Environmental Science and Pollution Research (2023) 30:38955–38969

https://doi.org/10.1007/s11356-022-24753-4

RESEARCH ARTICLE

Phytogenotoxicity of thymol and semisynthetic thymoxyacetic acid

in pre/post emergence of model plants and weeds
Carlos Eduardo de Oliveira Roberto1 · Patrícia Fontes Pinheiro2 · Thammyres de Assis Alves3 ·
Josimar Aleixo da Silva1 · Milene Miranda Praça‑Fontes3,4 · Taís Cristina Bastos Soares1,3,5

Received: 9 March 2022 / Accepted: 10 December 2022 / Published online: 1 January 2023
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Herbicides are commonly used to control weed. However, some plants are resistant to such products. To identify less harmful
herbicides, it is crucial to search for different mechanisms of action. Thymol is an easily acquired allelopathic compound,
capable of producing its respective semisynthetic derivative, thymoxyacetic acid. The aim of this study was to determine the
effects of thymol and thymoxyacetic acid molecules as bioherbicides in greenhouse at the concentration of 3 mmol ­L−1 in
pre- and postemergence applications in five species: Amaranthus viridis L., Cucumis sativus L., Lactuca sativa L., Eleusine
indica L., and Sorghum bicolor L. The initial seedling development and DNA changes were analyzed. These molecules were
contrasting with the solvent, in the negative control, and with the glyphosate, in the positive control, promoting phytogeno-
toxic activities. The toxic effect of thymoxyacetic acid was more effective in preemergence and thymol’s in postemergence.
We also observed a reduction in the germination speed index and root growth with a negative correlation to the increase
in potassium leaching. Damage to the root and shoot of the seedlings was verified at the DNA level, and the phytotoxicity
of the plants treated with the herbicide glyphosate was similar to the plants treated with the natural molecules tested. The
bioherbicidal effect of thymol and thymoxyacetic acid exacerbates the reduction of the environmental impact caused by the
disordered and increased use of residual pesticides.

Keywords Natural phenol · Semisynthetic phenoxyacetic acid · Phytotoxic · Genotoxic · Electrical conductivity · Potassium
leaching

Introduction
Responsible Editor: Gangrong Shi
Herbicides can be selected to manage weed species in order
* Milene Miranda Praça‑Fontes to provide an increase in productivity and cost reduction in
milenemiranda@yahoo.com.br cultivated areas (Yang and Zhu 2013). Nevertheless, they
may represent up to 45% of the production costs in several
1
Postgraduate Program in Agronomy at the Federal crops (ANVISA 2012; Moraes 2019). In Brazil, glyphosate
University of Espírito Santo. Alto Universitário, S/N. ZIP:
29.500‑000, Alegre, ES, Brazil is the most widely used active ingredient in herbicides for
2 different crops, followed by 2,4-D, which occupies the sec-
Department of Chemistry, Federal University of Viçosa,
Avenida Peter Henry Rolfs, S/N, Campus Universitário, ZIP: ond place in sales with 57 thousand tons (IBAMA 2020).
36570‑900, Viçosa, MG, Brazil Herbicides are applied in agricultural areas in the pre-
3
Postgraduate Program in Genetics and Breeding, Federal and postharvest period and stand out because they repre-
University of Espírito Santo. Alto Universitário, S/N. ZIP: sent 61.2% of the total use of pesticides in Brazil (Bom-
29.500‑000, Alegre, ES, Brazil bardi 2017). Most of these synthetic compounds present
4
Department of Biology, Federal University of Espírito high biological activity, are toxic products, and can cause
Santo. Alto Universitário, S/N. ZIP: 29.500‑000, Alegre, ES, cancer in animals and mutations in different species of liv-
Brazil ing beings. However, less than around 10% of the products
5
Department of Pharmacy and Nutrition, Federal University sprayed reach their target (Bohner et al. 2013). The exces-
of Espírito Santo. Alto Universitário, S/N. ZIP: 29.500‑000, sive application of pesticides triggers ecological imbalances,
Alegre, ES, Brazil

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38956 Environmental Science and Pollution Research (2023) 30:38955–38969

promoting agricultural pressure with the selection of resist- toxic potential for humans and animals. These compounds
ant weed biotypes, reducing the local biodiversity of flora must have specific sites of action (highly selective) and have
and fauna, and leading to the instability of ecosystems (Silva a short half-life in the environment, leaving less residue in the
et al. 2012; Belchior et al. 2017). soil, water, or plant (Kraehmer et al. 2014; Souto et al. 2021).
The use of the same herbicides has contributed to the There are natural compounds that have proven phytotoxic
selection of resistant biotypes of some spontaneous spe- activity and that can be used to obtain semisynthetics. For
cies, bearing enormous economic losses with their man- example, the phenolic monoterpene thymol which can found
agement. These resistant weeds arise from individuals that in the essential oils of plants such as thyme, rosemary, pep-
have genetic modifications within the same population, with per, peppermint, and oregano, among others, has shown the
the ability to survive and propagate after exposure to lethal activity in inhibiting the germination and root growth of
doses of the herbicides (Leal et al. 2012). plants (Pinheiro et al. 2015; Escobar et al. 2020).
The herbicides with the active ingredient glyphosate Due to the demand for the new herbicides, which are less
N-(phosphonomethyl) glycine (Fig. 1) are the most sold in polluting and toxic to living organisms and to humans, in
the world (Kudsk and Streibig 2003). This product is clas- this work, the compounds with herbicidal potential from
sified as a nonselective herbicide, applied in the postemer- natural phenols were synthesized. Taking into account the
gence period, and presents a systemic mechanism of action. structure of the commercial herbicide 2,4-Dichlorophenoxy-
In 2017, glyphosate was the best-selling active ingredient acetic acid (2,4-D), thymoxyacetic acid was synthesized
in herbicides in Brazil, with more than 173 thousand tons from thymol (natural phenol). The structures of the com-
sold (Bombardi 2017). Some invasive plant species have pounds are shown in Fig. 2.
presented resistance to glyphosate, mainly due to the use of Thymol molecules and their semisynthetic derivative,
transgenics in the crops, as Eleusine indica L. (capim pé-de- thymoxyacetic acid, were cytotoxic, genotoxic, and muta-
galinha), which in 2016 showed this behavior (Heap 2020). genic at different concentrations, inhibiting the germination
The search for alternative herbicides to control biotypes and the initial development of monocotyledonous (sorghum)
of weed resistant to commercial herbicides is of paramount and eudicotyledonous (lettuce) seedlings (Alves et al. 2018).
importance. One of the options to decrease the number Thus, this study was aimed at studying the effects of such
of resistant plants is the use of natural compounds in the molecules as the possible bioherbicides, at the concentration
development of new herbicides, the so-called bioherbicides of 3 mmol L ­ −1 with the applications in pre- and postemer-
(Dayan and Duke 2014). gence under greenhouse conditions, in five plant species,
The use of herbicides originated from natural compounds, Amaranthus viridis L. (weed), Cucumis sativus L. (cucum-
the semisynthetics, has been an alternative to maintain the ber), Lactuca sativa L. (eudicotyledonous model organism),
effectiveness in the use of herbicides with less polluting and Eleusine indica L. (weed), and Sorghum bicolor L. (mon-
ocotyledonous model organism). We evaluated the initial
seedling development and changes at DNA level.

Material and methods

Plant material

Five plant species were used for the tests, three eudicotyle-
donous (A. viridis L, C. sativus L., and L. sativa L.) and two
Fig. 1  Chemical structure of the active ingredient glyphosate
monocotyledons (E. indica L. and S. bicolor L.). The seeds
N-(phosphonomethyl) glycine
of L. sativa var. Monica from the Feltrin brand (lettuce),

Fig. 2  Chemical structure of

the natural phenol thymol,
its semisynthetic derivative
thymoxyacetic acid, and
2,4-dichlorophenoxyacetic acid
(2,4-D)

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C. sativus var. Caipira from TOP seed (cucumber), and S. shoot fresh mass (SFM), and shoot dry mass (SDM) of the
bicolor from BR seeds (sorghum) were purchased in spe- seedlings.
cialized stores. The weed seeds of A. viridis (caruru) and E. The GSI was determined according to Maguire (1962),
indica (capim pé-de-galinha) were collected at the Federal using the equation:
Institute of Espírito Santo (IFES), Campus, Alegre, Espírito
GSI = (G1∕N2) + (G2∕N2) + ⋯ + (Gn∕Nn)
Santo, Brazil, and identified at the botany laboratory of
CCAE-UFES, Alegre, Espírito Santo, Brazil, by the Doc- where G1, G2, Gn is equal to the number of germinated
tor in Botany Amélia Carlos Tuler, professor at the Center seeds and N1, N2, Nn corresponds to the number of days
for Biodiversity Studies-(CBIO) from the Federal Univer- after sowing.
sity of Roraima–(UFRR) Campus Paricarana, Boa Vista, On the twenty-first day after sowing, samples were
RR, Brazil, from the specialized bibliographies: Manual for taken from seedlings considered viable (formed roots and
the Identification and Management of Weeds in Sugarcane shoot) to evaluate the other variables. The root and shoot
Crops (Brighenti 2010) and Weed Identification and Control length (cm) was measured with the aid of a ruler. The
Manual (Lorenzi 2014). As they are common invasive spe- seedlings were separated into shoot and root, and then, the
cies, the samples of these species were not deposited in a fresh mass was weighed and stored in kraft paper bags and
collection in the herbarium. dried in an oven at 72 °C for 72 h for the quantification of
dry biomass, which was measured by an analytical balance
General experimental procedures (0.0001 g).

Six seeds of each species were sown into tubes of 115 c­ m3,
containing 150 g of autoclaved sand, with three replica- Electrical conductivity and potassium leaching
tions for each treatment, water, and solvent-acetone (2%, v
­v−1) + Tween (0.05%, v v­ −1), as negative controls (C −) and The methodology proposed by Prete (1992) was followed,
glyphosate (0.05%, v ­v−1) as positive control (C +). Thymol with adaptations, to determine the electrical conductivity
and thymoxyacetic acid were used at the concentration of (EC) and the potassium leaching (KL) in the seeds, before
3 mmol ­L−1. This concentration was chosen because it pre- and after treatment with thymol and thymoxyacetic acid.
sented the best results as described by Alves et al. (2018). Four samples of 50 seeds were separated, for each plant
Thymol (used in the tests), acetone, and diethyl ether species, and the masses were measured (0.001 g accuracy).
(used in the reactions) were purchased from Sigma Then, these samples were soaked into 75 mL of distilled
Aldrich®. The thymoxyacetic acid was obtained through water, for each treatment (inside of 180 mL plastic cups),
the same methodology proposed by Alves et al. (2018). and placed into a ventilated oven at 25 °C for 5 h. The solu-
For the preemergence test, sand soaked with 10 mL of tions, without the seeds, were poured into another recipient,
solution was used at the moment of sowing, according to and the conductivity reading was performed with a conduc-
Dias et al. (2009). In the present study, the protocol was tivity meter. The results were expressed in µS c­ m−1 ­g−1 of
adapted for the tubes in a greenhouse. For the postemergence sample. Immediately after reading the electrical conduc-
test, the leaves were sprayed on the 18th day after the seed- tivity, the potassium leached into the solution was read by
ling emergence, which were exposed for 4 consecutive days a flame photometer. The results of the amount of leached
to the effect of the molecules. 2.5 mL of the solution was potassium√were expressed in ppm, these data were trans-
sprayed each day, totaling 10 mL of solution. formed to x + 1.
The experiment was carried out for 21 days, with daily
measurements of the germination speed index (GSI) until
the germination was established. At the end of the experi- Genotoxicity assessment
ment, root length, shoot length, and fresh and dry mass were
measured. For the molecular analyzes, roots were separated The molecular analyses used to assess the genotoxicity of the
from the shoot of the seedlings, immediately stored into a tested compounds were performed using the ISSR markers.
polystyrene box with liquid nitrogen, and kept in a freezer The DNA extraction was performed using the Doyle
(− 30 °C) until the DNA extraction stage. and Doyle (1990) protocol with modifications proposed by
IAC (Agronomic Institute). The extraction buffer for each
Phytotoxicity assessment sample consisted of 0.28 mL of 5 mol ­L−1 NaCl; 0.1 mL
of Tris–HCl pH 8.0, 1 mol ­L−1; 0.03 mL EDTA pH 8.0,
The variables evaluated in the phytotoxicity test were as fol- 500 mmol ­L−1; 10 mg of PVP; 0.4 mL of 5% CTAB; and
lows: germination speed index (GSI), root length (RL), shoot 4 µL of β-mercaptoethanol and ultrapure water to reach the
length (SL), root fresh mass (RFM), root dry mass (RDM), volume of 1 mL. Then, they were stored at 4 °C, and their

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concentrations were quantified by the NanodropTM® device seeds of lettuce exposed to thymol and thymoxyacetic acid
(Thermo Fisher Scientific). at a concentration of 3 mmol ­L−1 showed limitations in GSI,
After the quantification and analysis of the DNA quality, RL and SL, when compared with positive control plants
the samples were diluted to a concentration of 25 ng µL−1. (glyphosate). For the other characteristics, such as RFM,
The DNA amplification reactions (polymerase chain reac- SFM, RDM, SDM, EC, and KL, the inhibitory effects were
tion (PCR)) were performed using the ISSR primers synthe- similar or superior to the commercial herbicide, glyphosate
sized at the Biotechnology Laboratory of British Columbia (Table 1).
University (Vancouver, British Columbia, Canada)—“UBC The thymoxyacetic acid provided a 70% decrease in GSI
primer set 100/9”—809, 810, 812, 827, 836, 842, 864, 868, in lettuce seeds, when compared to the positive control (C +),
and 884, previously selected to assess the damage induced glyphosate. This represents an 89% reduction in comparison
by treatments in the DNA of the roots and shoot of the spe- to the negative control (C −), distilled water, what makes the
cies. The PCRs were performed with a final volume of 20 thymoxyacetic acid more effective for the GSI inhibition.
µL, containing 2 µL of 10X buffer; 1 µL of 50 mM MgCl; 2 According to Valerio et al. (2007), this delay on germination
µL of 10 mM dNTP; 0.2 µL of Taq 5 U µL−1; 2 µL of 2 µM is an efficient and sensitive criterion in the phytotoxicity
primer; 1 µL of BSA; 3 µL of DNA; and 8.8 µL of ultrapure analysis, showing thymoxyacetic acid as a potential pre-
water. The amplification reactions of the ISSR primers were emergent herbicide. For the lettuce seedlings, thymol stood
performed in a thermocycler, following the amplification out for presenting a similar effect to glyphosate, elucidating
conditions: initial denaturation of 94 °C for 5 min; 35 cycles its toxic effects in the initial development by inhibiting RL
of 94 °C-45 s, 50 °C-45 s, and 72 °C-1 min, and 30 s; and and SL, suggesting greater efficacy as a post-emergent bio-
final extension 72 °C for 7 min. The PCR products were herbicide (Table 1). The results obtained in this research
analyzed on 2% agarose gels, stained with ethidium bromide are in agreement with those found by Alves et al. (2018),
and photographed under UV light, using a photodocumenter who evaluated the effects of thymol and thymoxyacetic acid
(Bio-Rad®, Molecular Imager® Gel Doc ™ XR System). on the germination and initial growth of L. sativa L. and
S. bicolor. The authors found that at the concentrations of
Statistical analysis 3 mmol ­L−1, 1.5 mmol L ­ −1, 0.75 mmol L
­ −1, and 0.375 mmol
−1
­L , these compounds were able to reduce the germination
The completely randomized experimental design was used, percentage and, the higher their concentration, the greater
with three replications with six seeds each, and plots com- the inhibitory effects on germination.
posed of eighteen seeds, in a simple factorial scheme 5 × 5, Besides the inhibitory effects, on the germinative and
arranged as Factor 1-Species and Factor 2-Treatments. The vegetative growth, the growth of lettuce seedlings is more
data obtained with the phytotoxicity analyses were subjected sensitive to the treatments with thymoxyacetic acid and
to analysis of variance. The means were submitted to regres- thymol, especially if related to the shoot growth, when
sion analysis and Dunnett’s test at a significance level of 5%. compared to C − (Table 1). This may affect the speed and
The effects caused by the exposure of the treatments were germination time, which can cause necrotic roots, seedling
analyzed by the presence and/or absence of bands on the death, or abnormalities, as observed in the post-emergence
gels, in comparison with the negative controls, following the study on lettuce seedlings (Fig. 3).
coding 1 for the presence of band and 0 for the absence of Although significant differences in the germination speed
band. The simple coincidence dissimilarity index between index, root growth, and shoot growth were noted, Ferreira
the different treatments was obtained with the molecular data and Aquila (2000) pointed out that the phytotoxic effect
encoded for the ISSR primers. Then, the UPGMA (Average generally occurs on the germination speed, promoting a
Link Between Group) cluster analysis was performed, and reduction on this variable, because germination is less
the cutoff points of the generated cladograms were estimated sensitive to allelochemical compounds than to the growth
from the Mojema coefficient. All analyses were performed of the seedlings (Ferreira and Borghetti 2004).
using the GENES statistical program (Cruz 2013). The phytotoxic effects of the thymoxyacetic acid were
observed on the seeds and seedlings of caruru, verified on
the reduction of GSI and RL, with inhibition similar or
Results and discussion superior to that of the active compound glyphosate, for all
the evaluated parameters. Thymol also significantly inhibited
Phytotoxicity the germination and initial growth of caruru seedlings, in
comparison to C + (Table 2). This can be attributed to the
According to the macroscopic assessments, significant phy- direct effect of the compound, or to the reduced growth of the
totoxicity of thymol and thymoxyacetic acid on the seeds roots (Oliveira et al. 2004). The reduction in the growth of
and seedlings was observed (Tables 1, 2, 3, 4, and 5). The the roots may be related to the inhibition of protein synthesis

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Table 1  Phytotoxicity of thymol and thymoxyacetic acid on the initial development of L. sativa seedlings on the pre and postemergence assess-
ments
(g ­seedlings−1) (cm ­seedling−1) (Sm−1 ­g−1) (mg ­dm3)

Treatment Preemergence

GSI RFM SFM RDM SDM RL SL EC KL

Thymoxy- 0.50 ± 0.00c 1.20 ± 0.58bc 1.48 ± 0.21bc 1.57 ± 0.00bc 1.60 ± 0.00bc 0.86 ± 0.00 0.70 ± 0.00bc 5.93 ± 0.63 38.97 ± 2.39
acetic
acid
Thymol 1.50 ± 1.14c 1.42 ± 1.15bc 1.55 ± 0.61bc 1.60 ± 0.00bc 1.61 ± 0.03bc 0.43 ± 0.00 0.26 ± 0.01b 4.66 ± 0.32ab 38.51 ± 2.70
Distilled 4.66 ± 0.29a 1.71 ± 0.00a 1.65 ± 0.72a 1.62 ± 0.00a 1.62 ± 0.03a 11.46 ± 0.00a 1.33 ± 0.00a 4.67 ± 0.47a 2.08 ± 0.07a
water
Solvent 3.11 ± 0.98b 1.33 ± 0.58b 1.52 ± 0.20b 1.59 ± 0.00b 1.61 ± 0.00a 9.96 ± 0.00b 0.50 ± 0.00b 4.85 ± 0.13b 27.65 ± 1.91b
Glyphosate 1.72 ± 0.63c 1.34 ± 0.00c 1.53 ± 0.10c 1.59 ± 0.00c 1.61 ± 0.00a 7.43 ± 0.00c 0.80 ± 0.00c 3.29 ± 0.76c 25.82 ± 0.25c
Treatment Postemergence
RFM SFM RDM SDM RL SL
Thymoxy- 1.07 ± 0.04b 1.15 ± 0.03b 1.02 ± 0.00abc 1.02 ± 0.01abc 12.20 ± 0.82abc 1.50 ± 0.20abc
acetic
acid
Thymol 1.06 ± 0.03bc 1.1 ± 0.01 1.01 ± 0.00ac 1.02 ± 0.00abc 1.03 ± 0.21 0.6 ± 0.20
Distilled 1.30 ± 0.01a 1.19 ± 0.03a 1.01 ± 0.01a 1.02 ± 0.01a 13.56 ± 1.36a 1.80 ± 0.20a
water
Solvent 1.05 ± 0.01b 1.15 ± 0.04b 1.02 ± 0.01b 1.02 ± 0.01b 12.43 ± 0.67b 1.63 ± 0.38b
Glyphosate 1.04 ± 0.01c 1.18 ± 0.02c 1.01 ± 0.01c 1.03 ± 0.01c 10.96 ± 1.38c 1.23 ± 0.25c

*
Means followed by letter a on the columns are statistically equal to distilled water, means followed by letter b are statistically equal to the sol-
vent, and means followed by letter c statistically differ from the negative control and from the solvent according to Dunnett's test (P > 0.05)
GSI germination speed index, RL root length, SL shoot length, RFM root fresh mass, RDM root dry mass, SFM shoot fresh mass of seedlings,
SDM shoot dry mass of seedlings, EC electrical conductivity, KL potassium leaching

Table 2  Phytotoxicity of thymol and thymoxyacetic acid on the initial seedling development of A. viridis L., on the pre- and postemergence assessments
(g ­seedlings−1) (cm ­seedling−1) (Sm−1 ­g−1) (mg ­dm3)

Treatment Preemergence

GSI RFM SFM RDM SDM RL SL EC KL

Thymoxy- 0.17 ± 0.10 1.41 ± 0.00ac 1.55 ± 0.01ac 1.60 ± 0.00ac 1.61 ± 0.00ac 0.46 ± 0.75bc 0.40 ± 0.52bc 7.34 ± 0.60abc 8.61 ± 0.22
acetic
acid
Thymol 0.63 ± 0.16abc 1.23 ± 0.00bc 1.49 ± 0.00bc 1.58 ± 0.00bc 1.60 ± 0.00c 0.63 ± 0.15bc 0.43 ± 0.15bc 6.64 ± 0.58abc 8.62 ± 0.60
Distilled 0.65 ± 0.13a 1.44 ± 0.00a 1.56 ± 0.00a 1.60 ± 0.00a 1.61 ± 0.00a 8.00 ± 1.65 a 1.00 ± 0.20a 6.47 ± 0.11a 2.18 ± 0.08a
water
Solvent 0.58 ± 0.06b 1.14 ± 0.00b 1.46 ± 0.00b 1.56 ± 0.00b 1.6 ± 0.00b 0.63 ± 0.21b 0.33 ± 0.15b 6.52 ± 0.50b 6.78 ± 0.38b
Glypho- 0.67 ± 0.29c 1.35 ± 0.00c 1.53 ± 0.00c 1.59 ± 0.00c 1.61 ± 0.00c 2.16 ± 0.72c 0.90 ± 0.10c 6.50 ± 0.19c 6.03 ± 0.27c
sate
Treatment Postemergence
RFM SFM RDM SDM RL SL
Thymoxy- 1.01 ± 0.00b 1.03 ± 0.00bc 1.10 ± 0.00abc 1.01 ± 0.00abc 3.86 ± 2.85abc 1.70 ± 0.38ab
acetic
acid
Thymol 1 .00 ± 0.00b 1.01 ± 0.00ab 1.01 ± 0.00abc 1.00 ± 0.00abc 2.50 ± 1.32abc 1.56 ± 0.72ab
Distilled 1.06 ± 0.00a 1.02 ± 0.00a 1.05 ± 0.00a 1.02 ± 0.00a 6.10 ± 0.85a 2.00 ± 0.26a
water
Solvent 1.02 ± 0.00b 1.02 ± 0.00b 1.01 ± 0.00b 1.01 ± 0.00b 4.70 ± 2.93b 1.33 ± 0.38b
Glypho- 1.06 ± 0.00c 1.01 ± 0.00c 1.02 ± 0.00c 1.00 ± 0.00c 1.17 ± 0.25c 0.50 ± 0.20c
sate

*
Means followed by letter a on the columns are statistically equal to distilled water, means followed by letter b are statistically equal to the sol-
vent, and means followed by letter c statistically differ from the negative control and from the solvent according to Dunnett’s test (P > 0.05)
GSI germination speed index, RL root length, SL shoot length, RFM root fresh mass, RDM root dry mass, SFM shoot fresh mass of seedlings,
SDM shoot dry mass of seedlings, EC electrical conductivity, KL potassium leaching

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Table 3  Phytotoxicity of thymol and thymoxyacetic acid on the initial development of C. sativus seedlings on the pre- and postemergence
assessments
(g ­seedlings−1) (cm ­seedling−1) (Sm−1 ­g−1) (mg ­dm3)

Treatment Preemergence

GSI RFM SFM RDM SDM RL SL EC KL

Thymoxy- 0.41 ± 0.14 1.79 ± 0.01 1.67 ± 0.44 1.63 ± 0.01 1.62 ± 0.07 2.91 ± 1.04 2.46 ± 0.47c 2.07 ± 0.08 3.29 ± 0.02
acetic
acid
Thymol 1.67 ± 0.48c 2.06 ± 0.04abc 1.75 ± 0.06abc 1.65 ± 0.00abc 1.62 ± 0.01abc 15.70 ± 2.38ab 3.16 ± 0.21ac 2.72 ± 0.08abc 3.11 ± 0.07
Distilled 3.72 ± 0.42a 2.29 ± 0.04a 1.51 ± 0.24a 1.67 ± 0.02a 1.70 ± 0.06a 14.23 ± 1.75a 3.93 ± 0.32a 2.70 ± 0.09a 1.47 ± 0.01a
water
Solvent 3.26 ± 0.33b 2.28 ± 0.52b 1.71 ± 0.66b 1.64 ± 0.07b 1.63 ± 0.08b 13.56 ± 0.65b 3.63 ± 0.31b 2.73 ± 0.02b 1.61 ± 0.06b
Glypho- 2.03 ± 0.22c 2.30 ± 0.04c 1.81 ± 0.03c 1.61 ± 0.01c 1.60 ± 0.01c 11.86 ± 0.57c 2.53 ± 0.15c 2.74 ± 0.06c 2.12 ± 0.02c
sate
Treatment Postemergence
RFM SFM RDM SDM RL SL
Thymoxy- 1.16 ± 0.04a 1.26 ± 0.01 1.06 ± 0.01 1.04 ± 0.01 11.93 ± 0.86abc 2.70 ± 0.92abc
acetic
acid
Thymol 1.11 ± 0.04 1.15 ± 0.02 1.05 ± 0.03b 1.03 ± 0.01 11.90 ± 1.71abc 2.33 ± 0.47abc
Distilled 1.12 ± 0.04a 1.23 ± 0.02a 1.10 ± 0.00a 1.05 ± 0.00a 11.94 ± 3.97a 2.10 ± 0.53a
water
Solvent 1.34 ± 0.02b 1.53 ± 0.05b 1.07 ± 0.01b 1.09 ± 0.01b 14.26 ± 1.94b 2.93 ± 0.25b
Glypho- 1.28 ± 0.05c 1.32 ± 0.03c 1.11 ± 0.04c 1.06 ± 0.01c 14.00 ± 0.20c 1.93 ± 0.90c
sate

*
Means followed by letter a on the columns are statistically equal to distilled water, means followed by letter b are statistically equal to the sol-
vent, and means followed by letter c statistically differ from the negative control and from the solvent according to Dunnett's test (P > 0.05)
GSI germination speed index, RL root length, SL shoot length, RFM root fresh mass, RDM root dry mass, SFM shoot fresh mass of seedlings,
SDM shoot dry mass of seedlings, EC electrical conductivity, KL potassium leaching

Table 4  Phytotoxicity of the phenol thymol and its acid derivative thymoxyacetic on the initial seedling development of E. indica, in the pre-
and postemergence assessments
(g ­seedlings−1) (cm ­seedling−1) (Sm−1 ­g−1) (mg ­dm3)

Treatment Preemergence

GSI RFM SFM RDM SDM RL SL EC KL

Thymoxy- 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 12.28 ± 0.66 15.25 ± 1.03
acetic
acid
Thymol 0.19 ± 0.08abc 1.06 ± 0.00bc 1.43 ± 0.00bc 1.56 ± 0.00bc 1.5 ± 0.00abc 0.83 ± 0.21abc 0.70 ± 0.20ac 8.71 ± 0.76abc 13.97 ± 0.71
Distilled 0.31 ± 0.06a 1.15 ± 0.00a 1.56 ± 0.01a 1.57 ± 0.00a 1.7 ± 0.01a 1.53 ± 0.49a 0.70 ± 0.10a 8.61 ± 1.46a 8.11 ± 0.61a
water
Solvent 0.18 ± 0.07b 1.09 ± 0.00b 1.44 ± 0.01b 1.56 ± 0.00b 1.6 ± 0.01b 2.23 ± 3.26b 0.26 ± 0.12b 9.00 ± 1.06b 12.46 ± 0.68b
Glyphosate 0.18 ± 0.07c 1.09 ± 0.00c 1.44 ± 0.00c 1.56 ± 0.00c 1.5 ± 0.00c 1.03 ± 0.38c 0.73 ± 0.21c 8.33 ± 0.44c 9.56 ± 0.14c
Treatment Postemergence
RFM SFM RDM SDM RL SL
Thymoxy- 1.02 ± 0.01bc 1.02 ± 0.00bc 1.03 ± 0.00bc 1.03 ± 0.00bc 0.66 ± 0.15c 0.93 ± 0.15c
acetic
acid
Thymol 1.01 ± 0.00bc 1.01 ± 0.00bc 1.01 ± 0.00bc 1.02 ± 0.00bc 0.60 ± 0.10c 0.56 ± 0.25c
Distilled 1.3 ± 0.00a 1.5 ± 0.01 a 1.80 ± 0.00a 1.50 ± 0.01a 2.37 ± 0.25a 1.60 ± 0.10a
water
Solvent 1.05 ± 0.00b 1.01 ± 0.01b 1.28 ± 0.00b 1.02 ± 0.00b 1.56 ± 0.74b 1.63 ± 0.47b
Glyphosate 1.01 ± 0.00c 1.01 ± 0.00c 1.01 ± 0.00c 1.03 ± 0.00c 0.56 ± 0.15c 0.80 ± 0.10c

*
Means followed by letter a on the columns are statistically equal to distilled water, means followed by letter b are statistically equal to the sol-
vent, and means followed by letter c statistically differ from the negative control and from the solvent according to Dunnett’s test (P > 0.05)
GSI germination speed index, RL root length, SL shoot length, RFM root fresh mass, RDM root dry mass, SFM shoot fresh mass of seedlings,
SDM shoot dry mass of seedlings, EC electrical conductivity, KL potassium leaching

13
 Table 5  Phytotoxicity of thymol and thymoxyacetic acid on the initial development of S. bicolor seedlings, on the pre- and postemergence experiments
(g ­seedlings−1) (cm ­seedling−1) (Sm−1 ­g−1) (mg ­dm3)

Treatment Preemergence
GSI RFM SFM RDM SDM RL SL EC KL
Thymoxy- 1.61 ± 0.98c 2.07 ± 0.02 1.75 ± 0.03 1.66 ± 0.01 1.63 ± 0.01 4.81 ± 1.30 1.90 ± 1.21abc 2.36 ± 0.06 2.48 ± 0.05
acetic
acid
Thymol 2.78 ± 1.12abc 2.37 ± 0.02c 1.83 ± 0.03c 1.68 ± 0.01c 1.64 ± 0.03bc 10.70 ± 0.89c 1.53 ± 0.47bc 2.78 ± 0.08abc 2.17 ± 0.03
Distilled 3.86 ± 0.19a 3.41 ± 0.07a 2.10 ± 0.09a 1.76 ± 0.05a 1.66 ± 0.11a 15.06 ± 1.72a 3.60 ± 0.53a 2.69 ± 0.05a 1.47 ± 0.02a
water
Solvent 3.61 ± 0.97b 3.12 ± 0.04 b 2.02 ± 0.03b 1.74 ± 0.04b 1.65 ± 0.01b 15.40 ± 1.22b 2.73 ± 0.25b 2.72 ± 0.03b 1.49 ± 0.01b
Environmental Science and Pollution Research (2023) 30:38955–38969

Glypho- 1.34 ± 0.32c 2.82 ± 0.04c 1.95 ± 0.01c 1.72 ± 0.01c 1.65 ± 0.01c 11.00 ± 2.93c 2.56 ± 1.14c 2.68 ± 0.03c 1.42 ± 0.00c
sate
Treatment Postemergence
RFM SFM RDM SDM RL SL
Thymoxy- 1.54 ± 0.11b 1.26 ± 0.03c 1.12 ± 0.05abc 1.06 ± 0.01ac 15.00 ± 1.50abc 3.60 ± 0.59bc
acetic
acid
Thymol 1.50 ± 0.06b 1.24 ± 0.00ab 1.10 ± 0.05abc 1.06 ± 0.01ac 14.83 ± 0.67abc 3.16 ± 0.26abc
Distilled 1.89 ± 0.50a 1.25 ± 0.02a 1.14 ± 0.19a 1.07 ± 0.01a 16.23 ± 1.66a 4.73 ± 0.60a
water
Solvent 1.49 ± 0.12b 1.26 ± 0.01b 1.08 ± 0.07b 1.08 ± 0.01b 15.26 ± 0.74b 3.83 ± 0.25b
Glypho- 1.81 ± 0.26c 1.24 ± 0.01c 1.16 ± 0.11c 1.07 ± 0.01c 14.23 ± 1.00c 3.50 ± 1.04c
sate
*
Means followed by letter a on the columns are statistically equal to distilled water, means followed by letter b are statistically equal to the solvent, and means followed by letter c statistically
differ from the negative control and from the solvent according to Dunnett’s test (P > 0.05)
GSI germination speed index, RL root length, SL shoot length, RFM root fresh mass, RDM root dry mass, SFM shoot fresh mass of seedlings, SDM shoot dry mass of seedlings, EC electrical
conductivity, KL potassium leaching

13
38961
38962 Environmental Science and Pollution Research (2023) 30:38955–38969

Fig. 3  Healthy and normal

lettuce seedling in the control
with water (A) and abnormal
seedling with evidence of death
caused by thymoxyacetic acid
after 2 days of exposure (B)

and to the rearrangement of the cell membranes (Seth et al. seeds in the greenhouse, which can be explained due to the
2007). significant increase in electrical conductivity and potassium
According to the increase in KL, the GSI of caruru seeds leaching. The high values obtained by the EC and KL analy-
decreased, suggesting that thymoxyacetic acid and thymol sis indicate that thymoxyacetic acid and thymol negatively
negatively affected the rate of rearrangement of the cell affected cell membranes and GSI, with these parameters
membranes, allowing the leaching of mineral salts, sugars, being negatively correlated (Table 4 and Fig. 6D).
proteins, and other compounds of the seed (Table 2). Another characteristic observed for the seedlings of
Thymol and thymoxyacetic acid interfered negatively and capim pé-de-galinha was the reduction in the accumulation
markedly on the germination and growth of cucumber seed- of fresh matter from the roots and shoot for all the treat-
lings. The reduction in the fresh and dry mass of cucumber ments, in comparison to C − . This significant difference
seedlings treated with thymoxyacetic acid in preemergence observed in the fresh mass of the seedlings is probably due
and with thymol in postemergence was also observed. The to differences in the water content absorbed and retained
total length of these seedlings was significantly reduced in the green tissues, which can be influenced by the other
and the RL of the seedlings treated with thymoxyacetic treatments (Table 4).
acid showed the lowest values in greenhouse, 11.32 times The results for S. bicolor indicated the existence of con-
shorter when compared to C − . Thymoxyacetic acid was siderable phytotoxicity of thymol and thymoxyacetic acid
more efficient than glyphosate for the variables GSI, RFM, on the physiological quality of seeds and seedlings. Azirak
RL, EC, and KA (Table 3). and Karaman (2008) verified this inhibitory effect on ger-
The thymoxyacetic acid affected the normal development mination and growth caused by thymol, in another weed
of preemergent cucumber seedlings. Several changes were species, B. decumbens, demonstrating its potential use in
observed, such as root reduction, leaf burning, radicle necro- pre and post-emergence as a bioherbicide. The thymoxy-
sis, absence of absorbent hair, and poorly developed shoot acetic acid was the treatment that most affected GSI and
(Fig. 4). The direct effect of the compound may have caused RL in preemergence. These effects were even more accen-
that, or it could have been a consequence of the reduced root tuated in the initial growth of these seedlings compared to
growth (Oliveira et al. 2004). The abnormal seedlings almost C + (Table 5). These drastic changes in RL show the strong
never complete their development, and the reduction of root compound phytotoxicity (Oliveira et al. 2011).
hair can hinder the absorption of nutrients by the root, caus- Table 5 shows a decrease in all the variables analyzed on
ing developmental deficiencies (Ferreira and Aquila 2000). the treatments, when compared to C − . When these variables
According to Aires et al. (2005) and Maraschin-Silva and are compared to C + , the reduction caused by the use of thy-
Aquila (2006), the allelochemical compounds affect the ini- mol and thymoxyacetic acid for RL, SL, and GSI is superior
tial growth, reflecting in the atrophy of the seedling parts, as to the inhibition caused by the active compound glyphosate.
the darkening that shows the morphological and anatomical Similar effects of the thymoxyacetic acid were observed
changes caused by phytotoxins. when compared to the active compound glyphosate, such as
For the monocotyledonous capim pé-de-galinha, the thy- leaf burning, reduction of the roots, and shoot’s system of the
moxyacetic acid totally inhibited the preemergence of the sorghum seedlings (Fig. 5). Pinheiro et al. (2015), studying

13
Environmental Science and Pollution Research (2023) 30:38955–38969 38963

Fig. 4  Healthy and normal

cucumber seedling in the con-
trol with water (A) and abnor-
mal seedling with reduced root,
leaf burning, radicle necrosis,
absence of absorbent hair and
poorly developed shoot, caused
by thymoxyacetic acid after
21 days of exposure (B)

the effect of the essential oil of P. amboinicus and its major oxygen species, with the consequent modification in the
compounds, carvacrol and thymol, concluded that the tested lipids of the membrane. According to Ferreira and Aquila
compounds had an effect on the germination of both lettuce (2000), the alteration on the germination of the seeds can
and sorghum. However, the results on lettuce, as well as in affect the permeability of the membranes, the transcription
the present study, were more expressive. Alves et al. (2018), of the DNA and the translation of the proteins, the function-
studying the action of thymoxyacetic acid, found that, just ing of the secondary messengers, the breathing, the seques-
like 2,4-D, it has a selective character, since it had an effect tration of oxygen, and the conformation of the enzymes and
only on eudicotyledons, differing from the results obtained receivers. Thus, thymol and thymoxyacetic acid, in the stud-
in this study with effects also on monocotyledons. ied concentration, interfered negatively on the growth of two
Although, in this study, thymol and thymoxyacetic acid monocot and three eudicot species studied. The germination
have shown inhibitory effects on both germinative and speed index was reduced for all the species and is directly
vegetative growth in monocots and eudicots, such results related to the increase in potassium leaching of the treated
were more expressive for eudicotyledonous species (lettuce, seeds (Fig. 6).
caruru, and cucumber). For monocots (capim pé-de-galinha Therefore, the results shown in Fig. 6 are related to the
and sorghum), despite the significant variables, they are ability of the tested allelochemicals to weaken the integrity
less representative in the effects of the tested compounds. of the cell membrane and to cause changes from electrical
Amri et al. (2013) reported that, in general, eudicotyledons conductivity and increase potassium leaching. Monoterpe-
are more sensitive to monoterpenes such as thymol than nes act on the phospholipids of the membranes, changing
monocotyledons. their composition, as a response mechanism when these
Zunino and Zygadlo (2004) studying thymol found out plants are subjected to water and oxidative stress, causing
that it also inhibited the growth of corn (monocotyledon- several disturbances in physiological and biochemical pro-
ous). This inhibition resulted from the production of reactive cesses (Zunino and Zygadlo 2004; Amri et al. 2013).

13
38964 Environmental Science and Pollution Research (2023) 30:38955–38969

Fig. 5  Sorghum seedlings under

similar effects caused by the
active compound glyphosate
(A) and thymoxyacetic acid
with 4 days of exposure (B)

The emergence of the seedlings was reduced for all the during the imbibition process, the release of cytoplasmic
species in the greenhouse, due to the increase in potassium solutes occurs in the liquid medium, in an intensity that is
leaching (Fig. 6). Such results can be attributed to the action proportional to the state of disorganization in which the
of the allelochemicals, which act directly in the processes of membranes are found (Marchi and Cicero 2002).
cell and membrane degradation (Qian et al. 2009), affecting Potassium is a nutrient highly demanded by the plants,
the vigor of the seeds (Silva et al. 2011). and its nutrition is substantial for vegetative growth and
One of the results of the membrane integrity loss is the development, acting on several metabolic functions such as
leakage of electrolytes and loss of selectivity of the mem- photosynthesis, pH stabilization, osmotic regulation, ionic
brane. According to Anazetti and Melo (2007), the leakage absorption, and protein synthesis. However, the excess of
of electrolytes and ion exchange determine the imbalance this mineral in the soil or substrate results in a reduction of
of cellular pH and reduce the concentration of ions. This water absorption by the plants’ roots and impairs the absorp-
variation can inhibit the synthesis of ATP and increase the tion of calcium and magnesium by competitive inhibition
production of reactive oxygen species, which can cause the (Faquin 2005).
rupture of membranes leading to cell necrosis and germina- The results of the present study indicated the existence
tion reduction. The electrical conductivity test, on the other of a phytotoxicity of thymol and thymoxyacetic acid on
hand, determines all the aspects of the seeds vigor that can the physiological quality of the seeds of the five species,
influence the establishment of seedlings under field condi- observed from Pearson’s correlation test. Table 6 shows
tions. This test is based on the vigor of the evaluated seeds that for lettuce, sorghum, caruru, and capim pé-de-galinha
and is closely related to the integrity of the cell membrane (weeds) with the increase in KL, the GSI was reduced, evi-
system. Thus, when the seeds are immersed in the treatments denced by the negative correlation where these variables

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Environmental Science and Pollution Research (2023) 30:38955–38969 38965

Fig. 6  GSI reduction of the 5

species studied and its relation-
ship with the increase in electri-
cal conductivity and potassium
leaching of these seeds. A:
lettuce, B: caruru, C: cucum-
ber, D: capim Pé-de-galinha,
E: sorghum, TA: thymoxy-
acetic acid, TM: thymol, DW:
distilled water, SV: solvent, GF:
glyphosate

The treatment of C − with distilled water reduced the

Table 6  Pearson’s correlation coefficients between germination speed
index (GSI), electrical conductivity (EC), and potassium leaching electrical conductivity and the potassium leaching in the
(KL) from treatments and seed germination in greenhouse solution, contributing to the increase on the germination
speed index of the seeds of the five studied species. This
Lettuce Caruru Cucumber Pé-de-Galinha Sorghum
effect is justified by the correlation that exists between these
GSI-KL − 0.80* − 0.85* − 0.60 ns − 0.84* − 0.84* variables, since on seeds treated with herbicidal solutions,
GSI-EC 0.50 ns − 0.84* − 0.79* − 0.75* − 0.63 ns there was an increase in electrical conductivity, potassium
EC-KL − 0.08 ns
0.96* 0.93* 0.96* 0.38 ns leaching, and, as a consequence, a reduction of the GSI
ns (Table 6 and Fig. 6).
Not significant. *Significant at 5% probability level, by the t-test

Genotoxicity

move to opposite directions. With the increase in electrical The molecular analyses reinforced and complemented the
conductivity, there was also a reduction in the GSI for the analyses in the phytotoxic tests. The mutagenic effects of
weeds studied presenting a strong and positive correlation thymol and thymoxyacetic acid were detected, and the
(close to one) between EC and KL, elucidating that with changes were verified in the DNA. The dissimilarity values
the increase in EC, the increase in KL occurs (Table 6). found between these molecules and the formation of distinct
The increase in EC values may be related to the reduction groups in relation to the negative controls and the similarity
in GSI values and the increase in KL, since these attrib- with the positive control can be seen in Figs. 7 and 8.
utes of seed vigor are related to changes in the fluidity and On the molecular test, thymol and thymoxyacetic acid
permeability of cell membranes (Abu-Romman et al. 2010; showed a toxic character similar or superior to that observed
Hussain and Reigosa 2011). in the herbicide glyphosate, since they caused changes in

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38966 Environmental Science and Pollution Research (2023) 30:38955–38969

Fig. 7  UPGMA cluster of eudicotyledonous species for all the treat- TA-S: thymoxyacetic acid shoot, DW-R: distilled water root, DW-S:
ments, using ISSR primers. A: prelettuce, B postlettuce, C: precar- distilled water shoot, S-R: solvent root, S–S: solvent shoot, T-R: thy-
uru, D: postcaruru, E: precucumber, F: postcucumber, G-R: glypho- mol root, T-S: thymol shoot
sate-root, G-S: glyphosat shoot, TA-R: thymoxyacetic acid root,

the DNA of roots and shoot of the seedlings. The proxim- was observed that the water treatment clustered both shoot
ity of individuals treated with such molecules differed from and root, while glyphosate promoted a high change in the
groups of individuals treated with negative controls, water root and formed an isolated and more dissimilar group
and solvent, both for eudicotyledons and monocotyledons among all the tested compounds. Thymol also promoted a
(Figs. 7 and 8), reinforcing the effects previously described significant change in the root, which was also grouped in
on the phytotoxicity analyzes. another isolated group. When analyzing the shoot in rela-
Thymol was more effective in eudicotyledons, according tion to thymol, it did not cause many molecular changes, as
to the molecular analysis, evidenced by the higher values of it was grouped with the solvent treatment (negative control)
dissimilarity between the DNA of root and shoot, in rela- (Fig. 7A, B).
tion to the negative controls, as observed in macroscopic For caruru in preemergence, no difference was observed
analyzes in postemergence. The same was verified for thy- between the treatments with thymoxyacetic acid, water,
moxyacetic acid, but the differences in DNA between shoot glyphosate, thymol, and solvent for the roots, while the shoot
and root for this compound were smaller, remaining in the of the negative controls formed isolated groups (Fig. 7C).
same group (Figs. 7 and 8). This smaller group might be formed due to the low ampli-
When assessing the DNAs of the roots and shoot of let- fication of the markers for the species. For the postemer-
tuce, both in pre- (Fig. 7A) and postemergence (Fig. 7B), it gent treatment, when the shoot was evaluated, thymol and

13
Environmental Science and Pollution Research (2023) 30:38955–38969 38967

Fig. 8  UPGMA cluster of monocot species for all the treatments, moxyacetic acid shoot, DW-R: distilled water root, DW-S: distilled
using ISSR primers. A: prelettuce, B postlettuce, C: precaruru, D: water shoot, S-R: solvent root, S–S: solvent shoot, T-R: thymol root,
postcaruru, E: precucumber, F: postcucumber, G-R: glyphosate-root, T-S: thymol shoot
G-S: glyphosat shoot, TA-R: thymoxyacetic acid root, TA-S: thy-

glyphosate formed a single group with 100% similarity (positive control) (Fig. 7E). Contrary to what happened in
between treatments. The same was observed for the use of preemergence, when thymol was used in postemergence
compounds in the roots, which formed a group, but with a for cucumber, an effect on the root was observed, similar
20% dissimilarity between the compounds, and a genetic to glyphosate (Fig. 7F).
distance of approximately 0.04. The solvents did not differ in The shoot of the monocots was severely affected by the
the root’s treatments, forming a group with 100% similarity, use of thymoxyacetic acid, as there was the formation of an
the treatments with solvent for the shoot were also clustered isolated group, containing only this treatment, with a dissim-
within that same group. Thymoxyacetic acid formed an iso- ilarity of 0.14 with the other groups. However, no mutagenic
lated group with shoot and root (Fig. 7D). effects of any of the active ingredients were observed when
When evaluating cucumber in preemergence, the forma- evaluating postemergence roots for capim pé-de-galinha,
tion of three large groups was observed. The first group since all treatments obtained 100% similarity with the posi-
comprises only the thymoxyacetic acid, with 100% simi- tive and negative controls (Fig. 8B).
larity between the DNAs of the root and shoot, suggesting For the analyses with sorghum in preemergence, it was
that the performance of the active principle for this spe- found that thymoxyacetic acid does not cause different
cies is similar in both parts of the plant, showing its toxic changes in shoot and root, as they obtained 100% similar-
effect, when compared with the macroscopic results, in ity and formed an isolated group. Another compound that
which the normal development of cucumbers’ seedlings deserves to be highlighted is thymol, which could cause
was affected. On the second group, the similarity between effects that led to the differentiation of the root, which
the treatments with water and solvent was evidenced, for received other treatments, forming a single group (Fig. 8C).
both shoot and roots. The presence of the root treated with For postemergency treatments, three large groups were
thymol was observed, which might suggest that the muta- formed; thymol promoted greater differentiation in the roots
genic effect of thymol in the roots in pre-emergence is and was grouped together with thymoxyacetic acid (shoot
almost null for this species, due to the proximity to the and root), while the shoot was grouped together with the
negative controls. However, its effect on the shoot is quite negative controls. This allows us to infer that the effect of
pronounced, considering that it was grouped in the third thymol is more pronounced on the roots than on the shoot
large group, together with the treatment with glyphosate of sorghum (Fig. 8D).

13
38968 Environmental Science and Pollution Research (2023) 30:38955–38969

Thymol showed more divergence between the DNAs Pinheiro, Thammyres de Assis Alves, Josimar Aleixo da Silva, Milene
of roots and shoot, differently from thymoxyacetic acid, Miranda Praça-Fontes, Taís Cristina Bastos Soares. The first draft of
the manuscript was written by Carlos Eduardo de Oliveira Roberto
in which in most cases the shoot and root were grouped and all authors commented on previous versions of the manuscript. All
together. authors read and approved the final manuscript.
The correlation between macroscopic and molecular
analyses was verified through the changes observed in the Funding The authors thank the “Conselho Nacional de Desenvolvi-
mento Científico e Tecnológico” (CNPq, Brasília—DF, Brazil) for
DNA caused by the compounds. The ISSR molecular mark- funding (number: 408775/2018–1) and for the research grant to Milene
ers allowed the detection of genotoxicity caused by thymol Miranda Praça-Fontes, Patrícia Fontes Pinheiro and Taís Cristina Bas-
and thymoxyacetic acid. tos Soares; the “Coordenação de Aperfeiçoamento de Pessoal de Nível
The phytotoxic action of thymoxyacetic acid may be due Superior” (CAPES), for the financial support (Finance Code 001)
and “Fundação de Amparo à Pesquisa e Inovação do Espírito Santo”
to its similarity to the herbicide 2,4-D, due to the presence of (FAPES, Vitória – ES, Brazil) for funding (grant 484/2021;2021-ZK8PJ
the Ar–O-CH2COOH group in the molecule. When auxinic and Cooperação CAPES/FAPES – PDPG) and for providing under-
herbicides are applied, they can be absorbed by roots and graduate research and master’s scholarships to the authors of this work.
leaves, being translocated by both phloem and xylem (sys-
Data availability The datasets generated during and/or analyzed dur-
temic). The translocation of these substances in monocots ing the current study are available from the corresponding author on
is more restricted than in sensitive eudicots. Thus, in broad reasonable request.
leaf species, auxinic herbicides can induce metabolic and
biochemical changes, triggering the plant death (Song 2014). Declarations
The phytotoxic action of thymol may be expressed to several
Ethics approval Not applicable.
factors. Araniti et al. (2020) found that plants treated with thy-
mol presented osmotic stress, supposedly due to the increase in Consent to Participate Not applicable.
­H2O2 (hydrogen peroxide). Furthermore, there was an increase
in the content of osmoprotectors (galactinol and proline), an Consent for publication Not applicable.
increase in light sensitivity, as well as a lower efficiency of PSII Competing interests The authors declare that they have no conflict
with a reduction in photosynthetic activity. Briefly, the phyto- of interest.
toxicity caused by thymol may be involved with the osmotic
and oxidative stress, which when combined may result in the
impediment of the normal development of the plant.
References
Abu-Romman S, Shatnawi M, Shibli R (2010) Allelopathic effects of
Conclusions spurge (Euphorbia hierosolymitana) on wheat (Triticum durum).
American-Eurasian J Agric and Environ Sci 7:298–302
Aires SS, Ferreira AG, Borghetti F (2005) Efeito alelopático de folhas
Thymol and its thymoxyacetic acid showed phytotoxic and e frutos de Solanum lycocarpum A. St.-Hil. (Solanaceae) na ger-
genotoxic activities for the studied species. minação e crescimento de Sesamun indicum L. (Pedaliaceae) em
The toxic effect of thymol and thymoxyacetic acid was solo sob três temperaturas. Acta Bot Bras 19:339–344. https://d​ oi.​
org/​10.​1590/​S0102-​33062​00500​02000​17
nonselective, similar to that of the active compound glypho- Alves TA, Pinheiro PF, Praça-Fontes MM, Andrade-Vieira LF, Corrêa
sate, turning these compounds to a good choice as nonselec- KB, Alves TA, Cruz FA, Lacerda Júnior V, Ferreira A, Soares TCB
tive bioherbicides for weed control. However, the toxic effect (2018) Toxicity of thymol, carvacrol and their respective phenoxy-
of thymoxyacetic acid is more effective in preemergence and acetic acids in Lactuca sativa and Sorghum bicolor. Ind Crop Prod
114:59–67. https://​doi.​org/​10.​1016/j.​indcr​op.​2018.​01.​071
thymol’s in postemergence. Amri I, Hamrouni L, Hanana M, Jamoussi B (2013) Reviews on phyto-
Thymol and thymoxyacetic acid promote damages to the toxic effects of essential oils and their individual components: news
roots and shoot, according to the observed changes at the approach for weeds management. Int J Appl Biol Pharm 4:96–114
DNA level of both monocots and eudicots. Anazetti MC, Melo OS (2007) Morte Celular por Apoptose: uma visão
bioquímica e molecular. Metrocamp Pesquisa 1:37–58
Molecular analyses showed the similarity of UPGMA ANVISA (2012) Agência Nacional de Vigilância Sanitária. Seminário
clusters of the plants treated with the active compound de mercado de agrotóxico e regulação
glyphosate and the tested molecules thymol and thymoxy- Araniti F, Miras-Moreno B, Lucini L, Landi M, Abenavoli MR (2020)
acetic acid, exacerbating the herbicidal potential of these Metabolomic, proteomic and physiological insights into the poten-
tial mode of action of thymol, a phytotoxic natural monoterpenoid
molecules. phenol. Plant Physiol Biochem 153:141–153. https://​doi.​org/​10.​
1016/j.​plaphy.​2020.​05.​008
Azirak S, Karaman S (2008) Allelopathic effect of some essential
Author contribution All authors contributed to the study conception oils and components on germination of weed species. Acta
and design. Material preparation, data collection and analysis were Agric Scand - B Soil Plant Sci 58:88–92. https://​doi.​org/​10.​
performed by Carlos Eduardo de Oliveira Roberto, Patrícia Fontes 1080/​09064​71070​12283​53

13
Environmental Science and Pollution Research (2023) 30:38955–38969 38969

Belchior DCV, Saraiva AS, López AMC, Scheidt GN (2017) Impac- ica-​da-​regul​acao-e-​preve​ncao-​da-​captu​ra-​regul​atori​a&​catid=​
tos de agrotóxicos sobre o meio ambiente e a saúde humana. 419:​2019&​direc​tory=1. Acessed 02 march 2022
Cad Ciênc Tecnol 34:135–151 Oliveira LM, Voltolini JC, Barbério A (2011) Potencial mutagênico dos
Bohner TOL, Araújo LEB, Nishijima T (2013) O impacto ambiental poluentes na água do rio Paraíba do Sul em Tremembé, SP, Brasil,
do uso de agrotóxicos no meio ambiente e na saúde dos trabal- utilizando o teste Allium cepa. Ambiente & Água-an Int J Appl Sci
hadores rurais. Rev Elet Cur Dir UFSM 8:329–341 6:90–103. https://​doi.​org/​10.​4136/​ambi-​agua.​176
Bombardi LM (2017) Geografia do uso de agrotóxicos no Brasil e Oliveira SCC, Ferreira AG, Borghetti F (2004) Efeito alelopático de
conexões com a União Europeia. FFLCH-USP folhas de Solanum lycocarpum A. St.-Hil. (Solanaceae) na ger-
Brighenti AM (2010) Manual for the identification and management of minação e crescimento de Sesamum indicum L. (Pedaliaceae)
weeds in sugarcane crops. Juiz de Fora: Embrapa Dairy Cattle. 150 p sob diferentes temperaturas. Acta Bot Bras 18:401–406. https://​
Cruz CD (2013) GENES: a software package for analysis in doi.​org/​10.​1590/​S0102-​33062​00500​02000​17
experimental statistics and quantitative genetics. Acta Sci Agron Pinheiro PF, Costa AV, Alves TA, Galter IN, Pinheiro CA, Pereira
35:271–276. https://​doi.​org/​10.​4025/​actas​ciagr​on.​v35i3.​21251 AF, Oliveira CMR, Fontes MMP (2015) Phytotoxicity and
Dayan FE, Duke SO (2014) Natural compounds as next-generation Cytotoxicity of Essential Oil from Leaves of Plectranthus
herbicides. Plant Physiol 166:1090–1105. https://​doi.​org/​10.​ amboinicus, Carvacrol, and Thymol in Plant Bioassays. J Agr
1104/​pp.​114.​239061 Food Chem 63:8981–8990. https://​d oi.​o rg/​1 0.​1 021/​a cs.​j afc.​
Dias LC, Demuner AJ, Valente VM, Barbosa LC, Martins FT, 5b030​49
Doriguetto AC, Ellena J (2009) Preparation of achiral and chiral Prete CEC (1992) Condutividade elétrica do exsudato de grãos
(E)-enaminopyran-2, 4-diones and their phytotoxic activity. J Agric de café (Coffea arabica L.) e sua relação com a qualidade da
Food Chem 57:1399–1405. https://​doi.​org/​10.​1021/​jf802​805f bebida. http://​www.​sbica​fe.​ufv.​br/​handle/​12345​6789/​392
Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Qian H, Xu X, Chen W, Jiang H, Jin Y, Liu W, Fu Z (2009)
Focus 12:39–40 Allelochemical stress causes oxidative damage and inhibition
Escobar A, Pérez M, Romanelli G, Blustein G (2020) Thymol bioac- of photosynthesis in Chlorella vulgaris. Chemosphere 75:368–
tivity: A review focusing on practical applications. Arab J Chem 375. https://​doi.​org/​10.​1016/j.​chemo​sphere.​2008.​12.​040
13(12):9243–9269. https://​doi.​org/​10.​1016/j.​arabjc.​2020.​11.​009 Seth CS, Chaturvedi PK, Misra V (2007) Toxic effect of arsenate
Faquin V (2005) Nutrição mineral de plantas. Lavras, Minas Gerais. and cadmium alone and in combination on giant duckweed
Ferreira AG, Aquila MEA (2000) Alelopatia: uma área emergente da (Spirodela polyrrhiza L.) in response to its accumulation. Envi-
ecofisiologia. Braz J Plant Physiol 12:175–204 ron Toxicol 22:539–549. https://​doi.​org/​10.​1002/​tox.​20292
Ferreira AG, Borghetti F (2004) Germinação: do básico ao aplicado. Silva L, Silva A, Queiroz M, Lima C, Rocha P, D’antonino L (2012)
Artmed Porto Alegre. Action of Eleusine coracana in the remediation of soils con-
Heap I (2020) The International Herbicide-Resistant Weed Database. taminated with picloram. Planta Daninha 30:627–632. https://​
Hussain MI, Reigosa MJ (2011) Allelochemical stress inhibits doi.​org/​10.​1590/​S0100-​83582​01200​03000​19
growth, leaf water relations, PSII photochemistry, non-photo- Silva VS, Cândido ACS, Muller C, Laura VA, Faccenda O, Hess SE,
chemical fluorescence quenching, and heat energy dissipation SC, Peres MTLP, (2011) Potencial fitotóxico de Dicranopteris
in three C3 perennial species. J Exp Bot 62:4533–4545. https://​ flexuosa (Schrad.) Underw. (Gleicheniaceae). Acta Bot Bras
doi.​org/​10.​1093/​jxb/​err161 25:95–104. https://​doi.​org/​10.​1590/​S0102-​33062​01100​01000​12
IBAMA (2020) Relatórios de comercialização de agrotóxicos. http://​ Song Y (2014) Insight into the mode of action of 2,4-dichlorophe-
www.​ibama.​gov.​br/​agrot​oxicos/​relat​orios-​de-​comer​ciali​zacao-​ noxyacetic acid (2,4-D) as an herbicide. J Integr Plant Biol
de-​agrot​oxicos. Acessed 02 march 2022 56(2):106–113. https://​doi.​org/​10.​1111/​jipb.​12131
Kraehmer H, Laber B, Rosinger C, Schulz A (2014) Herbicides as Souto AL, Sylvestre M, Tölke ED, Tavares JF, Barbosa-Filho JM,
weed control agents: state of the art: I. Weed control research and Cebrián-Torrejón G (2021) Plant-Derived Pesticides as an
safener technology: the path to modern agriculture. Plant Physiol Alternative to Pest Management and Sustainable Agricultural
166(3): 1119–1131. https://​doi.​org/​10.​1104/​pp.​114.​241901 Production: Prospects. Applications and Challenges Molecules
Kudsk P, Streibig JC (2003) Herbicides – a two-edged sword*. Weed 26:4835. https://​doi.​org/​10.​3390/​molec​ules2​61648​35
Res 43:90–102. https://​doi.​org/​10.​1046/j.​1365-​3180.​2003.​00328.x Valerio ME, García JF, Peinado FM (2007) Determination of phy-
Leal UA, Silva GN, Karam D (2012) Otimização dinâmica multiobjetivo totoxicity of soluble elements in soils, based on a bioassay
da aplicação de herbicida considerando a resistência de plantas danin- with lettuce (Lactuca sativa L.). Sci Total Environ 378:63–66.
has. Embrapa Milho e Sorgo-Artigo em periódico indexado (ALICE). https://​doi.​org/​10.​1016/j.​scito​tenv.​2007.​01.​007
Lorenzi H (2014) Weed Identification and Control Manual: No-till- Yang DT, Zhu X (2013) Modernization of agriculture and long-term
age and Conventional Planting, 7ª. Plantarum Institute for Flora growth. J Monet Econ 60:367–382. https://​doi.​org/​10.​1016/j.​
Studies, Nova Odessa, SP jmone​co.​2013.​01.​002
Maguire JD (1962) Speed of germination—Aid in selection and eval- Zunino MP, Zygadlo JÁ (2004) Effect of monoterpenes on lipid oxi-
uation for seedling emergence and vigor. Crop Sci 2:176–177. dation in maize. Planta 219:303–309. https://​doi.​org/​10.​1007/​
https://​doi.​org/​10.​2135/​crops​ci1962.​00111​83X00​02000​20033x s00425-​004-​1216-7
Maraschin-Silva F, Aquila M (2006) Potencial alelopático de espé-
cies nativas na germinação e crescimento inicial de Lactuca Publisher's note Springer Nature remains neutral with regard to
sativa L. (Asteraceae). Acta Bot Bras 20:61–69. https://​doi.​org/​ jurisdictional claims in published maps and institutional affiliations.
10.​1590/​S0102-​33062​00600​01000​07
Marchi J, Cicero S (2002) Procedimentos para a condução do teste de Springer Nature or its licensor (e.g. a society or other partner) holds
condutividade elétrica em sementes. Informativo Abrates 12:2 exclusive rights to this article under a publishing agreement with the
Moraes RF (2019) Agrotóxicos no Brasil: Padrões de uso, política da author(s) or other rightsholder(s); author self-archiving of the accepted
regulação e prevenção da captura regulatória. https://​www.​ipea.​ manuscript version of this article is solely governed by the terms of
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