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Toxicity of cyhalothrin lambda on erythrogram and renal parameters aenuated by Lavendula stoechas essenal oil in rabbit Toxicidad de la cihalotrina lambda sobre el hemograma los parámetros renales atenuados por el aceite esencial de Lavendula stoechas en conejo Soumaya Boubsil 1.2* , Hadjer Djemil 1.3 , Cherif Abdennour 2 1* Department of Biology, Faculty of Natural Life and Sciences, Mohamed Cherif Messaadia University, Souk Ahras, 41000, Algeria. 2 Laboratory of Animal Ecophysiology, Badji Mokhtar University, Annaba 23000, Algeria 3 Laboratory of the aquac and terrestrial ecosystems, University of Mohammed Cherif Messaadia, Souk Ahras , 41000, Algeria. * Corresponding author’s e-mail: s.boubsil@univ-soukahras.dz Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido : 14/10/2024 Aceptado: 18/12/2024 Publicado: 28/02/2025 hps://doi.org/10.52973/rcfcv-e35531 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 6 ABSTRACT Among the pyrethroid inseccides used in Algeria against a wide range of agricultural pests is lambda-cyhalothrin (LCT). The aim of this study was to invesgate the beneﬁcial eﬀects of Lavendula stoechas essenal oil on the hematotoxicity and nephrotoxicity of the inseccide lambda cyhalothrin in rabbits (Cuniculus Lepus). Twenty-seven (27) healthy local rabbits, with an average weight of 1.8 kg, were divided into three groups of nine each: the ﬁrst group considered as control, the second group treated with (100 mg/kg/BW) (LCT) and the third group treated with a mixture of (100 mg/kg/BW) (LCT) and (500 mg/ kg/BW) Lavandula s. essenal oil (LS.EO). The doses tested occurred daily in the diet over a 4-week period. Blood analysis revealed significant variations in the haemogram between the treated and control groups. A highly significant increase was recorded in the white blood cell, lymphocyte count in the LCT-treated group compared with the control group (P<0.001), highly and very highly significant decreases were observed in Red Blood Cell (RED), Hematocrit (HCT) and Hemoglo- bin(HGB) levels in the LCT-treated group compared with the control (P<0.001; P<0.0001), with the same result obtained for erythrocyte indices . Biochemical analysis of renal parameters showed a highly and very highly significant increase in urea and creatinine concentrations respectively in the LCT-treated group versus the control (P<0.001; P<0.0001). In contrast, the addition of lavendula essential oil improved the previous hematological and biochemical alterations. Key words: Rabbit; lambda cyhalothrin; Lavendula s essential oil; biological parameter RESUMEN Entre los inseccidas piretroides ulizados en Argelia contra una amplia gama de plagas agrícolas se encuentra la lamb- da-cialotrina (LCT). El objevo de este estudio fue invesgar los efectos beneﬁciosos del aceite esencial de Lavendula stoe- chas sobre la hematotoxicidad y nefrotoxicidad del inseccida lambda cihalotrina en conejos (Cuniculus Lepus). Veinsiete (27) conejos locales sanos, con un peso medio de 1,8 kg, fueron divididos en tres grupos de nueve cada uno: el primer grupo considerado como control, el segundo grupo tratado con (100t mg/kg/BW) (LCT) y el tercer grupo tratado con una mezcla de (100 mg/kg/BW) (LCT) y (500 mg/kg/BW) aceite esencial de Lavandula s (LS.EO). Las dosis probadas se suministraron dia- riamente en la dieta durante un periodo de 4 semanas. Los análisis de sangre revelaron variaciones signiﬁcavas en el he- mograma entre los grupos tratados y de control. Se registró un aumento altamente signiﬁcavo en el recuento de glóbulos blancos y linfocitos en el grupo tratado con LCT en compara- ción con el grupo de control (P<0,001), se observaron descen- sos altamente y muy altamente signiﬁcavos en los niveles de RBC, HCT y HGB en el grupo tratado con LCT en comparación con el control (P<0,001 ; P<0,0001), obteniéndose el mismo resultado para los índices eritrocitarios. El análisis bioquími- co de los parámetros renales mostró un aumento altamente y muy altamente signiﬁcavo de las concentraciones de urea y creanina respecvamente en el grupo tratado con LCT frente al control (P<0,001 ; P<0,0001). Por el contrario, la adición de aceite esencial de lavándulas mejoró las alteraciones hema- tológicas y bioquímicas anteriores. Palabras clave: Conejo; lambda cihalotrina; aceite esencial de Laván- dula s.; parámetros biológicos

Lambda cyhalothrin toxicity in rabbit / Boubsil et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Pescides are becoming increasingly important as a result of various epidemics in various countries around the world [1]. The main purpose of pescides is to control harmful organisms (animals, plants, fungi) but they can also regulate plant growth, have defoliang properes, or improve the storage or trans- port of crop products. The use of these products is constantly increasing throughout the world. According to world experts, the demand for pescides is such that their producon prac- cally doubles every ten years. Around 2 million tons of pescides are used internaonally, of which 47.5% are herbicides, 29.5% inseccides, 17.5% fungicides and 5.5% other pescides [2 , 3]. Pescides pose a health risk because the majority of the human populaon remains acvely or passively exposed to them. The exposure paern is also a maer of concern as it determines the exposure dose [4]. The abundance of these products and the small quantities applied have two consequences: the need to study these molecules in order to understand what happens to them in the environment and the difficulty of detecting and measuring the active substances which are found in low concentrations [5]. As a result, the extensive use of these products and the presence of their metabolites in the body can develop pathologies and environmental disturbances. The main pathologies suspected to result from exposure to pescides are cancers, reproducve and developmental prob- lems, weakening of the immune system, hormonal and neuro- logical disorders [6 ,7]. Natural products are a formidable alter- nave for combang the toxicity of pescides in order to reduce the negave impact on human health and the environment. The move towards green chemistry processes and the ongoing need for new tools with original modes of acon represent an arac- ve area of research that deserves parcular aenon. Unlike a chemical, remedy whose mode of acon the synthec molecule on a speciﬁc receptor site determines.The most studied natural products are ascorbic acid (vitamin C) and α-tocopherol (vitamin E) together or with other natural products such as quercen [8] as well as, the therapeuc qualies of a medicinal plant, which are based on the interacons of all its diﬀerent components. Ar- omac plants have played a vital role in all mes and places, parcularly in tropical countries. First used in their raw state, then infused, digested, in the form of ointment, perfume or al- coholic extracts, they have always played a major role in the cul- ture of many peoples throughout the ages, whether religiously or medicinally. Aromac plants have healing and curave eﬀects [9]. The ﬁrst elements of photosynthesis are primordial metab- olites: sugars, fay acids and amino acids. They produce speciﬁc metabolites called essenal oils, which have therapeuc prop- eres. Essenal oils are molecules with an aromac core and a volale character, giving the plant a characterisc odor. They are found in secretory organs such as leaves (peppermint, basil), ﬂowers (lavender, ylang ylang), wood (Atlas cedar, white san- dalwood), roots (ginger, valerian, vever) and seeds (coriander, green anise, carrot) [10]. They are chemical messengers used by aromac plants to interact with their environment and play a role in protecng against excess light and aracng pollinang insects). Oils have great potenal in terms of their biological ac- vity: anoxidant, an-inﬂammatory, ansepc, anmicrobial, anviral, anfungal, bactericidal, antoxic, inseccidal and in- sect repellent, invigorang, smulang, calming, etc [11]. They are free radical scavengers and metal chelators. The use of es- senal oils has been the subject of much research over the last decade and has aroused considerable scienﬁc interest, reﬂect- ed in the number of studies into the eﬀecveness of essenal oils against the harmful eﬀects of various xenobiocs. The pur- pose of this study was to determine the relaonship between the use of Lavendula steochas essenal oils and their anoxida- ve eﬀects in the treatment of hematological and renal issues brought on by long-term exposure to a pescide. MATERIALS AND METHODS Characteriscs of the Ouled Bechih study area The Ouled Bechih study area is located in north-eastern Al- geria, in the Machroha locality (36° 21’24‘’ N, 7° 50’83‘’ E) and the Bouhadjar locality (36° 30’12‘’ N, 8° 06’20‘’ E) (FIG.1). The Ouled Bechih forests cover an area of 6,582 hectares and pro- vide a rich diversity of species [12]. Climac factors have a major impact on a region’s biodiversity. The climate of the Souk Ahras region is conducive to the development of extensive forest areas on very hilly terrain, where slopes can exceed 20%, known as the agri-forest zone and covering a vast area. FIGURE 1. Geographical locaon of the study area (Ouled Bechih in Machroha forest-Souk Ahras. Northeast of Algeria) Compound tested KARATEKA® is a commercial name, mul-crop foliar insec- cide formulated from the acve ingredient lambda cyhalothrin (LCT), which belongs to the family of synthec pyrethroids. This combinaon provides a very broad spectrum of eﬃcacy against aphids and many other pests. It came from the Souk Ahras Agri- cultural Directorate (Algeria). Lavendula steochas used Lavender harvesng takes place during ﬂowering, between March and May. The aerial parts of Lavendula stoechas were collected in March 2024 in the Ouled Bechih region of the El-machroura Mountains, Souk Ahras, Algeria. The idenﬁcaon was carried out by Doctor Kei Louisa, botanist at the botany laboratory of the Faculty of Natural and Life Sciences at Mo- hammed Cherif Messaadia University, and then deposited in the botany laboratory herbarium. Aerial parts underwent drying at room temperature (20-25°C) for 14 d. 2 of 6

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Extracon of Lavandula stoechas essenal oils by hydrodisl- laon The plant is air-dried, away from light, to preserve the integ- rity of the molecules and prevent alteraon and reproducon by microorganisms. Next, 100 g of the dried aerial part of the plant got into a double 1000 mL ﬂask saturated with 530 mL dislled water, which is brought to the boil for 3 h, adding dislled wa- ter from me to me to prevent the mixture from drying out. The essenal oil-ﬁlled vapor passing through the condenser is condensed and collected in a clean bole. The process of hydro- disllaon, which includes the use of a Clevenger, F77, JOANLAB apparatus, is a way to extract essenal plant oils using water vapor. This method releases the volale components of plant cells, which are then condensed and dissipated. The goal is to increase yield by ulizing various hydromodulary raos (plant/ water) and disllaon mes while examining the eﬀects of var- ious parameters on the quality and chemical makeup of the ob- tained essenal oils. Animals A total of 27 local male rabbits (Cuniculus Lepus) aged 5 to 6 months and weighing 1.8 kg (Olba, WS-20 digital scale, French), were housed individually in galvanised baeries at room tem- perature (22 - 26 °C) and relave humidity (60 ± 5%). Food and water served ad libitum. The experimental protocol was in ac- cordance with the Naonal Guidelines for the Appropriate Care and Use of Animals in Laboratory Research established by Mo- hammed Cherif Messaadia University (Algeria). Experimental design The rabbits entered three groups of 9 animals each. Group I: nine rabbits kept as controls Group II: nine rabbits exposed to(100mg/kg/d)lambda cya- halothrin (LCT) [13] Group III: nine rabbits received both (100 mg/kg/d) lambda cyhalothrin (LCT) and (500 mg/kg/d) Lavandula stoechas [14] essenal oil (LS.EO). The doses tested took place daily in the diet over a 4-week period. Blood Specimens Hematological parameters Complete blood counts (CBC) performed on ancoagulated samples using Ethylene diamine tetra acec acid (EDTA) tubes. Data recorded in the following come from the use of automat- ic hematology analyzer (BK-6310.Biobase, China): white blood cells (WBC); red blood cells (RBC); hemoglobin (HB); mean cor- puscular volume (MCV); mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentraon (MCHC). Renal biomarkers The blood sample then underwent centrifugaon at 10000 g for 10 minutes (min) by using centrifuge (Rona 380, 380R and 380RHK, HETTICH®, French) to obtain plasma, to measure urea and creanine by using an automatic biochemistry analyzer (ARCHITECT ci4100) supplied with commercial kits (Spinreact, Spain). Stascal analysis Stascal signiﬁcance was assessed using ANOVA analysis with Tukey’s mulple comparison test aﬅer detecon of a nor- mal distribuon of the data and an appropriate P. P<0.0001 con- sidered signiﬁcant. RESULTS AND DISCUSSION Hematological parameters According to TABLE I, the results show a highly signiﬁcant in- crease in the WBC rate, the percentage of lymphocytes and the platelet rate in the group treated with LCT compared with the control and the group treated with LCT+L.S.EO. Concerning the parameters following red blood cells, hemoglobin, hematocrit, MCH, MCHC and MCV the values indicate a very highly signif- icant decrease in the group treated with lambda- cyhalothrin compared to the control group and the group treated with the mixture of lambda-cyhalothrin and Lavandula s essenal oil. On the other hand, the group receiving the mixture of insec- cide and essenal oil recorded signiﬁcant, highly signiﬁcant and non-signiﬁcant diﬀerences for the following parameters: red blood cells, MCV and MCH; white blood cells, lymphocytes and hematocrit as well as platelets and MCHC respecvely com- pared to the group treated with lambda cyhalothrin. 3 of 6

Lambda cyhalothrin toxicity in rabbit / Boubsil et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Cyhalothrin lambda is a broad-spectrum pyrethroid insecticide that induces physiological, biochemical and behavioural disturbances in laboratory animals, particularly with regard to body growth rate, the haematopoietic system, the kidneys and the liver. A complete blood count is an essen- al test for detecng changes in the various components of the blood. Blood count parameters provide important informaon about health and can help in the diagnosis and monitoring of various diseases and disorders. The results of the present study show a signiﬁcant increase in white blood cell count, lympho- cyte and platelet counts in the LCT and LCT+LS.EO treatment groups, compared with the control group. These results are consistent with the work of indicated reported a signiﬁcant in- crease in these parameters when rabbits were exposed to lamb- da-cyhalothrin [15]. Other authors concluded that the increase in the value of these parameters could be due to a pathological response, indicang that the body’s immune system was at- tempng to overcome and eliminate the toxic substances. With regard to the following parameters: red blood cells, hemoglobin and hematocrit, the data showed highly signiﬁcant reducons in the LCT-treated rabbit group compared with the control. This result is in agreement with that others study who highlighted a decrease in these parameters [16], which could be due to re- duced erythropoiesis or altered heme biosynthesis in the bone marrow [17]. In parallel was reported that hemoglobin concentraon and hematocrit signiﬁcantly reduced aﬅer LCT injecon in rabbits and those pyrethroids generally [18], induce hemolysis, which ulmately leads to a reducon of Red Blood Cells in the circu- laon [19]. Furthermore, these results are not consistent with those obtained by Azaﬁarimanga et al. [13] who found no sig- niﬁcant variaon in hemoglobin and hematocrit concentraons and red blood cell counts in the LCT-treated groups compared with the control group. The importance of erythrocyte indices (MCH, MCHC, and MCV) on the hemogram is to characterize the type of anemia. In general, their decrease indicates the presence of microcyc anemia (reduced globule size) and hypochromic anemia due to a lack of iron, heme and globin [20]. In the present study, the results of the erythrocyte indices (MCV, MCH, MCHC) showed highly signiﬁcant decreases in the LCT-treated group compared with the control group. Our results are comparable with those reported, who found that the increase or decrease in these in- TABLE I. Variaon in the levels of some hematological parameters in rabbits in the diﬀerent experimental batches (X ± SD) compared with the control. Groups Markers control LCT LCT+LS.EO Observation WBC (10³/µL) 6,79±0,97 a 11,11±1,45 b 10,87±1,73 a a** b=NS c=** Lymphocytes (%) 33,60±4,56 a 47,64±3,99 b 45,62±4,46 a a** b=NS c= ** RBC (10 6 /µl) 4,94±0,61 a 3,22±0,30 b 4,68±0,51 a a*** b=NS c=* HGB (g/dl) 10,64±1,40 a 7,37±0,53 b 8,94±0,63 a a*** b=NS c=* HCT (%) 34,57±4,96 a 24,22±2,67 b 35,02±4,09 a a** b=NS c=** PLT (10³/µl) 699±42,35 a 966,66±93,0 b 755,7±39,60 ab a*** b=NS c =NS MCH (pg) 25,10±1,55 a 20,11±1,44 b 23,76±1,32 a a** b=NS c= * MCHC (g/dl) 36,08±1,46 a 25,35±2,67 b 27,01±2,00 ab a** b=NS c=NS MCV (fl) 75,70±4,65 a 60,41±2,55 b 73,37±7,36 a a** b=NS c= * LCT: 100mg/kg/d of lambda-cyhalothrin ; LCT+LS.EO: 100mg/kg/bw of lambda-cyhalothrin+500 mg/kg/bw of Lavandula stoechas essenal oil NS: No signiﬁcant P<0.05; *: Signiﬁcant P<0.01; **: Highly signiﬁcant, P<0.001; ***: Very highly signiﬁcant p<0.0001. a= Control x LCT; b= Control x LCT+LS.EO; c= LCT x LCT+LS.EO 4 of 6

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico dices could be due to the increased acvity of the bone mar- row and the deﬁciency of certain hemopoiec factors [15 , 18]. Contrary to the conclusion the other study who noted no change in MCV, MCHC and MCH in rabbits treated orally with LCT [21]. Addional treatment with Lavendula essenal oil signiﬁcant- ly improved the disrupon of erythrocyte indices caused by the pescide. According to Baha et al. [22] Lavandula essenal oil oﬀers prospects for various biological applicaons, in parcu- lar for its anoxidant acvity.in vitro and in vivo studies on the synergisc eﬀect of lavender’s bioacve components with other molecules could prove an eﬀecve alternave for treang pa- thologies linked to oxidave stress. According to the results ob- tained by Ireola et al. [23], treatment with Lavandula essenal oil in rabbits exposed to inseccides brought erythrocyte index levels closer to normal values. Biochemical parameters The results of the renal funcon parameters shown in TA- BLE II, where urea levels showed a highly signiﬁcant and a very highly signiﬁcant increase in the LCT and LCT+LS.EO groups com- pared with the control. Creanemia showed a very highly sig- niﬁcant and another signiﬁcant increase in the LCT and LCT+LS. EO treated groups respecvely compared to the control group. TABLE II. Evaluaon of some kidney funcon parameters in rabbits in the diﬀerent experimental batches (X ± SD) compared with the control. Groups Markers control LCT LCT+LS.EO Observation Urea (g/L) 0,25±0,03 a 0,44±0,15 b 0,22±0,05 a a* b=NS c=** Creatinine (U/L) 5,89±0,02 a 8,58±0,79 b 7,52±0,80 b a*** b= * c= NS LCT: 100mg/kg/bw of lambda-cyhalothrin; LCT+LS.EO:100mg/kg/bw oﬂambda-cyhalothrin+500mg/kg/bw of Lavandula stoechas essenal oil NS: No signiﬁcant<0.05; *: Signiﬁcant p<0.01; **: Highly signiﬁcant, p<0.001; ***: Very highly signiﬁcant p<0.0001. a= Control x LCT; b= Control x LCT+LS.EO; c= LCT x LCT+LS.EO The toxicity of lambda cyhalothrin on renal parame- ters appears in table 2. Increased blood urea, creatinine concentrations are a specific and sensitive indicator of impaired renal function due to oxidative stress. Nadia et al. [24] and Aramjoo et al. [25] reported results similar to those obtained in this study. According to Eraslan et al. [26], the increase in urinary parameters is linked either to significant protein degradation, or to the toxic effect of pesticides on the kidneys, whose blood filtration capacity has been reduced. However, no significant difference and a signiﬁcant diﬀerence were found in creatinine and urea concentrations respecvely between the study groups compared with the control group [27]. Debabsa et al. [28] demonstrated that elevated plasma urea levels could indicate a decrease in glomerular ﬁltraon. Furthermore, the combined treatment with LCT+LS.EO in the present study showed renal funcon values almost similar to those of the control group. Essenal oil of Lavandula stoe- chas used for its potenal to reduce the toxicity of cyhalothrin lambda in male rabbits thus, the anoxidant properes of Lavendula stoechas extracts are mainly due to the presence of phenolic constuents, which could have a protecve eﬀect against nephrotoxicity induced by LCT administraon [22]. CONCLUSION The present study has demonstrated that exposure to lamb- da-cyhalothrin, a potent toxic inseccide, induces cytotoxic and hematological disorders, parcularly on renal biochemical markers and various hemogram indices reﬂecng a metabolic disorder. In the light of these observaons, further research on the mechanism of organ ssue damage with diﬀerent doses is required to beer assess the dose-eﬀect relaonship. In par- allel, prevenve supplementaon with lavender, essenal oil known for its anoxidant acvity due to the synergisc eﬀect of its bioacve components may prove a promising soluon against inseccide toxicity. Conﬂict of Interests The authors declare no conﬂict of interest regarding the publicaon of this manuscript. BIBLIOGRAPHICS REFFERENCES [1] Greenlee AR, Arbuckle TE, Chyou PH. Risk factors for fe- male inferlity in an agricultural Region. Epidemiology. 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