Arsenic Toxicity in Fish: Sources and Impacts

Abstract

Arsenic has become a major toxicological concern due to its rising concentrations in aquatic bodies. It is added to the water either by natural sources including weathering of rocks, sediments, volcanic eruptions and aquifers, or by anthropogenic sources including herbicides, wood preservatives, metal smelting, drugs, pesticides, burning of coal, agriculture runoff and petroleum refining processes among others. The untreated and uncontrolled discharge of arsenic by industries into the natural water bodies poses serious threat to aquatic fauna by deteriorating water quality and making it unsuitable for fishes. Fish is an important bioindicator of aquatic bodies and excessive arsenic concentration causes its bioaccumulation in fish organs and muscles. This deposited arsenic in the fish imposes serious damage to physiology, biochemical disorders such as poisoning of gills, livers, decrease fertility, tissue damage, lesions, and cell death. It also enters in the cell and produces reactive oxygen species which increases the level of stress which further concentrates the oxidative enzymes and cortisol levels in fish. The uncontrolled discharge of arsenic and its devastating impact on fish diversity is a major concern for aquaculture progress and economic stability. This, along with its other implications is the scope of this chapter.

1. Introduction

Heavy metals are gaining attention as significant pollutants due to their toxicity problems in ecosystem at different levels [1]. Some of these metals are commonly known as pollutants that include arsenic, cadmium, copper, nickel and lead which pose threats of serious nature to aquatic environment and to the living organisms in the aquatic ecosystem [2]. Arsenic is a metalloid element that is abundant in the aquatic environment as a result of both natural and anthropogenic processes. It is a significant and ubiquitous environmental contaminant that causes health issues to all living organisms [3]. Arsenic in herbicides, fungicides, pesticides and rodenticides is the significant source of environmental contamination [4]. Arsenic mainly enters into the environment through two channels: (a) natural activities, and (b) man-made activities. Natural activities such as volcanic eruption, forest fires and weathering of rocks add a significant amount of arsenic in aquatic environment. While, man-made activities, such as different industries including paint, pharmaceutical, pesticide, detergent and electronic industries are the main source of arsenic discharge in water bodies [5]. Besides all these sources, the smelting and mining operations along with the domestic and agriculture run-off continuously add arsenic in natural waters [6]. In water, arsenic trivalent, arsenite is oxidized in water in the presence of dissolved oxygen and converted into arsenate that remain intact in sediments for long period of time and pose serious threats to aquatic fauna. Arsenic toxicity has been reported in many countries including China, Pakistan, Bangladesh, India and other South Asian countries along with many parts of the United States [7]. According to IARC, three chemical forms of arsenic are present: organic arsenic, inorganic arsenic and arsenic gas [8]. Fish is most sensitive bioindicator of pollution and cannot be safe from harmful impacts of these pollutants [9]. These have potential to induce biochemical and physiological changes which ultimately effect overall behavior, growth pattern and ultimately leads to death [10, 11]. Arsenic can enter in to fish body through oral cavity with contaminated food and absorption through skin and gills. Arsenic hasa tendency to accumulate in fish tissues and organs and cause serious damages to gills, gastrointestinal tract, kidneys, heart, brain and other organ. Such damages alter fish behavior, homoeostasis, hematology and biochemical mechanisms [12].

2. Sources of arsenic

In natural environment, arsenic is a common crystalline metalloid having characteristics of both metal and non-metal. It is the 14th and 20th abundant element in saltwater and earth crust respectively [13] Arsenic contamination occurs due to both natural (such as volcanic eruptions, rock weathering) and anthropogenic activities (such as the production of alloys, pesticides, glass, and medicinal items, etc.) [14].

2.1 Natural sources of arsenic

The presence of arsenic in natural water is influenced by the aquifer’s local geology, hydrogeology and geochemical properties. Climate change and human activities also play a part and influence its presence. Natural sources of arsenic in water have been attributed to a variety of natural geochemical processes including oxidation of arsenic-bearing sulphides, desorption of arsenic from (hydro)oxides (e.g., iron, aluminum, and manganese oxides), reductive dissolution of arsenic-bearing iron (hydro)oxides, release of arsenic from geothermal water, and as well as leeching from sulphides.

2.1.1 Anthropogenic sources

Nonferrous metal mining and smelting, fossil fuel processing, combustion, wood preservation, pesticide production and its application in agricultural fields, municipal and industrial waste disposal and incineration are the main anthropogenic activities that may release arsenic into the environment [15, 16]. The majority of anthropogenic arsenic is released into the soil, primarily through pesticides or solid wastes. A significant amount, however, is also released into the air and water [17]. Arsenic, in its soluble forms, enters into the ground water and water bodies through runoff and leeching [18].

Mining tailing contain a significant amount of arsenic in the form of arsenopyrite, arsenian pyrite, arsenates and in association with iron oxyhydroxides. Arsenic can be produced by roasting arsenopyrite which is the most abundant ore mineral of arsenic, and by smelter dust of some metals such as gold, copper and lead [19]. Arsenic is found in approximately 11 million tonnes of copper and lead resources worldwide. In 2007, the total global production of arsenic trioxide was 59 thousand tonnes [20]. Arsenic is a highly toxic mineral found in the earth’s crust that can enter the food chain through soil, water and plants. The main anthropogenic sources of arsenic in Canada are smelter and base-metal refinery facilities as well as thermal and power-generation stations. It was estimated that the Canadian base-metal smelters and refineries released approximately 15 tonnes of arsenic per year in liquid effluent, 310 tonnes in the atmosphere and 770 tonnes in solid waste [21]. The majority of this emissions (almost 90%) came from coal-fired power or thermal power-generation stations, as well as smelter and metallurgical facilities. Arsenic concentrations have been found to be elevated in the vicinity of these sources.

Another significant anthropogenic source of arsenic is the widespread use of arsenical wood preservatives. Chromated copper arsenate (CAA) is one of the most common wood preservatives used worldwide on large scale containing 34 percent arsenic content [22]. It has been estimated that a considerable amount of CAA (7800 to 78,000 mg/kg) remained in the treated wood [23]. Leaching of preservative components from in-service treated wood has thus been a source of arsenic in the environment. Furthermore, widespread arsenic contamination around wood preservation occurs as a result of raw material handling, spills, sludge deposition and dripping from freshly impregnated wood or rain water leaching from impregnated wood piles, particularly under low pH conditions, at these sites [24]. Exposure to sunlight and weathering both increase the rate of leaching from the treated wood. As a result, elevated arsenic levels have been found in soils surrounding treated woods [25, 26]. Arsenic is a widely used component in pesticides and most commonly available as lead arsenate, calcium aresenate, magnesium arsenate, zinc arsenate, arsenite and Paris green. These are commonly used in apple orchard of Canada [6].

2.1.2 Arsenic species in air

Methylated form of arsenic is mainly common in atmosphere. Arsenate is predominated form of arsenic and is most likely present near smelters, volcanic eruptions and coal burning sites [3]. Peat and landfills are major sources of volatile arsine in air. Some volatile species of arsenic are emitted into the air due to microbial activities in soil and water bodies which further oxidize and reactive with atmospheric sulfur oxide and ozone [6, 27]. This atmospheric arsenic reaches soil and water bodies by snow, dry deposition and through rain fall. Some of the atmospheric arsenic particles also combine with dust and may then be inhaled or ingested by living organisms [28]. In rural areas, the arsenic concentration in rain and snowfall are comparatively low (0.00003 mg/L), while its concentration around coal burning industries is relatively high (0.0005 mg/L) (Figure 1) [29].

Figure 1.

Sources and routes of arsenic to approach water bodies. Arsenic has two main sources to enter water bodies: Anthropogenic and geogenic. Addition of arsenic in surface water bodies is by direct discharge. Gaseous arsenic from different sources also backs its way in to the water bodies through precipitation. Arsenic in soil and water enters the ground water through leeching.

3. Impacts of arsenic on fish

3.1 Prevalence of arsenic in the environment

Arsenic-exposed organisms may absorb arsenic by ingestion, inhalation and penetration through skin or mucous membranes which allows it to enter the cells mainly by active transport [30]. Inhaled arsenic induces severe biochemical and physiological changes such as poisoning, impaired growth and reproduction, immune system abnormalities, cell and tissue damage, oxidative stress and apoptosis in aquatic organisms.

3.2 Factors affecting toxicity of arsenic

3.2.1 Arsenic speciation

The toxic effect of Arsenic in the aquatic ecosystem depends on its form such as inorganic or organic and its level of oxidation [5]. Arsenite (As-III), arsine (As-III), Arsenate (AsV) and arsenic are the four oxidation states of arsenic [6]. Inorganic arsenic (iAs); As-III and As-V are prevalent forms in aquatic habitats. As-III is limited in extremely reduced conditions with a low redox potential while As-V is stable in oxygen-rich environments [31]. Inorganic metallic compounds are typically more hazardous than organometallic compounds. Likewise, inorganic Arsenic is more soluble in water than organic Arsenic it, thus, can accumulate in tissues more quickly [32]. Arsenic is converted into less- or non-toxic metabolites like arsenocholine (AsC) and arsenobetaine (AsB) for excretion when it is ingested by living organisms [33].

3.2.2 Biotic and abiotic factors

Many biotic and abiotic factors like exposure time, arsenic speciation, water temperature, pH, organic content, phosphate concentration, suspended particles, and presence of other chemicals and toxicants significantly alter the toxic and other effects of arsenic on aquatic life [34]. Median survival time of fishes usually reduces as the temperature and arsenic concentration increases. It is helpful to identify different arsenic species when investigating for arsenic exposure because they vary in their origin and toxicity [35].

By the biomethylation process, harmful inorganic arsenic is transformed into less toxic pentavalent (Met-A) forms such as monomethylarsonate (MMAv), dimethylarsenate (DMAv) and trimethylarsine (DMAIII). Nevertheless, monomethylarsenite (MMAIII), dimethylarsenite (DMAIII) and trimethylarsonic oxide (TMAOv) are more hazardous than inorganic arsenic and are produced through biomethylation. In general, AsB (arsenobetaine), arsenocholic (AsC), and DMAA make up around 85 to 90% of the arsenic found in edible parts of marine fish, whereas iAs (inorganic arsenic) species make up about 10%. Little is known about the types of arsenic found in freshwater fishes, but what is available indicates that AsB and DMAA are the dominant arsenic species in freshwater fishes [36].

3.2.3 Bioaccumulation

The process by which some toxic chemicals (heavy metals including arsenic and other toxicants) present in the environment accumulate in living organisms is known as bioaccumulation. Their absorption is considered passive, with diffusion gradients formed by metal adsorption or binding to tissue and cell surfaces [37]. Arsenic accumulation in tissue will depend on the rates at which various organs take in and eliminate arsenic [38]. Every organism metal concentration is determined by a variety of mechanisms, including its intake, excretion, storage, and transformation. Bioaccumulation differs among metal species and in fish species because of variations in their permeability, metabolic rates and the amount and types of metal binding ligands present at the organismic surface. Although accurate quantification of all these activities may not be necessary, understanding their proportional role in the overall pattern of metal turnover is sometimes the only way to evaluate tissue residue data.

The inhabitants of aquatic environments, such as fish, are unable to escape the negative impacts of arsenic [39]. The amount of toxic metal found in various fish organs is used as an indicator for the ecosystem metal contamination. This is thought as an important approach for highlighting the significance of higher metal levels in aquatic organisms [40]. Arsenic has a multidimensional impact on fish as they bioaccumulate in various tissues and can harm their immunological, respiratory, digestive, excretory, reproductive, neurological, and endocrine systems.

Mostly, arsenic accumulates in all of the fish critical organs. The most common site for the highest concentrations of arsenic in fish are the liver, kidney, and gills [41] as well as rarely the gut [42]. To determine the impact of arsenic contaminated water, several studies have been carried out. The gills and liver of tilapia have shown to accumulate significantly more arsenic thus the gill was noted to be most significantly impacted organ [43]. Great Slave Lake, Northwest Territories, Canada inhabitant fish arsenic concentration were determined in the gill, liver, muscle and skin of fishes Clarias gariepinus and Labeo umbratus. The liver was noted to be the most suspected organ [44]. In another study, the bioaccumulation of arsenic in the gill, liver, and intestine of freshwater fish Oreochromis niloticus was evaluated. The most affected organs among those mentioned above were the liver and gills. As the liver is the primary metabolic organ and the gills are always in constant contact with the environment, the gills were shown to be the most affected.

4. Effect of arsenic on behavioral changes

Fish exposed to chemicals exhibit quantifiable behavioral changes that provide unique information that cannot be discovered using traditional toxicological techniques [45]. The relationship between behavior and an organism physiology, ecology, and environment offers a special point of view [46]. Even a tiny quantity of some toxicants can make fish behave abnormally due to impaired sensory sensitivity. Numerous abnormal behaviors, including erratic movement, fast opercula movement, jumping out of the test medium, lateral swimming and loss of balance were observed by exposure to sodium arsenate [47]. Within a few minutes of exposure, especially at higher concentrations, 2.250 mgL⁻¹ of sodium arsenate, the treated fish began to exhibit their first obvious responses. However, depending on the concentrations in the exposure medium, fish exposed to low concentrations, i.e., below 0.08 mgL⁻¹ of sodium arsenate, had no or little behavioral alterations. Neurotoxic effects and sensory system irritability were the root causes of the aberrant behaviors. The fish avoidance responses to arsenic are indicated by their jumping and back-and-forth movements. The excessive mucus secretion was likely caused by sodium arsenate directly irritating the skin. The dysfunction of the nervous system may be the cause of lateral swimming and loss of balance [48].

5. Effect of arsenic on fish major organs and organ system

5.1 Effects on organs

Arsenic is hazardous to fish organs, including the skin, liver, kidney, lung, gastrointestinal tract and muscles [49]. Among them liver and kidneys, are essential organs in vertebrates that carry out detoxification processes, protein synthesis, homeostasis and excretion of nitrogenous waste respectively. Acute and sub-acute effects of arsenic may involve many organ systems particularly respiratory, cardiovascular, gastrointestinal, neurological and hematological systems. At 9.64 mgL⁻¹ waterborne arsenic and 43.1–60 μgg⁻¹ dietary arsenic concentration exposure anemia, liver degeneration and gallbladder inflammation were noted [50].

5.1.1 Skin

Fish skin serves as its outermost protective barrier. Because of the presence of club and mucous cells in the tissue, the skin and gill tissue of fish are keratinized and have a mucous covering [51]. It is susceptible to several water-dissolved toxins because of constant contact. Skin of Clarias batrachus (L.) exposed to 1 mgL⁻¹ water born sodium arsenate induce significant damage, including enormous tear and wear, sloughing of epithelial cells and degeneration of club cells, whose contents seep out onto the body surface. This result in altered epidermal histomorphology. Mostly during exposure times, the mucous cells exhibit significant hyperplasia and hypertrophy [52]. Fish gills and skin develop a thick coating of slime as a defensive strategy. The protein, DNA and RNA contents of the epidermis also fluctuate periodically and independently [1]. With increased exposure time, damage to the epidermis becomes more prominent, and in certain areas, the majority of the cells in the bottom layer of the epidermis lost their integration and cell borders with adjacent cells [50]. Skin lipid and glycogen levels gradually decliness a result of exposure to arsenic, Hetero pneustisfossilis L. has been found to exhibit both hypo- and hyperpigmentation [53].

5.1.2 Gills

Gas exchange, ion control, and excretion of metabolic waste products are the three primary functions performed by gills. Consequently, by serving as a first barrier, gills can significantly contribute to the body defense against hazardous chemicals by reducing the number of poisonous compounds that are taken up by other organs [54]. Gills serve as the initial sites where waterborne contaminants are concentrated because of their constant exposure to the outside environment. One of the first signs of toxicant poisoning is respiratory discomfort. Fish are particularly susceptible to the toxicity of arsenic due to the high rate at which it is absorbed through the gills [55]. The presence of arsenic in gill tissue indicates that the gills were in direct touch with arsenic-contaminated water. The majority of research on arsenic toxicity in freshwater fish has focused on the effects of arsenic intake through the gills and nutritional absorption by fish that feed on benthic organisms [56]. Fish, upon arsenic exposure, show difficult breathing because coagulated mucus blocks the gills, and fish suffer direct damage from arsenic ions to their blood vessels which led to vascular collapse in the gills and anoxia [57]. After being exposed to sodium arsenite, Oreochromis mossambicus experienced the lifting of the lamellar epithelium, desquamation, edema, necrosis, aneurism, fusion of the secondary lamellae and hyperplasia of epithelial cells in the gills. Damage to gill tissues observed as a result of arsenic exposure can reduce oxygen consumption and also impaired osmoregulatory system. Hence, injury to the gills may reduce their ability to breathe effectively, altering the metabolic processes necessary for the growth and survival of fish [34].

5.1.3 Liver

By synthesizing proteins, detoxifying metabolites and aiding in digestion, the liver plays a significant role in metabolic regulation. Fish liver plays a crucial role in the absorption, bioaccumulation, biotransformation and elimination of arsenic [32]. Liver is the main target organ of arsenic poisoning. Arsenic is efficiently metabolized in fish tissue, particularly in the liver and gut and it tends to accumulate in fish such as the green sun fish and the tilapia mossambica. Its accumulation and detoxification lead to changes in the liver, including eosinophilic granules in the cytoplasm of hepatocytes, nuclear vacuolation, nuclear hypertrophy, and irregularly shaped nuclei [35]. The liver is very important for regulating metabolism through detoxification in the cytoplasm, brownish-yellow granules indicative of bile stagnation was seen. Melano-macrophages were recognized as compact cell clusters with granules that had a dark yellowish color. The severity of cellular rupture, bile stagnation and pyknotic nucleus increase with an increase in arsenic dosage [36]. The liver of the tilapia (O. mossambicus) exhibited congestion, focal lymphocytic and macrophage infiltration, cloudy swelling, shrinkage of hepatocytes, focal necrosis, vacuolization and dilation of sinusoids, vacuolar degeneration, and nuclear hypertrophy when exposed to various concentrations of arsenic. Chromosome fragmentation and the activation of certain proteins are brought on by sodium arsenite in the liver [37]. Clarius batracus exposed to arsenic had lower liver protein and glycogen levels and higher lipid contents [28]. In low-dose (50 μgL⁻¹) exposure to sodium arsenite, zebra fish liver proteome showed gender-specific responses [38]. With non-lethal exposure to arsenic, severe degenerative reactions in Channa punctatus hepatopancreas were seen [39]. In liver tissue of C. batrachus and C. punctatus hepatic pathologies were seen in the form of congestion, cloudy hepatocyte swelling, karyolysis, vacuolar degeneration and nuclear hypertrophy dilatation of sinusoids [40].

5.1.4 Kidney

Kidney along gills is primary pathways for waste excretion in fish body. When fish are exposed to arsenic contaminants, histopathological alterations occur. Upon arsenic exposure, kidney enzyme, glutathione decreases [40]. Upon non-lethal doses (3.8 mgL⁻¹ and 7.6 mgL⁻¹ arsenic) exposure C. punctatus (Bloch) suffer from shrinkage of glomerulus and increase in the Bowman’s space consequently rise in urine quantity. Abnormalities in the renal tubule, such as necrotic and apoptotic cells, less intratubular space and more brush border cells with a higher height also occurred [41]. Arsenic accumulates in the head kidney of C. batrachus upon chronic exposure to arsenic, decreasing the number of head kidney macrophages and the head kidney somatic index while increasing hemosiderin accumulation. Head kidney macrophage showed significant endoplasmic reticulum, chromatin condensation, and a lack of nuclear membrane structural integrity. A considerable amount of superoxide anions and decreased generation of pro-inflammatory ‘IL-1 β like’ factors functioning as immunotoxic to fish and impairment in humoral response were detected [42]. In C. batrachus glomerulus shrinkage, vascular congestion, and raptured bowman’s capsule were seen [43].

5.1.5 Gastrointestinal tract

The gastrointestinal system is the main pathway for dietary arsenic intake and absorption. Arsenic is delivered to the body organs via the circulatory system after being absorbed by the digestive tract [44]. Dietary exposure to arsenic has been demonstrated to damage the mucosal lining of the lake whitefish gastrointestinal system, causing mucosal sloughing and increased mucosal production [32]. Gastrointestinal disorders might result from acute arsenicexposure. Although the gastrointestinal effects are most noticeable immediately after ingesting arsenic, they can also develop with chronic exposure through other means. The primary gastrointestinal lesion appears to be increased small blood vessel permeability, which results in fluid loss and hypotension [45]. Exposure to a high concentration of arsenic, 20 mgL⁻¹, displayed disorganized, and consequent fusion of mucosa, lamina propria and edema, damaged serosa and degeneration [46]. Different Fish species exposed to various arsenic species included lake whitefish, walleye, northern pike, Esoxlucius, white sucker, C. commersoni, and longnose sucker (Catostomus catostomus). The concentrations of total arsenic and most arsenic species were maximum in the gastrointestinal tract in comparison to concentrations in liver and muscle [47]. The effects of arsenicals on tyrosine absorption by the winter flounder Pseudopleuronectesamericanus. Their findings showed that arsenicals inhibit Na-dependent tyrosine uptake [48].

5.1.6 Brain

Brain is extremely sensitive to arsenic because of its high rate of polyunsaturated fatty acids, oxygen consumption and extremely high rate of oxygen free radical formation without correspondingly large levels of arsenic [49]. It is thought that the arsenic poisoning altered the important biochemical components of the Labeo rohita brain tissues, such as proteins, lipids and nucleic acids, as indicated by the considerable variations in absorbance levels between the control and arsenic-intoxicated brain tissues [50]. The decrease in protein content upon arsenic exposure may be caused by decreased protein synthesis and by metal elements greater affinity for certain protein amino acid residues, which is regarded as the primary biochemical marker for early detection of stress. The conversion of protein into amino acid residues to expand the amino acid pool may possibly be the cause of the decreasing protein content. The modification of behavioral indicators and brain endonucleotidase activity is likewise caused by arsenic [51].

5.1.7 Muscles

The muscles, which make up to 80% of the fish itself, are what give the fish its swimming propulsion. The fish may move in any direction due to the muscle numerous orientations of arrangement (myomeres). Fish muscle, which makes up the majority of its bulk, is the part that people often eat [52]. The least amount of arsenic accumulated in the muscles across all experimental groups compared to other soft tissues. Muscle tissue does not directly come into touch with toxicants, so it is active detoxification site. As a result, arsenic is not transferred from other tissues to muscles. The least amount of arsenic has been found in the muscle of the Mugil cephalus [1]. Content of arsenic in muscle tissue increased significantly as fish grow. While continuous exposure to arsenic may change the size of muscle fibers, higher concentrations of metals were found in younger fish, which is typically due to the short residence times of these metals within the fish, associated with the increased metabolic rates in comparison to older organisms [53]. As exposure might start molecular alterations during embryogenesis that seem to cause abnormal muscle development [54]. H. fossilis fish exposed to 7 and 20 mgL⁻¹ of arsenic had degeneration in their muscle bundles, specific regions of necrosis, atrophy, and vacuolar degeneration [46]. Fish muscle sampling revealed that arsenic speciation was not stable under various storage and sample preparation conditions.

5.1.8 Gonads

Fish reproduction was thought to be a reliable predictor of endocrine disruption caused by chemical substances, especially arsenic, in aquatic environments [55]. An earlier monitoring study in the Mekong Delta of Vietnam found a link between arsenic accumulation and gonad development in the catfish, Pangasianodonhypophthalmus [56]. Furthermore, after inducing spermatogenesis with human chorionic gonadotropin (hCG) through the synthesis of 11-KT, exposure of the testis to high concentrations of arsenic (100 μgL⁻¹) led to the production of reactive oxygen species (ROS) in the testis, which ultimately led to the apoptosis of germ cells. These results suggest that a low dose of arsenic decreases spermatogenesis by inhibition of steroidogenic enzyme activity and expression, whereas a large dose of same substance triggers oxidative stress-mediated germ cell death. In freshwater perch exposed to arsenite, ovarian degeneration was observed.

6. Biochemical and physiological changes

In aquatic medium, toxicants often exhibit their effects at the cellular or molecular level, which causes significant alterations in biochemical markers. Heavy metal pollution also has an impact on the body primary building blocks, such as lipids, proteins, and carbohydrates, which are crucial for building the body and generating energy [58]. Among these blood glucose level utilized as an indicator of environmental stress and showed how carbohydrate metabolism changed in the presence of hypoxia and stress. When Indian catfish, C. batrachus, were exposed to sub-lethal arsenic concentrations i.e., 0.50 μM, substantial cytological alterations and changes in serum biochemical markers were detected [59].

6.1 Carbohydrate

When fish are under stress, carbohydrates are main and immediate energy sources whereas, protein is spared. Changes in the levels of glucose, lactic acid and glycogen are among the effects of arsenic stress on fish carbohydrate metabolism. Among this blood glucose level was utilized as an indicator of environmental stress and showed how carbohydrate metabolism changed in the presence of hypoxia and stress [58]. Three important Indian carps, Catla catla, L. rohita and Cirrhinus mrigala were subjected to sub-lethal levels of toxic metals, including arsenic, and the results suggest that arsenic has a hyperglycemic impact [60]. Thus, in response to arsenic exposure significant glycogenolysis occur, which would raise blood sugar levels. As a result, a rise in blood glucose during sub-lethal exposure may be caused by decreased insulin secretion or gluconeogenesis to meet the increased metabolic demands imposed by arsenic stress. The large drop in plasma glucose levels after acute therapy may be due to arsenic-induced hypoxic conditions, which cause an excessive amount of stored carbs to be consumed.

6.1.1 Protein

Due to anoxic or hypoxic conditions, which increase carbohydrate consumption, heavy metal stress that affects glucose levels indicates a change in energy requirements and expenditure. As glycogen stores run short, tissue proteins use the process of deamination of amino acids to supply keto acids. Thus, a study of serum protein composition is needed to understand how energy requirements and expenditure change under metal stress. In order to determine fish overall nutritional status, estimation of total protein, albumin, and globulin in serum is of great diagnostic significance [61]. The ratio of albumin to globulin is a helpful measure for monitoring changes in the relative proportion of serum protein. Furthermore, hepatic tissue necrosis may cause a reduction in protein synthesis [62]. In Cyprinus carpio subjected to arsenic for 72 hours, serum albumin first decreased from 2 to 4 hours and then increased over the course of 72 hours whereas serum protein and globulin levels initially increased sharply from 2 to 20 hours and then decreased by 72 hours. When C. gariepinus was given arsenic, the serum total protein, albumin, and globulin levels rose [63].

6.1.2 Lipid

Lipid bilayers make up biomembranes, which also have different kinds of protein embedded in or attached to them. All biomembranes mostly consist of phospholipids. For the biomembrane to function properly, the lipid component composition must be maintained. Biomolecules including stored lipids, proteins, and carbs assist fish in dealing with stress. When a fish is under acute stress or toxicity, stored glycogen heals it; however, when a fish is exposed to arsenic continuously, the degree of the stress increases and lipids and proteins begin to play a role. Either the oxidation process or gradual saturation can mobilize lipids to supply the energy demand [64]. The integrity of the cell membrane is maintained by phospholipids and cholesterol. High-density lipoproteins (HDL), which are good cholesterol, and low-density lipoproteins (LDL), which are bad cholesterol, both exist in the body and aid in the removal of harmful cholesterol from the blood. The HDL cholesterol level should be as high as possible. As compared to LDL cholesterol, very-low-density lipoprotein (VLDL) is similar in that it is mostly made up of lipids with little protein. HDL is the primary serum lipoprotein in rainbow trout, followed by LDL and VLDL [65]. The primary form of reserve lipids, triacylglycerols, are mobilized prior to phospholipids during starvation. It is well-recognized that arsenic alter lipid levels. Hence, lipid profile analysis also functions as a biomarker for fish health. During arsenic intoxication serum total lipid levels in C. gariepinus significantly rose from 0.95 to 2.27 g/L [66]. Total cholesterol levels increased in C. carpio after 32 days of exposure to a combination of various metals [67]. As compared to fish from the reference population, C. gariepinus from Egypt’s EL Rahawy drain, which is heavily contaminated with heavy metals (Cu, Fe, Pb, Cd, Mn, and arsenic), showed a considerable increase in blood total lipids, cholesterol, and triglyceride level [64].

7. Effect on oxidative status and other enzymes

Enzymes are biological macromolecules that regulate an organism metabolism. Much work has been conducted on how arsenic exposure affects the enzyme activity of certain fish [68]. Arsenic is absorbed through the gills, it has the potential to disturb the antioxidant system and impact the body reactions to oxidants by boosting glutamate cysteine ligase activity and glutathione levels Glucose-6-phosphate dehydrogenase (G6PDH) was significantly increased in fish gills upon exposure of arsenic, which modified antioxidant responses to an arsenic pro-oxidant challenge. As the produced nicotinamide adenine dinucleotide phosphate (NADPH) is an important element for the H₂O₂-scavenging pathway of cells [69] and for glutathione metabolism, there is indication that glucose-6-phosphate dehydrogenase, a major enzyme of the pentose phosphate pathway, has an important function in antioxidant systems [70]. Effect of sodium arsenate on L. rohita liver and muscle tissue and enzyme level was assessed and a significant decrease in acid phosphatase, glutamate-oxaloacetate transaminases, glutamate pyruvate transaminase and alkaline phosphatase enzyme activities were observed which revealed that arsenic causes disruptions in body metabolism [71]. Upon exposure of two non-lethal doses, an increase in the activity of antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GP_x) and catalase was noted in Indian cat fish, C. batrachus, however, after exposure, there was a significant drop in glutathione reductase (GR) activity, demonstrating the oxidative stress in fish. As a result of the excessive H₂O₂ generation, arsenic toxicity in C. batrachus may be targeted at the peroxisomal metabolizing enzymes. Reactive oxygen species production is linked to arsenic toxicity, which can seriously harm or damage the neurological system [72]. Enzyme activities are known as sensitive biochemical markers and are frequently used in aquatic toxicology to evaluate the health of the organism [73]. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and are two of the many enzymes that are evaluated, and they are frequently used to identify tissue damage brought on by toxicants [74]. C. batrachus exposed to arsenic at sub-lethal concentrations higher level of aspartate aminotransferase and alanine aminotransferase was noted [59]. Toxicity is linked to the creation of reactive oxygen species, due to its capacity to bind to SH groups, arsenic can inhibit the activity of numerous enzymes, including those involved in cellular glucose absorption, gluconeogenesis and glutathione synthesis, fatty acid oxidation (77). Many blood serum enzymes have been used as markers for hepatic dysfunction and injury. ALT and AST are enzymes that employed, and they are frequently used to identify tissue damage induced by toxicants [74]. In L. rohita exposed to arsenic, found a considerable reduction in liver ALT and AST which implies a major decline in the structure and function of cell organelles such the endoplasmic reticulum and the mitochondria [71]. Also, it was hypothesized that the cells utilize the phosphate-like substance, arsenate, for energy and signaling. Arsenic has the ability to inhibit the synthesis of energy and normal cell signaling by replacing phosphate in enzymes or signaling proteins (77). Delta aminolevulinic acid dehydratase (ALAD) levels were assessed to record the disruption in hemoglobin production and erythrocyte alterations. Arsenic exposure decreased the activity of ALAD in the blood by 62%. 2,3-Dimercaptosuccinic acid andALAD levels could be returned to control levels by using DMSA alone or in combination with N-acetylcysteine just the mixture of the two was able to raise RBC glutathione levels in those who were not exposed to arsenic [75].

8. Hematological changes

The hematopoietic system is influenced by short- and long-term arsenic exposure. Hematological profiles of fish are commonly used to detect the environmental contamination in aquatic ecosystem [76]. Different fish blood parameters are used to determine the effects of sub-lethal arsenic. Hematological and biochemical examinations of blood parameters in fish exposed to pollutants are crucial for determining the animal structural and functional status [77]. Red and white blood cell formation may be reduced as a result of arsenic exposure [78]. Leukocyte numbers were reduced as a result of chronic arsenic exposure which had an impact on the structure of the head kidney [50]. Arsenic induces changes to hematological markers and oxidative stress in the fish liver [79]. Numerous studies [80, 81, 82] have noted an anemic state of the fish during acute and sub-lethal treatment, which led to a low level of hemoglobin (Hb) in the arsenate-treated fish. Another potential explanation is that the toxicity of arsenic may inhibit erythropoiesis due to its effect on membranes. It was also that the fish exposed to toxicants had a lower amount of red blood cells [83]. In Oryctolaguscuniculus, that had been exposed to waterborne arsenic, a decrease in hemoglobin and packed cell volume was noted [84]. WBC count in O. mykiss exposed to arsenic was found to have decreased significantly due to reduction in lymphocytes [84]. It is commonly stated that changes in leukocyte counts upon exposure to contaminants may be linked to a decline in the fish nonspecific immunity. The control of immunological function by leukocytes occurs in a wide range of species, and the development of more WBCs in animals under stress suggests a defense mechanism against stress [85]. Chronic arsenic exposure changed the shape of the head kidney and was linked to a decrease in leukocytes [43]. The formation of reticular tissues, edematous growth, and lower leukocyte counts in the kidney and spleen, together with the establishment of an unusual lymphocyte population, indicate that arsenic probably impacts the process of lymphopoiesis and blast development in C. batrachus. Another cause could be because arsenic effect prevents WBCs from maturing and releasing themselves from tissue reservoirs. Moreover, exposure of arsenic in C. carpio led to reduced numbers of granulocyte, hemoglobin, erythrocyte and hematocrit when compared to the control group [86].

9. Immunotoxic effects

Arsenic as an immunotoxic substance has an impact on a variety of immunological responses, including altering co-receptor expression, lowering delayed hypersensitivity reactions, reducing mitogen-activated T-cell proliferation, production of freer intracellular Ca²⁺and releasing lymphokines [87]. The primary immune-competent fish organs are the head kidney, spleen and thymus [88]. The head kidney macrophages (HKM) are essential for the activation of fish innate immunity, and arsenic-induced macrophage mortality is going to impair the immunological system of the exposed fish. Arsenic accumulates in fish liver and kidney and it can impair the fish immune system by decreasing the synthesis of antibodies and cytokines [89]. Sub-lethal fish become immunocompromised and vulnerable to infections as a result of arsenic exposure, which alters the functional arms of their innate and acquired immune systems [90]. In fish, adaptive immunity develops later, therefore the impact of ecotoxins on innate immunity may be more substantial [91]. In zebra fish, the system expressed crucial antiviral genes and generated enough tumor necrosis factor (TNF-α) to fall within the range of arsenic [92]. Arsenic has a significant impact on the immune system in fish, with the two immunologically significant organs, the head kidney and spleen, responding to its toxic effects in various ways. Arsenic caused a drop in both T- and B-lymphocytes cell responses in the head kidney and spleen, although its effects seem to be more prominent on the B-cells. The phagocytic capability of macrophages was similarly impacted by fish exposure to different arsenic concentrations, which helped in the spread and duration of bacterial and viral infections [42].

10. Cytogenotoxic effects

Arsenic is recognized as a possible sulfhydryl-reactive substance that may bind to and aggregate many cell surface proteins [93]. Arsenic increases nitric oxide generation at the level of transcriptional activation along with inducing ribosylation, polyadenosine diphosphate, DNA strand breaks, depletion of nicotinamide adenine dinucleotide and the development of micronuclei, like other oxygen radical-producing stressors [94]. Cell death may be caused by the accumulation of cellular proteins, the generation of reactive oxygen species, or the stimulation of protein tyrosine kinases by arsenic [36]. Furthermore, denaturing of biological enzymes and changing gene regulation are toxic consequences of inorganic arsenic. For the purpose of researching the cytotoxicity of various arsenic compounds, fish cell lines may be used as sensitive substitutes for entire fish. In JF cells, arsenite may cause apoptosis by the induction of oxidative stress, however in TO-2 fish cell lines, it disrupts the cell cycle without the induction. Arsenic may impair cell division by disrupting the spindle apparatus [95]. In addition, it causes sister chromatid exchange, the formation of micronuclei, DNA-protein crosslinking, and different types of mutations [96]. In reaction to arsenic, a duration- and dose-dependent increase in the formation of micronuclei in the gill cells of Danio rerio was reported [97]. It is known to prevent DNA repair [98] and even worsen the effects of other mutagenic substances [59], making fish more susceptible to a variety of disorders. There are abundant evidences thatarsenic can interfere with gene expression, especially due to its impact on signal transduction [59]. The DNA-binding abilities of the transcription factors NFkβ, and AP-1, i.e., ‘activator protein 1’, were demonstrated to be affected by low arsenicconcentrations, which increased gene expression and induced cell proliferation [99]. High concentrations of arsenic, however, may reduce NFjβ activation, restrict cell growth and cause apoptosis [100]. The co-mutagenicity and possibly the co-carcinogenicity of arsenic may be primarily mediated by the repair inhibition. Arsenic exposure may lead to DNA hypomethylation as a result of ongoing methyl depletion, which supports abnormal gene expression (Figure 2).

Figure 2.

Impact of arsenic on different organs of fish. Induction of oxidative stress, inhibition of fish growth, weakness of the immune system, reduced fecundity rate, damaging of the cell and tissues and enhancement of the cell death through apoptosis are the effects of arsenic toxicity in fish.