**3. General effects of heavy metals**

Heavy metals have been used for a variety of purposes since before recorded history, and they have been vital to the advancement and prosperity of civilizations. Animal populations may be subjected to dangerous amounts of heavy metals at levels very close to those experienced by humans [20, 21]. A substantial number of animals found in heavily metal-polluted areas ingest metal-affected grasses, feed materials, vegetables, and rice plants in addition to contaminated drinking water, which is another potential source of exposure to heavy metals [1, 17]. Heavy metals are widely used and naturally found in the environment, which exposes both humans and animals to them to varying degrees [1, 5]. In addition, because metals are ubiquitous and have been there throughout life's evolution, organisms must contend with them because they may be harmful [22]. Heavy metal traces, including copper (Cu), cobalt (Co), manganese (Mn), iron (Fe), and zinc (Zn), are necessary for a variety of vital physiological processes, including the regulation and operation of several enzyme systems, oxygen and electron transport, hormone synthesis, antioxidant defense, immunity, and fertility [1, 5]. In addition to negatively affecting growth and physiological processes, a lack of essential metals can also make non-essential heavy metals more hazardous [1, 5]. However, even necessary metals will turn hazardous with prolonged contact [14]. Lead (Pb), cadmium (Cd), and mercury (Hg) are examples of toxic heavy metals that are dangerous even at very low doses and have no known biological benefits [23]. Non-essential toxicant metals frequently imitate essential metals to enter the body and potentially disturb important cellular processes [24]. The bioaccumulation of hazardous metals can also be explained by this. Furthermore, because detoxification systems cannot break down an atomic species into a subcomponent with lower toxicity, the elemental nature of metals influences their biotransformation and toxicity [25]. Since metals are elements, their indestructibility and bioaccumulation together lead to a significant concern for metal as a toxicant [14, 22]. The level of exposure, type of heavy metal and its form, age, sex, physiological and nutritional health of the exposed animal, and method of poisoning all affect how toxic heavy metals are to animals [26].

The majority of metals are concentrated in the liver and kidney, along with other essential organs, where they can have toxic or non-toxic effects such as oxidative stress, immunotoxicity, cardiotoxicity, teratogenicity, enzyme inhibition, birth abnormalities, and endocrine disruption [27]. Due to the wide range of chemical characteristics and toxic endpoints, the precise chemical basis of metal toxicity is poorly understood, and there is no universal mechanism for all dangerous metals [22]. It is true that heavy metals cannot degrade into other substances since they are elements, their forms, however, can be converted to free metal ions, which will modify their biological availability, activity, and toxicity [28]. Metals in their ionic form can interact with biological systems and toxicological targets in a variety of ways, making them chemically very reactive [14]. This can lead to a variety of toxic effects and damage to different organs, such as the kidney, nervous system, liver, respiratory system,


### **Table 2.**

*Sources and toxicological side-effects of some heavy metals.*

endocrine and reproductive systems, and gastrointestinal tract (**Table 2**) [11, 25, 27]. Targets for heavy metals typically include biological molecules, macromolecules, membranes, or organelles, and interactions between free metal ions and these targets are what cause toxicity [29]. Hazardous metals commonly act by inhibiting enzymes, subcellular organelles, interactions with DNA that cause mutagenesis and cancer, covalent alteration of proteins, displacement of other essential metals-dependent proteins, and the production of free radicals [28, 29]. The toxic metals are grouped into four based on their toxic effects: metals (copper and iron) acting as Fenton reaction catalysts and contributing to the production of free radicals and oxidative stress; metals (nickel, cadmium, and chromium) linked to cancer; metals (aluminum, lead, and tin) linked to neurotoxicity; and generally toxic metals like mercury [30]. However, a growing body of research indicates that most heavy metals can cause oxidative stress in a variety of animal species, including buffalo [31]. This can have an impact on the oxidative stress quotient [25]. Free radical overproduction and oxidative stress damage biomolecules, subcellular structures, and even entire cells, such as neurons, which not only compromise immunity but can result in a variety of illnesses [30]. Oxidative stress has a significant impact on farm animals' ability to produce and reproduce, and it may result in significant financial losses [30, 31]. The endocrine systems of animals may be disrupted by toxic heavy metals, which can also affect animal reproduction and productivity [27]. Heavy metal-induced co-selection of antibiotic resistance genes (ARGs) has emerged as a new environmental concern as there is mounting evidence that heavy metals might affect antibiotic resistance [32]. It has been discovered that the presence of heavy metals in the environment, such as arsenic, copper, and zinc, even at low levels increases bacterial resistance to tetracycline [33–35]. Due to their usage in feed and exposure to heavy metal contamination in the environment, livestock and the systems used to produce them are seen as a major source of heavy metals [9]. As a result, the environment around cattle may be contaminated with heavy metals and antibiotics, which could increase the fast-expanding worry over antibiotic resistance [35]. The vulnerabilities of both humans and animals as a result of compound resistance are highlighted by the confirmed positive link between heavy metal resistance and coexisting methicillin-resistant *Staphylococcus aureus* (MRSA) [35, 36]. Mercury-poisoned animals cannot produce meat, liver, or kidneys fit for human consumption [18]. Depending on the type of mercury poisoning, milk might also be dangerous [37].

### **3.1 Ruminants**

Cattle, particularly young calves, are more susceptible to heavy metals [25, 37]. Owing to their natural curiosity, licking habits, and indiscriminate eating habits, cattle can ingest lead-bearing objects present in their environment as domestic, industrial, or agricultural waste and suffer from accidental acute lead poisoning [37]. Contamination of vegetation and pastures nearby secondary lead smelters (battery recycling units) and lead-zinc smelters [17] was the source of acute lead toxicity in cattle and buffaloes and subclinical toxicity in goats affecting the essential trace mineral profile [27]. The liver and kidneys of the fetus of a lead-poisoned pregnant heifer contained 0.425 and 4.84 ppm of lead, respectively, which were 72% and 84% of the lead concentrations in the respective organs of the dam [25, 37]. Most findings indicate a comparatively higher tolerance in sheep and goats to toxic metals like lead and cadmium than cattle [25, 38–40]. Sheep excrete higher concentrations of lead, chromium, and nickel in their excrement than cows [41]. This may be a reason for the comparatively higher lead concentrations in the milk of ewes than cows [42]. Sheep mostly show subacute toxicity that simulates signs of lead toxicity in adult cattle [37, 41, 42]. Goats, though comparatively more tolerant to lead (chronic toxicity dose of 400 mg per kg body weight) than cattle and sheep, can also exhibit cumulative lethal toxicity with predominant signs of CNS involvement following long-term exposure to lead [25, 27]. Cattle might accidentally absorb lead-containing things from their environment, such as household, industrial, or agricultural trash, due to their natural curiosity, licking tendencies, and indiscriminate feeding habits. This can result in acute lead poisoning [37, 43]. Acute lead toxicity in cattle and buffaloes, as well as subclinical toxicity in goats affecting essential trace mineral profiles, was caused by contamination of vegetation and pastures near secondary lead smelters (battery recycling units) and lead-zinc smelters [44, 45]. The majority of research shows that sheep and goats are more tolerant than cattle to hazardous metals like lead and cadmium. Compared to cows, sheep produce more lead, chromium, and nickel in their excretions [27, 39, 46]. This could explain why sheep milk has somewhat higher lead amounts than cow milk [45]. Most sheep exhibit subacute toxicity, which mimics adult cattle's lead toxicity symptoms [37, 45]. Even though they are more tolerant of lead than cattle and sheep (chronic toxicity dose: 400 mg per kg body weight), goats can nevertheless develop cumulative fatal toxicity, with CNS involvement being the main symptom after prolonged lead exposure [27, 47]. Despite the fact that unintentional acute or chronic poisoning from organic mercury or inorganic compounds can occur in domestic animals, mercury poisoning is uncommon [48–50]. It's possible that phenyl-mercury, which is present in the treated grains as organic mercury, is a more frequent cause of chronic cumulative poisoning [50, 51]. Only when massive amounts of grains are fed to cattle over long periods of time does clinical disease develop. Accidental administration of mercury-containing medications and licking or cutaneous absorption of mercuric oxide-containing skin dressings can also result in sporadic occurrences of poisoning in horses [37, 52]. Mercury poisoning is rare in domestic animals, but accidental acute or chronic poisoning can occur following ingestion of organic mercury or inorganic compounds [53]. Oral ingestion of organic mercury present in the form of phenyl-mercury in the treated grains may be a more common source of cumulative chronic poisoning [47]. However, a ration containing up to 10% of treated grains was not harmful; even feeding a single large amount of grain was incapable of causing toxicity in ruminants [49]. The clinical illness may occur in cattle only when large amounts of grains are fed for long periods [51]. Sporadic cases of

poisoning in horses can occur due to accidental administration of drugs containing mercury and licking or cutaneous absorption of skin dressings containing mercuric oxide [27, 37]. In a study, mercury levels were found to be significantly higher in the blood (7.410 g/kg), milk (4.750 g/kg), and urine (2.80 g/kg) of nursing cows raised within a 5-kilometer radius of a thermal power plant [37]. The exposed cows' hemoglobin levels were significantly lower, and their blood urea nitrogen, serum creatinine, albumin, and serum glutamate oxaloacetate transaminase values were all higher, showing effects of mercury on animal health [54, 55]. The study came to the conclusion that long-term exposure of the cows to fly ash mercury may have an effect on the human population, either directly or indirectly through the food chain [55]. While chronic administration of inorganic mercuric chloride (0.8 gm/kg body weight for 14 weeks) in horses caused mercury toxicity, toxic effects in sheep can be evident with an intake of 17.4 mg/kg body weight [37]. Due to the frequent discharge of cadmium and lead together from many industrial sources, the clinical symptoms of spontaneous poisoning commonly combine the two metals [48]. Cadmium levels in feed greater than 50 mg/kg dry matter are linked to toxicity in cattle and sheep [56]. Large amounts of cadmium accidentally consumed can harm the liver and induce acute nephrotoxicity [57]. In animals, chronic intake is linked to metal accumulation, particularly in the kidneys, liver, lungs, and testes [25, 58]. Inappetence, weakness, weight loss, poor hoof keratinization, dry, brittle horns, matting of the hair, and keratosis are a few examples of clinical symptoms in cattle [59]. Significant necropsy abnormalities included degenerative alterations in most organs and hyperkeratosis of the stomach epithelium [37, 59]. Cattle and buffaloes from an industrial area have been observed to have vascular degeneration and necrotic alterations in the liver, kidneys, and lungs and frequently have tissue cadmium levels above 2 ppm [25, 58, 60]. Anemia, nephropathy, and bone demineralization were the results of experimentally poisoning sheep with 2.5 mg of cadmium per kg of body weight [27, 59, 61]. Congenital flaws, stillbirths, and abortion are further potential impacts [61]. The ruminant species that is most vulnerable to chronic copper poisoning is the sheep, and poisoning cases in sheep have been reported all over the world [62, 63]. Contrarily, cattle were thought to be significantly more tolerant of copper accumulation in the past, and up until recently, copper poisoning in cattle was very rare [64]. On the other hand, copper poisoning and the mortality it causes are on the rise everywhere in the world, especially in dairy cattle [64, 65]. Since ruminants have poor homeostatic control over copper absorption, which makes them more sensitive, they have evolved mechanisms for storing excess copper in the liver by decreasing copper in the liver [64, 66]. However, when exposed to copper levels greater than those required for health, they are unable to manage their excretion skills and could become copper toxic [66]. Acute poisoning may happen from consuming 20–100 mg of copper per kilogram of body weight in sheep and young calves and 200–800 mg of copper per kilogram of body weight in mature cattle [67]. Chronic copper poisoning in sheep may occur with daily consumption of 3.5 mg of copper per kg when their grazing pasture contains 15–20 ppm copper (DM) and low levels of molybdenum [25, 68, 69]. Goats have substantially higher copper requirements (15–25 mg per kg, DM) than sheep (640 mg per kg, DM) and can tolerate a far higher dietary copper intake than sheep [69]. Goats' great tolerance to copper may be due to low hepatic absorption [67]. Goats may be able to tolerate a higher concentration of the copper antagonist molybdenum compared to sheep and cattle [66]. The clinical symptoms of acute copper poisoning that are most frequently seen are anorexia, stomach pain, diarrhea, dehydration, unsteadiness, salivation, and collapse before death, which typically

occurs within 24 hours. Animals that survive acquire icterus and dysentery [70]. Calves that survive for three or more days are known to have ascites, hydrothorax, hemoglobinuria, head pushing, opisthotonus, aimless roaming, bruxism, circling, and ataxia [71]. A haemolytic disease is chronic poisoning [72]. Sheep that are affected exhibit anorexia, thirst, sadness, jaundice, haemolytic anemia, and hemoglobinuria, as well as accelerated breathing and heart rate [73]. Sheep also exhibit nervous indications such as sadness, blindness, and tetraparesis [25, 74].
