Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 06/12/2024 Aceptado: 19/02/2025 Publicado: 09/04/2025 hps://doi.org/10.52973/rcfcv-e35587 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 7 Detecon of plascs parcles in equine blood by Scanning Electron Microscopy Detección de parculas pláscas en sangre de equinos mediante Microscopía Electrónica de Barrido ¹University of Guayaquil – Faculty of Veterinary Medicina and Zootechnics – Ecuador. ²Fauna, Conservaon and Global Health Research Group - Amazon Regional University (IKIAM); Km 7 Via Muyuna - Muyuna Parish CP 150102 Tena - Ecuador. ³Army Producon and Supply Unit (REMONTA); Pichincha - Ecuador. ⁴Naonal University of Rosario, Faculty of Veterinary Sciences – Argenna. *Corresponding autor: darwin.yanez@ikiam.edu.ec ABSTRACT The study was conducted in the province of Guayas, located in the coastal region of Ecuador. The researchers analysed blood samples from 30 horses of different breeds (purebred, pony and mixed breeds) to detect the presence of micro- and nanoplascs (MPs and NPs). Blood smear and scanning electron microscopy (SEM) techniques were used to idenfy and quanfy plasc parcles in randomly selected animals aged between 2 and 12 years, with a body weight (BW) between 100 and 380 kg and a body condion score (BCS) between 5 and 6 (on a scale of 1 to 9), fed on natural grass and balanced supplements. The results did not show the presence of MPs, but NPs were idenfied in the blood smear of all animals, with an average of 51 parcles per field of 1700 square microns (µm²) at a depth of 5 micrometres (µm) and an average size of 426.33 nanometres (nm). No significant difference was found in the number or size of NP parcles between the sexes (females and males) (P=0.288); a greater presence of NPs was observed in younger horses (P<0.040). The pure-blood breed had a larger size of plasc parcles (P < 0.020) and the crossbreeds had a greater amount of NP parcles (P < 0.010) compared to other breeds. The research concludes that NPs are present in equine blood, highlighng the ability of these contaminants to enter the body and potenally cause adverse health effects. In parcular, younger animals showed a higher presence of NPs in blood, suggesng that the effects of exposure may be more severe in the early stages of life. Key words: Plasc parcles; young animals; scanning electron microscopy RESUMEN El estudio se llevó a cabo en la provincia del Guayas, ubicado en la región Costa del Ecuador, donde se analizaron muestras de sangre de 30 equinos de diversas razas (Pura Sangre, Poni y Meszos) para detectar la presencia de micro y nanopláscos (MPs y NPs), con animales de entre 2 y 12 años de edad, un peso corporal (PC) de entre 100 y 380 Kg, y una condición corporal (CC) de 5 y 6 (en escala del 1 al 9), seleccionados aleatoriamente, alimentados con paszales naturales y suplementos balanceados, se ulizó técnicas de fros sanguíneo y microscopía electrónica de barrido (MEB) para idenficar y cuanficar las parculas pláscas, los resultados no mostraron la presencia de MPs, pero se idenficaron NPs en el extendido de sangre de todos los animales, con un promedio de 51 parculas por campo de 1700 micrómetros cuadrados (µm²) a una profundidad de 5 micrómetros (µm), y un tamaño promedio de 426,33 nanómetros (nm), no se evidenció significancia en la candad o tamaño de las NPs entre los géneros (hembras y machos) (P=0,288), se observó una mayor presencia de NPs en equinos de menor edad (P<0,040), en la Raza pura sangre, las parculas pláscas encontradas fueron de mayor tamaño (P<0,020), y los Meszos presentaron mayor candad de NPs (P<0,010), en comparación con otras razas, la invesgación concluye que las NPs están presentes en la sangre de los equinos, lo que deja en evidencia la capacidad de estos contaminantes para ingresar al organismo y potencialmente causar efectos adversos en la salud, en parcular, los animales más jóvenes mostraron mayor presencia de NPs en sangre, lo que sugiere que los efectos de la exposición podrían ser más severos en las primeras etapas de vida. Palabras clave: Parculas pláscas; animales jóvenes; microscopía electrónica de barrido Israel Culcay-Troncozo 1 , Darwin Yánez-Avalos 2 * , Johana Delgado-Lozada 2 , Miltón Montalvo-Lozada 2 , Raul Díaz-Albuja 3 , Pablo Marini 4

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Growing concern about plasc parcles has led to an increase in research into their presence, distribuon and effects on ecosystems and living organisms. Microplascs are plasc parcles less than 5 millimetres in size, while nanoplascs are those with a diameter of less than 100 nanometres. These parcles originate both from the fragmentaon of larger plascs through processes such as ultraviolet radiaon, mechanical degradaon and biological weathering [1], and from industrial products that contain these materials in parculate form, such as cosmecs, personal care products and synthec texles [2]. The presence of plasc parcles in aquac and terrestrial ecosystems has been widely documented, with detrimental effects on both fauna and flora. These pollutants not only affect organisms that accidentally ingest them, but can also be absorbed by biological ssues, causing toxic effects at the cellular level. It has been observed that micro- and nanoplascs can induce a number of adverse physiological responses, such as oxidave stress, alteraons in the immune system, DNA damage, and dysfuncon of the reproducve and metabolic systems [3 , 4]. A key part of the rural and agricultural ecosystem, it is esmated that 39 million donkeys (Equus asinus), 40.5 million horses (Equus caballus), and 12.3 million mules ((Equus asinus X Equus Caballus), live in developing countries, making up over 85% of the world’s equids. In these countries, they are primarily used as labour, pack animals, oſten performing tasks in harsh and impoverished condions for long hours of the day [5]. In addion, exposure in animals consuming natural roadside pastures, a common resource in rural Ecuador, as plascs are found in many environments, equines may be exposed through their diet, so it is crucial to study how these contaminants affect various species in nature [6]. The presence of microplascs and nanoplascs provides insight into the dispersion of these contaminants in various species, not just those that directly enter the human food chain [7]. Exploring the existence in terms of accumulaon and effects of micro- and nanoplascs can reveal more about the biological suscepbility of different animal groups [8]. The results may alert us to the long-term effects of plasc exposure on the health of animals and, consequently, on the ecosystems in which they live [9]. The effects on living organisms, especially large terrestrial mammals, have emerged as a serious challenge due to their wide distribuon and complex effects on ecosystems [10]. Plascs are a major global pollutant, with large quanes being released into the oceans every day due to mass producon, overuse of this resilient material and poor environmental management [11]. The effects of plasc parcles on human and animal health have caused global concern, which calls for a sound toxicological approach using appropriate methods to further invesgate and understand the health problems caused by these pollutants [12]. This problem also affects developed countries, where large quanes of plasc waste are generated [13]. Therefore, understanding the magnitude of the situaon and the importance of disseminang informaon about plascs in the body is crucial to raise awareness and priorise public health [14]. The aim of this work was to detected the presence of micro- and nanoplascs in a selected area of equine (Equus caballus) blood smears using scanning electron microscopy. MATERIALS AND METHODS Bioethical aspects The criteria for scienfic research with animals established by the Naonal Commission for Scienfic and Technological Research (Fondecyt-Conicyt, Chile) [15], were applied in the development of this study. Locaon of the study The present study was carried out in the coastal region of Ecuador, in the province of Guayas, located in the south-east of the country. Animals from the EQUIMAS research centre, located on the coast with the following coordinates: latude: -2.272346 and longitude: -80.144874 [16]. Experimental design The coastal region, province of Guayas, has 16,138 horses [17], for this study 30 horses from the EQUIMAS Equestrian Centre were used, of the following breeds: Thoroughbred, Poni and Meszos, with an age between 2 and 12 years, a body weight (BW) between 100 and 380 kg, and a body condion (BW) between 5 and 6 (on a scale of 1 to 9) [18]. Horses were randomly selected, fed with balanced supplements and natural grasses based on Brachiaria decumbens (17,585 kg DM/ha/year and PC 7-12%), Brachiaria brizantha (26,970 kg DM/ha/year and PC 8-14%) [19]. Sample collecon The animals were placed in a suitable area and muzzled with a rope to immobilise them. Biosafety standards were followed [15]. Blood collecon equipment was carefully prepared to preserve the condion of the samples. In addion, the use of materials such as plasc syringes was excluded, Vacutainer tubes were used for blood collecon with minimal vacuum necessary to reduce the contact of the plasc-coated needle with the blood, which helps to reduce the release of microplascs and transfer of contaminants [20]. The lateral region of the neck, where the jugular vein is located, was palpated and the site was disinfected with coon wool and alcohol. A vacutainer (Medlab, EDTA Glass-Tube, Ecuador) was used, the jugular vein was punctured at a 45-degree angle to the skin, the tube containing ethylenediaminetetraacec acid (EDTA) (Medlab, EDTA Glass-Tube 5mL, Ecuador) was placed and 5 mL of blood was withdrawn. The needle was withdrawn and pressure was applied to the puncture site to stop the bleeding. The blood was homogenised with gentle agitaon. Samples were idenfied and stored at 4°C (Medicalpro, Transport refrigerator for blood samples, Ecuador) from the me of collecon to the Public Health Research Instute (INSPI) laboratory in Guayaquil [21]. Blood smear The samples collected were processed in the laboratory of the Public Health Research Instute (INSPI) in Guayaquil. A drop of blood was taken with a glass capillary (Medical Supplies, Haematology Glass Capillaries, Ecuador), the drop of blood was placed in the centre of a clean glass slide (Medical Supplies, Glass Slides, Ecuador), with another slide (in a gentle and controlled manner), the drop of blood was spread, forming a thin and uniform layer, taking care that the smear was homogeneous, 2 of 7

Detecon of plascs parcles in equine blood / Culcay et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico avoiding the cells to break or agglunate. The aim was to obtain an even and thin smear, thus achieving the desired characteriscs. In addion, the corresponding code of 5 (five) smears/sample was placed on the slides and stored in special boxes (MedLab, Glass Petri dish, Ecuador) that had previously been subjected to heat drying [22]. Sample drying The samples were kept for four days (d) in a controlled environment chamber, free of contaminaon, at a temperature of 24 to 25°C (room temperature) (BINDER, ED115, Germany), aſter which the slides were placed (Medical Supplies, Glass Slides, Ecuador), in an oven for 24 h at a temperature of 40°C, 24 h at a temperature of 60°C and 24 h at a temperature of 80°C. From day 4 to d 6 they were kept at a temperature of 100°C. The plates were then subjected to 150°C for 3 h to complete the Crical Point Dryer (CPD) phase without bubble formaon or deformaon, which is essenal to preserve the structural integrity of the parcles [23]. Metallizaon of samples for SEM The coang process was carried out by spuering with gold for 20 s using the (JEOL, JPC1200 Fine Counter, Japan), which consists of pumping metal onto the surface of the sample using an electric current. This produced a very thin layer (5 to 10 nm thick), which allowed observaon without electrostac charging of the sample and also increased the contrast of the images to obtain high quality microphotographs [24]. Implementaon of Posive and Negave Controls in the Detecon of Microplascs The posive control consisted of the inclusion of samples containing known plasc parcles, such as polyethylene or polypropylene microplascs in ultrapurified water, to verify the ability of analycal techniques, such as scanning electron microscopy (SEM), to detect these parcles. This control allowed validaon of the efficacy and sensivity of the detecon process, ensuring that the methods used were appropriate for idenfying plasc contaminants [25 , 26]. On the other hand, the negave control involved the use of samples without plasc parcles, such as contaminant-free ultrapure water, to detect any cross-contaminaon during the analysis process. The negave control samples were observed under SEM to confirm that the plasc parcles found in the experimental samples were not the result of handling or collecon equipment [27]. Sample mounng and scanning electron microscope (SEM) observaon The plates were placed in the SEM sample chamber, which is maintained at a high vacuum. The (SEM) (JEOL, JSM-7001F, Japan), emits a beam of electrons into the vacuum chamber, which is focused on the surface of the sample, producing electrical signals as secondary electrons. For imaging, the resulng electrical signal was amplified, then processed and digised by the microscope soſtware to produce a three- dimensional grey-scale image of the sample based on the surface topography and parcle size present in the samples [28]. Analysis by SEM confirmed the presence of plasc parcles in the blood samples, using the JSM IT500 version 1.300 soſtware integrated with the SEM, counng the parcles on the erythrocytes at a depth of 5 μm and measuring the plasc parcles in a field of 1700 μm² of the blood smear, excluding possible contaminaon by scanning the upper parts of the smear and the outer areas of the smear. The results were recorded by INSPI-cerfied technicians. Variables • Presence of nanoplascs / field of 1700 square microns (µm²). • Size of nanoplascs in the nanometre (nm) range. • Presence of nanoplascs / Field of 1700 square micrometres (µm²) according to sex, age and race. Stascal analysis The data obtained for each study variable were first tested for normality and then subjected to parametric or non- parametric tests as appropriate [29]. The data were analysed using generalised linear mixed models with parcle size and quanty as connuous variables and sex, age and race as discrete variables [30]. RESULTS AND DISCUSSION No microplascs were found, but the presence of nanoplascs was detected in all animals, with a result of 1530 NPs, with an average of 51 NPs/field of 1700µm². TABLE I and FIG. 1 show the results of the presence and quanty of nanoplasc parcles in a field of 1700 (µm²). TABLE I. Amount of nanoplasc parcles present in equine blood spread in a 1700μm2 field Parcles/Nanoplascs Sample Average TOTAL Size 30 426.33nm 12790.16nm Quanty 30 51un 1530um Nanometers (nm) and Unites (um) Ingeson of nanoplascs is a common route of exposure for many animals. Marine and terrestrial animals have been shown to accidentally ingest plascs by ingesng contaminated food or drinking water containing plasc parcles. In the case of horses, nanoplascs are likely to be ingested via the diet, specifically by consuming grass or water contaminated with plascs present in the environment [31]. This may occur in rural and agricultural areas where plasc contaminaon is more prevalent due to the use of plasc packaging and other pollung products. Similarly, to Prata et al. 2022 [32] presented results from a study in which 18 cats (Felis catus) and 17 dogs (Canis lupus familiaris) from urban areas were analysed and microplascs were found in kidney, lung, liver and blood clot samples, which was aributed to high levels of urban polluon. As well as the presence of nanoplascs in sheep (Ovis aries) liver ssue [33]. 3 of 7

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 1. Size measurement of five plasc nanoparcles present in erythrocytes of equine, with an average of 426.33 nm, by SEM. erythrocytes, with an average of 426.33 nm Shows the results of the parcle size of nanoplascs found in equine blood, where the number of parcles was counted in a field of 1700 µm² with a depth of 5 µm. In addion, a representave number of parcles (five parcles) were measured and marked with their respecve size in nanometres (nm) (TABLE ll). TABLE II. Parcle size of nanoplascs in equine blood idenfied by SEM over an area of 1700 µm² Number of parcle Sample Size/nm TOTAL /nm P 1 30 395.8 a 11872.8 0.288 2 30 425.0 a 12750.8 0.288 3 30 416.7 a 12500.0 0.288 4 30 455.2 a 13656.2 0.288 5 30 439.0 a 13171.0 0.288 Similar leers do not show significant differences, (P=0.288). The average parcle size of nanoplascs was 426.33 nm. It should be noted that microplascs are fragments smaller than 5 mm and nanoplascs are those smaller than 100 nm [34]. Two main sources of origin for the impact of microplascs and nanoplascs on the environment were idenfied: primary origin, which are intenonally produced for direct use (cosmecs, etc.), and secondary origin, which are plascs that degrade over the years and break down into micro and nanoparcles of the same size [35]. Bilal et al. [36] observed the presence of nanoplascs in a group of poultry (Gallus gallus domescus), with the possible sources of contaminaon being the feed supplied and the farm environment. Oxidave stress was associated with exposure to nanoplascs and higher rates of inflammatory bowel disease were found compared to healthy animals in the absence of nanoplascs. The gender variable has been used as a reference, where the result between females and males did not differ significantly in terms of average size and presence of nanoplasc parcles [37]. In a study that detected the presence of polyethylene, polyvinyl chloride and polypropylene in breast milk samples analysed, it was suggested that exposure and absorpon of residues not only reach different parts of the body, but can even be transmied through breaseeding (TABLE lll) [37]. TABLE III. Nanoplascs in equine blood by gender (female and male), over an area of 1700 µm² Variables Female (8/30) Male (22/30) t Prob. Sign. Average size/nm 425.09 426.79 -0.06 0.48 ns Nanoplascs in the field at 1700 µm². 59.00 48.09 0.54 0.30 ns Nanometres (nm), Square microns (µm²), Probability (Prob.), Significance (sign), No signif- icant difference (ns) In addion, microplascs have been idenfied in the placentas of mammals, in which at least several types of plasc waste have been found, with polypropylene predominang in the chorioamnioc membranes, highlighng the importance and urgency of assessing the risks that microplascs may pose during pregnancy and the effects this may have on the foetus and the mother [38]. Plasc parcles such as bisphenol A, phthalates and polychlorinated biphenyls have been linked to inferlity as endocrine disrupng chemicals and acute exposure can cause low ferlity and reproducve problems in farm animals [39]. One of the characteriscs of certain plasc parcles is lipophilicity, which means an affinity for lipid-rich ssues such as the epididymis and testes, which could facilitate transfer to semen, which is associated with semen quality in terms of volume, sperm count, molity and morphology [40]. According to TABLE IV, the presence of a higher amount of plasc parcles is evident in the younger animals. Since, there is a significant difference in inverse relaon between the age of the horse and the amount of NPs, respecvely. 4 of 7 TABLE IV. Nanoplascs in equine blood by age (2 - 12 years), in a 1700 µm² field Variables Intersect (a) Regression (b) Correlaon ( r ) Determinaon (r^2) Prob. Average size/nm 386.70 6.23 0.24 0.06 0.21 Nanoplascs in the field at 1700 µm². 93.68 -6.70 0.38 0.142 0.040 Nanometres (nm), Square microns (µm²), Probability (Prob), Significant difference (P<0.040)

Detecon of plascs parcles in equine blood / Culcay et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The presence of plasc parcles observed at early ages suggests that most of the effects are not exerted by the plasc parcles but by their metabolites, which originate in the liver [41]. In rats, it has been found that higher doses than those to which humans are normally exposed can cause severe disrupon to the developing male reproducve system. In addion, the presence of these parcles and other addives may cause toxicity, carcinogenicity and mutagenicity, as these compounds have been found in high concentraons in urine and breast milk, which is the first food of all mammals in the early stages of life [42]. The problem with plasc nanoparcles is that they cause injury aſter ingeson of plasc fragments, affecng the digesve system, leading to starvaon and even physiological damage ranging from oxidave stress to carcinogenesis [43]. In addion, the accumulaon of microplasc parcles in the body has a prolonged period of storage, parcularly in liver ssue, causing liver disease and metabolic problems [44]. In FIG. 2, the amount of nanoplascs is significantly related to a first order model, F=4.63; = (P<0.040), the value of R2=0.14; indicang that 14% of the change in the score of the amount of nanoplascs in blood is related to the age of the horse. FIGURE 2. Relaonship between equine age and the presence of nanoplascs in blood In resource-poor urban and rural areas, living condions and the environment are more exposed to polluon, including that from the degradaon of plasc products and their fragmentaon into smaller parcle sizes, such as nanoplascs (EPA, 2023). In these cases, although exposure to nanoplascs cannot be directly related to breed, this can be argued according to the results shown in TABLE V, Thoroughbred horses have a larger size of plasc parcles and half-bloods have a larger number of plasc parcles, as they are more exposed to these pollutants due to the condions of their environment [45]. TABLE V. Nanoplascs in equine blood by breed, observed over an area of 1700 µm² Variables Pure blood Meszo Ponny Prob. Average/nm 471.74 a 404.81 b 393.06 b 0.02 Nanoplascs in the field at 1700 µm². 23.60 b 69.89 a 18.00 b 0.01 Nanometres (nm), Square microns (µm²), Probability (Prob), Different leers present significant difference (P<0.020), (P<0.010) CONCLUSION The results obtained show the presence of nanoplascs in the blood of equines tested by blood smears at a depth of 5 µm, with an average of 51 parcles per 1700 µm² field. These findings suggest that nanoplascs have the ability to penetrate the body of animals, raising concerns about their potenal to cause long-term adverse health effects. In parcular, younger animals had higher levels of nanoplascs in their blood, suggesng that the effects of exposure may be more pronounced at earlier stages of development. As no differences in the presence or size of plasc parcles were observed between females and males, the results indicate that nanoplasc contaminaon affects equines across the board, regardless of sex. ACKNOWLEDGEMENTS The authors would like to thank the Public Health Research Instute (INSPI) - Guayaquil, and the Fauna, Conservaon, and Global Health Research Group (FASGLOBAL) of the Regional Amazon University IKIAM. Conflict of interests The authors declare no conflict of interest regarding the publicaon of this manuscript. BIBLIOGRAPHIC REFERENCES [1] Allen BS, Materić D, Allen D, Macdonald A, Holzinger R, Le-Roux G. An early comparison of nano to microplasc mass in a remote catchment’s atmospheric deposion. J. Hazard Mater. Adv. [Internet]. 2022; 7:100-104. doi: hps://doi.org/pcxp [2] Schröter L, Ventura N. Nanoplasc toxicity: insights and challenges from experimental model systems. Small. [Internet]. 2022; 18(31):220. doi: hps://doi.org/pcxq [3] Sáenz C. Microplascs in the biota of Andean rivers. [Undergraduate thesis]. Quito- Pichincha. Ecuador; University of the Americas;2020 [cited 02 dic 2024]. 66 p. Available in: hps://goo.su/0ZG [4] Castellanos W, Paytan L, Curasma J. Ecotoxicological influence of microplascs on the haematological profile of fistulated Brown Swiss cale. Rev. Cient. Cienc. Ing. [Internet]. 2023; 3(2):48-56. doi: hps://doi.org/pcxw [5] Burn CC, Dennison TL, Whay HR. Relaonships between behaviour and health in working horses, donkeys, and mules in developing countries. Appl. Anim. Behav. Sci [Internet]. 2010; 126(3-4):109-118. doi: hps://doi.org/ cpjnh2 [6] Holzer M, Mitrano DM, Carles L, Wagner B, Tlili A. Important ecological processes are affected by the accumulaon and trophic transfer of nanoplascs in a freshwater periphyton-grazer food chain. Sci Total Environ .[Internet]. 2019; 646: 1048-1056. doi: hps:// doi.org/g49j [7] Long Y, Zhang Y, Zhou Z, Liu R, Qiu Z, Li J, Wang W, Li X, Yin L, Wen X. Are microplascs in livestock and poultry manure an emerging threat to agricultural soil safety?. Environ Sci Pollut Res. [Internet]. 2024; 31(10):11543–11558 doi: hps://doi.org/gtm7dz 5 of 7

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico [8] Marcelino RC, Cardoso RM, Domingues ELBC, Gonçalves RV, Lima GDA, Novaes RD. The emerging risk of microplascs and nanoplascs on the microstructure and funcon of reproducve organs in mammals: A systemac review of preclinical evidence. Life Sci. [Internet]. 2022; 295:120404. doi: hps://doi.org/gpzkgg [9] Masciarelli E, Casorri L, Di Luigi M, Beni C, Valenni M, Costanni E, Aielli L, Reale M. Microplascs in Agricultural Crops and Their Possible Impact on Farmers’ Health: A Review. IJERPH . [Internet]. 2025; 22(1):45. doi: hps:// doi.org/pcxx [10] Liu M, Liu J, Xiong F, Xu K, Pu Y, Huang J, Zhang J, Pu Y, Sun R, Cheng K. Research advances of microplascs and potenal health risks of microplascs on terrestrial higher mammals: a bibliometric analysis and literature review. ENV GEOCH H. [Internet]. 2023; 45(6):2803- 2838. doi: hps://doi.org/gv6vg4 [11] De La Torre GE. Micropláscos en el medio marino: una problemáca que abordar. Rev. Sci. Tecnology. [Internet]. 2019[citado el 02 dic 2024]; 15(14):27-37. Disponible en: hps://goo.su/8C30W [12] Ham DH, Choi JS, Choi JH, Park WT. Microfluidic thrombosis analysis system: possibilies and limitaons. Micro Nano Syst. Le. [Internet]. 2023;11:17. doi: hps://doi.org/pcx2 [13] García S. Referencias históricas y evolución de los pláscos. Rev. Iberoam. Polym. [Internet]. 2009[cited Nov. 12 2024]; 10(1):71-80. Disponible en: hps://goo. su/lKre [14] Celi-Simbaña SS, Andrade-Mora DS, Loza-Pavón SJ, Bermeo-Sierra TI. Micropláscos, un problema de salud pública emergente. Rev. Inf. Sci. [Internet]. 2023; 102:4- 9. doi: hps://doi.org/pcx3 [15] Gallo C, Gimple J, Villaroel R, Goméz-Lopéz C, Méndez G, Saavedra-Sotomayor MA. Bioethics Advisory Commiee, Fondecyt-Conicyt: Bioethical aspects of animal experimentaon: FONDECYT-CONICYT. [Internet]. 2009 [cited Nov 28 2024]; 23-27 p. Available in: hps://goo. su/Qnbo8la [16] Gobierno Provincial Autónomo Descentralizado del Guayas (GPADG). Plan de Desarrollo y Ordenamiento Territorial de la Provincia del Guayas. Guayaquil; 2021 [cited December 2 2024]. Available in: hps://goo. su/5rYlA [17] Instuto Nacional de Estadísca y Censos (INEC). Resultados Nacionales con Resúmenes Provinciales CNA. [Internet]. 2000 [cited December 2 2024]. Available in: hps://goo.su/RcjCr4 [18] Henneke DR, Poer GD, Kreider JL, Yeates BF. Relaonship between condion score, physical measurements and body fat percentage in mares. Equine Vet. J. [Internet]. 1983; 15(6):371–372. doi: hps://doi: hps://doi.org/ d3bw7b [19] León R, Bonifaz N, Guérrez F. Pastos y forrajes del Ecuador: Siembra y producción de pastos.1th. ed. Universidad Politécnica Salesiana; 2018. [20] Rochman CM, Hoh E, Kurobe T, Teh SJ. Ingested plasc transfers hazardous chemicals to fish and induces hepac stress. Scienfic Reports. [Internet]. 2013; 3:3263. doi: hps://doi.org/gf7cz3 [21] De Castro T, Valdez L, Rodríguez M, Benquet N, Rubianes E. Decline in assayable progesterone in bovine serum under different storage condions. Trop. Anim. Health Prod. [Internet]. 2004; 36:381–384. doi: hps://doi.org/ ſt5cqq [22] Andrade-Becerra R, Pérez-Rubiano C, Vargas-Avella J. Manual de práccas de laboratorio en Microbiología Veterinaria. 1th. ed. Tunja: UPTC; 2023. 25 p. doi: hps:// doi.org/pcz7 [23] Chandra J, George N, Narayanankuy SK. Isolaon and characterizaon of cellulose nanofibrils from arecanut husk fibre. Rev. Carbohydr. Polym. [Internet]. 2016; 142:158–166. doi: hps://doi.org/ggczwc [24] Reyes-Zambrano SJ, Lecona-Guzmán CA, Guérrez-Miceli FA, Santana-Buzzy N, Slas-Flores I, Tzec-Simá M, Barredo- Pool FA, Ruiz-Lau N, Ávila-Miranda ME. Scanning electron microscopy and enzymac analysis in Agave americana during Fusarium oxysporum infecon. Mex J Phytopathol. [Internet]. 2020; 38(3):408–419. doi: hps://doi.org/ pcz8 [25] Lusher AL, Welden NA, Sobral P, Cole M. Sampling, isolaon, and idenficaon of microplascs ingested by fish and invertebrates. Analycal Methods. [Internet]. 2017; 9(9):1346-1360. doi: hps://doi.org/gf428t [26] Van-Cauwenberghe L, Devriese L, Galgani F, Robbens J, Janssen CR. Microplascs in sediments: A review of techniques, occurrence and effects. Mar. Environ. Res. [Internet]. 2015; 111:5-17.doi: hps://doi.org/ggrmr2 [27] Mithun MH, Bin-Shaikat MF, Sazzad SA, Billah M, Salehin S, Foysal AM, Jubayer A, Islam R, Anzum A, Rahman- Sunny A. Microplascs in Aquac Ecosystems: Sources, Impacts, and Challenges for Biodiversity, Food Security, and Human Health - A Meta-Analysis. Angiotherapy. [Internet]. 2024; 8(11):100035.doi: hps://doi.org/pcz9 [28] Kumar A, Negi YS, Choudhary V, Bhardwaj NK. Characterizaon of cellulose nanocrystals produced by acid hydrolysis from sugarcane bagasse as agro-waste. J. Mater Phys. Chem. [Internet]. 2014; 2(1):1–8. doi: hps://doi.org/gjjkxb [29] Dagnino J. Biostascs and epidemiology. Analysis of variance and correlaon. Rev. Chil. Anest. [Internet]. 2014; 43(2):306-310. doi: hps://doi.org/pc2b [30] INFOSTAT. Stascal analysis for research and management. Infostat. [Internet]. 2020[cited December 12 2024]. Available in: hps://goo.su/zzVz4f [31] Wright SL, Rowe D, Thompson RC, Galloway TS. Microplasc ingeson decreases energy reserves in marine worms. Curr. Biol. [Internet]. 2013; 23(23):2186– 2190. doi: hps://doi.org/p92 [32] Prata JC, Patrico-Silva AL, Da-Costa JP, Dias-Pereira P, Carvalho A, Fernandes AJ, Da Costa FM, Duarte AC, Rocha-Santos T. Microplascs in internal ssues of companion animals from urban environments. Animals. [Internet]. 2022; 12(15):1979. doi: hps://doi.org/pc2c [33] Bahrani F, Mohammadi A, Obaradaran S, De La Torre GE, Ramavandi B, SaeedI R, Tekle-Röering A. Accumulaon of microplascs in edible ssues of livestock (Cow and sheep). SSRN [Internet]. 2023; 4:6-10. Preprint. doi: hps://doi.org/pc2d 6 of 7

Detecon of plascs parcles in equine blood / Culcay et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico [34] Arthur CM, Baker J, Bamford H. Proceedings of the Internaonal Research Workshop on the Occurrence, Effects, and Fate of Microplasc Marine Debris; 2008 September 9-11; University of Washington Tacoma, Tacoma, WA, USA. Group; 2009. p 530. Available in: hps://n9.cl/zsnqox [35] Lacava JE, Schmaedke A, Denaro MA, Bernabeu PR, Gargarello RM. Evaluaon of the presence of microplascs on the beaches of the Río de la Plata: Buenos Aires City and surroundings. Rev. AIDIS Ing. Cienc. Ambient. [Internet]. 2022; 15(3):1163-1173. doi: hps:// doi.org/pc2f [36] Bilal M, Taj M, Ul-Hassan H, Yaqub A, Shah MIA, Sohail M, Rafiq N, Aque U, Abbas M, Sultana S, Abdali U, Arai T. First Report on Microplascs Quanficaon in Poultry Chicken and Potenal Human Health Risks in Pakistan. Toxics. [Internet]. 2023; 11(7):612. doi: hps://doi.org/ gwnxcx [37] Ragusa A, Svelato A, Santacroce C, Catalano P, Notarstefano V, Carnevali O, Papa F, Rongiole MCA, Baiocco F, Draghi S, D’Amore E, Rinaldo D, Giorgini E. Plascenta: First evidence of microplascs in human placenta. Environ. Int. [Internet]. 2021; 146:106-274. doi: hps://doi.org/ghqv [38] Amato-Lourenço LF, Carvalho-Oliveira R, Ribeiro-Júnior G, Dos Santos-Galvão L, Augusto-Ando R, Mauad T. Presence of airborne microplascs in human lung ssue. J. Hazard Mater. [Internet]. 2021; 416:124-126. doi: hps://doi.org/gj6frf [39] Urli S, Corte-Pause F, CrociatI M, Baufeld A, Monaci M, Stradaioli G. Impact of microplascs and nanoplascs on livestock health: An emerging risk for reproducve efficiency. Animals. [Internet]. 2023; 13(7):1132. doi: hps://doi.org/pc2g [40] Grechi N, Ferronato GA, Devkota S, Ferraz MAMM. Microplascs are detected in bull and dog sperm and polystyrene microparcles impair sperm ferlizaon. Biol. Reprod. [Internet]. 2024; 111(6):1341-1351. doi: hps://doi.org/pc2h [41] Talsnes CE, Andrade AJ, Kuriyama SN, Taylor JA, Vom- Saal FS. Components of plasc: experimental studies in animals and relevance for human health. Philos. Trans. R. Soc. B. [Internet]. 2009; 364(1526):2079–2096. doi: hps://doi.org/bqsrn9 [42] Blackburn K, Green D. The potenal effects of microplascs on human health: What is known and what is unknown. Ambio. [Internet]. 2022; 51(3):518-530. doi: hps://doi.org/gm28cb [43] Wang W, Guan J, Feng Y, Nie L, Xu Y, Xu H, Fu F. Polystyrene microplascs induced nephrotoxicity associated with oxidave stress, inflammaon, and endoplasmic reculum stress in juvenile rats. Front. Nutr. [Internet]. 2023; 9:1059660. doi: hps://doi.org/pc2j [44] Ying-Yong CQ, Valitaveel S, Tang BL. Toxicity of microplascs and nanoplascs in mammalian systems. Int J Environ Res Public Health. [Internet]. 2020; 17(5):1509. doi: hps://doi.org/gjf83k [45] Deng Y, Zhang Y, Lemos B, Ren H. Tissue accumulaon of microplascs in mice and biomarker responses suggest widespread health risks of exposure. Sci. Rep. [Internet]. 2017; 7:46-87. doi: hps://doi.org/f945n3 7 of 7