An appraisal of traditional knowledge of plant poisoning of livestock and its validation through acute toxicity assay in rats

Background: Kashmir Himalaya hosts the most diverse and rich flora in the world, which serves as grazing land for millions of small ruminants in the area. While most plant species are beneficial, some can be poisonous, causing economic losses and animal health issues for livestock. Consequently, this study is the first comprehensive report on the traditional phyto-toxicological knowledge in District Muzaffarabad and the assessment of its authenticity through experimental studies in rats. Methods: The data regarding traditional knowledge was gathered from 70 key respondents through semi-structured interviews, which was quantitatively analyzed and authenticated through plant extract testing on Wistar female rats and comparison with published resources. Results: A total of 46 poisonous plant species belonging to 23 families and 38 genera were reported to be poisonous in the study area. Results revealed that leaves were the most toxic plant parts (24 species, 52.1%), followed by the whole plant (18 species, 39.1%), stem (17 species, 36.9%), and seeds (10 species, 21.7%). At the organ level, liver as most susceptible affected by 13 species (28.2%), followed by the gastrointestinal tract (15 species, 32.6%), nervous system (13 species, 8.2%), dermis (8 species, 17.3%), renal (7 species, 15.2%), respiratory (4 species, 8.7%), cardiovascular system (3 species, 6.5%), and reproductive system (2 species, 4.3%). The poisonous plant species with high Relative frequency citation (RFC) and fidelity level (FL) were Nerium oleander (RFC, 0.6; FL, 100), Lantana camara (RFC, 0.6; FL, 100), and Ricinus communis (RFC, 0.6; FL, 100). Experimental assessment of acute toxicity assay in rats revealed that Nerium oleander was the most toxic plant with LD50 of (4,000 mg/kg), trailed by Ricinus communis (4,200 mg/kg), L. camara (4,500 mg/kg), and Datura stramonium (4,700 mg/kg); however, other plants showed moderate to mild toxicity. The major clinical observations were anorexia, piloerection, dyspnea, salivation, tachypnea, constipation, diarrhea, tremor, itchiness, and dullness. Conclusion: This study showed that numerous poisonous plants pose a significant risk to the livestock industry within Himalayan territory, leading to substantial economic losses. Consequently, it is of utmost importance to conduct further comprehensive studies on the phytotoxicity of plants.


Introduction
Plants serve as the primary food source for herbivores and are also used for the treatment of many diseases (Elshafie et al., 2023).Nonetheless, some plants are also known for their toxicity in ruminants due to the presence of toxic compounds (Dubey et al., 2018).These toxins can have detrimental effects on the ruminants, leading to economic losses such as health deterioration, low productivity, deformed offspring, and even death (Kebede et al., 2015;Paim et al., 2023).
Ethnoveterinary studies involve the exploration and documentation of traditional knowledge and practices related to using plants, minerals, and other natural resources for managing animal health and wellbeing (Teuscher and Lindequist, 2023).These studies have gained significance due to their potential to offer sustainable and cost-effective solutions for livestock healthcare in rural and resource-limited areas.However, alongside the benefits, it is essential to address the potential risks associated with the use of plantbased remedies, including the risk of poisoning (Uprety et al., 2022).
Although the livestock are selective feeders while grazing or browzing in rangelands, occasional livestock poisonings are widespread worldwide in free-range systems due to poisonous plants (McKenzie, 2020).These cases may occur when preferred plant species are scarce during dry seasons, forcing animals to browse perennial shrubs and bushes rich in toxic secondary metabolites (Oladosu et al., 2016), causing production loss, morbidity, and mortality in animals (Dubey et al., 2018).The poisonous plants may sometimes be eaten accidentally (Nielsen et al., 2019).In addition, nomads and villagers may not be familiar with potentially toxic plants during hay making (Majeed et al., 2020).Thus, the most important factors that expose ruminants to plant poisoning are feed shortage, nutritional deficiency, and sudden exposure to plants (Stegelmeier et al., 2013).
Plant toxins might be synthesized within the plant itself, produced by microorganisms associated with animals, or simply incorporated and accumulated by the plant (Onyeyili et al., 2018).Plants can be categorized into different groups based on their poisonous principle (Oladosu et al., 2016).These toxic properties tend to be chemically identical or even similar within a single group of related plant species (Botha and Penrith, 2008), the nature of these toxins changes with respect to their origin and surrounding environmental conditions (Desta, 2019).The primary toxic compounds that serve as defense mechanisms in plants against herbivores include tannins ( Kara and Sürmen, 2019), phenolics (Stevenson et al., 2017), alkaloids (Matsuura and Fett-Neto, 2015), phytohemagglutinins (Dubey et al., 2018), terpenes (Dearing and Weinstein, 2022), cyanogenic glycosides (Gershenzon, 2017), and oxalates (Karabourniotis et al., 2020).
Plant poisoning has the potential to impact a wide range of body systems.Numerous factors contribute to the toxicity of plants in livestock (Botha and Penrith, 2008).Among ruminants, the severity of plant toxicity can differ based on the animal species or breed, age, gender, and overall health status (Leong et al., 2017).Further, plant growth conditions, developmental stages, specific parts of the plant, and consumption amounts are all critical factors that influence the outcome (Dixit, 2007).The poisoning is also influenced by various chemical factors like size of particles, solubility, toxicity, rate of absorption and excretion, tendency to bind with body tissues or fluids, and absence of established metabolic processes (Khan et al., 2018).Diagnosis of plant-induced livestock poisoning involves considering the animals' history, observed clinical symptoms, postmortem findings, signs of plant consumption through grazing or browsing, and the presence of toxic plants in the gastrointestinal tract (Botha and Penrith, 2008).
The Muzaffarabad division in Azad Kashmir is rich in plant diversity, including a wide variety of potentially toxic plant species.These plants can be a threat to both animal health and the livestock industry in the region.Prior to our research, no comprehensive study had been conducted to identify and describe the native poisonous plants in Azad Jammu and Kashmir.Therefore, this study represents a pioneering effort to bridge this research gap by identifying these poisonous plants and thoroughly examining the harmful impacts of plant poisoning on animal wellbeing.The information regarding indigenous poisonous plants was collected from livestock holders through periodic surveys and validated through existing literature.Additionally, the accuracy of knowledge held by livestock keepers and available in published reports was assessed by testing the toxicity of selected poisonous plants through acute toxicity assay in Wistar female rats.

Ethics approval and consent to participate
Formal consent was received from informants regarding data collection and publication, and the Participatory Rural Appraisal (PRA) approach, as mentioned in the Kyoto Protocol, was applied with the consent of the informant.Additionally, the study was approved by the Ethical committee and Board of Advance Studies and Research, Sindh Agriculture University Tandojam, in its 144th meeting under serial number DAS/937/2022.

Study area
Muzaffarabad is the capital of Azad Jammu and Kashmir, Pakistan (Figure 1).It is located in the western Himalayan range, between latitude 34o03′-34o35′N and longitude 73o23′-73o45′E, with a land area of around 1,642 km 2 .The study area exhibits a wide range of elevations, with the lowest point located in the southern Kohala locality at 582 m above sea level (m.a.s.l.)The highest points in the region are found at the summits of Makra Mountain and Neela Ganja Mountain, which reach heights of 3,819 m and 4,473 m, respectively.The variation in elevation across the study area has important implications for a range of ecological processes, including temperature gradients, precipitation patterns, and vegetation distribution.It also significantly impacts human activities such as agriculture and transportation infrastructure.
The bordered by the Muzaffarabad district with Hattian Bala district to the east, Neelum district to the northeast, Hazara division in Khyber Pakhtunkhwa (KPK) to the north and northwest, Bagh district to the south, and Murree hills in Punjab to the southwest.The topography is distinguished by a scenic landscape, with mountains ranging from subtropical valleys to the fascinating alpine zones of the Himalayas.Natural beauty abounds in the region, with dense forests, swiftly flowing rivers, and meandering streams.The Jhelum and the Neelum are the two notable rivers in the area (Khan et al., 2018).
The climate in Muzaffarabad district is subtropical highland.Temperatures range from −2-40 °C between January and August.The average annual rainfall ranges between 1,000 and 1,300 mm, with the majority falling between May and August (around 680 mm).Winds blow from west to east during the day and from southeast to north at night.The wind speed is higher in the afternoon than in the morning.The snow line is approximately 1,200 m above sea level in winter and approximately 3,300 m. a.s.l. in summer (Qasim et al., 2010;AJK, 2015).

Relative frequency citation (RFC)
The data gathered was subjected to quantitative analysis using the Index of RFC, employing the formula provided below: The RFC functions as a gauge for the particular importance of each species, gauged by how often they are mentioned in citations.The FC represents the proportion of survey participants who brought up the use of a specific species, divided by the total number of participants (N).It is worth mentioning that the analysis does not consider the various ways the species are used (Majeed et al., 2020).

Fidelity level (FL)
FL was assessed for frequently mentioned diseases or conditions using the approach outlined by Hoffman and Gallaher (2007).This indicates the percentage of individuals who identified a particular species as the cause of poisoning compared to the overall number of individuals who acknowledged the role of plant poisoning in affecting livestock (Khunoana et al., 2019).

FL
Np N * 100 In this context, Np is the count of individuals who associated a particular livestock ailment with plant poisoning, whereas N represents the overall count of individuals who witnessed disease symptoms or indicators in their livestock due to plant poisoning.

Priority ranking test
A priority ranking test was used to rank the top poisonous plants.In this test, 15 selected key respondents were asked to rank the plants based on the toxicity in the study area by following a method of Martin (1995).

Validation of folk knowledge through published scientific literature
For authenticity of the data obtained from informants on poisonous plants, a comprehensive literature review was conducted by systematically analyzing research papers from various electronic-based journals and books reporting information related to poisonous plants, their toxicity, and the symptoms of toxicity (Figure 2).Multiple search engines were utilized for a literature search, including Web of Science, Google Scholar, ResearchGate, PubMed, SciFinder, Scopus, Chemical Abstracts Services, books, dissertations, and technical reports.The search was performed using specific keywords, such as "poisonous plants," "lethal dose LD 50 ," and "toxic compounds." In every research article, we gathered information about various characteristics of each plant.These included their scientific names, family classification, names used locally, lifeform, the specific toxic plant parts, type of disease and animal affected, recommended dosage and levels of toxicity, specific toxin, and mechanism of action, clinical symptoms, susceptible organ and species.
Only those articles were selected that presented original research, were written in English, had undergone a peer-review process, and had full text available online through various scientific search engines.A total of 129 documents were ultimately selected for inclusion in this review, covering the period from 1981 to March 2023.In addition to academic research articles, this review's basic framework was put together by consulting a range of books about local plants, broadcast news sources, and print media.Online botanical resources such as www.theplantlist.org,www.plantoftheworldonline.org, www.gbif.org,www.jstor.org,and www.tropics.org(accessed on 18 June 2023) were also used to gather data on other names for plants and their geographic distribution.

Poisonous plant selection and preparation of extracts for acute toxicity assays
The 12 poisonous plants for toxicity assays were selected on the basis of a ranking based on a literature review, a survey from stakeholders, and the abundance of plants in the selected area.Poisonous plant leaves were collected from various areas in Muzaffarabad.Leaves were shade-dried at room temperature, and an electrical homogenizer was used to grind them to a powder.The ratio of the dried part's (1.5 kg) weight to the original weight (4 kg) was calculated and then kept in glass bottles for further use in the experiment.The 120 g powder material was poured into a transparent glass bottle with 800 mL of 95% ethanol and soaked for 3 days.The glass bottle was labeled with a plant name.Further, the soaked material was filtered using a muslin cloth or filter paper.A rotary evaporator was used to remove the solvent (ethanol) from the flask.However, the extract was dried more thoroughly in the hot air oven at 65 °C and stored at 40 °C.
Selection and housing of animals (rats) for the experiment Wistar female rats, aged 8-12 weeks, weighing 125-150 g, were obtained from DOW University of Health Sciences (DUHS), Karachi.These rats were maintained in the animal house of the Department of Veterinary Parasitology, Sindh Agriculture University, Tandojam.The animals were maintained under standard environmental conditions of a temperature of 24 °C ± 10 °C, a 12 h dark-light cycle, free access to drinking water, and a standard pelleted diet.

Screening of selected plants for toxicity by limit test
The limit test was performed as per guidelines of the Organisation for Economic Cooperation and Development (OECD) 423.The purpose of the limit test was to determine the toxicity of themostly reported plants, selected on the basis of the survey and literature review, by initially testing a dose of 2,000 mg/kg body weight and, as no deaths occurred in rats, finally a higher dose of 5,000 mg/kg body weight was tested showing higher signs of toxicity.

Preparation of doses and administration of treatments
The animals were weighed before the experiment (Table 1), and doses were calculated as per body weight.The ethanol extract of each plant was weighed, dissolved in 0.1 mL dimethylsulfoxide (DMSO), and then water was added to make a final volume of 1 mL.Rats were deprived of food and water 2 h before the experiment.The doses were orally administered to rats with the help of a 16-gauge gavage needle.The animals were examined for mortality and apparent changes at specific intervals, including the first, sixth, 12th, and 24th hour.Subsequently, the animals were monitored once a day for 2 days.
Initially, for testing the dose of 2,000 mg/kg body weight, 36 rats were divided into 12 groups, each having three rats.Regular tap water was given to the control group, while 10% DMSO was added to water for the sham control group.The remaining treatment groups received a single oral dosage of various plant extracts  (2,000 mg/kg body weight), as shown in Table 1.Following the OECD's recommendations, the experiment was repeated using a new set of three rats for every different plant extract because no fatalities were observed, as outlined in Table 2.
Next, the 5,000 mg/kg body weight dose was tested.For this experiment, 30 rats were divided into 10 groups, each having three rats.The control group received regular tap water, while the sham control group was given 10% DMSO in water.Plant extracts (5,000 mg/kg body weight) were administered as a single oral dose for the remaining treatment groups.If the first animal survived, two further animals were dosed.If only one of the three animals died, the LD 50 value was expected to exceed 5,000 mg/kg.If both animals died, then dosing proceeded at 2,000 mg/kg.If all three animals survived, the maximum dose administered was considered to be the LD 50 value.

Determination of lethal dose 50 (LD 50 ) through up-and-down method
Plant extracts causing mortality in rats at the dose of 5,000 mg/kg body weight were further tested using the up-and-down procedure following OECD test guidelines 425 (OECD, 2001) to determine the LD 50 .For this purpose, doses of 2,500, 3,000, 3,500, 4,000, 4,500, and 5,000 mg/kg were applied, respectively.The plants were classified into various toxicity grades as per the toxicity standards of OECD (Table 2).LD 50 below 5,000 mg/kg is classified as highly toxic, while LD 50 above 5,000 mg/kg is considered harmless (Ea et al., 2021).
The LD 50 was calculated using the arithmetic method by (Alui and Nwude, 1982) using the formula LD 50 = LD 100 -(Dd × Md)/N; where LD 100 = dose that caused 100% mortality; N = number of animals per group; Dd × Md = dose difference (Dd) multiplied by mean death (Ea et al., 2021).LD 50 is considered highly toxic if it is below 2,000 mg/kg, whereas it is considered safe if it exceeds 5,000 mg/kg (Hayes and Loomis, 1996).

Data analysis
The results were presented as the averages and the standard deviation (SD).The statistical significance was assessed through a one-way analysis of variance (ANOVA) followed by the post hoc least-significant difference (LSD) test.Results with p-values lower than 0.05 were regarded as significant.

Demography
To collect data on plant poisoning affecting livestock across different areas of Muzaffarabad district, 70 participants were interviewed, consisting of 30 males and 40 females.This group encompassed various roles, including Gujjars (farmers), Bakkarwals (nomads), laboratory professionals, veterinarians, livestock holders, artificial inseminators, dairy owners, milk vendors, traditional healers, and agricultural experts.Notably, all traditional healers and veterinarians were of the male gender.The age spectrum of the respondents ranged from 20 to 80 years.Among the respondents, the predominant proportion, approximately 67.14%, lacked formal education, while the remaining individuals held masters or Ph.D. degrees (Table 3).

The LD 50 of various plants, according to literature survey
As depicted in Table 6, R. communis, was the most toxic plant in the light of literature, out of 12 plants that were also tested for toxicity in the current study, with LD 50 of less than 20 mg/kg.This plant showed different symptoms in livestock, such as general body weakness, loss of appetite, difficulty in breathing, mild fever, abdominal pain, cramps, piloerection, miscarriage, neurological issues, depression, acute purgation, and sudden diarrhea.L. camara, on the other side, with LD 50 157.37mg/kg, caused liver damage, bile flow obstruction, hair erection, light sensitivity, and accumulation of bile in the liver.N. oleander with an LD 50 157.37mg/kg causes bulging of the eyeballs, stomach pain, salivation, emesis, motion, piloerection, irregular heartbeat, weakness, and sluggishness.E. helioscopa, with LD 50 of 1,211 mg/kg, caused acute dermatitis when animals encounter milky sap, piloerection, frothing in the mouth and throat, purgation, weight loss, and D. viscosa LD 50 1,250 mg/kg caused nausea, vomiting, and shivering in animals.The rest of the plants showed LD 50 > 2,000 mg/kg, which, according to OECD 423, is less toxic.

Limit test for determination of plant LD 50
The female Wistar rats orally administered ethanol extract at a dose of 2,000 mg/kg body weight showed no signs of toxicity or mortality for all 12 plants tested for toxicity (Table 6; Figure 7).However, when a 5,000 mg/kg body weight dose was applied, signs of toxicity, mortality, and a significant reduction in body weight were found in various selected poisonous plants, namely, N. oleander, R. communis, L. camara, and D. stramonium.Moreover, the mortality of animals was observed within 10-24 h with severe clinical manifestations, i.e., anorexia, water intake, piloerection, dyspnea, salivation, tachypnea, constipation, diarrhea, tremors, itchy nose, and dullness.The severe toxic effect caused by N. oleander was followed by R. communis, L. camara, and D. stramonium, but other plants showed moderate to mild clinical manifestations.For the determination of the exact LD 50 of each plant, extracts were tested through the Up-and-Down method.The LD 50 for poisonous plants was found to be 4,000, 4,200, 4,500, and 4,700 mg/kg for N. oleander, R. communis, L. camara, and D. stramonium, respectively (Table 7).

Pearson's correlation
Pearson's correlation coefficient was computed to assess the relationship between the level of reported fidelity and RFC.The outcome reveals a substantial correlation between the FL and RFC variables, with a correlation coefficient of r = 0.9 (Figure 8).

Discussion
In our ecosystem, plants are essential for food, shelter, and aesthetic beauty (Saha et al., 2022).Nevertheless, not all plants are harmless when they interact with animals.Certainly, many plant species contain toxic compounds that can cause animals from mild irritation to severe illness or even death (Koshariya et al., 2023).Understanding plant toxicity in animals is critical for veterinarians, cattle holders, nomads, and wildlife managers to ensure the health and safety of both domestic and wild animals (Gupta, 2012;McKenzie, 2020).Our study area selection was motivated by the rich plant biodiversity and the lack of research on poisonous plants and their toxicity to livestock.As far as we know, no research has been conducted on plant phytotoxicity in District Muzaffarabad and its surroundings.A comprehensive study on the phytotoxicity of plants in this region is extremely important because this area has a significant number of small ruminants compared to other parts of Azad Jammu and Kashmir.It is crucial for farmers to be able to identify which plants are toxic for livestock and to take the necessary precautions to avoid them in order to prevent livestock from consuming them.

Demographic dynamics
In this study, 70 key informants interviewed were selected from both genders.Our informant selection is counter to the majority of ethno-veterinary studies, where men predominate.In the study area, women typically keep and care for animal herds, which can be explained by the ethnotoxicology study's nature, cultural differences, and traditional roles in which women are responsible for household

Plant diversity
This study reported 46 poisonous plants from 23 families and 38 genera.Asteraceae, Fabaceae, Ranunculaceae, Solanaceae,   Euphorbiaceae, and Convolvulaceae were the most dominant families.Herbs were the most prevalent form of life, followed by shrubs and trees.Asteraceae has many species, including Bidens bipinnata, Senecio vulgaris, Ageratum conyzoides, Xanthium indicum, and P. hysterophorus.Asteraceae has been reported as a dominant plant family by many researches (Sema et al., 2018;Kefifa et al., 2020).There is a significant variation in floristic diversity within plant families depending on climate and study area (Dansou et al., 2021).This difference could also be explained by the diversity of socio-cultural clusters that differ from one nation to another since the utilization of natural plant resources for healing intentions might be linked with the knowledge of these socio-cultural groups.The Asteraceae, Solanaceae, Fabaceae, and Euphorbiaceae stand out as some of the most significant families of poisonous plants.Many researchers have previously documented the plants in these families as primarily responsible for plant poisoning (Wink, 2013;Parihaar et al., 2014;Diaz, 2015).Toxins such as alkaloids such as atropine, hyoscyamine, and scopolamine (Baloch et al., 2017), cynogenic glycosides like anthocyanins and anthrachinone (Randrianarivo et al., 2014;Serrano, 2018), saponin including both terpenoid (Randrianarivo et al., 2014) and steroids (Umair et al., 2017;Dusemund et al., 2018), and cyanogenic glucosides (Junior et al., 2022) make these families toxic.

Toxic plant parts and their toxicity
In the present study, phytotoxicity was confined to the entire plant or limited to some parts.The most toxic plant parts were the leaves, followed by the whole plant.The most susceptible systems to plant toxicity were the gastrointestinal tract, affected by 15 species; the liver affected by 13 species, and the nervous system affected by 13 species.It has been widely shown that leaves can be used for both therapeutic and grazing reasons.Leaves are metabolically dynamic plant parts (Alexiades, 1996).This is due to the fact that leaves are extremely nutrient-rich plant components that animals can readily chew through or swallow, unlike seeds and fruits (Matsuura et al., 2016).According to our research, most of the toxicity in livestock was produced by plants rich in cyanogenic chemicals, glycosides, saponins, and alkaloids.Animals exposed to alkaloids experience severe toxicity, which damages their liver and affects their neurological system (Adibah and Azzreena, 2019).In the majority of cases, they are lethal and cause depression, miscarriage, shivering, nausea, and immobility (Langford and Boor, 1996;Vetter, 2000;Baloch et al., 2017).Conversely, glycosides can induce paralysis, agitation, weakness, burning, tremors, and vision loss (Nguyen et al., 2020;Kharchoufa et al., 2021;Chen et al., 2023).Saponins can cause skin inflammation, liver issues, agitation, burning of skin, and necrosis, leading to kidney failure in livestock (Tadesse et al., 2017).Poisonous herbs and shrubs pose the greatest threat to grazing animals (Baloch et al., 2017;Bricarello et al., 2023).As in our case, both life forms were commonly available in the study area for cattle, whereas tree forage was typically offered to them by herders under food shortage.
Relative frequency of citation and fidelity level of plants N. oleander showed maximum relative frequency of citation (RFC) values (0.6), followed by L. camara (0.6), D. stramonium (0.4), R. communis (0.6), S. surattense (0.5), A. indica (0.5), L. cordifolius (0.5), P. hysterophorus (0.5), E. helioscopa (0.4), M. philippinensis (0.4), J. adhatoda (0.4), Dodonaea viscosa (0.3), and B. variegata (0.3).For instance, if a researcher wanted to assess the phytotoxicity potential of these plants, they might choose to plant N. oleander, L. camara, or R. communis due to their high RFC values.Alternatively, a gardener looking for low-maintenance landscaping options might choose A. indica or L. cordifolius, as they still have relatively high RFC values.However, it is important to note that high RFC values do not always indicate desirable qualities.For example, P. hysterophorus has a high RFC value but is considered an invasive species in many parts of the world, causing harm to native plants and ecosystems.Similarly, D. stramonium is highly toxic and can be dangerous if ingested or handled improperly.Plants with high RFC value had a reputation for being poisonous among the local inhabitants of the region.These plants have the potential to serve as the foundation for subsequent evaluation of the assessment of phytochemical profiles and further toxicological studies (Panter et al., 2011).It is crucial to prioritize the conservation of these plants to ensure the sustainable use of these resources, as they also have therapeutic properties (Mukheriee and Wahile, 2006).Notably, N. oleander, L. camara, R. communis, D. stramonium, Solanum surattense, and A. indica were among the highly toxic species.Plants exhibiting higher FL are considered suitable for further ethnotoxicological research (Bibi et al., 2014).Plant fidelity level shows the degree of reliance and consistency of local communities on specific plant species for various purposes (Chaachouay et al., 2022).It also highlights the cultural, medicinal, and economic significance of plants within a given society (Leonti, 2022).The level of plant fidelity can vary widely among different cultures and regions, depending on factors such as traditional knowledge, the availability of resources, and environmental conditions (Pratama et al., 2021).

Experimental acute toxicity
The current study aimed to assess the toxicities of poisonous plants in District Muzaffarabad, AJK.To achieve this, Wistar rats were subjected to a limit test following OECD guidelines to determine the toxicity of the plant extracts.At a dose of 5,000 mg/kg body weight, N. oleander, R. communis, L. camara, and D. stramonium caused toxicity and mortality.In contrast, other plants showed moderate to mild clinical manifestations.Additionally, mortality of animals was observed within 10-24 h with severe clinical manifestations, i.e., anorexia, water intake, piloerection, dyspnea, salivation, tachypnea, constipation, diarrhea, tremor, itching nose, and dullness.
There is a large diversity in methodology and purposes of various experimental studies whereby the toxicity of plants has been studied.Studies use diverse methods of preparing plant extracts, doses, routes of administration, animal species, etc., which result in variable toxicity reported as LD 50 of various plants.Bandara et al. (2010) and Ceci et al. (2020) reported that the clinical signs of oleander toxicity are basically the same in all species.For the determination of the exact LD 50 of each plant through the fine-tuning method, LD 50 was considered poisonous plants.N. oleander with 4,000 mg/kg, followed by R. communis, L. camara, and D. stramonium, were found highly poisonous, indicating that their LD 50 is less than 5,000 mg/kg body weight for four plants.In the case of the N. oleander plant extract, different researchers have reported varying LD 50 values (Ceci et al., 2020;Shridhar, 2022).The lethal doses of various dried N. oleander leaves vary depending on the animal species (LD 50 ).Cattle are more susceptible than small ruminants, according to the LD 50 , which is 50 mg/kg for cattle, 110 mg/kg for goats, and 250 mg/kg for sheep (Oryan et al., 1996).The LD 50 of oleander for mice is 4,000 mg/kg, according to research conducted by others.While several studies came to the semi-quantitative conclusion that 3-10 oleander leaves could be deadly, the LD 50 of oleander leaves for cattle is 50 mg/kg.According to reports, the LD 50 of oleander for sheep is 250 mg/kg.However, there is disagreement on the lethal dose of oleander (Shridhar, 2022).According to the toxicity and lethality indicators associated with the type of toxin present in the plant's aerial parts, the lethal dose of Thevetia peruviana for rats and mice is 447 mg/kg body weight (Ea et al., 2021).Systematic overview of acute toxicity in rats determined through limit test showing number of rats, dose concentration, and lethalities.Different susceptibilities may be due to experimental conditions and methodological differences.In the case of N. oleander plant extract, the LD 50 values reported by different researchers may have resulted from differences in the experimental conditions and methodology used.For example, the use of different animal species, routes of administration, and types of extracts can affect the LD 50 values reported.Additionally, differences in the preparation of an extract, like the concentration and purity of the extract, can also contribute to differences in LD 50 values.Various clinical manifestations, such as diarrhea, abdominal pain, irregular heartbeat, seizures, coma, and death, were observed in rats receiving treatments.According to Prasad et al. (2016), the signs of toxicity in rats that appeared after oral administration of the ethanol extract of the plant's aerial parts included tremor, dyspnea, tachypnea, severe hypothermia, muscular relaxation, urinary retention, generalized seizures, and death.
In the current study, the LD 50 of the R. communis plant was determined to be 4,200 mg/kg.Similarly, Tewari et al. ( 2005) reported a rat oral LD 50 of 4,560 mg/kg for R. communis.According to Das et al. (2000), the LD 50 of R. communis was found to be 4,750 mg/kg body weight in rats.Additionally, in disagreement with the current study, Polito et al. (2019) reported LD 50 value of 2,000 mg/kg for an intraperitoneal administration of a ricin-containing fraction of R. communis seed extract in mice.Murade et al. (2021) determined the oral LD 50 of R. communis leaves in mice to be 89.3 mg/kg.The oral LD 50 of R. communis leaves in rats was reported to be 131.1 mg/kg by Riaz et al. (2012).
When administered intravenously, the LD 50 of ricin in rats is roughly 1.78 mg/kg, and when administered orally, it is roughly 21.8 mg/kg (Worbs et al., 2011).The authors further reported that the rats are particularly susceptible to the toxic effects of ricin, and even small doses can result in severe illness or death.Lethargy, weakness, loss of appetite, diarrhea, seizures, and respiratory failure are all possible signs of ricin poisoning in rats.On the other hand, Sadashiv (2011) reported that mice given an ethanol extract of the aerial parts of the R. communis plant did not exhibit significantly different behaviors.
In the present study, the LD 50 (4,500 mg/kg) was observed for L. camara, which is in line with the study of Sharma et al. (2007).On the other hand, Jawonisi and Adoga, (2017) also reported LD 50 greater than 5,000 mg/kg body weight in the rats.Kumar et al. (2016) reported an LD 50 value of 2,526 mg/kg for an aqueous extract of L. camara leaves administered intraperitoneally in rats.However, Pandeya et al. (2022) reported an LD 50 value of 2,891 mg/kg for an ethanolic extract of L. camara leaves administered orally to mice.In the current study, we observed some signs of toxicity such as loss of body weight, tremors, piloerection, diarrhea, and food and water consumption in rats administrated with L. camara.Mukherjee et al. (2010) tested the toxicity of L. camara leaves in rats and found symptoms of toxicity, such as weight loss, decreased food intake, and altered liver function.The rats also exhibited neurological symptoms like tremors, convulsions, and paralysis.Lantadenes is the toxic principle in L. camara leaves (Bevilacqua et al., 2011;Pour et al., 2011).Similarly, Sharma et al. (2007) observed that the main hepatotoxic compound is lantadene A, a pentacyclic triterpenoid.
The LD 50 for D. stramonium was found to be 4,700 mg/kg in the present study.Murtala et al. (2023) determined that the LD 50 was greater than 2,000 mg/kg.According to Lian et al. (2022), typical lethal doses of Datura seeds ranging from 50 to 100 may have atropine.In the experiment, clinical manifestations such as decreased activity and lethargy, ataxia (loss of coordination), tremors or seizures, salivation and lacrimation (excessive salivation and tearing), mydriasis (dilated pupils), respiratory depression or difficulty breathing, diarrhea, coma, or death were observed (Friedman, 2004).D. stramonium could trigger tachycardia, difficulty breathing, convulsions, and a decrease in locomotor activity in the presence of high levels of alkaloids such as atropine, hyoscamine, and scopolamine (Ogunmoyole et al., 2019).S. surratense is known to contain toxic compounds such as chaconine and solanine (Parvez et al., 2019).In the current study, symptoms of toxicity, such as gastrointestinal upset, vomiting, diarrhea, and even more severe symptoms, such as seizures and respiratory depression, were observed in our experiment.
In the present study, the LD 50 of L. cordifolius was determined through a limit test and is greater than 5,000 mg/kg.Khan et al. (2018) reported several bioactive compounds from L. cordifolius, including cicutoxin, chlorogenic acid, rutin, and quercetin.The limit test results showed that LD 50 exceeded 5,000 mg/kg.These findings imply that L. cordifolius is reasonably safe because it takes a high dose to be fatal.Several toxic substances, including diterpenes, triterpenes, phorbol esters, and ingenol esters, are known to be present in the plant species Euphorbia helioscopia, which is a member of the Euphorbiaceae family.The milky sap of the plant contains the diterpene ester known as helioscopinolide, which is the most notable toxic substance in this plant (Morita et al., 2014).According to the study of Aleksandrov et al. (2019), rats at an oral dose of 1,000 mg/kg showed signs of toxicity such as lethargy, decreased appetite, and diarrhea.Our results depicted that P. hysterophorus extract administrated in to rats resulted in an LD 50 of 4,900 mg/kg.According to Narasimhan et al. (1984), P. hysterophorus extract has a toxic LD 50 of 1,500 mg/kg in mice.It contains the active ingredient in Parthenium, a sesquiterpene lactone called parthenin.Parthenin causes animal toxicity, Pearson's correlation showing the linear relationship between the percentage of informants who mentioned a certain poisonous plant species (FL) and the citing percentage of informants for a particular plant species (RFC).
including hepatotoxicity, nephrotoxicity, and neurotoxicity (Khaket et al., 2015).We also reported LD 50 of A. indica greater than 5,000 mg/kg.According to reports, these plants contain glycosides such as arisaematoside, oxalate crystals that can cause mouth pain and diarrhea (Basu et al., 2014).Desai et al. (1988) observed restlessness and hyperactivity, convulsions and tremors, lethargy, depression, reduced food intake, weight loss, diarrhea, and impaired coordination and balance caused by A. indica.
The current study also reported Mallotus philippensis with LD 50 greater than 5,000 mg/kg.According to Thakur et al. (2005), this plant at a 2,000 mg/kg dose caused mortality in all the animals within 24 h.In addition, Gangwar et al. (2014) reported that M. philippinensis administered orally to mice at a dose of 1,000 mg/kg caused death in 50% of the animals within 24 h.
The different behavior of plant poisoning reported in our experimental and already reported studies can be attributed to plant nature, growth stages, geographic location, environmental conditions, the abundance of species, and the species and breed of livestock are just a few of the variables that can affect livestock variation (Mensah et al., 2019;Griffiths et al., 2021).The complex interactions between the chemical makeup of plants and the metabolic function of animals cause variations in plant toxicity (Jung et al., 2020).Plant species have different levels of toxins that can affect livestock differently.Poisonous plants can be classified by active ingredients (cyanide, nitrate, oxalate, pyrrolizidine alkaloids, glycosides, etc.), target organs and systems (cardiotoxic, hepatotoxic, neurotoxic, teratogenic plants, etc.), or family and genus (Ohlsen et al., 2022).

Conclusion
It is evident from the present study that poisonous plants pose a serious threat to livestock in Muzaffarabad.Plants with active ingredients such as cyanide, nitrate, oxalate, pyrrolizidine alkaloids, and glycosides resulted in gastrointestinal, neurological, hepatotoxic, and dermatological disorders in livestock.N. oleander, L. camara, R. communis, D. stramonium, S. surattense, A. indica, L. cordifolius, and P. hysterophorus were the highly poisonous species reported from the study area.However, other species exhibited mild to moderate symptoms.The study also shows different plant poisonings in nature can be explained by the complex interactions between plants and animals, as well as their nature, growth stages, geographic location, environmental conditions, and livestock species.In order to gain a deeper understanding of traditional knowledge related to poisonous plants and their effects on animal health, future research needs to be conducted in appropriate experimental conditions involving relevant livestock species.Cattleholders should also be informed about toxic plant hazards to reduce the risk of poisoning and provided with appropriate management strategies.Establishing diagnostic laboratories equipped with advanced analytical techniques can help in the early diagnosis and treatment of plant-poisoned animals, reducing mortality and economic losses.The findings of this study emphasize the need for continuous plant research and monitoring.

FIGURE 1
FIGURE 1Map of the study area showing study sites.

FIGURE 2
FIGURE 2Flow chart showing how information was retrieved from different resources.

FIGURE 3 Floristic
FIGURE 3Floristic diversity showing lifeform spectra of the study area.

FIGURE 4
FIGURE 4Shows dominant plant families and the distribution (number) of poisonous plants within each family recorded from the study area.

FIGURE 5
FIGURE 5Percentage of poisonous plant parts.

FIGURE 6
FIGURE 6Number of poisonous plants causing different system/organ disorders in livestock of the study area.

TABLE 1 Experimental
design for determination of plant toxicity by Limit test showing different groups of animals orally administrated with different concentrations of extract (treatments).

TABLE 2
(Teke and Kuete, 2014)city of substances based on LD 50 and dose ranges according to Organization for Economic Cooperation and Development (OECD) guidelines(Teke and Kuete, 2014).

TABLE 3
Demographic profile of the selected informants from the study area showing their gender, categories, and educational background percentages.

TABLE 4
Enlisted poisonous plants with their toxic parts, susceptible animals, affected organs, and their phytotoxicity reports by informants.

TABLE 4 (
Continued) Enlisted poisonous plants with their toxic parts, susceptible animals, affected organs, and their phytotoxicity reports by informants.

TABLE 4 (
Continued) Enlisted poisonous plants with their toxic parts, susceptible animals, affected organs, and their phytotoxicity reports by informants.

TABLE 5
List of poisonous plants showing Relative Frequency Citation (RFC) and Fidelity Level (FL).RFC shows the citing percentage (FC) of informants for a particular plant species while FL shows the percentage of informants who mentioned a certain poisonous plant for toxicity in livestock in the study area.

TABLE 5 (
Continued) List of poisonous plants showing Relative Frequency Citation (RFC) and Fidelity Level (FL).RFC shows the citing percentage (FC) of informants for a particular plant species while FL shows the percentage of informants who mentioned a certain poisonous plant for toxicity in livestock in the study area.

TABLE 6
List of highly poisonous plants showing informant scores and their ranking obtained from priority ranking test and acute toxicity assessment of these plants through a Limit test.In the Limit test, 0, 1, 2, and 3 show the number of mortalities in rats.Tested LD 50 shows the lethality of these plants in rats obtained through the current experimental study, which is further compared with LD 50 reported in different literature.

TABLE 7
Clinical symptoms of toxicity in different groups of rats treated with control, sham control, and poisonous plant extract.Where (−) shows no symptoms, (+) shows mild symptoms, (++) shows moderate symptoms, and (+++) shows severe symptoms.