COMPARATIVE ASSESSMENT OF SORBIC AND BENZOIC ACID VIA EXPRESS BIOTEST
Рубрики: RESEARCH ARTICLE
Аннотация и ключевые слова
Аннотация (русский):
Negative physiological and biochemical effects of chronic and subchronic doses of benzoates and sorbates may pose a certain risk to human health. Identifying new biomarkers responsible for the body’s response to these compounds could provide significant details in determining the mechanism of their toxicity. To assess comparatively physiological, cytological, cytogenetic, and biochemical parameters in onion roots cells we used an Allium test. The roots were previously treated with sorbic and benzoic acids. The study recorded the dose-dependent toxic effect of these preservatives on the root mass growth. The EC50 values obtained for benzoic and sorbic acids (10 mg/L and 110 mg/L respectively) were significantly lower than the regulated concentrations prescribed by the standards for their content in certain types of food products. With an increase in concentrations of these acids, the mitotic index of meristematic cells decreased in experimental groups compared to control groups. The data obtained confirmed the necessity of estimating the mitotic index when choosing onion for the Allium test. The necessity resulted from the fact that low proliferative activity could cause false positive results. Sorbic and benzoic acids in concentrations below the corresponding EC50 increased the frequency of chromosomal aberrations in apical meristematic cells of the roots compared to control. Thus, benzoic and sorbic acids had reliable mitodepressive and genotoxic effects on the dividing cells of onion roots. The study explored the dynamics of lipid oxidation biomarker accumulation (malon dialdehyde, MDA) after exposure to benzoic and sorbic acids. The toxic effect of benzoic acid appeared not to be associated with oxidative damage to root cell lipids, whereas sorbic acid in concentrations from 20 to 200 mg/L resulted in a multiple increase in MDA concentration in the test samples compared to control. At the same time, lipid peroxidation showed a higher level of sensitivity compared to other indicators of this test. Further, the data obtained on the toxic influence of sorbic and benzoic acids can be used in express methods to assess food and ecological security of these acids.

Ключевые слова:
Food preservatives, Allium cepa, biotesting, lipid peroxidation toxicity, cytogenetic analysis, biomarkers
Текст
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INTRODUCTION
Food preservation has remained a problem
throughout the human history. It is caused by the activity
of environmental microorganisms and enzymatic
reactions in the products during their production
and storage [1, 2]. About a third of the population
in the developed countries are estimated to suffer
from diseases transmitted through food especially
falsified [3]. Food safety is directly related to the
development of chemicals that prevent or slow down the
spoilage of these products.
Sorbic and benzoic acids, as well as their salts, are
known to be widely used as food preservatives. Their
production is steadily increasing. These acids are
contained in some fruits, berries, dairy products. Sorbic
acid is an unsaturated fatty acid and is used only as a
preservative in food, animal feed, tobacco, cosmetics
and pharmaceuticals. It is metabolized like normal
fatty acids, so this acid was assumed to have no side
effects. Benzoic acid is a synthetic additive, used as a
preservative and antioxidant. It is excreted by the human
body through the kidneys.
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There are numerous data on the health safety of
these compounds in regulated food products. Recently,
however, there are more discussions on the necessity to
develop scientific approaches to studying mechanisms of
their toxicity [4, 5]. The interest in this problem is due
to by the detected adverse effects of the chronic and
subchronic benzoate and sorbate intake by both animals
and humans. Thus, adding benzoic acid to pig feed
increased the liver enzymes activity and changed the
blood formula negatively, eventually damaging the liver
and spleen, respectively [6].
In vitro studies of human erythrocytes demonstrated
that sodium benzoate reduced the level of key
metabolic enzymes of amino acids (aspartate and
alanine aminotransferase) and alkaline phosphatase
significantly [7]. There is strong evidence that attention
deficit and hyperactivity syndrome in children and
anxiety conditions in rats could be associated with
high doses of sodium benzoate [8–9]. Other researchers
showed that sodium benzoate caused numerous negative
physiological and biochemical changes in mice and rats.
The changes included reducing the mass of reproductive
organs and embryos and the level of sex hormones in
mice [10]. As for human blood cell culture, sorbic acid
demonstrated the inhibitory effect on biochemical
reactions in the activated immune response [11].
However, the mechanism of toxicity for these
preservatives is still unclear. In addition, creating a new
algorithm for assessing food safety is debated a lot. The
algorithm especially concerns foods containing several
food additives because of their potential additive and
synergetic effect of toxicity [12]. It is yet to be found out
if the food preservatives may exert increased activity in
people with specific diseases or genetic defects.
To rise up to the challenge, it is necessary to go
beyond standard toxicity tests to identify molecular
biological protection mechanisms and to identify
biomarkers responsible for the body reaction to the
effects of chemical compounds. All the more so, as
modern methodology and instrumentation system are
able to tackle these complex problems. New approaches
should not only monitor and evaluate toxic effects, but
also result in the adequate test systems for modelling
detoxification and metabolism of food preservatives in
the human body. It is important to develop new model
systems. They should be simple to execute, cheap, and
able to simulate the reactions of the human body, both
on the physiological and molecular levels, with the
maximum available accuracy.
In this aspect, the special interest is given to the
work on a comprehensive assessment of biomarkers
of neurotoxicity and antioxidant enzymes activity in
daphnia under the influence of food sweetener sucralose.
It is due to the evidence that Gammarus zadachi and
Daphnia magna crustaceans exposed to this sweetener
altered their swimming behavior [13]. The tests were
carried out on these organisms to compare the activity
of acetylcholinesterase (AChE), lipid peroxidation
enzymes, and the ability to absorb oxygen radicals
(ORAC assay) in them. The authors observed the
stimulating effect of sucralose on the activity of AChE
and lipid peroxidation, but not on the antioxidant
capacity (ORAC). In humans, an increased AChE
activity was also associated with neurodegenerative
diseases such as Alzheimer’s disease, Parkinson’s
disease, multiple sclerosis, and restless legs syndrome.
It is important to note that the data obtained in this work
are consistent with those in other experimental studies
on human cell cultures and vertebrates. However, plant
test systems are also of interest, in particular Allium
cepa L. onion roots (Allium test).
Traditionally, the Allium test has been used
as a bioindicator in numerous studies on toxicity,
cytotoxicity, and genotoxicity of various chemical
compounds. It is recommended by WHO experts
as a standard for the cytogenetic monitoring of the
environment. Recently, it has been increasingly used
to assess the genotoxic potential of medicinal plants,
food additives, and even ionizing radiation [14–17]. The
Allium test was an excellent eukaryot model in vivo.
It was one of the few direct methods for measuring
damages in biological systems after exposure to
various toxicants and mutagenes. Its main advantages
include the following characteristics. First, the apical
meristematic root cells can show constant mitotic
division. Second, the roots may incubate directly
with the object being tested. Third, these cells have
large chromosomes, which allows a comprehensive
analysis of DNA damage. In addition, the test indicators
were shown to be more sensitive than the models on
microorganisms, cell cultures, and even animals [15, 18].
Allium cepa was also presented as an effective test
object in studying the reaction of plant cell biomarkers
to chemical toxicants of different nature. It is known
that chemical pollutants can induce the formation of
active forms of oxygen. In its turn, oxygen can activate
enzymes of peroxidation and result in damaging
various biological molecules, including lipids. Thus,
it was found that herbicide glyphosate and copper
salts significantly increased lipid peroxidation in plant
cells [19, 20]. In our opinion, the Allium test can help
significantly expand our knowledge of the mechanisms
of damage to biological systems of eukaryotes, including
the damage after exposure to sorbic and benzoic acids.
Moreover, no information was found on an effect of
these preservatives on physiological and biochemical
parameters in the meristematic cells of onion roots.
The aim of the research was to compare changes in the
mass growth, activity of lipid peroxidation enzymes,
cytological and cytogenetic parameters of Allium cepa
roots after treatment with sorbic and benzoic acids.
STUDY OBJECTS AND METHODS
In the research we used such preservatives as sorbic
acid (Alfa Aesar by Thermo Fisher Scientific) and
benzoic acid (Alfa Aesar by Thermo Fisher Scientific).
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Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 125–133
Allium cepa onion bulbs (Stuttgarter sort) of the same
size (2.5–3 cm in diameter) and mass (5–7 g) were
selected as a test organism. Dry scales were removed
from the bulbs before incubation. Preliminarily, the
germination was conducted in 15 mL test tubes with
bottled water for 2 days in the dark at 25°С.
The bulbs with roots over 1 cm long were selected
for further studies. Before treatment with benzoic and
sorbic acid solutions, the average mass of the roots was
determined in a separate group of the control bulbs.
Then the bulbs were transferred to the solutions of
these acids in the bottled water and incubated for 2 or
3 days depending on the purpose of the experiment.
After the incubation, the roots were cut off, dried with
filter paper, and weighed [21]. The EC50 value was
determined by the concentration of the preservative,
which retarded the root mass growth by 50% compared
to control, considering the average mass of the roots
before treatment with acids. For cytogenetic analysis,
the apical meristematic cells of the roots were stained
with acetoorcein (1 g of orcein dye was diluted in
50 mL of 45% CH3COOH). The roots were placed in a
70% solution of ethyl alcohol for the long-term storage.
Next, instant squash preparations were obtained, the
analysis of which was carried out with the help of a light
microscope Axioskop 40, Zeiss.
The lipid oxidation level was determined by the
concentration of malon dialdehyde (MDA) in the onion
roots [22]. The sample weight of approximately 0.25 g to
the fourth decimal place was measured in a 15 mL test
tube. Then 1 mL of trichloroacetic acid solution (Merck,
Germany), concentration of 200 g/dm3, was added. The
mixture was thoroughly stirred with a glass stick. Then
the stick was washed with 3 mL of the same solution of
trichloroacetic acid. The tubes were tightly corked and
centrifuged at 1000 g and 4°C for 15 min. One milliliter
of supernatant was transferred to a clean 15 mL test
tube. Four milliliters of thiobarbituric acid solution
(0.5 g of thiobarbituric acid (Diaem, Russia)) was added
to 100 mL of trichloroacetic acid solution (200 g/dm3).
The test tubes were closed and placed in a water bath
at 95°С for 30 min. Then the test tubes were pulled out
and cooled in an ice bath. The cooled solutions were
centrifuged at 1000 g and 20°C for 10 min.
The spectrophotometric detection was performed
with the obtained solutions at 600 and 532 nm. The
MDA content was calculated according to the formula:
where ABS532 is the absorption value at 532 nm;
ABS600 is the absorption value at 600 nm;
K is the dilution factor;
Ke is the molar coefficient of extinction;
l is a beam path length, cm;
mwt is the weight of the sample, g.
The statistical processing of the results was carried
out in Microsoft Excel and Statistica programs (v. 12).
In the paper, the analysis of average values by Student’s
criterion with Fisher’s angular transformation was used
for comparative estimation of percentages.
RESULTS AND DISCUSSION
The macroscopic parameters were studied and
comparatively evaluated, particularly, for the levels
of mass growth in the onion roots after treatment with
benzoic and sorbic acid solutions. According to the
literature review, the macroscopic parameters appeared
more sensitive in comparison with the cytological and
cytogenic parameters [23]. This conclusion seemed
logical because these parameters reflected the final
effect of all disorders in the plant cells. In this work,
when calculating the growth of root mass, the average
weight of roots was subtracted both in control and
experimental samples before their treatment with
preservatives solutions. Thus, the EC50 overstatement
error was eliminated in these samples. In the
preliminary experiments the solutions of preservatives
were used with the concentrations not exceeding the
permissible levels for some food products, namely,
1 g/L and 2 g/L. Death of the roots was observed after
2 days of incubation. Therefore, we reduced the range
of acid concentrations significantly. As a result, the root
growth and dose-dependent toxic effects were observed
during the same incubation period (Figs. 1 and 2). The
roots in the samples remained white and unchanged in
shape throughout the incubation. However, there were
statistically significant differences between the control
and test samples, namely, when treated with benzoic
acid at concentrations of 0.01 (P < 0.1); 0.05 (P < 0.05);
0.1 (P < 0.05) and 0.2 g/L (P < 0.05) and with sorbic
acid at concentrations of 0.02 (P < 0.1); 0.1 (P < 0.05);
0.2 (P < 0.05) and 0.3 g/L (P < 0.05). EC50 was 10 mg/L
for benzoic acid and 110 mg/L for sorbic acid. Thus,
these values differed significantly from the domestic
regulatory norms on the content of these food additives
in certain types of food.
As far as we know, this is the first study in which
EC50 values were identified for these preservatives in
the Allium test. At the same time, cyanobacteria with
Figure 1 Root mass growth inhibition after treatment with
sorbic acid (n = 10). * P < 0.05, ** P < 0.1
Sorbic acid concentration, mg/L
𝑥𝑥 􀵌
𝐴𝐴𝐵𝐵𝑆𝑆􀍷􀍵􀍴 − 𝐴𝐴𝐵𝐵𝑆𝑆􀍸􀍲􀍲 ∗ 𝐾𝐾
𝐾𝐾𝑒𝑒 ∗ 𝑙𝑙 ∗ 𝑚𝑚𝑤𝑤𝑡𝑡
0
30
60
90
0 50 100 150 200 250 300 350
Root mass growth, %
Sorbic mg/l
0
20
40
60
80
100
0 Root mass growth, %
50
100
150
200
250
300
MDA concentration, μmol/g
of wet weight
EC50 **
*
*
*
*
*
× × ×
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Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 125–133
EC50 from 9 mg/L were the most sensitive to benzoic
acid in similar studies using different organisms living
in water when treated for 14 days. In molluscs, fish
and amphibians, EC50/LC50 values were determined
within 100–1291 mg/L for 24–96 h [24]. The results
of these studies confirmed the high sensitivity of the
macroscopic parameters in the Allium test.
However, the studies on the toxicity of benzoic and
sorbic acids using the Allium test focused mainly on
the microscopic indicators reflecting the peculiarities of
cell division and chromosomal aberrations occurring in
its process, and EC50 was not determined. At the same
time, the tested concentrations of preservatives were
usually much higher than the EC50 values we found. So
the exposure of the roots to the preservative solutions in
these studies usually did not exceed several hours [25,
26]. We believe that such experimental conditions are
suitable only for acute toxicity testing. They are totally
unacceptable for the study of subchronic and chronic
consequences of negative effects, especially at the
biochemical level. The last aspect should be considered
the most interesting in the case of food preservatives.
Therefore we believe that the Allium test scheme
previously proposed for environmental monitoring
did not lose its relevance for studying the toxic effects
of these preservatives. The Allium test included a
comparative analysis of macro- and microindicators at
concentrations of toxicants within their EC50 [23].
The mitotic index is one of the microindicators in the
Allium test which is used as an indicator of the level of
cell proliferation. It is known that the dose-dependent
deviation of the mitotic index in the experimental
samples compared to the control values, both increasing
and decreasing, indicates cytotoxicity of the tested
chemical. In our previous study, the mitotic index of
cells of their meristem was decreasing significantly with
an increase in concentrations of preservatives (Table 1).
In testing highly toxic doses of sorbic acid (from 1 to
2 g/L), the mitotic index decreased only slightly when
the concentration of this acid increased [26]. This data
confirmed the previous assumption that there may be
difficulties in interpreting the research results due to the
high concentration of preservative.
The cytogenetic analysis was carried out on the
squash preparations of the apical meristematic cells of
onion roots obtained in the previous study. The analysis
determined the accumulation dynamics of chromosomal
aberrations when the concentrations of sorbic and
benzoic acids were increased. According to Table 1,
when acid concentrations increased, the proportion of
mitosis pathologies also increased, peaked, and then
decreased. It is noteworthy that the highest percentage
of chromosomal aberrations coincided with acid
concentrations close to the corresponding EC50 of these
preservatives. The drop in chromosomal aberrations at
high concentrations of acids is probably associated with
a significant decrease in the number of divisible cells in
the meristematic cells of roots.
The types of major chromosomal aberrations
detected in the experiment are shown in Fig. 3. The
analysis of the data allows us to conclude that stickness
of chromosomes in metaphase and chromosomes with
laggard in anaphase make the main contribution to the
spectrum of chromosomal aberrations. These anomalies
account respectively for aberrations ranging from 23.8%
to 70% (for sticky metaphase) and from 13.6% to 45.2%
(for chromosome with laggard). Also, there were the
following aberrations of the mitosis process detected in
micropreparations: C-mitosis, multiple fragmentation
of chromosomes, change in the spatial orientation of
chromosomes at the metaphase stage in cells. The least
observed anomalies included bridges and fragments
(about 2%, depending on the concentration of the tested
substances).
It seems remarkable to consider the whole spectrum
of aberrations. The most numerous anomalies found
while analyzing biomaterial can be due to the effects
Figure 2 Root mass growth inhibition after treatment with
benzoic acid (n = 10). * P < 0.05, ** P < 0.1
Table 1 Mitotic index and chromosomal aberrations
in meristematic cells of onion roots after exposure to benzoic
and sorbic acids (n = 10)
Acid concentration,
g/L
Mitotic
index, %
Chromosomal aberrations
based on the total
number of cells, %
Benzoic acid
0.01 12.02 ± 0.48c 0.88 ± 0.14a
0.02 10.20 ± 0.49a 1.00 ± 0.16a
0.1 6.89 ± 0.33a 0.50 ± 0.09b
0.2 0.75 ± 0.42a 0.05 ± 0.04a
Sorbic acid
0.02 9.63 ± 0.42a 0.95 ± 0.14a
0.1 6.81 ± 0.38a 0.79 ± 0.12a
0.2 1.52 ± 0.16a 0.10 ± 0.04a
0.3 0.00 ± 0.00a* 0.00 ± 0.00a*
Control 12.97 ± 0.48 0.30 ± 0.08
а P < 0.05, b P < 0.1, c P < 0.15, * 6503 cells were observed
Benzoic acid concentration, mg/L
􀍷􀍵􀍴 − 𝐴𝐴𝐵𝐵𝑆𝑆􀍸􀍲􀍲 ∗ 𝐾𝐾
𝑒𝑒 ∗ 𝑙𝑙 ∗ 𝑚𝑚𝑤𝑤𝑡𝑡
300 350
mg/l
0
20
40
60
80
100
0 50 100 150 200 250
Root mass growth, %
Benzoic acid concentration, mg/l
0
4
8
12
16
Контроль 0,01 0,05 0,1 0,2
MDA concentration,
μmol/g of wet weight
Benzoic acid concentration, g/l
0
20
40
60
80
100
120
Контроль MDA concentration, μmol/g
of wet weight
0.2 0.3 0.4 0.5 0.6
acid concentration, g/l
0
100
200
300
Контроль 0,02 0,2
MDA concentration, μmol/g
of wet weight
Sorbic acid concentration, g/l
EC50
*
*
*
*
** ** **
** *
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Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 125–133
of mitotic spindle disorder and changes in the surface
of chromosomes. The aberrations occur in this group
probably due to the influence of the tested substance on
the proteins regulating the work of the mitotic spindle in
the cell [27]. On the other hand, bridges, fragments, and
micronuclei are associated with clastogenic aberrations
(arising from the fracture of the chromosome and
anomalies of the further molecular genetic processes,
unequal translocation or inversion of the chromosome
segments). In the study [28], the analysis of genotoxicity
of sodium benzoate (in concentrations from 20 to
100 mg/kg) discovered the prevalence of aberrations
related to mitotic spindle disorders and changes in the
surface of chromosomes. These are the main types of
agglutination and C mitosis disorders. The clastogenic
effect of the factor was not recorded at all for this
indicator.
On the other hand, studies with high concentrations
of sodium benzoate exposed a much wider spectrum
of chromosomal aberrations. The aberrations included
agglutination and fragmentation of chromosomes, their
reduction, the formation of binuclear cells, chromosomal
bridges and other disorders [29]. According to data [30],
treating cells with sorbic acid resulted in the
chromosomal aberrations associated with mitotic spindle
disorder. Clastogenic aberrations were not detected.
Similar data were obtained in the study of the effects of
sorbic acid on the formation of micronuclei in cells [31].
This study recorded reliable mitodepressive
and genotoxic effects at very low concentrations of
preservatives (10 and 20 mg/L for benzoic and sorbic
acids, respectively). It is important to note that the data
obtained were consistent with the results on genotoxicity
of these acids and their salts for human and animal
cell culture. The results were published in a number of
papers, describing the exposure to both low and high
doses of these preservatives. Thus benzoic acid caused
sister chromatid exchange, chromosomal aberrations,
and micronuclei formation in human lymphocyte
cells [32]. Other researchers demonstrated the genotoxic
effect of sodium sorbate on Chinese hamster cells, as
well as clastogenic, mutagenic and cytotoxic effects
of sodium benzoate on the cell culture of human
lymphocytes [29, 33].
MDA concentration is commonly used as an
indicator of lipid peroxidation when the tissues are
exposed to chemical toxicants. MDA was measured in
the onion roots of the control and experimental groups
obtained in our previous study. In the experimental
groups of onion roots this biomarker analysis showed
Figure 3 Stained preparations of meristemic cells of oninon
roots: (a, b) fragmentation in anaphase; (c, d) fragments
of chromosomes in anaphase; (e) C-mitosis; (f) anaphase
with laggards; (g) fragmentation in metaphase; (h) sticky
metaphase, without pathologies: (i) prophase;
(j) metaphase; (k) anaphase; (l) telophase
Figure 5 Effects of different doses of sorbic acid on MDA
level in roots after 2 days of incubation (n = 10). * P < 0.05
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
(j) (k) (l)
Figure 4 Effects of different doses of benzoic acid on MDA
level in roots after 2 days of incubation. * P < 0.05, ** P < 0.1
Control
Control
g/L
g/L
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a significant dose-dependent increase in the MDA
levels (by 760%) compared to the control samples. At
the same time benzoic acid did not have a significant
effect on the process (Figs. 4 and 5). Since sorbic acid
can be subjected to partial hydrolysis, MDA was
measured in a solution of sorbic acid (0.2 g/L) after
4 days of incubation, but its content exceeded only by
57% compared to the values in the control samples of
onion roots.
Since the biomarkers of oxidative stress usually
showed a two-phase response rather than a linear
response, we expanded the range of sorbic acid dilutions
and increased the period of incubation with acids up to
3 days to identify the dynamics of MDA biosynthesis.
Indeed, this pattern of the two-phase response was
confirmed again [13]. With an increase in sorbic acid
concentration, the level of this biomarker increased
evenly at first, reached its peak, and then dropped
dose-dependent until it reached MDA value in the
control samples (Fig. 6). Like in the previous study,
the maximum concentration of MDA was recorded for
sorbic acid at 200 mg/L. The concentration was above
that in the control group by almost 2000%, i.e. lipid
oxidation level also increased with exposure time.
As far as we know, these are the first experiments
to study the dynamics of lipid oxidation biomarker
generation in the Allium test after exposure to benzoic
and sorbic acids. Both acids reduced root growth and
the mitotic index of apical meristematic cells. However,
these negative phenomena were accompanied by the
simultaneous increase in MDA only in the case of sorbic
acid. These results are consistent with the data on the
treatment of wheat seeds with benzoic acid [34]. This
study did not detect any change in lipid peroxidation
activity different from control when treated with low
concentrations of this preservative.
In the case of benzoic acid, its toxic effects were
probably not associated with oxidative damage to lipids.
In addition, the study showed the protective reaction in
the plant cell to benzoic acid in concentrations of 1 to
10 mM. The reaction was accompanied by an increased
activity of glutamate and malate dehydrogenase,
enzymes activating catabolic and metabolic processes
[35]. In the current study, both the MDA level and
root mass growth increased with an increase in the
concentration of sorbic acid from 20 to 200 mg/L
(Figs. 5 and 6). Thus, there was a clear correlation
between the physiological index and MDA, the latter
being even more sensitive.
According to the literature, the meristematic cell
mitotic index in the control samples when using the
Allium test is both close to our result (12.97 ± 0.48) and
well below it [20, 36, 37]. This indicator could change
depending on the quality of the batch of onions, its
variety, and storage conditions. However, the question
remained whether there was a dependence between the
Figure 6 Effect of different doses of sorbic acid on MDA level in roots after 3 days of incubation (n = 10). * P < 0.05, ** P < 0.1
Control
Table 2 Inhibition of the root mass growth and the mitotic
index after treatment with sorbic acid (n = 9)
Sorbic acid
concentration,
g/L
Root mass
growth, g
Mitotic
index, %
Chromosomal
aberrations based
on the total number
of cells, %
0.02 0.19 ± 0.07 6.84 ± 0.40a 0.29 ± 0.09
0.2 0.05 ± 0.02a 0.00 ± 0.00a* 0.00 ± 0.00a*
Control 0.27 ± 0.04 7.97 ± 0.89 0.15 ± 0.06
a P < 0.05, *1748 cells were observed
Figure 7 Effect of different doses of sorbic acid on MDA level
in roots after 2 days of incubation (n = 9). * P < 0.05, ** P < 0.1
Control
g/L
g/L
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initial level of the mitotic index and the ability of root
cells to fully respond to the effects of toxicants.
To this end, another study was conducted to
examine the toxic effect of sorbic acid solutions at low
and high concentration on onions with a small part of
meristematic dividing cells in control. In this study, the
miotic index in the control samples (Table 2) was 40%
lower than that obtained in the previous study (Table 1).
The conditions of the Allium test in the previous and the
current study did not differ. The comparative analysis
showed the following negative trends in the results.
First, the roots in the experimental groups became soft
and acquired a yellowish hue after 2 days of incubation
with the acid. Second, there was no gain in the root
mass compared to control (Table 2) when treated with
a high-concentration acid solution (0.2 g/L), whereas in
the previous study the gain was 25%, and roots did not
change the color (Fig. 1). Similar negative changes were
recorded at the biochemical level in MDA measurement
(Fig. 7). The lipid oxidation activity in these samples
compared to the previous study was significantly higher
both in the test and control samples.
According to the obtained results, it seems advisable
to select batches of bulbs before the Allium test. This
selection is necessary as the low values of the mitotic
index may result in false positive results, both in terms
of EC50 estimates and biochemical indicators.
CONCLUSION
The results of this study showed that sorbic and
benzoic acids caused toxic effects in the roots of Allium
cepa. These preservatives affected the physiological,
biochemical, cytological, and genetic characteristics of
the plant system. Treating onion roots with these acids
in concentrations of 1 and 2 g/L, which are acceptable
for some food products, was so highly toxic as to lead
to their death. When concentrations of these acids
decreased, EC50 limits for benzoic and sorbic acids were
shown to be 10–20 and 20–100 mg/L, respectively.
These concentrations of preservative solutions induced a
50% retardation in root growth, a significant decrease in
the mitotic index, especially in the case of sorbic acid,
and almost a triple increase in chromosomal disorders.
Thus, these preservatives at very low concentrations
gave a chronic and subchronic toxic effect. Based
on the conducted studies, it is necessary to use the
concentration of food preservatives within their detected
EC50 values to assess these toxicity indicators in the
Allium test. If these conditions are met, it is possible to
simulate the processes of detoxification and metabolism
for these compounds, both at the cellular level and the
whole organism.
Therefore, it can help gain a better understanding
of the biological actions of these agents. Indeed, the
negative effects found under these conditions for sorbic
acid, but not benzoic acid, were correlated with the lipid
oxidation biomarker. In this regard, we believe that the
study of this biomarker can provide valuable information
for monitoring and predicting early effects of sorbic acid
on animal and human cells. Yet, it is probably necessary
to study the role of catabolic processes to determine the
molecular mechanisms of activation of enzymes with
benzoic acid [35].
CONFLICT OF INTEREST
The authors state that there is no conflict of interest.

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