The use of selected biomarkers, fatty acid and histopathology features to detect the possible toxic effects of the penconazole-containing fungicide Topas on marine clam siphons (Ruditapes decussatus)

Introduction

The aquatic ecosystem serves as a primary sink for potentially dangerous chemicals released from industrial and domestic sources. Among these contaminants, pesticide use has skyrocketed in recent years. Approximately 2×10⁹ kg are utilized annually (Kalliora et al. 2018Kalliora C., Mamoulakis C., Vasilopoulos E., et al. 2018. Association of pesticide exposure with human congenital abnormalities. Toxicol. Appl. Pharmacol. 346(March): 58-75. https://doi.org/10.1016/j.taap.2018.03.025
), but only 0.1% of the applied pesticides reach the target pests raising public concerns about potential hazardous effects on non-target creatures (Hart and Pimentel 2002Hart K.A., Pimentel D. 2002. Environmental and economic costs of pesticide use. In: Pimentel D, editor. Encyclopedia of pest management. New York: Marcel Dekker; p. 237-239. https://doi.org/10.1201/NOE0824706326.ch99
).

Coastal ecosystems such as lagoons are complex and dynamic, with constantly changing environmental circumstances (Kamel et al. 2014Kamel N., Burgeot T., Banni M., et al. 2014. Effects of increasing temperatures on biomarker responses and accumulation of hazardous substances in rope mussels (Mytilus galloprovincialis) from Bizerte lagoon. Environ. Sci. Pollut. Res. 21: 6108-6123 https://doi.org/10.1007/s11356-014-2540-5
). Some Mediterranean lagoons in Tunisia have become the most extensively changed and threatened habitats, owing primarily to urbanization and agricultural and industrial releases (Barhoumi et al. 2014Barhoumi B., Clérandeau C., Le Menach K., et al. 2014. Pollution biomonitoring in the Bizerte lagoon (Tunisia), using grass goby, Zosterisessor ophiocephalus (Teleostei, Gobiidae) as a bioindicator species. Mar. Environ. Res. 101: 184-195. https://doi.org/10.1016/j.marenvres.2014.07.002
), which reach shorelines via precipitation, irrigation or groundwater flux (Surfrider 2020Surfrider. 2020. Chemical pollution of the ocean: the pesticide issue. Retrieved October 3, 2024, from https://www.surfrider.eu/learn/news/pollution-chimique-ocean-question-pesticides
), making them an extremely useful system for ecotoxicological monitoring. At the local level, various studies have been conducted to assess the environmental influence associated with aquatic pollution in Tunisia, indicating different sources of pollutants and causes of pesticide contamination (López Martinez 2024López Martínez R. 2024. Study of the establishment of an allocated zone for aquaculture (AZA) in the Lagoon of Bizerte, Tunisia. 112 pp.
). Indeed, in the Bizerte lagoon system, Necibi and Mzoughi (2020)Necibi M., Mzoughi N. 2020. Distribution of organochlorine pesticides in sediment cores from the Bizerte Lagoon (Tunisia). Int. J. of Environ. Anal. Chem. 100(10): 1118-1132. https://doi.org/10.1080/03067319.2019.1650173
reported contamination of sediments by pesticides, manifested by high concentrations of organochlorine pesticide pollutants. In addition, several authors have reported that the uncontrolled use of pesticides and fertilizers on agricultural land has increased the nitrate content in Tunisian aquatic ecosystem pollution (Grünberger et al. 2024Grünberger O., Hamdi R., Lagacherie M., et al. 2024. Pesticide contamination pattern of surface water in an urban-agricultural mediterranean watershed (Wadi Guenniche, Bizerte Lagoon, Northern Tunisia). J. Environ. Sci. Health. Part B. 59(8): 521-539. https://doi.org/10.1080/03601234.2024.2375905
).

Penconazole (PEN) is a typical sterol demethylation inhibitor of triazole fungicide that controls numerous pathogens in crops such as fruits, vegetables and tea plants (Husak et al. 2017Husak V.V., Mosiichuk N.M., Storey J.M., et al. 2017. Acute exposure to the penconazole-containing fungicide Topas partially augments antioxidant potential in goldfish tissues. Comp. Biochem. Physiol. Part - C. 193: 1-8. https://doi.org/10.1016/j.cbpc.2016.12.003
). Several characteristics make it persistent in soil and water, such as high chemical stability and low biodegradability (Wang et al. 2011Wang C., Wu Q., Wu C., et al. 2011. Application of dispersion-solidification liquid-liquid microextraction for the determination of triazole fungicides in environmental water samples by high-performance liquid chromatography. J. Hazard. Mater. 185(1): 71-76. https://doi.org/10.1016/j.jhazmat.2010.08.124
). Accordingly, there is concern about the extensive application of triazole fungicides and their possible detrimental effects on non-target organisms in both terrestrial and aquatic ecosystems resulting from spray drift and surface runoff (Konwick et al. 2006Konwick B.J., Garrison A.W., Avants J.K., et al. 2006. Bioaccumulation and biotransformation of chiral triazole fungicides in rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 80: 372-381. https://doi.org/10.1016/j.aquatox.2006.10.003
). However, little is known about PEN availability in surface water. For example, the study of Dalvie et al. (2003)Dalvie M.A., Cairncross E., Solomon A., et al. 2003. Contamination of rural surface and ground water by endosulfan in farming areas of the Western Cape, South Africa. Environ Health. 2(1): 1. https://doi.org/10.1186/1476-069X-2-1
showed that surface waters in the Western Cape, South Africa, contain less than 2 μg L^-1 of this fungicide.

High concentrations of triazole fungicides cause various toxic outcomes, including carcinogenicity, reproductive toxicity and hepatotoxicity in mammals (Peffer et al. 2007Peffer R.C., Moggs J.G., Pastoor T., et al. 2007. Mouse liver effects of cyproconazole, a triazole fungicide: role of the constitutive androstane receptor. Toxicol. Sci. 99: 315-325. https://doi.org/10.1093/toxsci/kfm154
). In addition, they are principally considered to affect lipid biosynthesis and metabolism pathways (Hermsen et al. 2011Hermsen S.A.B., Pronk T.E., van den Brandhof E.J., et al. 2011. Chemical class-specific gene expression changes in the zebrafish embryo after exposure to glycol ether alkoxy acids and 1,2,4-triazole antifungals. Reprod. Toxicol. 32(2): 245-252. https://doi.org/10.1016/j.reprotox.2011.05.010
). An LC₅₀ (median lethal concentration) of 20.55 mg L^-1 for paclobutrazol has been reported for zebrafish (Danio rerio) (Ding et al. 2009Ding F., Song W.H., Guo J., et al. 2009. Oxidative stress and structure-activity relationship in the zebrafish (Danio rerio) under exposure to paclobutrazol. J. Environ. Sci. Health. - Part B Pesticides, Food Contaminants, and Agricultural Wastes. 44(1): 44-50. https://doi.org/10.1080/03601230802519652
). The acute toxicity of difenoconazole on zebrafish was 1.17 mg L^-1 for larvae, 1.45 mg L^-1 for adult fish, and 2.34 mg L^-1 for embryos (Mu et al. 2013Mu X., Pang S., Sun X., et al. 2013. Evaluation of acute and developmental effects of difenoconazole via multiple stage zebrafish assays. Environ. Pollut. 175: 147-157. https://doi.org/10.1016/j.envpol.2012.12.029
). The hepatic antioxidant enzymes, the RNA/DNA ratio and haematological and plasma biochemical parameters are affected in rainbow trout (Oncorhynchus mykiss) after exposure to 0.5 mg L^-1 of propiconazole (Li et al. 2010aLi Z.H., Zlabek V., Grabic R., et al. 2010a. Effects of exposure to sublethal propiconazole on the antioxidant defense system and Na+-K+-ATPase activity in brain of rainbow trout, Oncorhynchus mykiss. Aquat. Toxicol. 98(3): 297-303. https://doi.org/10.1016/j.aquatox.2010.02.017
). A previous study also detected gene expression changes in zebrafish embryos after treatment with 4 mg/L of flusilazole (Hermsen et al. 2011Hermsen S.A.B., Pronk T.E., van den Brandhof E.J., et al. 2011. Chemical class-specific gene expression changes in the zebrafish embryo after exposure to glycol ether alkoxy acids and 1,2,4-triazole antifungals. Reprod. Toxicol. 32(2): 245-252. https://doi.org/10.1016/j.reprotox.2011.05.010
). According to the harmonized classification and labelling approved by the European Union, PEN is highly toxic to aquatic organisms with long-lasting effects (hazard statements H400 and H410) (European Chemicals Agency (ECHA), 2023European chemicals agency (ECHA) 2023. Retrieved 23 May 2024 from https://echa.europa.eu/substance-information/-/substanceinfo/100.060.231
). However, the impact of PEN on bivalves, namely clams, is quite limited. Only the work of Yoloğlu (2019)Yoloğlu E. 2019. Assessment of Na⁺/K⁺ -ATPase, Mg²+-ATPase, Ca²+-ATPase, and Total-ATPase Activities in Gills of Freshwater Mussels Exposed to Penconazole. Comm. J. Biol. 3: 88-92. https://doi.org/10.31594/commagene.632082
reported the assessment of Na⁺/K⁺-ATPase, Mg²⁺-ATPase, Ca²⁺-ATPase, and total-ATPase activities in gills of freshwater mussels exposed to PEN.

In addition to their varying harmfulness, triazole fungicides are also known to induce oxidative stress, one of the main mechanisms of toxicity associated with these xenobiotics (Monserrat et al. 2007Monserrat J.M., Martinez P.E., Geracitano L.A., et al. 2007. Pollution biomarkers in estuarine animals: critical review and new perspectives. Comp. Biochem. Physiol. - Part C 146: 221-234. https://doi.org/10.1016/j.cbpc.2006.08.012
). Indeed, PEN has been displayed to increase production of reactive oxygen species (ROS), prompting alterations in the intracellular redox status and inducing oxidative damage to cellular macromolecules, as observed in invertebrates and vertebrate species (Chaâbane et al. 2016Chaâbane M., Ghorbel I., Elwej A., et al. 2016. Penconazole alters redox status, cholinergic function, and membrane-bound ATPases in the cerebrum and cerebellum of adult rats. Hum. Exp. Toxicol. 36(8): 854-866. https://doi.org/10.1177/0960327116672911
, Yoloğlu 2019Yoloğlu E. 2019. Assessment of Na⁺/K⁺ -ATPase, Mg²+-ATPase, Ca²+-ATPase, and Total-ATPase Activities in Gills of Freshwater Mussels Exposed to Penconazole. Comm. J. Biol. 3: 88-92. https://doi.org/10.31594/commagene.632082
). In this line, it is well documented that lipids and their most common components, fatty acids (FAs) are particularly susceptible to the oxidative reactions of ROS (Pamplona 2008Pamplona R. 2008. Membrane phospholipids, lipoxidative damage and molecular integrity: a causal role in aging and longevity. Biochim. Biophys. Acta. 1777(10): 1249-1262. https://doi.org/10.1016/j.bbabio.2008.07.003
). Though lipids and FAs have been demonstrated to be potent proxies for contamination-induced stress in animals (Signa et al. 2015Signa G., Di Leonardo R., Vaccaro A., et al. 2015. Lipid and fatty acid biomarkers as proxies for environmental contamination in caged mussels Mytilus galloprovincialis. Ecol. Indic. 57: 384-394. https://doi.org/10.1016/j.ecolind.2015.05.002
), no scientific research has yet been released regarding PEN’s impact on the composition of FAs in bivalves.

Molluscs, particularly bivalves, have assumed a major role in assessing levels of contaminants worldwide due to characteristics such as their sedentary behaviour, filter-feeding practices, high filtration rate and capacity to concentrate contaminants (Chalghmi et al. 2016Chalghmi H., Bourdineaud J.P., Haouas Z., et al. 2016. Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon. Arch. Environ. Contam. Toxicol. 70(2), 241-256. https://doi.org/10.1007/s00244-015-0185-0
). The clam Ruditapes decussatus (Linnaeus, 1758), one of the plentiful bivalves on the Tunisian coasts (Hamza-Chaffai et al. 2003Hamza-Chaffai A., Pellerin J., Amiard J.C. 2003. Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabès (Tunisia). Environ. Int. 28(7): 609-617. https://doi.org/10.1016/S0160-4120(02)00102-2
) living in muddy sand deposits of coastal areas, is commonly used and judged as an effective sentinel species, specifically in confined coastal environments (Costa et al. 2013Costa P.M., Carreira S., Costa M.H., et al. 2013. Development of histopathological indices in a commercial marine bivalve (Ruditapes decussatus) to determine environmental quality. Aquat. Toxicol. 126(2013): 442-454. https://doi.org/10.1016/j.aquatox.2012.08.013
). Given the aforementioned factors, it is quite important to investigate the damage produced by Topas 100 EC to the antioxidant defence system, histoarchitecture, and FA profiles in the siphons of R. decussatus, which represent the primary routes of toxicants to enter its body at different concentrations (4, 40 and 400 µg L^-1). Hence, based on the current study, using the battery of cellular and biochemical markers along with histological analysis proves useful for assessing Topas 100 EC contamination in marine invertebrates.

Material and methods

Sampling method and experimental design

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Clams were collected from a site that is far from anthropogenic activities within the Bizerte lagoon (37°11'20.4"N 9°51'16.2"E), located on Tunisia’s North coast in the western Mediterranean Basin (Ghribi et al. 2020Ghribi F., Richir J., Bejaoui S., et al. 2020. Trace elements and oxidative stress in the Ark shell Arca noae from a Mediterranean coastal lagoon (Bizerte lagoon, Tunisia): are there health risks associated with their consumption? Environ. Sci. Pollut. Res. 27(13): 15607-15623. https://doi.org/10.1007/s11356-020-07967-2
). To minimize differences in biochemical responses, organisms with similar shell length (3.86±0.39 cm) and weight (8.75±2.33 g) were collected in October 2021. R. decussatus specimens were carried straight to the laboratory at the Faculty of Science of Tunis in aerated tanks holding saltwater, and then acclimated for 72 hours in an aquarium containing 35 L of seawater with constant aeration (temperature 19±2°C, salinity 36±1, oxygen 6.2 mg/L and a 12 light to 12 dark photoperiod). The sampling area’s properties were used to maintain salinity, temperature and pH during the acclimation and experimental periods (Ghribi et al. 2020Ghribi F., Richir J., Bejaoui S., et al. 2020. Trace elements and oxidative stress in the Ark shell Arca noae from a Mediterranean coastal lagoon (Bizerte lagoon, Tunisia): are there health risks associated with their consumption? Environ. Sci. Pollut. Res. 27(13): 15607-15623. https://doi.org/10.1007/s11356-020-07967-2
). Following acclimation, the R. decussatus specimens were separated into four groups of 30 individuals each and transferred to 25 L experimental aquaria in a duplicate design (n=15) under controlled circumstances, as described in the present study.

Before exposure, the Topas 100 EC stock solution (Syngenta, Bâle, Switzerland) containing penconazole (active ingredient of 100 g L^-1) was prepared in seawater. We diluted the solution to obtain three concentrations (4, 40, and 400 µg L^-1). The Topas-exposed and non-exposed groups were designed as follows (Fig. 1):

• Group I (control): unexposed R. decussatus kept in Topas-free water.

• Group II (D1): R. decussatus exposed to Topas dose of 4 µg L^-1 for 96 hours.

• Group III (D2): R. decussatus exposed to Topas dose of 40 µg L^-1 for 96 hours.

• Group IV (D3): R. decussatus exposed to Topas dose of 400 µg L^-1 for 96 hours.

Fig. 1.  Experimental design of Ruditapes decussatus exposure to Topas.

The D1 concentration used in this study was based on the PEN concentration found in surface waters (2 µg L^-1) in the Western Cape, South Africa (Dalvie et al. 2003Dalvie M.A., Cairncross E., Solomon A., et al. 2003. Contamination of rural surface and ground water by endosulfan in farming areas of the Western Cape, South Africa. Environ Health. 2(1): 1. https://doi.org/10.1186/1476-069X-2-1
). To ascertain whether R. decussatus specimens are tolerant to elevated concentrations of this pesticide, D2 and D3 were selected to ensure a detectable impact of PEN.

The saltwater was replaced every 24 hours to ensure water quality, and Topas concentrations were restored. Throughout the experiment, there was no mortality in either the control or the Topas-treated groups. The trials were carried out following ethical standards (EC Directive 86/609/EEC) and were authorized by the Ethical Committee of the Faculty of Sciences of Tunis.

R. decussatus specimens (n=15) were dissected on ice to obtain siphon tissues, then homogenized in 10% Tris-HCl buffer (100 mM; pH=7.4) containing 1 mM EDTA and 1 mM PMSF and centrifuged at 9000 × g for 20 min at 4°C (Centrifuge UNIVERSAL 320R, Hettich, Germany). The obtained supernatants were stored in Eppendorf tubes at −80°C for biomarker assays (Fouzai et al. 2020aFouzai C., Trabelsi W., Bejaoui S., et al. 2020a. Cellular toxicity mechanisms of lambda-cyhalothrin in Venus verrucosa as revealed by fatty acid composition, redox status and histopathological changes. Ecol. Indic. 108. https://doi.org/10.1016/j.ecolind.2019.105690
). Other gill specimens were homogenized using an Ultra-Turrax (T18: UGS 13396299, IKA, Germany) and then conserved at -20°C for FA analysis (n=6). For histological analysis, three specimens from each condition (n=3) were cleaned under running water, fixed in 10% buffered formalin solution and embedded in paraffin (Martoja and Martoja 1967Martoja R., Martoja-Pierson M. 1967. Initiation aux techniques de l’histologie animale, Masson: Paris, 345.
).

Results

Estimation of FRAP capacity and AChE activity

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As shown in Figure 2A, the antioxidant capacity measured by FRAP was increased significantly in all treated Topas groups (p<0.01). However, AChE activity decreased significantly by 74% and 65% in groups D2 and D3 during the treatment period (Fig. 2B).

Fig. 2.  (A) FRAP capacity and (B) acetylcholinesterase activity (AChE) in the siphons of untreated (control) and treated Ruditapes decussatus with different concentrations of Topas (4, 40 and 400 μg L^-1) for 96 hours. Values are expressed as means±SE (n=8). D1, D2 and D3 groups vs control group (CT): *p<0.05, **p<0.01, ***p<0.001.

Enzymatic antioxidant levels

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Table 2 and Figure 3 show the levels of enzymatic antioxidative responses in the siphons of R. decussatus clams. SOD activities were significantly incremented by the Topas treatment (+20 and +59%) at doses D2 and D3 when compared with the control group, while no significant change was observed at dose D1. For CAT activity, a significant enhancement was observed in siphon tissues of R. decussatus (p<0.001) exposed to all three concentrations of Topas when compared with the negative control group (Table 2). Additionally, Topas exposure led to a significant 44% increase in GPx activity in siphons of clams treated with dose D3 when compared with the control. For GST activity (p<0.001), a significant increase was recorded for all treated groups (Table 2).

Table 2.  Enzymatic and non-enzymatic parameters: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), metallothionein (MTs), glutathione reduced (GSH) and ascorbic acid levels in siphons of Ruditapes decussatus under exposure to graded doses of Topas (4, 40 and 400 μg L^-1) for 96 hours.

Parameters and treatment	Control	4 μg L-1	40 μg L-1	400 μg L-1	Fд /H∂	P
SODα	229.48±4.54a	246.37±9.01ab	275.11±10.73b	364.72±14.33c	22,39	<0.001
CATβ	10.12±0.47a	66.6±1.26b	24.25±0.73c	54.76±0.76d	29,09	<0.001
GPxλ	233.582±0.93a	257.11±5.05a	235.6±8.7a	336.28±17.38b	7,83	<0.001
GSTε	0.002±0a	0.007±0b	0.01±0.001c	0.01±0c	25,35	<0.001
MTsγ	0.99±0.06a	1.21±0.1a	1.13±0.04a	2.38±0.11b	20,08	<0.001
GSH£	12.55±0.66a	14.42±0.76a	15.59±1.4b	16.01±0.78b	2,7	0.0654
Ascorbic acid ¥	11.16±0.53a	13.61±1.26a	12.74±1.06a	26±2.3b	19,59	<0.001

Values are means ± standard error for eight clams in each group.
Superscript letters a, b, c and d indicate significant differences (p<0.05) between exposure concentrations.
^α unit/mg of protein
^β nmol of H₂O₂/min/mg of protein
^λ nmol of GSH/min/mg of protein
^ε mmol/min/mg of protein
^γ µmol of GSH/mg of protein
^£ µg/mg of protein
^¥ mmol/mg of protein
^д ANOVA F Test
^∂ Kruscal-Wallis H test

Fig. 3.  The star plots of biomarker responses (CAT, SOD, GPx, GST, MTs, GSH and AA) for the clam R. decussatus exposed to different concentrations of Topas (4, 40 and 400 µg L^-1) for 96 hours.

Fatty acid composition

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The FA profiles of the siphons are illustrated in Table 3. Twenty-four different FAs were identified for all specimens. The major FA class in the untreated (control) group was saturated fatty acids (SFAs) (up to 47.11% of total FAs), followed by polyunsaturated fatty acids (PUFAs) and then monounsaturated fatty acids (MUFAs) (with 43.03% and 9.87%, respectively, of total FAs) in siphons. Overall, compared with the control group, PUFAs increased significantly, whereas SFAs decreased significantly. Following the treatment of R. decussatus with Topas, the amount of n-6 PUFAs (16.73% of total PUFAs in the control group in siphons) increased significantly (p<0.001) in siphons. In addition, a similar trend was observed for those of n-3 PUFAs in doses D1 and D2 (24.35% of total PUFAs in the control group) but showed a significant depletion (p<0.05) at the highest dose exposure, D3. Our results revealed a significant increase in arachidonic acid (ARA, C20:4n-6) in the siphons treated with the doses D1 and D2 when compared with the control. For the levels of eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) in siphons, similar responses were observed, with a significant increase (p<0.01) of EPA at dose D2 and a significant increase of DHA (p<0.01) at doses D2 and D3. Consequently, there was a significant positive correlation between EPA and DHA levels in siphons. A non-methylene-interrupted dienoic (NMID) FA (C22:2i/2j) was found in our specimens and its level was greatly elevated in all treatment groups. Following the identification of all FAs, the data were utilized for a hierarchical cluster analysis using a heatmap and a dendrogram. This produced a group of samples of the control group that had been isolated from the three Topas doses (Fig. 4).

Table 3.  The fatty acid composition (%) in the siphon tissue of Ruditapes decussatus control (CT) and groups exposed to different doses of Topas (4, 40 and 400 μg L^-1) for 96 hours.

Fatty acids	Control	D1 (4 μg L-1)	D2 (40 μg L-1)	D3 (400 μg L-1)
C14: 0	24.15±0.88	21.21±1.85	5.51±0.48***	21.69±0.47*
C15: 0	1.26±0.12	1.13±0.17	0.81±0.09*	1.91±0.19*
C16: 0	11.57±0.6	7.58±0.68**	4.82±0.3***	7.79±0.22**
C18: 0	4.64±0.12	5.86±0.63	3.72±0.18**	5.74±0.27**
C20: 0	2.07±0.22	0.22±0.05***	2.09±0.16	0.85±0.09**
C22: 0	3.42±0.53	0.37±0.02**	5.56±0.25**	0.41±0.02**
∑SFA	47.11±0.31	36.38±0.46*	22.51±0.85***	38.39±0.47***
C15: 1	0.42±0.03	0.41±0.05	0.5±0.002*	1.2±0.09***
C16: 1	2.1±0.05	2.1±0.36	1.28±0.17**	1.09±0.11***
C18: 1	3.25±0.6	10.45±1.27**	2.85±0.29	6.52±0.18**
C20: 1	2.76±0.51	3.19±0.44	4.17±0.28*	4.1±0.33*
C22: 1	1.34±0.11	0.62±0.08**	2.59±0.12***	0.58±0.06**
∑MUFA	9.87±0.85	16.77±0.76*	11.39±0.26	13.49±0.27**
C18: 2n6 (LA)	6.95±0.68	3.95±0.36**	2.4±0.09**	2.6±0.41**
C18: 3n6	0.85±0.11	1.07±0.13	0.77±0.07	2.4±0.37**
C18: 3n3 (ALA)	3.17±0.16	2.34±0.22*	2.38±0.22*	1.09±0.11***
C18: 4n3	2.93±0.33	5.12±0.45**	3.79±0.09*	2.17±0.23
C20: 2n6	2.86±0.19	13.07±0.81***	3.89±0.18**	11.15±1.19**
C20: 3n6	0.76±0.07	2.41±0.11***	3.2±0.15***	1.1±0.16
C20: 4n6 (ARA)	2.22±0.27	3.38±0.38*	5.79±0.18***	2.56±0.34
C20: 5n3 (EPA)	3.68±0.23	4.41±0.34	5.11±0.23**	2.86±0.16*
2i/2j (NMID)	1.94±0.08	3.55±0.47*	8.33±0.09***	2.97±0.22**
C22: 2n6	2.22±0.23	1.88±0.17	7.15±0.47***	1.39±0.25*
C22: 5n6	0.87±0.1	0.34±0.05**	3.74±0.51**	0.51±0.06*
C22: 5n3 (DPA)	2.43±0.09	3.23±0.41	6.16±0.46***	2.56±0.35
C22: 6n3 (DHA)	12.14±0.51	10.36±0.85	13.39±0.06*	14.76±0.28**
∑PUFA	43.03±0.54	55.11±0.31**	66.09±0.87***	48.12±0.28***
n-3 PUFA	24.35±0.28	25.47±1.74	30.83±0.42***	23.45±0.18*
n-6 PUFA	16.73±0.6	26.1±0.88***	26.93±0.44***	21.7±0.5**
n-6/n-3 PUFA	0.69±0.03	1.03±0.04**	0.87±0.01**	0.93±0.03**

Values are expressed as means±standard error (n=6).
*p < 0.05; **p < 0.01; ***p < 0.001.
SFA, saturated fatty acid(s); MUFA, monounsaturated fatty acid(s); PUFA, polyunsaturated fatty acid(s); NMID, non-methylene-Interrupted dionic fatty acids (C22:2i + C22:2j); n-3 PUFA, omega 3 fatty acids; n-6 PUFA, omega 6 fatty acids; ARA, arachidonic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; SA, stearic acid; GLA, γ-linolenic acid.; LA, linoleic acid; ALA, α-linolenic acid. The bold values in Table 3 represent the sum of SFA, MUFA, and PUFA. This bold formatting is used to highlight these totals.

Fig. 4.  Heatmap depicting an overall comparison of the whole fatty acids data set in Ruditapes decussatus siphons against different concentrations of Topas (µg L^–1). The pink square border shows the fatty acids with the highest sensitivity when compared with the respective ones in the control. CT, control; D1, 4 µg L^–1; D2, 40 µg L^–1; D3, 400 µg L^–1.

Histopathological analysis

 ⌅

Histopathological observations showed significant lesions of the tissue analysed compared with specimens collected from the control tanks (Fig. 5). The siphons of the control group showed no morphological abnormalities and well-defined epithelial cells (Fig. 5A). In contrast, compared with the control group, exposure to Topas at different concentrations led to an increase in damage severity in a concentration-dependent manner. Contaminated siphons exposed to doses D1 and D2 of Topas showed changes such as rupture of epithelial cells (Fig. 5B and C). Dose D3 caused further and more severe histological damage ranging from lipofuscin granules to rupture, haemocyte infiltration, vacuolization and deformation of epithelial cells when compared with the control group (Fig. 5D).

Fig. 5.  Representative histological sections of the siphons of Ruditapes decussatus control (A) and exposed to different Topas concentrations (B, 4 µg L^-1; C, 40 µg L^-1; D, 400 µg L^-1) after 96 hours of exposure, stained with haematoxylin-eosin. EPC, epithelial cells; mt, muscular tissue; hi, haemocyte infiltration; lgd, lipofuscin granule degradation. Blue circles indicate rupture; red circles indicate vacuolization; arrowheads indicate deformation of epithelial cells. Scale bar=40 μm

Multivariate analysis

 ⌅

The PCA, applied to better elucidate the differential effects of the series of Topas concentrations, produced a two-dimensional pattern explaining 65.9% of the total variance, including factor 1 (43.7%) and factor 2 (22.2%), as shown in Figure 6. The PCA biplot of all the biochemical data depicted a clear separation between control and Topas-exposed clams. In this line, a strong correlation was noted between the oxidative stress biomarkers and doses D1, D2, and D3, as evidenced by an increase in FRAP, MDA, H₂O₂, AOPP, PCO, and GSH levels, as well as SOD, CAT, GPx, GST, AA and MT levels (Fig. 7). Overall, the PCA performed on the whole dataset highlighted the clear separation between the experimental groups, indicating activation of detoxification mechanisms. The relationships observed between the studied parameters were statistically confirmed using Pearson’s linear correlation analysis, as represented in Figure 7.

Fig. 6.  Principal component analysis (PCA) represented by two factors (F1=43.7% and F2=22.2%) and produced by biochemical variables (MDA, H₂O₂, PCO, CAT, GPx, SOD, GSH, AA, MT, AChE, PUFA, MUFA, SFA, ω6 (n-6 PUFA), ω3 (n-3 PUFA), ARA, EPA, DHA) in Ruditapes decussatus siphons of control (CT) and specimens treated with a series of Topas concentrations (D1, 4 µg L^-1; D2, 40 µg L^-1; D3, 400 µg L^-1) for 96 hours.

Fig. 7.  Correlation matrix for redox state biomarkers and fatty acid profiles. Each cell indicates Pearson correlation coefficient value, r, for a pair of biomarkers. Positive correlations (0<r<1.0) are displayed in blue and negative correlations (–1<r<0) in red. Colour intensity is proportional to the correlation coefficients.

Discussion

Health risks posed by emerging chemicals, including pesticides such as penconazole, are a growing global concern due to their widespread presence, increased exposure and substantial toxic effects (Chaâbane et al. 2018Chaâbane M., Elwej A., Ghorbel I., Chelly S., Mnif H., Boudawara T., Ellouze chaabouni S., Zeghal N., Soudani, N. 2018. Penconazole alters redox status, cholinergic function and lung’s histoarchitecture of adult rats: Reversal effect of vitamin E. Biomed. Pharmacother. 102: 645-652.
, Yoloğlu 2019Yoloğlu E. 2019. Assessment of Na⁺/K⁺ -ATPase, Mg²+-ATPase, Ca²+-ATPase, and Total-ATPase Activities in Gills of Freshwater Mussels Exposed to Penconazole. Comm. J. Biol. 3: 88-92. https://doi.org/10.31594/commagene.632082
). However, few studies have examined PEN’s distribution in non-target organisms such as mussels, crayfish and fish (Icoglu Aksakal and Ciltas 2018Icoglu Aksakal F., Ciltas A. 2018. Developmental toxicity of penconazole in Zebrfish (Danio rerio) embryos. Chemosphere. 200: 8-15. https://doi.org/10.1016/j.chemosphere.2018.02.094
, Yoloğlu 2019Yoloğlu E. 2019. Assessment of Na⁺/K⁺ -ATPase, Mg²+-ATPase, Ca²+-ATPase, and Total-ATPase Activities in Gills of Freshwater Mussels Exposed to Penconazole. Comm. J. Biol. 3: 88-92. https://doi.org/10.31594/commagene.632082
, Alkan Uçkun and Barım Öz 2020Alkan Uçkun A., Barım Öz Ö. 2020. Acute exposure to the fungicide penconazole affects some biochemical parameters in the crayfish (Astacus leptodactylus Eschscholtz, 1823). Environ. Sci. Pollut. Res. 27(28): 35626-35637. https://doi.org/10.1007/s11356-020-09595-2
). The current report offers valuable insights into the potential oxidative damage in the siphons and the FA composition of the bivalve Ruditapes decussatus exposed to the triazole fungicide Topas, which contains PEN. This study provides new tools for evaluating the sensitivity of clams to environmental pollution, grounded in scientific understanding of metabolic pathways linked to adaptation under stress from varying doses of Topas.

Following Husak et al. (2017)Husak V.V., Mosiichuk N.M., Storey J.M., et al. 2017. Acute exposure to the penconazole-containing fungicide Topas partially augments antioxidant potential in goldfish tissues. Comp. Biochem. Physiol. Part - C. 193: 1-8. https://doi.org/10.1016/j.cbpc.2016.12.003
, Topas enhanced generation of ROS such as superoxide radicals (O₂^•−), hydrogen peroxide (H₂O₂) and hydroxyl radicals (HO^•) and induced oxidative stress in fish. This finding was supported herein by a significant rise in H₂O₂ levels in the D3 group, possibly implying an aberration of the mitochondrial respiration chain. Using the Fenton/Haber-Weiss route, H₂O₂ reacts with free iron (Fe²⁺) to produce a more reactive and combative radical species, HO^• (Krumova and Cosa 2016Krumova K., Cosa G. 2016. Chapter 1: Overview of reactive oxygen species, in Singlet oxygen: applications in biosciences and nanosciences, Volume 1, pp. 1-21. https://doi.org/10.1039/9781782622208-00001
). This chemical is involved in the lipid peroxidation (LPO) process, resulting in loss of membrane integrity (Ayala et al. 2014Ayala A., Muñoz M.F., Argüelles S. 2014. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4- hydroxy-2-nonenal. Oxidative Med. Cell. Longev. 2014: 360438. https://doi.org/10.1155/2014/360438
). In agreement with this, the current study found that all Topas-treated clams developed LPO, showing that the accumulation of Topas in the organism resulted in oxidative toxicity that exceeded the antioxidant defence capacity of the clams and caused oxidative damage. In accordance with our results, it has been reported in various studies that MDA levels rise significantly in fish tissues due to pesticide use (Hatami et al. 2019Hatami M., Banaee M., Nematdoost Haghi B. 2019. Sub-lethal toxicity of chlorpyrifos alone and in combination with polyethylene glycol to common carp (Cyprinus carpio). Chemosphere 219: 981-988. https://doi.org/10.1016/j.chemosphere.2018.12.077
).

Furthermore, to protect themselves against stressful environmental conditions, a considerable change in the FA composition was noticed in all Topas-treated clams, following LPO (Trabelsi et al. 2019Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acid composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208. https://doi.org/10.1007/s11356-019-05492-5.
, Fouzai et al. 2020Fouzai C., Trabelsi W., Rabeh I., et al. 2020b. Fatty acid profile and antioxidant status in Venus verrucosa gills as λ-cyhalothrin toxic effects. Toxicol. Ind. Health. 36(11): 898-907. https://doi.org/10.1177/0748233720961219
b). The PUFAs (such as ARA, EPA and DHA) have been identified as essential components of all cell membranes and tissues. They are crucial components that not only determine the nutritional value of clams but also guarantee normal growth and development, and recently they have been used as a bio marker to determine xenobiotic impacts (Monroig and Kabeya 2018Monroig Ó., Kabeya N. 2018. Desaturases and elongases involved in polyunsaturated fatty acid biosynthesis in aquatic invertebrates: a comprehensive review. Fisheries. Sci. 84(6): 911-928. https://doi.org/10.1007/s12562-018-1254-x
). In addition, they influence membrane permeability, cell signals and several physiological pathways, as well as providing energy (Liu et al. 2015Liu J.J., Green P., John-Mann J., et al. 2015. Pathways of polyunsaturated fatty acid utilization: Implications for brain function in neuropsychiatric health and disease. Brain. Res. 1597: 220-246. https://doi.org/10.1016/j.brainres.2014.11.059
). The current findings revealed a clear alteration in the lipid fraction of treated R. decussatus siphons compared with the control values. There was a tendency for an increase in PUFAs, ARA, EPA, and DHA in Topas-treated groups compared with the control. This result could be interpreted as an adaptive response of clams against environmental stressors, where they expend effort and energy to scavenge the ROS overproduction and attempt to maintain the stability and fluidity of lipid membranes. Conversely, this investigation showed a highly significant decline in SFA amounts in all treated groups. These FAs may be used as an energy source due to their high caloric content. As a result, SFA levels may decrease following Topas exposure. Consistent with this, the findings described above showed that the PUFA levels showed an opposite tendency to the SFA levels. In this line, the increase in PUFAs may occur mainly at the expense of SFA levels. Indeed, the decrease in SFA amount may be due to its metabolization when the organism completes the elongation and the desaturation processes to synthesize the PUFAs, which showed a higher concentration in the treated groups. According to Yin et al. (2017)Yin X., Chen P., Chen H., Jin W. Yan X. 2017. Physiological performance of the intertidal Manila clam (Ruditapes philippinarum) to long-term daily rhythms of air exposure. Sci. rep. 7: 1-12. https://doi.org/10.1038/srep41648.
, these essential FAs improve the stress resistance of aquatic organisms. It is well known that ARA is naturally stored within lipid bodies in immune cells (Tallima and El Ridi 2018Tallima H., El Ridi R. 2018. Arachidonic acid: Physiological roles and potential health benefits - A review. J. Adv. Res. 11: 33-41. https://doi.org/10.1016/j.jare.2017.11.004
). There are many biological activities involved in its metabolism, including the regulation of innate immunity and the resolution of inflammation (Calder 2010Calder P.C. 2010. Omega-3 fatty acids and inflammatory processes. Nutrients 2(3): 355-374. https://doi.org/10.3390/nu2030355
). Indeed, from the significant increase in the n-6 PUFA/n-3 PUFA ratio it can be inferred that this pollutant has a pro-inflammatory effect. Consequently, the significant rise in ARA levels observed in Topas-treated siphons may reflect its involvement in the inflammatory cell response (Calder 2010Calder P.C. 2010. Omega-3 fatty acids and inflammatory processes. Nutrients 2(3): 355-374. https://doi.org/10.3390/nu2030355
) and highlight one of the defensive mechanisms of the molluscs to mitigate the harmfulness of Topas.

In addition, NMID FA (C22:2i/2j) can be synthesized de novo by bivalves and used to recover the more sensitive PUFAs such as DHA (Zhukova 1991Zhukova N.V. 1991. The pathway of the biosynthesis of non-methylene-interrupted dienoic fatty acids in molluscs. Comp. Biochem. Physiol. 100(4): 801-804. https://doi.org/10.1016/0305-0491(91)90293-M
). Their isolated double bonds can protect membrane phospholipids by slow auto-oxidation, contrary to the normal structure of PUFAs (Fokina et al. 2013Fokina N.N., Ruokolainen T.R., Nemova N.N., et al. 2013. Changes of blue mussels Mytilus edulis L. lipid composition under cadmium and copper toxic effect. Biol. Trace. Elem. Res. 154(2): 217-225. https://doi.org/10.1007/s12011-013-9727-3
, Signa et al. 2015Signa G., Di Leonardo R., Vaccaro A., et al. 2015. Lipid and fatty acid biomarkers as proxies for environmental contamination in caged mussels Mytilus galloprovincialis. Ecol. Indic. 57: 384-394. https://doi.org/10.1016/j.ecolind.2015.05.002
). To the best of our knowledge, this is the first study to evaluate Topas’s effect on NMID FAs. In the siphons of Topas-treated groups, NMID FAs were detected at higher levels than the control values, indicating that increased LPO had resulted in a loss of membrane fluidity. Taken together, these results indicate that Topas has toxic effects likely via affecting lipid metabolism. Our results were in line with those recorded in bivalves exposed to lead and acrylamide, respectively (Chetoui et al. 2019Chetoui I., Bejaoui S., Trabelsi W., et al. 2019. Exposure of Mactra corallina to acute doses of lead: effects on redox status, fatty acid composition and histomorphological aspect. Drug Chem. Toxicol. 45(1): 311-323. https://doi.org/10.1080/01480545.2019.1693590
, Trabelsi et al. 2019Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acid composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208. https://doi.org/10.1007/s11356-019-05492-5.
). Overall, the hierarchical cluster analysis confirmed the sensitivity of the FA composition of siphons to Topas exposure.

It has been reported in the present study that some PUFAS (ARA, EPA and NMID) involve a biphasic response characterized by enhancement at dose D2 followed by diminishment at dose D3 in siphon tissues of R. decussatus.

This pattern may reflect a general adaptive response to cope with stress or as part of the organism’s reaction to the stressor. The significant increase observed at dose D2 could result from changes in lipid metabolism or activation of enzymes involved in FA synthesis. However, at dose D3, the fungicide may disrupt normal enzyme function in FA synthesis and metabolism, causing a decrease in their levels. Thus, responses to chemical stress may vary with exposure time, dose and the vulnerability of the species (Cheung et al. 2001Cheung C.C.C., Zheng G.J., Li A.M.Y., et al. 2001. Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, Perna viridis. Aquat. Toxicol. 52: 189-203. https://doi.org/10.1016/S0166-445X(00)00145-4
).

As with lipids, other major functional components such as proteins, mainly those of the membrane, may be the target of ROS attack (Fokina et al. 2013Fokina N.N., Ruokolainen T.R., Nemova N.N., et al. 2013. Changes of blue mussels Mytilus edulis L. lipid composition under cadmium and copper toxic effect. Biol. Trace. Elem. Res. 154(2): 217-225. https://doi.org/10.1007/s12011-013-9727-3
). The generation of free radicals can cause structural and functional damage to proteins (Alderman et al. 2002Alderman C.J.J., Shah S., Foreman J.C., et al. 2002. The role of advanced oxidation protein products in regulation of dendritic cell function. Free Radical. Biol. Med. 32(5): 377-385. https://doi.org/10.1016/j.clinbiochem.2011.03.134
), as evidenced in our study by the significant increase in protein oxidation indicators, primarily AOPP and PCOs, in all treated clam siphons. According to our Pearson correlation, these findings are correlated with the H₂O₂ levels, thereby reflecting an excess of ROS production and protein oxidative damage in clam siphons.

Exposure of R. decussatus siphons to Topas may lead to irreversible and adverse changes at the cellular level in this soft body part. Consequently, 96 hours of treatment at higher Topas concentrations significantly improved FRAP capacity in siphon tissue. According to Llesuy et al. (2001)Llesuy S., Evelson P., Campos A.M., et al. 2001. Methodologies for evaluation of total antioxidant activities in complex mixtures. A critical review. Biol. Res. 34(2): 51-73. https://doi.org/10.4067/S0716-97602001000200009
, the FRAP assay has been established as a reliable measure of the system’s ability to control ROS-induced damage. Following this, our results clearly reflected the significant enhancement of FRAP to neutralize ROS damage (Fig. 2A). Based on the above results and to further prevent cellular oxidative damage, antioxidant enzymes such as SOD, CAT, GPx and GST play a crucial role in helping organisms adapt to stressful conditions by protecting against ROS overproduction and lipid peroxidation, thereby preventing cellular oxidative damage (Ullah et al. 2014Ullah R., Zuberi A., Ullah S., et al. 2014. Cypermethrin induced behavioral and biochemical changes in mahseer, Tor putitora. J. Toxicol. Sci. 39: 829-836. https://doi.org/10.2131/jts.39.829
). Accordingly, the overgeneration of H₂O₂ in exposed clams, especially at dose D3, may be due to the action of SOD, which was significantly higher under this dose. SOD can convert superoxide radical (O₂ ⁻) to H₂O₂ and molecular oxygen (O₂), consequently rendering the potentially harmful (O₂ ⁻) less hazardous (Rodríguez-Fuentes et al. 2015Rodríguez-Fuentes G., Rubio-Escalante F.J., Noreña-Barroso E., et al. 2015. Impacts of oxidative stress on acetylcholinesterase transcription, and activity in embryos of zebrafish (Danio rerio) following Chlorpyrifos exposure. Comp. Biochem. Physiol Part - C: Toxicology and Pharmacology. 172-173: 19-25. https://doi.org/10.1016/j.cbpc.2015.04.003
). Similar findings were reported in goldfish that were exposed to PEN (Husak et al. 2017Husak V.V., Mosiichuk N.M., Storey J.M., et al. 2017. Acute exposure to the penconazole-containing fungicide Topas partially augments antioxidant potential in goldfish tissues. Comp. Biochem. Physiol. Part - C. 193: 1-8. https://doi.org/10.1016/j.cbpc.2016.12.003
). The activity of the primary antioxidant enzyme CAT also increased in Topas-treated siphons, similar to findings in the liver of G. aculeatus under prochloraz contamination (Sanchez et al. 2008Sanchez W., Piccini B., Porcher J.M. 2008. Effect of prochloraz fungicide on biotransformation enzymes and oxidative stress parameters in three-spined stickleback (Gasterosteus aculeatus L.). J. Environ. Sci. Health. B 43 (1): 65-70. https://doi.org/10.1080/03601230701735151
) and in rainbow trout liver after long-term exposure to propiconazole (Li et al. 2010bLi Z.H., Zlabek V., Li P., et al. 2010b. Biochemical and physiological responses in liver and muscle of rainbow trout after long-term exposure to propiconazole. Ecotoxicol. Environ. Saf. 73: 1391-1396. https://doi.org/10.1016/j.ecoenv.2010.05.017
). To break down H₂O₂ into water (H₂O) and mitigate oxidative stress, the rise in GPx activity in all exposed specimens indicates a collective response from other enzymatic activities to protect cells from damage. The data of the present study are in accordance with those of Fouzai et al. (2020b)Fouzai C., Trabelsi W., Rabeh I., et al. 2020b. Fatty acid profile and antioxidant status in Venus verrucosa gills as λ-cyhalothrin toxic effects. Toxicol. Ind. Health. 36(11): 898-907. https://doi.org/10.1177/0748233720961219
, who demonstrated a significant increase in GPx activities in bivalves after exposure to lambda-cyhalothrin. Indeed, GST is best known for its ability to catalyse the conjugation of the reduced form of GSH to xenobiotic substrates for the purpose of detoxification (Jifa et al. 2006Jifa W., Yu Z., Xiuxian S., et al.2006. Response of integrated biomarkers of fish (Lateolabrax japonicus) exposed to benzo[a]pyrene and sodium dodecylbenzene sulfonate. Ecotoxicol. Environ. Saf. 65(2): 230-236. https://doi.org/10.1016/j.ecoenv.2005.08.002
).

The increased trend of GST in all groups treated in the present study confirms the activation of the cellular detoxification process to address oxidative injuries, a finding that aligns with similar results observed in the gills of PEN-exposed goldfish (Husak et al. 2017Husak V.V., Mosiichuk N.M., Storey J.M., et al. 2017. Acute exposure to the penconazole-containing fungicide Topas partially augments antioxidant potential in goldfish tissues. Comp. Biochem. Physiol. Part - C. 193: 1-8. https://doi.org/10.1016/j.cbpc.2016.12.003
).

Defence against damage can also be facilitated by non-enzymatic antioxidants, which form a primary system to limit free radical toxicity. As the most abundant low-molecular-weight thiol in cells, GSH plays a key role in preventing ROS-induced damage. In our study, the notable increase in GSH levels in Topas-treated R. decussatus likely reflects its active role in detoxification through thiol (-SH) groups in response to free radical accumulation. Additionally, the chemical composition of penconazole promotes direct conjugation with GSH, suggesting that the detoxification of Topas in siphons occurs via direct GSH conjugation, enhancing the hydrophilicity and excretion of xenobiotics (Sanchez et al. 2008Sanchez W., Piccini B., Porcher J.M. 2008. Effect of prochloraz fungicide on biotransformation enzymes and oxidative stress parameters in three-spined stickleback (Gasterosteus aculeatus L.). J. Environ. Sci. Health. B 43 (1): 65-70. https://doi.org/10.1080/03601230701735151
). Furthermore, the considerable rise in AA could be related to the increased amount of GSH in the siphons of Topas-treated clams. Following Krishnan et al. (2009)Krishnan N., Kodrík D., Kłudkiewicz B., et al. 2009. Glutathione-ascorbic acid redox cycle and thioredoxin reductase activity in the digestive tract of Leptinotarsa decemlineata (say). Insect. Biochem. Mol. Biol. 39: 180-188. https://doi.org/10.1016/j.ibmb.2008.11.001
, the recycling process of AA is aided by GSH, and the increase of this molecule and in particular AA counteracts oxidative damage. Our results were in line with those of Telahigue et al. (2020)Telahigue K., Rabeh I., Hajji T., et al. 2020. Assessment of the impacts of glyphosate and its commercial formulation Roundup® on the respiratory tree of the sea cucumber Holothuria forskali using a multivariate biomarker approach. Chemosphere. https://doi.org/10.1016/j.chemosphere.2020.129376.
describing the increase in the AA antioxidant in the sea cucumber Holothuria forskali exposed to glyphosate and its commercial formulation Roundup. Furthermore, the present findings corroborate with those recorded by Sayeed el al. (2003)Sayeed I., Parvez S., Pandey S., et al. 2003. Oxidative stress biomarkers of exposure to deltamethrin in freshwater fish, Channa punctatus Bloch. Ecotoxicol. Environ. Saf. 56(2): 295-301. https://doi.org/10.1016/S0147-6513(03)00009-5
in the fish Channa punctatus exposed to deltamethrin.

Given their molecular properties and their role in metal uptake, transport, storage and excretion, MTs play a major role in detoxification, homeostatic regulation of metals and protection against oxidative stress by acting as a metal-chelating agent for the excess of metals in the cells (Mao et al. 2012Mao H., Wang D.H., Yang W.X. 2012. The involvement of metallothionein in the development of aquatic invertebrate. Aquat. Toxicol. 110-111: 208-213. https://doi.org/10.1016/j.aquatox.2012.01.018
). Our results showed that exposure to Topas-induced MT synthesis in R. decussatus siphons reflected a high binding affinity between Topas and MTs. To explore data, the star plots served as a helpful visual tool that was applied in this study to combine multi-biomarker responses in the clams (CAT, SOD, GPx, GST, GSH, AA and MTs).

In addition to the non-enzymatic antioxidant discussed above, AChE activity is widely used as a neurotoxicity biomarker in bivalves (Smii et al. 2021Smii H., Khazri A., Ali M., et al. 2021. Titanium dioxide nanoparticles are toxic for the freshwater mussel Unio ravoisieri: Evidence from a multimarker approach. Diversity. 13(12): 1-17. https://doi.org/10.3390/d13120679
), as it terminates nerve impulses by catalysing acetylcholine hydrolysis. AChE is a primary inhibition target for pesticides, particularly biocides, organophosphorus pesticides and carbamates (Uluturhan et al. 2019Uluturhan E., Darılmaz E., Kontas A., et al. 2019. Seasonal variations of multi-biomarker responses to metals and pesticides pollution in M. galloprovincialis and T. decussatus from Homa Lagoon, Eastern Aegean Sea. Mar. Pollut. Bull. 141: 176-186. https://doi.org/10.1016/j.marpolbul.2019.02.035
). In our case, the results showed that Topas had destructive effects on esterase mechanisms by inhibiting AChE activities (Alkan Uçkun and Barım Öz 2020Alkan Uçkun A., Barım Öz Ö. 2020. Acute exposure to the fungicide penconazole affects some biochemical parameters in the crayfish (Astacus leptodactylus Eschscholtz, 1823). Environ. Sci. Pollut. Res. 27(28): 35626-35637. https://doi.org/10.1007/s11356-020-09595-2
). Topas concentrations showed a positive correlation with AChE inhibition in siphon tissues at doses D1 and D2. However, even at dose D3 AChE’s inhibitory impact decreased. This outcome may be explained by the findings of Ahammad Sahib et al. (1980)Ahammad Sahib I.K., Sailatha D., Ramana Rao K.V. 1980. Impact of malathion on acetylcholinesterase in the tissues of the fish Tilapia mossambica (Peters)—a time course study. J. Biosci. 2(1): 37-41. https://doi.org/10.1007/BF02703131
, who discovered that malathion’s inhibitory effect decreases over prolonged exposure due to acetylcholine accumulation in fish tissues from AChE inhibition caused by pesticide stress. Our results also align with those of Cravo et al. (2012)Cravo A., Pereira C., Gomes T., et al. 2012. A multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon, South Coast of Portugal. Mar. Environ. Res. 75: 23-34. https://doi.org/10.1016/j.marenvres.2011.09.012
, who reported AChE activity inhibition in R. decussatus from the Ria Formosa lagoon, Portugal, where various contaminants, including metals, PAHs and tributyltin, are present. Collectively, the multivariate analysis, including the PCA and Pearson correlation, performed on the biochemical data matrix has further highlighted the clear distinction between the control and Topas-treated groups. In addition, this analysis confirms the differential sensitivity/defensive response of R. decussatus clams to Topas exposure.

Histopathology is considered another effective method for keeping an eye on anthropogenic contamination. This study is the first to demonstrate the possible histological effects of Topas on R. decussatus siphons at gradual levels. The morphological state of R. decussatus siphons revealed the rupture of epithelial cells at doses D1 and D2. However, marked lipofuscin granules and vacuolization associated with haemocytes infiltration, rupture and deformation of epithelial cells were recorded at dose D3. The current histopathological findings could be attributed to ROS formation and the subsequent lipid peroxidation consequences, which can induce cell membrane rupture and disrupt membrane permeability and fluidity. The presence of abundant lipofuscin deposits and haemocyte infiltrations in the analysed tissues unequivocally indicate infiltrative inflammation in response to fungicide exposure (De Vico and Carella 2012De Vico G., Carella F. 2012. Morphological features of the inflammatory response in molluscs. Res. Vet. Sci. 93(3): 1109-1115. https://doi.org/10.1016/j.rvsc.2012.03.014
). Indeed, as mentioned above, lipofuscin aggregates confirm the course of inflammation of tissues as a result of the pro-phenoloxidase (PO) activating systems, a chain of immune mechanisms involved in phenomena such as recognition and encapsulation of foreign matter (Stara et al. 2021Stara A., Pagano M., Albano M., et al. 2021. Effects of long-term exposure of Mytilus galloprovincialis to thiacloprid: A multibiomarker approach. Environ. Pollut. 289(August): 117892. https://doi.org/10.1016/j.envpol.2021.117892
). Haemocyte infiltration is certainly related to an increase in haemolymph flow, facilitating the migration of defensive cells to the site of inflammation and penetrating the epithelium through the diapedesis process (Pagano et al. 2016Pagano M., Capillo G., Sanfilippo M., et al. 2016. Evaluation of Functionality and Biological Responses of Mytilus galloprovincialis after Exposure to Quaternium-15 (Methenamine 3-Chloroallylochloride). Molecules. 21(2): 1-12. https://doi.org/10.3390/molecules21020144
). These two reaction patterns were mainly documented in the siphon tissues of the specimens analysed, showing a dose-dependent exposure effect compared with the controls (see current results). Therefore, because of the lack of research targeting Topas toxicity and histopathological features in molluscs, our study makes an original contribution.

Conclusion

Our results suggest that Topas exposure could induce significant changes in the physiology of R. decussatus. It induces oxidative damage in clam siphons, as evidenced by an increase in lipid peroxidation and protein oxidation, in addition to perturbations in the enzymatic and non-enzymatic antioxidant status. Exposure to this fungicide also affected the cholinergic system, FA profiles and siphon histoarchitecture. The knowledge gained from our findings indicates for the first time that Topas is a potential neurotoxicant pesticide that exerts its neurotoxic effects via the generation of oxidative stress. Consequently, exposure to Topas should be carefully monitored.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request. Source data are provided in this paper.

Conflicts of interest

The authors have no conflict of interest to declare.

Research funding

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Ethical statement

All experiments were performed in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Authors’ contribution statement

Boutheina Ben Abdallah: Investigation, Methodology, Software, Formal analysis, Writing - original draft, Data curation, Visualization. Safa Bejaoui: Investigation, Conceptualization, Methodology, Resources, Data curation, Review and editing. Wafa Trabelsi: Methodology. Dalya Belhassen: Methodology, Software. Zeineb Khila: Methodology. Samir Boubaker: Methodology, Resources. Chayma Ben Fayala: Methodology. Nejla Soudani: Conceptualization, Supervision, Writing - review and editing, Validation.

References