydrogen 174
bonds. Similarly, to the above case, since the compound contains aromatic rings, the for- 175
mation of π-π interactions is probable, with HA containing more aromatic rings, com- 176
pared to FA, and as such, they have greater potential for π-π interactions. This is also the 177
case with hydrogen bonds. However, due to the increased content in aromatic rings, HA 178
is more hydrophobic compared to FA. Therefore, due to the hydrophobic nature of cyper- 179
methrin, hydrophobic interactions can also take place. This is also reported in a previous 180
study, where authors examined HA and FA coatings for mineral particles, so as to adsorb 181
cypermethrin [26]. According to the authors, the more hydrophobic the coating, the better 182
the extraction efficiency of cypermethrin. This is in line with the findings of the present 183
work. 184

185
Figure 3. % Extraction efficiency of cypermethrin using humic acid (HA) or fulvic acid (FA) ex- 186
tracted from different concentrations of lignite from South Field (SF) or Mavropigi (MP) mines; Bars 187
represent the average extraction efficiency of five replicate analyses; Statistically significant differ- 188
ences (p < 0.05) are depicted with small and capital letters for the same concentration is sorbent used. 189

3.3. Azoxystrobin sorption 190

Fungicides play an important role in agriculture. Azoxystrobin, one of the most com- 191
monly used fungicides, has several functional groups, and it is proved to participate in 192
complex metabolic pathways resulting in the formation of many metabolites. 193
Azoxystrobin can be transferred to the water bodies via leaching processes. It is also 194
known that residues of azoxystrobin can remain in the water, soil, or air for a long time, 195
rendering it a persistent environmental pollutant [27]. 196
Results for the extraction efficiency of azoxystrobin by HA and FA are presented in 197
Figure 4. Νo differences were recorded between the different HA and FA species. 198
Separations 2022, 9, x FOR PEER REVIEW 6 of 9

However, the performance of the two materials (HA or FA) was different. HA achieves a 199
better sorption (up to 40% when 2000 ppm were used), whereas FA achieved nearly 10%. 200
This difference can be attributed to the interactions between azoxystrobin and the lignite 201
extracts. Azoxystrobin is a molecule that contains three aromatic rings. Thus, it is capable 202
to interact with other compounds via π-π interactions. Since HA has multiple aromatic 203
rings, such interactions are more easily developed, resulting in enhanced extraction effi- 204
ciency. Moreover, extraction of azoxystrobin should be further achieved by the formation 205
of hydrogen bonds, that further enhance the extraction performance of the sorbent mate- 206
rials. 207

208
Figure 4. % Extraction efficiency of azoxystrobin using humic acid (HA) or fulvic acid (FA) extracted 209
from different concentrations of lignite from South Field (SF) or Mavropigi (MP) mines; Bars repre- 210
sent the average extraction efficiency of five replicate analyses; Statistically significant differences 211
(p < 0.05) are depicted with small and capital letters for the same concentration is sorbent used. 212

3.4. Leaching study 213

The HA and FA performances as sorbents materials, were not only examined in terms 214
of sorption efficiency, but also for their ability to leach the sorbed amount of each exam- 215
ined compound [28]. To this end, after the end of the extraction process, 40 mL of the 216
tested solution were discarded and 40 mL of water were added. The concentration of the 217
examined compounds was measured every 1 h for 4 h. From all tested conditions (a total 218
of 36 conditions), a minor leaching (5‒10% of the sorbed amount) of azoxystrobin was 219
recorded in the case of FA (both SF and MP) after 1 h and reached up to 25% after 3 h. No 220
further leaching was observed after 4 h total. The results suggest that azoxystrobin sorbed 221
from FA can leach back to the new solution over time. However, no leaching was recorded 222
for HA for azoxystrobin, suggesting that the sorption is more efficient. Moreover, no 223
leaching was observed in the cases of glyphosate and cypermethrin, highlighting the po- 224
tential of the materials to be used as sorbents. 225

3.5. Extraction from lake water 226

Environmental samples are complex matrices, comprised of many different compo- 227
nents. As such, the extraction efficiency of compounds from real samples may be compro- 228
mised, owing to the components of the samples. In order to examine whether the two 229
materials can be used for real life samples, their efficiency to extract the compounds from 230
Separations 2022, 9, x FOR PEER REVIEW 7 of 9

spiked (50 ppm) lake water was examined. To this end, 2000 ppm of FA were used for the 231
extraction of glyphosate and 2000 ppm of HA for the extraction of cypermethrin and 232
azoxystrobin. As can be seen in Figure 5, a minor decrease (3‒5%) in the extraction effi- 233
ciency was recorded in each case, However, no statistically significant differences (p < 234
0.05) were recorded for the extraction efficiencies between deionized water and lake wa- 235
ter. 236
In real life environmental samples, the concentration of the compounds is expected 237
to be lower. Therefore, we also examined the extraction efficiency of the materials, by 238
spiking lake water with 1 ppm of each compound. In all cases, the compounds were totally 239
extracted from the two materials, bespeaking excellent efficiency. The above corroborate 240
that the two materials can be used for the extraction of the compounds from lake water 241
samples. 242

243
Figure 5. % Extraction efficiency of the glyphosate (using 2000 ppm FA), cypermethrin (using 2000 244
HA) and azoxystrobin (using 2000 ppm HA) from deionized and lake water; Bars represent the 245
average extraction efficiency of five replicate analyses. Statistically significant differences (p < 0.05) 246
are depicted with small letters. 247

4. Conclusions 248
In this study, the sorption efficiency of three representative compounds belonging to 249
the class of H/I/F was examined. According to our results, HA and FA are promising 250
sorbent materials for the examined compounds, with HA achieving better sorption for cy- 251
permethrin and azoxystrobin, while FA was found to be more efficient for glyphosate. 252
Moreover, no leaching of the sorbed compounds was recorded. Also, the extraction effi- 253
ciency of the two materials was not compromised when tested in lake water. Although 254
further studies are needed to evaluate the performance of the sorbent materials to a wider 255
variety of compounds, the fact that they were able to sorb the active compounds, despite 256
the presence of other compounds (deriving from the commercial products) is highly 257
promising for future studies. 258

Author Contributions: Conceptualization, E.S.P., T.C., V.A. and S.I.L.; methodology, T.C. and S.I.L.; 259
software, T.C.; vali-dation, E.A.M, T.C., V.A., E.B. and S.I.L.; formal analysis, E.A.M. and T.C.; in- 260
vestigation, E.A.M., E.S.P, T.C. and V.A.; resources, S.I.L.; data curation, E.A.M., E.S.P, V.A. and 261
E.B.; writing—original draft preparation, E.A.M; writing—review and editing, E.A.M., E.S.P., T.C., 262
V.A., E.B. and S.I.L.; visualization, T.C.; supervision, T.C., V.A. and S.I.L.; project administration, 263
S.I.L.; funding acquisition, S.I.L. All authors have read and agreed to the published version of the 264
manuscript. 265

Funding: This research received no external funding. 266

Separations 2022, 9, x FOR PEER REVIEW 8 of 9

Institutional Review Board Statement: Not applicable. 267

Informed Consent Statement: Not applicable. 268

Data Availability Statement: Not applicable. 269

Conflicts of Interest: The authors declare no conflict of interest. 270

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