Biodiversity Journal, 2022, 13 (2): 329–338 https://doi.org/10.31396/Biodiv.Jour.2022.13.2.329.

338

Toxicity assessment of a binary nanometric mixture

(ZnO/Fe2O3) in Cornu aspersum (Müller, 1774) (Gastropoda
Helicidae)

Sana Besnaci1*, Mabrouka Bouacha2,Yamine Babouri1, Labiba Zerari1 & Samira Bensoltane1,3

1
Laboratory of Cellular Toxicology, Department of Biology, Faculty of Sciences, Badji Mokhtar University, Annaba
23000 Algeria
2
Laboratory of Biochemistry and Environmental Toxicology, Department of Biochemistry, Faculty of Science, Badji
Mokhtar University, Annaba 23000, Algeria
3
Department of Dental Surgery, Faculty of Medicine, Badji Mokhtar University, Annaba 23000 Algeria
*Corresponding author email: s.besnaci@yahoo.fr

ABSTRACT The effects of harmful mixtures on living organisms are of greater importance due to ex-
posure of these complex mixtures of contaminants in the environment. As nanoparticles
(NPs) released can potentially interact with many pollutants, and even other NPs in this
study, adult Cornu aspersum (Müller, 1774) (Gastropoda Helicidae) were used to estimate
a mixture effect of two NPs “Fe2O3 and ZnO” on the exchange of metabolites (carbohy-
drates, lipids, and proteins), and some oxidative stress biomarkers: Glutathione (GSH),
Glutathione-S-Transferase (GST), Catalase (Cat), and by performing hepatopancreas his-
tological sections of this gastropod after four weeks of treatment. During this period, snails
have ingested wheat flour containing the powder of this mixture at doses of 0, 1, 2, and 3
mg/g of wheat flour. The biochemical assays of metabolites reveal disturbances in metab-
olism by increasing the protein content and decreasing lipid and carbohydrate levels. The
decrease of GSH level, GST activity, and Cat activity proves that failure of the detoxifica-
tion system triggers oxidative stress. The histological study confirms the biochemical results
by the tissue lesions, which are very serious, and in a surprisingly dependent dose manner
with inflammations, necrosis, hypertrophies, degeneration of the connective tissue, and tu-
bular membranes.

KEY WORDS Cornu aspersum; nanoparticles; Fe2O3, ZnO; oxidative stress; histology.

Received 09.12.2021; accepted 23.03.2022; published online 24.04.2022

INTRODUCTION velopment of new innovative materials, systems,

and devices, the fabrication and processing of struc-
tures and materials on a nanoscale in various fields.
In the last decade, industrial use of metallic
oxide nanoparticles has recently found applications Such have given solutions to many environmental
in many fields such as consumer products, medi- problems like availability of clean water, nanocat-
cine, and the environment (Sengul & Asmatulu, alyst for hydrogen generation, dental materials,
2020). Nanotechnology includes the research, de- drug delivery systems, antimicrobial nanopowders,
330 SANA BESNACI ET AL.

nanosensors for the detection of pathogens or toxic ate organisms, doses and methods to evaluate their
materials, gas-separation nanodevices, and much environmental impact accurately, and understand the
more, especially in medicine and healthcare effects of these NPs mixtures.
(Kaushik, 2020 ; Mazari et al., 2021). Each tech- The objective of this study was to evaluate the
nology shows positive aspects but also creates prob- effects of a binary metallic oxide nanoparticles
lems and limitations (Khan et al., 2020). The use (MONPs) mixture on hepatopancreas metabolites
and disposal of NPs will lead to the intentional or rates, antioxidant defenses system changements,
accidental release of NPs into the environment, and histology damages on the snail Cornu aspersum
thereby increasing the environmental exposure of (Müller, 1774) (Gastropoda Helicidae).
these nanoparticles to humans and other species
(Feidantsis et al., 2020; Rather et al., 2021).
The released NPs can potentially interact with MATERIAL AND METHODS
numerous pollutants, including other NPs, leading
to biological effects (bioaccumulation and/or toxic- The land snail Cornu aspersum is a good
ity) that are poorly understood (Deng et al., 2017). bioindicator of terrestrial pollution. The snails were
Based on the individual behavior and toxicity of ma- collected from an uncontaminated site in the
terials it is very important to understand if it is pos- Annaba region (situated in the northeast of Algeria),
sible to predict the toxicity pattern made by their and they were raised in the following optimal envi-
mixture or a whole new toxicity pattern will appear ronmental conditions: photoperiod 18h light/24h,
due to potential interactions with biotic creatures (Li temperature (20 ± 2 °C), humidity 80 to 95%.
et al., 2015). Two types of MONPs were used: nano-Fe2O3
However, it is very difficult to evaluate and de- and nano-ZnO in an equal amount of mixture. They
tect the assessement of more than one chemical on were synthesized in the department of the physics
a contaminated environment (Besha et al., 2020). university of Annaba into two different laboratories.
Studies investigating mixture effects, rather than The first one in the laboratory of magnetism and
individual effects, present a more realistic reflection spectroscopy of solids (LMS2) by mechanical
due to the exposure of complex mixtures of con- grinding from the elemental powder hematite, for a
taminants in the environment (Ko et al., 2017). The final size of about 26nm. The second is in the lab-
evaluation of mixture effects is a difficult task in oratory of study and research of condensed states
environmental assessment. The mixture toxic effect (LEREC) via the co-precipitation method with a
can be defined as an additive, synergistic (>addi- crystallite size closed to 59 nm.
tive), or antagonistic (<additive) action based on the The forty snails were divided into four groups,
results of these models (Ko et al., 2018). in perforated plastic boxes, and the treatment was
Several studies have been done for the evaluation started (the cleaning of the boxes and the change of
of nanoparticles toxicity, most of them agree that zinc the food is carried out twice a week). Three increas-
oxide NPs is of a more remarkable toxic effect than ing doses were chosen from our mixture (nano-
several other NPs (TiO2, CeO2, NiO, CuO, Al2O3, Fe2O3in/nano-ZnO): 1, 2, and 3mg/g of food with
Fe3O4, MgO, SiO2, WO3) as well as iron oxide (Yu a control group that receives only wheat flour.
et al., 2016; Ko et al., 2018). Because of its applica- After four weeks, the hepatopancreas was recov-
tions “Fe2O3 nanometric” in several techniques and ered for biochemical assays of metabolites (total
products that go into diagnosing and treating diseases carbohydrates, total lipids and total proteins) stress
in humans (Luo et al., 2020; Hernández-Hernández parameters (GSH, and GST, and Cat), and histolog-
et al., 2020), it is very important to make these as- ical sections of the four groups.
sessments. The uses of nano zinc oxide have under- A portion of each hepatopancreatic sample (100
gone an increase in use, especially in the field of mg) is used in the protocol for extracting and mea-
cosmetology (Lee et al., 2020) because of its diffu- suring biochemical metabolites (Shibko et al.,
sion characteristics. Both ZnO and Fe2O3 NPs have 1966), carbohydrates according to the method of
effects on the embryonic stage through visible mal- Duchateau & Florkin (1959), lipids according to
formations and accumulations (Besnaci et al., 2016a; Goldsworthy et al. (1972) and proteins according
Amin et al., 2021). It is obligatory to adopt appropri- to Bradford (1976).
 Toxicity assessment of a binary nanometric mixture (ZnO/Fe2O3) in Cornu aspersum (Gastropoda Helicidae) 331

Another fragment of hepatopancreas (500 mg) The observed decrease in Cat is significant at the
of all the groups is ground in a phosphate buffer (pH first dose and very highly significant at the last two-
7.4) to serve for the other enzymatic and non-enzy- dose (Fig. 6).The control group presents a healthy
matic assays, the enzymatic activity of GST (glu- hepatopancreas in which all its tissue constituents
tathione-S-transferase) is dosed according to Habig are in a normal state, it appears formed by the jux-
et al. (1974) method, the enzymatic activity of Cat taposition of numerous tubules, the spaces which
(Catalase) according to Aebi and GSH (glutathione) separate them being occupied by a connective tissue
after deproteinization with the sulfosalicylic acid within which the hemolymph circulates. The lumen
according to Weckbecker & Cory (1988) technique. is bordered by a simple epithelium combining sev-
Three hepatopancreas were kept in 10% forma- eral cell types of various morphologies, which fall
lin for each batch. To make the histological study
on this organ, passing through the following proto-
col: fixation, inclusion, cutting, staining, and assem-
bly. In the end, microscopic observation is made
using a camera microscope.
The means ± standard deviation (SD) are calcu-
lated for each experiment group. The statistical analy-
sis used are the AV1 “one-way analysis of variance”
with two comparison tests that of Fisher and Tuckey.
All calculations were performed using Minitab analy-
sis and data processing software version 16.2.
Knowing that:
P≤0.05: Significant difference compared to the
control (*).
P≤0.01: Highly significant difference compared
to the control (**).
P ≤0.001: Very highly significant difference
compared to the control (***).

RESULTS

Results show variations between the treated

groups and the control one using Fisher and Tuckey
statistical tests for all the studied parameters and at
all used doses (C: Control, M1: mixture at the dose
mg/g, M2: mixture at a dose of 2 mg/g and M3:
mixture at a dose of 3 mg/g).
This difference was illustrated as a decrease in
the carbohydrate level in a highly significant man-
ner at the first dose and highly significant at the
other two doses of the nanometric mixture (Fig. 1).
The lipid level has also decreased in a very highly
significant manner in the first two doses and highly
significant in the third dose of the mixture (Fig. 2).
Conversely, the protein level significantly increased
at the three doses studied (Fig. 3). Figures 1–3. Rate of metabolites (μg/mg tissue) - Fig. 1:
The rate of GSH, GST, and Cat show a decrease Carbohydrates. Fig. 2: Lipids. Fig. 3: Proteins - in the hep-
atopancreas of C. aspersum after four weeks of exposure at
that is significant for GSH and highly significant for 0, 1, 2 and 3 mg/g of the nanometric mixture nano-
GST at the three doses of the mixture (Figs. 4, 5). Fe2O3/nano-ZnO.
332 SANA BESNACI ET AL.

A very small inter-tubular connective tissue with

some inflammatory infiltrates has also been ob-
served (Fig. 8).
Regarding the tissue of the group treated with
the 2mg/g dose of the nanometric mixture, we note
that the observations are the same noted in that of
the 1mg/g dose with a more retracted tubular lumen
(Fig. 9).
The observation of the tissue treated with the
third dose (3 mg/g) shows in addition to the same
observations noted in the first two doses an absence
of tubular light, necrosis, hypertrophies with in-
crease in number of excretory cells (Fig. 10).

DISCUSSION

Several studies were conducted to determine the

toxicity of nanoparticles on human health (Bara-
nowska-Wójcik et al., 2020; Bengalli et al., 2021) ,
and the environment (Kik et al., 2020; Martínez et
al., 2021). As said in the introduction, nanoparticles
as well as small molecules can interact in air, water
or soil, whether or not their combination had effects
and that is what we wanted to know about our mix-
ture ZnO/Fe2O3. Ko et al. (2018) evaluated the
toxic effects of individual and mixture NPs: ZnO,
NiO, CuO, TiO2 and Fe2O3 through the chloro-
phyll level of an alga, they found that the chloro-
phyll level is especially affected at different doses
of ZnO/Fe2O3 mixture.
In our work, the effect of this mixture on
metabolite rates was verified. From the results ob-
tained we can say that a great disturbance in
metabolic has been induced, firstly through the de-
Figures 4–6. Rates of hepatopancreatic stress parameters -
Fig. 4: GSH. Fig. 5: GST. Fig. 6: CAT - of C. aspersum crease of the carbohydrates level. With the same
after four weeks of treatment at 0, 1, 2 and 3 mg/g of the doses, the iron oxide NPs alone induced an increase
nanometric mixture nano-Fe2O3/nano-ZnO. in the carbohydrate content of C. aspersum’s hep-
atopancreas (Besnaci et al., 2016b). Chronic expo-
sure of Oreochromis niloticus (Linnaeus, 1758)
into three main categories: secretory cells, digestive
(Cichliformes Cichlidae) to metallic iron oxide NPs
cells and calcium cells. The cells that compose them
induced an increase in plasma glucose levels (Ates
are of various morphologies that present three types:
et al., 2016). On the other hand, Filippi et al. (2015)
digestive cell, calcium cell and excretory cell. Each found an increase in carbohydrates through the in-
tubule is surrounded by a tubular membrane that crease of each gluconeogenesis and glycogenolysis
forms a rigid wall between two tubules (Fig. 7). following a treatment of human modified hepato-
The tissue examination of the hepatopancreas of cytes by ZnO NPs. On several other biological
the snails treated with the 1 mg/g dose of the nano- models, fish (Lee et al., 2014, Khosravi-Katuli et
metric mixture shows a narrowing of the tubular al., 2018), prokaryotes (He et al., 2011) and even
lumen which led to a grouping of the tubular mass. unicellular ones (Khaldi & Grara, 2016) treatment
 Toxicity assessment of a binary nanometric mixture (ZnO/Fe2O3) in Cornu aspersum (Gastropoda Helicidae) 333

with ZnO NPs induces this increase in the carbohy- level (Zhu et al., 2008). In contrast, Fahmy et al.
drate rate. In our results, we can say that the mixture (2014) reported a decrease after treatment of B.
gave an opposite effect with respect to each of the alexandrina snails with nano-ZnO. While the results
separate NPs Fe2O3 and ZnO. This decrease in car- of Khaldi & Grara (2016) NPs ZnO show an increase
bohydrates is explained by the increase in energy in certain concentrations and a decrease in another
requirements following the detoxification reactions higher, so it is a question of dose. Our variation is ex-
after exposure to xenobiotics (ZnO, Fe2O3) and the plained by the detoxification system reactions and its
prolonged youth, which will therefore reduce the enzymes “stress proteins”. The presence of metals
reserves and energy inputs. may also induce the synthesis of proteins enabling to
The level of lipids also decreased in a very sig- connection between them (Zidar et al., 2009), their
nificant way at the three doses of the mixture, this transfer in granules corresponds to intracellular detox-
can be explained by the chemical stress caused by ification pathways described in soil invertebrates.
the xenobiotic (ZnO and Fe2O3 NPs). Besnaci et al., Metals tend to bind to cytoplasmic metalloproteins.
(2016b) also found this result with C. aspersum Metals‐metalloproteins can then be excreted in pellet
snails after treatment with nano- Fe2O3. The appli- form after lysosomal action (Pihan, 2001).
cation of nanoscale zinc oxide on another type of The oxidative stress caused by a particle is con-
snail (freshwater) Biomphalaria alexandrina (Ehren- sidered one of the most important mechanisms of
berg, 1831) (Gastropoda Planorbidae) revealed a de- toxicity of nanoparticles, especially for particles
crease on total lipids and total cholesterol contents in containing transition metals. Metals, such as Zn, Fe,
hemolymph and tissues (Fahmy et al., 2014). Cu and Mn are considered as transitional metals due
For the protein level, the results show an increase to their great participation in metabolic reactions,
at all three doses. Boucenna (2016), Besnaci et al. serving as cofactors for several enzymes (Saad,
(2016b) and Sidiropoulou et al. (2018) had also noted 2003; Sellami et al., 2017). In several cases, the ef-
this variation in C. aspersum after treatment with fects of predator stress were affected by the accu-
nanoscale iron oxide. Treatment with nanometric iron mulated metals, thus providing strong evidence for
oxide on rats also induced an increase in the protein stressor interactions.

Figures 7–10. Histological sections of the Cornu aspersum hepatopancreas. Fig. 7: control. Figs. 8–10: treated with the
nano-Fe2O3/nano-ZnO mixture, (a) tubule at 400-fold magnification (b) tissue organization at 100-fold magnification. L:
lumen; dc: digestive cells; cc: calcium cells; ec: excretory cells; ict: intertubular connective tissue; BM: basement membrane;
N: nucleos; (*L) retracted lumen; (#L) lumen absent; (arrow) tubular mass grouping; (##) inflammatory infiltrates; (**)necro-
sis; ( triangle) hypertrophy; (zig zag) high number of excretory cells; (star) connective tissue almost absent.
334 SANA BESNACI ET AL.

In order to understand the mechanism of our iron oxide that is also able to release free metal ions
nanoparticles mixture, we studied three markers of (Fahmy et al., 2014).
oxidative stress including total Glutathione concen- The inhibition of CAT activity was also noted,
tration, Glutathione-s-transferase activity and Cata- after exposure to ZnO/Fe2O3 NPs mixture in hep-
lase activity. atopancreas tissue of treated snails may be due to
The significant decrease noted at the three studied the enhancement of the peroxidation end product,
doses of our mixture of the rate of the three evalua- MDA, which is known to inhibit protein synthesis
tion parameters of oxidative stress (GSH, GST and and the activities of certain enzymes (Fahmy et al.,
CAT) indicated that there was a failure in the detox- 2014). Several nano zinc oxide studies have noted
ification system. Many metallic nanoparticles have this decrease, Panda et al. (2003) and Dimkpa et al.
been studied to exert their toxicity through oxidative (2012) explained the absence of increased CAT ac-
stress. Metallic nanoparticles can liberate free ions tivities with ZnO NPs by no-reactivity of these en-
in the cytoplasm upon surface oxidation (Kumar, zymes to Zn ions in wheat. Fahmy et al., (2014)
2006; Asharani et al., 2008; Dubey et al., 2015). report a decrease in Catalase activity at the level of
The results of our study confirmed that the de- the haemolymph in the snail B. alexandrina treated
crease in GSH content in digestive gland appears to with different doses of NPs of zinc oxide, they sug-
be a common response of mollusks to metal expo- gested at the end of the study that ZnO inhibits cata-
sure (Xiong et al., 2011; Ali et al., 2012; Fahmy, lase. Du et al. (2017) noted a decrease in Catalase
2014). Reduction in GSH after ZnO and Fe2O3 NPs in Danio rerio (F. Hamilton, 1822) (Cyprinidae) ze-
exposure could be due to its increased use in free brafish embryos after treatment with ZnO NPs. Ac-
radical scavenging. Several studies on the NPs of cording the results noted by sarkar and Sil, (2014)
zinc oxide and iron oxide have noted this variation. and Besnaci et al. (2019) after treatment with NPs
Following treatment with NPs of iron oxide, of Fe2O3, not only zinc inhibits Catalase activity.
Boucenna (2016) on C. aspersum and Sarker & Sil So the effect of the mixture is a synergistic effect
(2014) on cell cultures noted this decrease. Studies of each of the two NPs.
by Fahmy et al., (2014) and Falfushynska et al. Histological damages are one of the conse-
(2015) also reported a decrease in GSH level, the quences of oxidative stress, associated with behav-
first in B. alexandrina following treatment with ZnO ioral, physiological and also biochemical
NPs and the second in Unio tumidus Philipsson, disturbances. The histological study showed that the
1788 (Bivalvia Unionidae) treated with Zn NPs. mixture causes unambiguous tissue damage. Indeed,
GST is an enzyme that participates in the detox- histological examination of the hepatopancreas
ification process due to conjugation reaction be- highlights the appearance of lymphoplasmacytic in-
tween GSH and xenobiotics (Cummins et al., 2011). flammatory infiltrates at the lowest dose; this could
Thiol compounds, such as reduced and oxidized be a first biological response due to the presence of
GSH, represent the initial protective substances xenobiotics. The digestive gland is a very important
against heavy-metal ions and other pollutants. In ac- organ in gastropods, responsible for the production
cordance with our result, Escobar et al. (1996) and of enzymes, the absorption and storage of nutrients,
Sanzgiri et al. (1997) reported that the enhanced endocytosis and excretion of certain particles and
free-radical concentrations resulting from oxidative mainly involved in the detoxification of pollutants.
stress conditions can cause loss of GST enzymatic Histological and histochemical changes are used as
activity (Fahmy, 2014). It has been considered that biomarkers of xenobiotic exposure.
the low levels of oxidative stress induce the expres- The observed tissue damage shows the toxicity
sion of protective mechanisms, whereas larger doses of the metal nanoparticles studied and confirm the
result in the activation of pro-inflammatory mecha- biochemical results obtained. In the same direction
nisms, and cell death at the extreme levels (Nel et (evaluation of the toxicity of nano- Fe2O3 on C. as-
al., 2001). Freshwater snail Biomphalaria alexand- persum) (Besnaci et al., 2016b, Boucenna, 2016)
rina were treated with different doses of nano-ZnO, other studies noted the same observations: inflam-
and found a decrease in GST activity, which con- matory infiltrates, hypertrophies, and necrosis. ..etc.
firms the strong action of nano-zinc oxide with the NPs induce the release of ROS, disrupting the func-
release of ROS and especially in combination with tioning of organelles such as mitochondria and lyso-
 Toxicity assessment of a binary nanometric mixture (ZnO/Fe2O3) in Cornu aspersum (Gastropoda Helicidae) 335

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