Acta agriculturae Slovenica, 119/2, 1–9, Ljubljana 2023 doi:10.14720/aas.2023.119.2.2657 Original research article / izvirni znanstveni članek Peroxidase activity as a biochemical marker of insecticide use in vegetables Nassima SENANI 1 , Samia BEDOUHENE 1, 2 , Karim HOUALI 1 Received April 15, 2022; accepted April 12, 2023. Delo je prispelo 15. aprila 2022, sprejeto 12. aprila 2023 1 Analytical Biochemistry and Biotechnology Laboratory, Mouloud Mammeri University, Tizi-Ouzou, Algeria 2 Corresponding author, e-mail: samia.bedouhene@ummto.dz Peroxidase activity as a biochemical marker of insecticide use in vegetables Abstract: The insecticides use is important for crop im- provement and protection, but in excessive amounts, they would induce a dysfunction of metabolic enzymatic systems in plant tissues, leading to undesirable qualitative changes. In this context, we are interested in peroxidase (POD), an important enzyme in plant physiology but whose activity seems to be con- ditioned by the presence of insecticides in the soil. This work aims to study the impact of locally used insecticides (chlorpy- rifos and dimethoate) on the activity of POD in parsley, onion, celery and garlic grown in soils treated or not. POD extraction was performed using Tris-HCl buffer (pH 7.3); its activity was measured using the substrate o-dianisidine in the presence of H 2 O 2 . Our result showed that POD activity for insecticide treat- ed parsley, celery and onions increased by 30 % 127 % and 341 % respectively, however did not change significantly for garlic. Thus, the action of these chemicals is not trivial because they may alter non-target pathways, especially when doses are not adjusted accordingly. We found that insecticide stress would increase POD activity in all vegetables except garlic, which showed tolerance to insecticides. Our findings suggest that organic farming conditions could minimize peroxidase activ- ity in parsley, celery and onion. We add that overproduction of POD negatively affects the quality and reduces the shelf life of vegetables, thus would be a very interesting biomarker of in- secticide stress. Key words: peroxidase activity; enzymatic browning; in- secticides; oxidative stress; crop protection Aktivnost peroksidaze kot biokemični označevalec uporabe insekticidov v zelenjavi Izvleček: Uporaba insekticidov je pomembna za izboljša- nje in zaščito pridelkov, vendar bi v prevelikih količinah pov- zročili motnje v delovanju presnovnih encimskih sistemov v rastlinskih tkivih, kar bi povzročilo neželene kakovostne spre- membe. V zvezi s tem nas zanima peroksidaza (POD), ki je pomemben encim v fiziologiji rastlin, vendar se zdi, da njeno delovanje pogojuje prisotnost insekticidov v tleh. Namen tega dela je preučiti vpliv lokalno uporabljenih insekticidov (klorpi- rifos in dimetoat) na aktivnost POD v peteršilju, čebuli, zeleni in česnu, ki rastejo v tleh, obdelanih ali ne. Ekstrakcija POD je bila izvedena z uporabo pufra Tris-HCl (pH 7.3); aktivnost en- cima POD je bila izmerjena z uporabo substrata o-dianizidina v prisotnosti H 2 O 2 . Naši rezultati so pokazali, da se je aktivnost POD pri peteršilju, zeleni in čebuli, tretiranih z insekticidi, po- večala za 30 %, 127 % oziroma 341 %, pri česnu pa se ni bistve- no spremenila. Tako delovanje kemikalij ni nepomembno, saj lahko spremenijo neciljne poti, zlasti če odmerki niso ustrezno prilagojeni. Ugotovili smo, da stres zaradi insekticidov poveča aktivnost POD pri vseh vrtninah, razen pri česnu, ki je pokazal toleranco na insekticide. Naše ugotovitve kažejo, da bi lahko pogoji ekološkega kmetovanja zmanjšali aktivnost peroksidaze pri peteršilju, zeleni in čebuli. Dodajamo, da prekomerna pro- dukcija POD negativno vpliva na kakovost in zmanjšuje rok trajanja zelenjave, zato bi bila zelo zanimiv biomarker insekti- cidnega stresa. Ključne besede: aktivnost peroksidaz; encimsko porjave- nje; insekticidi; oksidativni stres; zaščita pridelkov Acta agriculturae Slovenica, 119/2 – 2023 2 N. SENANI et al. 1 INTRODUCTION Crops are exposed to a variety of diseases and pests that are responsible for important losses of yields and limited agricultural productivity worldwide. Food and Agriculture Organization (FAO) estimates that annually up to 40 percent of global crop production is lost to pests (FAO, 2021). Plant pathogens can be fungal, bacterial, viral, insects or nematodes and can damage plant parts above or below the ground and alter their quality (Pandit et al., 2022). In order to control these pathogens and pro- tect crops, the intensive agricultural systems rely heav- ily on the use of chemical pesticides. Nevertheless, the excessive application of pesticides has become a major cause of widespread ecological imbalances. Indeed, these chemicals resulted in serious problems of insecticide re- sistance, pest resurgence and pesticide residues accumu- lation in soil, water and plant tissues (Gull et al., 2019). Besides, pesticides may induce physiological variations in plants such as plant growth (Parween et al., 2015), germination (Fatma et al., 2018). Thus, processes of seed germination, cell division and elongation are changed (Gaspar et al., 1991). They also induce metabolic and en- zymatic dysfunctions and toxicities on cell membranes (Moriwaki et al., 2017). For instance, several studies have shown a varia- tion in peroxidase levels after treatment with insecticides (García-Hernández et al., 2005). The peroxidase enzyme (EC1.11.1.7) is an important antioxidant that plays a piv- otal role in plant growth and development (Breda et al., 1993). Peroxidases belong to a family of glycoproteins containing iron atoms as a prosthetic group and different quantities of carbohydrate residues (Van Huystee, 1987). Peroxidases are located mainly in the cell wall and in the vacuoles of plant cells; their location varies according to the age, species and developmental stage of the plant (Gaspar et al., 1982). Elevation in POD (peroxidase) activity has been linked to resistance to stress and self- defence mechanisms. Under stress conditions, the rate of respiration increases with upregulation in peroxidase enzyme activity (Aspinall & Paleg, 1981). High levels of POD in plants are involved in multiple deteriorating changes affecting flavor, texture, color and nutrition in processed fruits and vegetables (Bett-Garber et al., 2005). Therefore, knowledge about how they react is an impor- tant consideration in food technology. The use of insecticides is not trivial on the quality of plants and on human health, especially when their dos- age and treatment periods are not respected. Moreover, a major problem in Algeria is the unreasonable and ran- dom use of insecticides by farmers. In spite of the use of prohibited products such as DDT (dichloro-diphenyl- trichloro-ethane), the overdosing of insecticides and the non-respect of the life span of insecticides are alarming problems, which must be addressed seriously. Chlorpyrifos and Dimethoate are the most used in- secticides in Algeria, they are applied at 0.3-0.7 kg ha -1 and 1.5 liters of product/ha respectively on many crops: fruits and vegetables (beans, broccoli, cabbage, cauli- flower, peppers, potatoes, spinach, tomatoes) (Worthing & W alker, 1983). The half-life of chlorpyrifos ranges from 60 to 120 days and its persistence appears to be highly dependent on pH, climatic conditions and other soil factors, ranging from two weeks to more than a year. Dimethoate is rapidly absorbed and broken down in the plant by hydrolysis and oxidation (Menzie, 1969). Its half-life in plants varies from 2 to 5 days (Melnikov et al., 1977) and it disappears after an average of 30 days, de- pending on the plant species and the climatic conditions The aim of this study is to investigate the effects of insecticides on peroxidase activity in selected vegetables namely parsley, celery, garlic and onion bulbs. Parsley (Petroselinum crispum (Mill.) Fuss (Petroselinum sati- vum) a biennial herb is an important dietary source of vitamins and essential metals. Supplementation with parsley at sufficient levels can promote the levels of vita- mins and essential metals in the human body (Zhai et al., 2015). Celery (Apium graveolens L.) (also called krafes in northern Africa) belongs to the Apiaceae family. It grows annually or perennially throughout Europe and in tropi- cal and subtropical regions of Africa and Asia. Celery is considered the most widely used plant in traditional food and medicine because it contains compounds such as li- monene, selinene, furocoumarin glycosides, flavonoids, and vitamins A and C (Kooti et al., 2014; Al-Asmari et al., 2017; Li et al., 2019). Garlic (Allium sativum L.) is one of the oldest of all cultivated plants that has been used as a spice or food for over 400 years (Choi et al. 2007). On- ion (Allium cepa L.) is botanically included in the Ama- ryllidacea family and a variety of species are found across a wide range of latitudes and altitudes in Europe, Asia, N. America and Africa (Griffiths et al. 2002). Onion is widely used in all parts of the world as a flavoring vegeta- ble in various types of food. These vegetables represent the most important commercial crops and indispensable vegetables in Algeria and other countries thereby provide an important backdrop for evaluating the effects of insec- ticides in Algeria. 2 MATERIALS AND METHODS 2.1 CHEMICALS AND REAGENTS O-dianisidine and bovin serum albumin (BSA) were obtained from Sigma Aldrich. H 2 O 2 (30 % [v/v]) Acta agriculturae Slovenica, 119/2 – 2023 3 Peroxidase activity as a biochemical marker of insecticide use in vegetables was provided by Prolabo. All chemicals were of the best commercially available quality, and all solutions were prepared using deionized water. 2.2 SAMPLES Two groups of tissue samples from fresh parsley, celery, garlic, and onion were involved in this study. The first group was provided by a local farmer using chlorpy- rifos and dimethoate as insecticides. The second group was provided by a local organic farmer who does not use insecticides. Only uninjured plants were selected. 2.3 PREPARATION OF CRUDE EXTRACT Peroxidase enzyme extraction was carried out ac- cording to Diao et al. (2019). Five grams of each plant were mixed with an electric blender. The resulting mixture was homogenized with 30 ml of Tris-HCl buf- fer (50 mM, pH 7.3) containing 0.5 MCaCl 2 and 5 mM DTT, at 4 °C for 1 hour. After filtration, the extracts were centrifuged (14.000 g, 4 °C, 45 min). The supernatants containing the peroxidase were stored at -20 °C until use. 2.4 TOTAL PROTEIN CONCENTRATION Protein content of each extract was determined according to the spectrophotometric method of Lowry (1951). The reaction medium contains 3 ml of solution C and 20 µl of the extract; let it stand for 10 minutes in dark, at room temperature, then add 0.3 ml of Folin-Ciocalteu reagent diluted to half. After 15 minutes, absorbance is measured at 750 nm. Concentrations are expressed in grams per 100 grams of fresh matter (g 100 g -1 ) using the regression equation obtained with BSA. 2.5 ENZYME ASSAY Peroxidase activity was assayed according to the method of Bradely et al. (1982) modified by Bedouhene et al. (2020). The change in absorbance at 460 nm due to the oxidation of o-dianisidine in the presence of hydrogen peroxide (H 2 O 2 ) and enzyme extract at 25  °C was monitored using Jenway 6405 UV/VIS Spectrophotometer. A standard assay solution contained 15 mM o-dianisidine, 10 mM H 2 O 2 in sodium phosphate buffer pH 6.5 was prepared. Twenty-five microliters of the crude extract (contained peroxidase enzyme) were add- ed to the standards solution in total volume of 1 ml. The change of color is due to the oxidation of o-dianisidine in the presence of hydrogen peroxide (H 2 O 2 ). Kinetics of POD activity is followed by monitoring the change in absorbance at 470 nm per min (Abs/min). One enzyme unit (U) is defined as the amount of enzyme producing a 0.001 absorbance change per min under the assay condi- tions used. The readings were taken for every 1 min for 10 minutes and enzyme extract at 25 °C was monitored using Jenway 6405 UV/VIS Spectrophotometer. 2.6 DATA ANALYSIS The results were expressed as mean values with their standard deviations. The Two-way ANOVA analysis test was used to estimate the significance of the obtained data for each experiment. The Tukey-Kramer multiple- comparison test was used for analysis of the two sam- ple groups (treated versus untreated) results. Wherever differences are reported as significant, a 95 % confidence Figure 1: Oxidation of the molecular chromophore (o-dianisidine) by H 2 O 2 and peroxidase, and the resultant color change from colorless to brown Acta agriculturae Slovenica, 119/2 – 2023 4 N. SENANI et al. ent levels of POD activity. The level of POD activity was low in garlic treated with insecticides. This finding is sup- ported by the proteins contents results (Table 1). 3.2 COMPRAISON OF PEROXIDASE ACTIVITIES Peroxidase activities from parsley, celery, garlic and onion bulbsare summarized in Figure 3. Plant samples not subjected to insecticides show POD activities ranging from 201 to 2922, where parsley shows the highest activ- ity, followed by celery and garlic, onion shows the lowest concentration. Higher POD activities ranging from 777 to 3769 Umin -1 g -1 were observed in samples from insecti- cide-treated plants. Significantly the highest activity was found in insecticide-treated plant tissues from parsley with 3768.74 ± 141.59 Umin -1 g -1 and celery with 2680.81 level was used. The data analysis was performed using GraphPad Prism software version 5.01 (2010). 3 RESULTS 3.1 PEROXIDASEACTIVITY Activity was measured in extracts of treated and untreated vegetables with insecticides by spectropho- tometry using o-dianisidine as chromogenic agent and hydrogen peroxide (H 2 O 2 ) as substrate (Fig. 1). POD is an enzyme related to plant defence and plays an es- sential role in resistance to membrane damage, mainly through the enzymatic degradation of H 2 O 2 . Peroxidase activity was strongly elevated in treated vegetables versus untreated samples (Fig. 2). The four plants showed differ - Figure 2: Kinetics of peroxidase activity is followed by monitoring the change in absorbance at 470 nm per min (Abs/min)of crude vegetables extracts (parsley, celery, garlic and onion) treated with insecticides compared to crude vegetables extracts without insecticides (control samples). Data represent mean values ± standard deviation of three determinations Acta agriculturae Slovenica, 119/2 – 2023 5 Peroxidase activity as a biochemical marker of insecticide use in vegetables ± 373.66 Umin -1 g -1 (p< 0.001). Insecticide-treated onions showed lower activity, with a measurement of 776.99 ± 33.62 Umin -1 g -1 (p< 0.01). Samples derived from garlic did not show a significant increase in POD activity in in- secticide-treated 772.84 ± 67.25 Umin -1 g -1 (p>0.05) com- pared to the untreated samples that had POD activity of 1253.09± 232.84 Umin -1 g -1 . 4 DISCUSSION Use of insecticides leads to a dysfunction of meta- bolic enzyme systems in plant tissues, and negatively modifies certain physiological functions. In order to show the difference in tolerance behaviour and toxicity level among different vegetables selected against insecti- cide stress, the activity of the antioxidant enzyme peroxi- dase was evaluated. In this work, we compared POD activity in insecti- cide-treated and untreated parsley, onion, garlic, and cel- ery . The assessment of the oxidation of o-dianisidine in the presence of H 2 O 2 revealed that the four plants had signifi- cant differences (p<0.05). Our findings are comparable to those of two groups, Hemeda & Klein (1990) and Ponce et al. (2004). They reported differences in POD activity indifferent crude vegetable extracts. García-Hernández (2005) showed high activity of POD in peppers treated with insecticides. On the other hand, the application of insecticides on garlic did not show an increase in POD activity compared to the other plants studied; this could be explained by the fact that the analyzed part is the bulb and not the leaf part. Garlic is described as a biopesti- cide possessing other defense mechanisms apart from peroxidase, such as poly sulfides. Several studies have shown that garlic possess some insecticidal, fungicidal, acaricidal, nematocidal and bactericidal properties (La- lla et al., 2013; Nwachukwu & Asawalam, 2014). Garlic has received much interest in recent years with respect to environmental concerns about the use of chemically synthesized plant protection products and has been pro- posed as a green pesticide; a new and environmentally sustainable alternative for application in control pro- grams against various pest species. Indeed, this plant is equipped by evolution to defend itself against pathogens and pests (Mamduh et al., 2017; Wang et al., 2019). Phytotoxicity by excessive use of insecticide has been evaluated in some physiological traits in other cul- tivars and plants (Mousavizadeh & Sedaghathoor, 2011; Diao et al. 2011; 2019). García-Hernández (2005) report- ed that the highest insecticides rates caused alterations in the expression of peroxidase. The potential variation in peroxidase activity can be reflected in the growth and yield of plants, playing an important role in some stages of the metabolism, such as the auxin catabolism, and lignin formation (Fang & Kao, 2000). Peroxidase is in- volved in detoxification of xenobiotic a defense system of plants (Çördük, 2016; Lubos et al., 2011), its increase in plants is thought to be a response to stress, especially when the levels of H 2 O 2 which is its substrate is high. The expression of each peroxidase isoform, is linked to the physiological status and the stress of developing condi- tions in a plant (Lobarzawsky et al., 1991). Hajjar et al. (2018) were able to identify many isoforms of POD us- ing electrophoresis and spectrophotometric approaches. Additionally, they found that each isoform is activated depending on the chemical structure and properties of the insecticide. Vegetables Organs Total protein (g 100 g -1 ) in untreated plant Total protein (g 100 g -1 ) in treated plant Parsley Leaves 1.02 ± 0.011 1.23 ± 0.001 * Celery Leaves 0.26 ± 0.001 0.91± 0.014* Garlic Bulbs 1.03 ± 0.009 0.73 ± 0.007 Onion Bulbs 0.15 ± 0.001 0.32 ± 0.003* Table 1: Proteins contents in crude vegetables extracts untreated and treated with insecticides Data represent mean values ± standard deviation of three determinations. * Means are significantly different (p< 0.05) Figure 3: Peroxidase activity of crude vegetable extracts treated or un- treated with insecticides. Error bars indicates the standard deviation of determinations. Differences were considered significant at p< 0.05. (ns > 0.05, **p < 0.01, ***p < 0.001) Acta agriculturae Slovenica, 119/2 – 2023 6 N. SENANI et al. Chlorpyrifos and dimethoate are organophospho- rus insecticides with a large spectrum activity. Their mechanism of action is to inhibit cholinesterase, which is the cause of potential toxicity in humans (Gupta, 2016; Dhiraj et al., 2020; Nazam et al., 2020). The excessive use of insecticides can underlie health problems in humans; ranging from minor problems(e.g., eye irritation, skin ir- ritation, skin sensitization) (Damalas & Eleftherohorinos, 2011) to neurotoxicity or cancer (Foster & Brust, 1995; Yadav et al., 2019).Exposure to organophosphate insec- ticides leads to depression of plant growth and nitrogen metabolism (Parween et al., 2011). The highest exposure of the Algerian consumer to pesticide residues through consumption of raw fruit and vegetables was found to be (42 %) for chlorpyrifos (Mebdoua et al., 2017). Fatma et al. (2018) showed a significant decrease in seed germination of Allium cepa in the presence of these insecticides, and the effects were enhanced with increas- ing their doses. Thus, seed germination, a primary physi- ological process of plant growth, is strongly influenced by environmental stress. Stunting of plant growth at higher concentrations of applied pesticides indicates a reduc- tion in cell division, cell elongation, and conversion of indole-3 acetic acid to various photo-oxidative products, as these compounds function as potent auxin antagonists (Tevini & Teramura, 1989). Plants possess a complex antioxidant system including enzymes such as catalase (CAT; EC.1.11.1.6), peroxidase (POD; EC. 1.11.1.7), and superoxide dismutase (SOD; EC. 1.15.1.1) to miti- gate and repair ROS damage (Pandey & Rizvi, 2010). There are several evidences of insecticide degradation by high activity of oxidoreductase enzymes which reflects the level of toxicity and also the ability to combat stress (Dong et al., 2007; Yildiztekin et al., 2015; Singh et al., 2015). Several studies have showed that spraying of crops with organophosphorus insecticides was associated with a remarkable stimulation in peroxidase activity (Garcia- Hernandez et al., 2005). Hajjar et al. (2018) found that that the highest level of increase in peroxidase activity was recorded at 20 days after spraying tomato plants with organophosphorus insecticides compared with untreated plants. Furthermore, the effects in interaction and re- sponse of peroxidase activity relied significantly on two factors; the insecticide and the dose. The effect of insecti- cides depended on their formulations and physicochem- ical properties (vapor pressure and solubility), climatic conditions (temperature, humidity, and sunlight), plant characteristics (genus and species), location of their ap- plications and importantly the number and doses applied (Heshmati et al., 2020). García-Hernández et al. (2005) showed that insecticides applied at low doses did not cause significant differences in peroxidase activity com- pared to the control without insecticides, but a higher dose significantly increased peroxidase activity. Similar trends have also been reported in studies related to physi- ological injury by insecticides in hot pepper (Atale et al., 1995; García- Hernández et al., 2000). Furthermore, the results obtained here are consistent with the hypothesis reported by García-Hernández et al. (2005), who report- ed that insecticide-induced stress influences antioxidant enzymatic activity. The impact of regulated expression of peroxidase in plants has a direct effect on their shelf life. Indeed we noticed that the shelf life of parsley and celery that have not been treated with insecticides is relatively longer than that of treated vegetables. Furthermore, the external morphology of insecticide treated vegetables is altered to appear less shiny. Some farmers apply insecticides in concentrations that are higher than the recommended amount to control resistant pests, occasionally reporting better control, but the yields are reduced and may have undesirable conse- quences. In general, the manufacturer’s recommended application protocol does not have a negative effect on the plants, and some reports showed that there are cer- tain insecticides that act as growth stimulants when ap- plied at low doses (Ahemad & Khan, 2012; Singh et al., 2015; Yang et al., 2020). Other studies have shown that the excessive use of fertilizers, inappropriate irrigation, and exploitation of metal resources can lead to salt stress to a large extent (Shrivastava & Kumar, 2015; Gull et al., 2019). Under these circumstances, plants are likely to face biotic and abiotic stresses more frequently and si- multaneously. The action of commercial chemicals is not trivial because they modify non-target physiological pathways, especially when the doses are not adapted. Work from this study suggests that insecticide stress influences anti- oxidant enzyme activity and supports that organic farm- ing conditions minimize peroxidase activity and enzyme browning in parsley, celery and onion. We conclude that POD is a very interesting biomarker of insecticide stress, and that overproduction of POD negatively affects their quality and shelf life. 5 CONCLUSION Our study showed a significant increase in peroxi- dase activity on samples from conventional agriculture. These results represent an alarming report on the exces- sive and unreasonable use of insecticides by farmers, which is why it is important to inform farmers about the danger of these practices. Indeed, the use of chemicals to control pests can be useful on the one hand, but on the other hand can present many risks for human health. In Acta agriculturae Slovenica, 119/2 – 2023 7 Peroxidase activity as a biochemical marker of insecticide use in vegetables this perspective, the evaluation of peroxidase enzymatic activity could be a reliable tool for the evaluation of the physiological stress resulting from the application of in- secticides and will help to prevent the loss of antioxidant potential as well as the quality of vegetables, including the commonly used aromatic plants such as parsley and celery. Thus, we recommend through this study to re- duce doses by combining biopesticides and by producing long-term resistant varieties, we also underline the im- portance of peroxidase which seems to be an interesting marker of insecticide-induced stress. Finally, additional and further studies are required to determine the doses of pesticides that do not significantly influence peroxi- dase activity. 6 ACKNOWLEDGEMENTS Funding was provided by the General Direction of research and development technologies/Ministry of Higher Education and Research Sciences DGRSDT/ MERS (ALGERIA). We are also grateful to Dr. Achouak Arfaoui for careful reading of the manuscript. 7 REFERENCES Ahemad, M., & Khan, M.S. (2012). Effects of pesticides on plant growth promoting traits of Mesorhizobium strain MRC4. Journal of the Saudi Society of Agricultural Sciences, 11, 63- 71. https://doi.org/10.1016/j.jssas.2011.10.001 Al-Asmari, A.K., Athar,M.T., & Kadasah, S.G. (2017). An Updated Phytopharmacological review on medicinal Plant of Arab region: Apium graveolens Linn. Pharmacog- nosy Reviews, 11, 13-18. https://doi.org/10.4103/phrev. phrev_35_16 Atale, A. S., Narkhede, M. N., & Atale, S. B. (1995). Effects of some agrochemicals on meiotic cell division in chilli. Jour- nal of Maharashtra Agricultural Universities, 20, 195-197. Aspinall, D., & Paleg, L.G. (1981). Proline accumulation: Physi- ological aspects, In: Paleg, L.G. and Aspinall, D. (Eds.), The Physiology and Biochemistry of Drought Resistance in Plants (pp. 205-241).Sydney, Academic Press. Bedouhene, S., Dang, P .M., Hurtado-Nedelec, M., & El-Benna J. (2020). Neutrophil degranulation of Azurophil and spe- cific granules. Methods in Molecular Biology, 2087, 215-222. https://doi.org/10.1007/978-1-0716-0154-9_16 Bett-Garber, K.L, Lamikanra, O., Lester, G.E., Ingram, D.A., & Watson, M.A. (2005). Influence of soil type and stor- age conditions on sensory qualities of fresh-cut cantaloupe (Cucumis melo). Journal of the Science of Food and Agricul- ture, 85, 825-830. https://doi.org/10.1002/jsfa.1970 Bradely, P.P., Priebat, D.A., Christensen, R.D., & Rothstein, G. (1982). Measurment of cutaneous inflammation; sestima- tion of neutrophil content with an enzyme marker. The Journal of Investigative Dermatology, 78, 206-209. https:// doi.org/10.1111/1523-1747.ep12506462 Breda, C., Buffard, D., Van Huystee, R.B., & Esnaul, R. (1993). Differential expression of two peanut peroxidase cDNA clones in peanut plants and cells in suspension culture in response to stress. Plant Cell Reports, 12, 268-272. https:// doi.org/10.1007/BF00237133 Choi, M. K., Chae, K.Y., Lee, J.Y., & Kyung, K. H. (2007). Antimicrobial activity of chemical substances derived from S-alk (en) yl-L-cysteine sulfoxide (alliin) in gar- lic, Allium sativum. Food Science and Biotechnology, 16, 1-7.2092-6456(eISSN). Çördük, N., Akıncı, N., Kaya, N., Yücel, G., & Akı, C. (2016). Effects of dodine on total protein content and peroxidase activity in ViciafabaL. Sakarya University Journal of Sci- ence, 20(3), 627-633. https://doi.org/10.16984/saufen- bilder.22241 Damalas, C. A., & Eleftherohorinos, I. G. (2011). Pesticide ex- posure, safety issues, and risk assessment indicators. Inter- national journal of environmental research and public health, 8(5), 1402-1419. https://doi.org/10.3390/ijerph8051402 Dhiraj, S., Jitesh, K., Paramjeet, K., & Priti, B. (2020). Toxicity, natural and induced degradation of chlorpyrifos. The Jour- nal of the Chilean Chemical Society, 65, 4807-4816. http:// dx.doi.org/10.4067/S0717-97072020000204807  Diao, M., Kone, O. H., Ouedraogo, N., Bayili, R. G., Bassole, I. H. N., & Dicko, M. H. (2011). Comparison of peroxidase activities from Allium sativum, Ipomoea batatas, Raphanus sativus and Sorghum bicolor grown in Burkina Faso. African Journal of Biochemistry Research, 5, 124-128. http://www. academicjournals.org/AJBR Diao, M., Dembele, R.H., Konate, K., & Dicko, M. H. (2019). Etude comparative des peroxydases de dix (10) plantes su- périeures couramment rencontrées au Burkina Faso. The International Journal of Biological and Chemical Sciences, 13, 2533-2545. https://doi.org/10.4314/ijbcs.v13i6.9 Dong, K .(2007). Insect sodium channels and insecticide resistance. Invertebrate Neuroscience, 7, 17 https://doi. org/10.1007/s10158-006-0036-9 Fang, W . C. H., & Kao, C. H. H. (2000). Enhanced peroxidase activity in rice leaves in response to excess iron, copper and zinc. Plant Science, 158, 71-76. https://doi.org/10.1016/ s0168-9452(00)00307-1 FAO. (2021). Scientific review of the impact of climate change on plant pests–A global challenge to prevent and mitigate plant pest risks in agriculture, forestry and ecosystems. Rome. FAO on behalf of the IPPC Secretariat. https://doi.org/10.4060/ cb4769en Fatma, F., Verma, S., Kamal, A., & Srivastava, A. (2018). Phyto- toxicity of pesticides mancozeb and chlorpyrifos: correla- tion with the antioxidative defence system in Allium cepa. Physiology and Molecular Biology of Plants, 24(1), 115-123. https://doi.org/10.1007/s12298-017-0490-3. Foster, R. E., & Brust, G. E. (1995). Effects of insecticides ap- plied to control cucumber beetles (Coleoptera: Chrysomel- idae) on watermelon yields. Crop Protection, 14, 619-624. https://doi.org/10.1016/0261-2194(95)00071-2 García-Hernández, J. L., Troyo-Diéguez, E., Jones, H., Nolasco, H., & Ortega-Rubio, A. (2000). Efectos de la aplicación de Acta agriculturae Slovenica, 119/2 – 2023 8 N. SENANI et al. Biochemical, Molecular, and Physiological Aspects of Plant Peroxidases. Univeristy of Geneve, Geneve, Italy, 207p. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein Measurement with the phenol reagent. The Journal of Biological Chemistry, 193, 265-275. https://doi. org/10.1016/S0021-9258(19)52451-6 Lubos, E., Loscalzo, J., & Handy, D.E. (2011). Glutathione per- oxidase-1 in health and disease: from molecular mecha- nisms to therapeutic opportunities. Antioxidant and Re- dox Signaling, 15(7), 1957-97. https://doi.org/10.1089/ ars.2010.3586 Mamduh, Z., Hosseininaveh, V., & Khalil Talebi-Jahromi, A. (2017).Side effects of garlic extract on the life history pa- rameters of the predatory bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae). Crop Protection, 100, 65-72. https://doi.org/10.1016/j.cropro.2017.05.029 Mebdoua, S., Lazali, M., Ounane, S. M., Tellah, S., Nabi, F., & Ounane, G. (2017). Evaluation of pesticide residues in fruits and vegetables from Algeria. Food Additives & Con- taminants: Part B.Surveillance, 10, 91-98. https://doi.org/10 .1080/19393210.2016.1278047 Melnikov, Yu. A. (1977). Some applications of the greens function method in mechanics. International Journal of Solids and Structures, 13(11), 1045-1058. https://doi. org/10.1016/0020-7683(77)90075-0 Menzie, Calvin M., & United States. (1969). Bureau of Sport Fisheries and Wildlife. Metabolism of pesticides / by Calvin M. Menzie Bureau of Sport Fisheries and Wildlife Wash- ington, D.C. Moriwaki, H., Yamada, K., & Nakanishi, H. (2017). Evaluation of the interaction between pesticides and a cell membrane model by surface plasmon resonance spectroscopy analysis. Journal of Agricultural and Food Chemistry, 65(26), 5390- 5396. https://doi.org/10.1021/acs.jafc.7b01895. Mousavizadeh, S. J., & Sedaghathoor, S. (2011). Peroxidase ac- tivity in response to applying natural antioxidant of essen- tial oils in some leafy vegetables. Australian Journal of Crop Science, 5, 494-499. Nazam, N., Lone, M. I., Hamid, A., Qadah, T., Banjar, A., Alam, Q., ……M., Ahmad, W . (2020). Dimethoate Induces DNA Damage and Mitochondrial Dysfunction Triggering Apop- tosis in Rat Bone-Marrow and Peripheral Blood Cells. Tox- ics, 8, 80. https://doi.org/10.3390/toxics8040080 Nwachukwu, I. D.,& Asawalam, E. F. (2014). Evaluation of freshly prepared juice from garlic (Allium sativum L.) as a biopesticide against the maize weevil, Sitophilus zea- mais (Motsch.) (Coleoptera: Curculionidae). Journal of Plant Protection Research, 54(2), 132-138. https://doi. org/10.2478/jppr-2014-0021 Parween, T., Jan, S., Mahmooduzzafar, S., Fatma, T., & Hameed Siddiqui, Z. (2016). Selective effect of pesticides on plant. A review. Critical Reviews in Food Science and Nutrition, 56(1), 160-179. https://doi.org/10.1080/10408398.2013.78 7969 Pandey, K. B., & Rizvi, S. I. (2010). Markers of oxidative stress in erythrocytes and plasma during aging in humans. Oxi- dative Medcine and Cellular Longevity, 3(1), 2-12. https:// doi.org/10.4161/oxim.3.1.10476 Pandit, M.A., Kumar, J., Gulati, S., Bhandari, N., Mehta, P., insecticidasorganofosforados sobre el rendimiento (y sus parámetros) en ají (CapsicumannuumL. cv. Ancho San Luis). Phyton, 67, 113-120. http://www.redalyc.org/articu- lo.oa?id=60912502019 Garcia-Hernandez, J. L., Nolasco, H., Troyo-Dieguez, E., Muril- lo-Amador, B., Flores-Hernandez, A., Orona-Castillo, I., & Valdez-Cepeda, R. D. (2005). The effect of insecticides on peroxidase activityin hot pepper plants (Capsicum annum L.). The Revista Chapingo Serie Horticultura, 11(1), 129- 133. http://www.redalyc.org/articulo.oa?id=60912502019 Gaspar, T. C., Penel, C., Thorpe, T., & Greppin, H. (1982). Per- oxidases 1970-1980. Asurvey of their biochemical and physi- ological roles in higher plants. Université de Genève, Centre De Botanique, Genève. Gaspar, T., Penel, C., Hagage, D., & Greppin, H. (1991). Per- oxidases in plant growth, differentiation and development processes, pp. 249-280. In: Biochemical, Molecular, and Physiological Aspects of Plan Peroxidases. Univeristy of Geneve, Geneve, Italy. Griffiths, G., Trueman, L., Crowther, T., Thomas, B., & Smith, B. (2002). Onions-a global benefith to health. Phytotherapy Research, 16, 603-615. https://doi.org/10.1002/ptr.1222 Gull, A., Lone, A. A., & Wani, N. U. I. (2019).Biotic and abi- otic stresses in plants. In: De Oliveira A (ed) Abiotic and biotic stress in plants. Intech Open, London. https://doi. org/10.5772/intechopen.85832 Gupta, P.K. (2016). Chapter 17 - Toxic effects of pesticides (agrochemicals). In Fundamentals of Toxicology. Essential Concepts and Applications (pp. 85-202). Academic Press. https://doi.org/10.1016/B978-0-12-805426-0.00017-2 Hajjar, M. J., Alsaikhan, M. S., & Soliman, A. M. (2018). The potential effect of organophosphorus insecticides on per- oxidase enzyme activity in tomato plants (Solanum lycoper- sicum L.) grown in al-hassa, Saudi Arabia. Bionature, 38, 48-57. Hemeda, H. M., & Klein, B. P. (1990). Effects of naturally occurring antioxidants on peroxidase activity of veg- etable extracts. Food Science, 55, 184-186. https://doi. org/10.1111/j.1365-2621.1990.tb06048.x Heshmati, A., Komaki, H. A., Nazemi, F., & Mousavi Khaneghah, A. (2020). Persistence and dissipation behav- ior of pesticide residues in parsley (Petroselinum crispum) under field conditions. Quality Assurance and Safety of Crops & Foods, 2(3), 55–65. https://doi.org/10.15586/qas. v12i3.755 Kooti, W., Ali-Akbari, S., Asadi-Samani, M., Ghadery, H., & Ashtary- Larky, D. (2014). A review on medicinal plant of Apium graveolens. Advanced Herbal Medicine, 1(1), 48-59. Lalla, F. D., Ahmed, B., Omar, A., & Mohieddine, M. (2013). Chemical composition and biological activity of Allium sativum essential oils against Callosobruchus maculates. Journal of Environmental Science Toxicology and Food Tech- nology, 3(1), 30-36. https://doi.org/ 10.9790/2402-0313036. Li, S., Li, L., Yan, H., Jiang, X., Hu, W., Han, N., & Wang, D. (2019). Antigouty arthritis and antihyperuricemia prop- erties of celery seed extracts in rodent models. Molecular Medicine Reports, 20, 4623-4633. https://doi.org/10.3892/ mmr.2019.10708. Lobarzawsky, J. H., Greppin, H., Penel, C., & Gaspar, T. (1991). Acta agriculturae Slovenica, 119/2 – 2023 9 Peroxidase activity as a biochemical marker of insecticide use in vegetables Katyal, R., ……Kaur, J. (2022). Major biological control strategies for plant pathogens. Pathogens, 11, 273. https:// doi.org/10.3390/pathogens11020273 Ponce, A. G., Delvalle, C. E., & Roura, S. L. (2004). Natural es- sential oils as reducing agents of peroxidase activity in leafy vegetables. LWT-Food Science and Technology, 37, 199-204. https://doi.org/10.1016/j.lwt.2003.07.005 Shrivastava, P.,& Kumar, R. (2015). Soil salinity: A serious en- vironmental issue and plant growth promoting bacteria as one of the tools for its alleviation. Saudi Journal of Bio- logical Sciences, 22(2), 123-131. https://doi.org/10.1016/j. sjbs.2014.12.001 Singh, S., Gupta, R., & Sharma, S. (2015). Effects of chemical and biological pesticides on plant growth parameters and rhizospheric bacterial community structure in Vigna radia- ta. The Journal of Hazardous Materials, 30, 102-110. https:// doi.org/10.1016/j.jhazmat.2015.02.053 Tevini, M., & Teramura, A. H. (1989). “UV-B effects on terres- trial plants”. Photochemistry and Photobiology, 50(4), 479- 487. http://dx.doi.org/10.1111/j.1751-1097.1989.tb05552.x Van Huystee, R.B. (1987). Some molecular aspects of plant peroxidase biosynthetic studies. Annual Review of Plant Physiolologie, 38, 205-219. https://doi.org/10.1146/annurev. pp.38.060187.001225 Wang, H., Wu, Y., Liu, X., Du, Z., Qiu, Y., Song, J., ……Li, X. (2019). Resistance and clonal selection among Allium sa- tivum L. germplasm resources to Delia antiqua M. and its correlation with allicin content. Pest Management Science, 75(10), 2830-2839. https://doi.org/ 10.1002/ps.5478 Worthing, C. R., & Walker, S. B. (1983). The pesticide manual. 7 th ed. Croydon. The British Crop Protection Council. 695 p. Yadav, H., Sankhla, M. S., & Kumar, R. (2019). Pesticides-in- duced carcinogenic and neurotoxic effect on human. Fo- rensic Research & Criminology International Journal, 7, 243- 245. https://doi.org/10.15406/frcij.2019.07.00288 Yang, K. J., Lee, J., & Park, H. L. (2020). Organophosphate pes- ticide exposure and breast cancer risk: A rapid review of human, animal, and cell-based studies. International Jour- nal of Environmental Research and Public Health, 17, 5030. https://doi.org/10.3390/ijerph17145030 Yildiztekin, M., Kaya, C., Tuna, A. L., & Ashraf, M. (2015). Oxidative stress and antioxidative mechanisms in tomato (Solanum lycopersicu L.) plants sprayed with different pes- ticides. Pakistan Journal of Botany, 47(2), 717-721. https:// doi.org/10.21448/ijsm.450830 Zhai, Q., Narbad, A., & Chen, W . (2015). Dietary strategies for the treatment of cadmium and lead toxicity. Nutrients, 7, 552-571. https://doi.org/10.3390/nu7010552