R E V I E W
An Overview of the Adverse Effects of Heavy Metal Contamination on Fish Health
Mehjbeen Javed1•Nazura Usmani1
Received: 13 February 2016 / Revised: 5 March 2017 / Accepted: 3 May 2017 ÓThe National Academy of Sciences, India 2017
Abstract Rapid industrialization results in the production of huge amounts of solid and/or liquid wastes, which is usually discharged into the nearby water bodies, leading to the damage of the important ecosystems and seafood products. Therefore, the present overview aims to highlight the issue of pollution of aquatic ecosystems and fish health.
Heavy metals are widely used in every industrial applica- tion; therefore, they form the core group of pollutants of any industrial discharge. Some of the heavy metals such as Fe, Mn, Co, Ni, Cu, Zn and Cr are essential as they form the cofactor for many of the enzymes and also needed in metabolic activities. On the contrary, their exceeding amount is also detrimental to both animals and human beings. Based on the current review, it has been observed that to monitor the health of indicator organism (fish), battery of bioassays or biomarkers are required. In addition to this rationale of using the few selected parameters such as condition indices, bioaccumulation, blood biochemistry, marker enzymes of tissue damage, oxidative stress, geno- toxicity and histopathology in describing, the aquatic pol- lution has also been emphasized. All these parameters are significantly affected by heavy metals and hence proved as useful tools in biomonitoring or toxicity assessment stud- ies. Since fishes are consumed by large mass of population
due to their high protein and polyunsaturated fatty acid content, human health is also under danger.
Keywords Heavy metalsBioaccumulation Oxidative stress GenotoxicityHistopathology
Introduction
Water is essential for all forms of life. Seas and oceans contribute approximately 97%, while the freshwater resources consist only 3% of the entire water reserve of the earth. About 68.7% of the freshwater is locked up in gla- ciers and ice caps on poles, 30.1% in groundwater, 0.3% in surface water bodies and 0.9% in other forms [1]. So the amount of freshwater on Earth is limited, but its quality is always under suspect as reported by Global Analysis and Assessment of Sanitation. Now, water has a key role in sustaining ecological balance. Moreover, it is not only the main component of the biosphere but also a major part of the living organisms [2]. Life cannot be sustained more than few days without water, while an inadequate supply of water may change the pattern of distribution of organisms as well as of human beings. The widespread scarcity, the gradual destruction and the aggravated pollution of the water resources also lead to degradation of ecosystem.
Nowadays, water quality issues are gaining recognition as river waters are getting heavily polluted at many places.
Moreover, groundwater quality, at many places, is beginning to deteriorate to cause serious implications on the supply of water for drinking, irrigation and industrial use as all of them are important determinants of public health. The level of natural contaminants and chemical pollutants is high and also is increasing at several places. Environmental pollution became all the more hazardous as the urban life became more Significance statement This overview highlights the issues of
surface water pollution and fish health due to heavy metals. It includes the major polluted water bodies of the world and their source of pollution. In addition, it also emphasizes upon the relevance of biomarkers which can suitably be used to assess the fish health.
& Mehjbeen Javed
1 Aquatic Toxicology Research Laboratory, Department of Zoology, Aligarh Muslim University, Aligarh, Uttar Pradesh 202002, India
DOI 10.1007/s40011-017-0875-7
and more prevalent. Rather, it has increased parallel to the industrial development. In the second half of twentieth cen- tury, increasing environmental pollution due to rapid indus- trialization and population growth has caused natural resources to become more polluted so that destruction of ecosystem became an acute issue. The effluents discharged from the industries into the water bodies contain many toxic compounds like phenols, oils, pesticides, heavy metals, xenobiotics and polyaromatic hydrocarbons. These effluents affect the physicochemical parameters of water such as tem- perature, pH, dissolved oxygen, total solids, dissolved solids and suspended solids. These parameters are often employed to assess the water quality.
In addition, the heavy metals form the core group of pollutants in the industrial and daily life activities. The exceeding contents of heavy metals like Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd and Pb were also reported in several freshwater resources so that the available water has been rendered unsafe for domestic consumption, irrigation, and industrial needs. In a nut shell, this degradation of water quality has led to water scarcity for the human consumption. Figure1 illustrates the impact of heavy metal-loaded industrial waste on general health of fish and fish consumers. Table1 shows some of the water bodies of the world polluted by some point source of pollution.
A Brief Introduction of Selected Heavy Metals
Heavy metals, in general, apply to a group of metals and metalloids with atomic density greater than 4 g/cm3 or 5 times or more, greater than water [3]. However, being a heavy metal has little to do with density but concerns mainly with the chemical properties. Electronegativity (power of the elements to attract and accept electrons in a compound formation) of metals also has some bearing on their ecological effects with respect to toxicity to aquatic organisms. If the electronegativity is more, the toxicity will also be more [4]. Heavy metals are unbiodegradable and persist in the environment for a quite long time even after the source has been removed. Therefore, heavy metals are considered most dangerous in the toxicological studies.
The present review focuses on the metals most fre- quently found in surface water, and these include 24Cr,
25Mn,26Fe,27Co,28Ni,29Cu, and30Zn. No one is denying the fact that these are essential metals, but when their concentrations exceed a threshold concentration in the aquatic ecosystem, they act as pollutants and create stress in fish. These metals, belonging to first transition series of the periodic table, are known to stimulate the production of reactive oxygen species (ROS) in the living systems. This production of ROS accounts for the toxicity.
Chromium (24Cr52.01)
It is one of the most common ubiquitous pollutants in the environment and does not occur naturally in the pure metallic form. This element is present in?1,?2,?3,?4,
?5 and?6 oxidation states. However, its divalent (Cr2?), trivalent (Cr3?), and hexavalent (Cr6?) oxidation states are stable. Cr2? is a rare form; however, Cr3? and Cr6? are most common. It is the hexavalent form of Cr, which is allowed to cross biological membranes of aquatic organ- isms [5]. In natural waters, the concentration of Cr is low and is within the range of between 1 and 2lg/L [6].
Several industrial applications like chrome plating, electric furnaces, corrosion inhibitors, burning of coal, fuel addi- tive, petrochemical fertilizers, discharges from manufac- turing processes of Fe and steel industries and cooling towers are responsible for releasing Cr.
Manganese (25Mn54.94)
It exists in several oxidation states like ?2, ?3, ?4,?5,
?6 and?7. The most stable oxidation states of Mn are?2,
?3, ?4 and ?7. Soluble ionic divalent Mn2? exists in water in the presence of lesser concentration of O2at low pH [7]. It is released into the environment by several industrial applications like steel industries, emission from welding rods, fuel additive, dry cell batteries, alloys, electric coils and the burning of fossil fuels.
Iron (26Fe55.85)
It also exists in several oxidation states like ?2,?3,?4,
?5 and?6. However, the most stable states that exist in solution are the ferrous (Fe2?) and ferric (Fe3?); the latter is the most common form found in surface waters [7]. The main source of increased iron concentrations in the aquatic environment is the mineral, iron and steel processing industrial runoff, alloys, acid mine drainage, chemical industries, dye industries, fertilizers, organic chemicals, metal processing, food canneries, tanneries, titanium dioxide production and petrochemicals.
Cobalt (27Co58.94)
It exists in ?2, ?3 and ?4 oxidation states. However, (Co2?) and (Co3?) are its most stable forms. The main sources of increased cobalt concentrations in the aquatic environment are electroplating, preparation of drying agents, production of catalysts, turbine making, alloys, high-speed tools, cobalt metal powders utilized as an indicator of humidity and also those used in painting porcelain.
Nickel (28Ni58.7)
It exists in?2, ?3 and?4 forms. However, Ni2? is the most stable. This divalent ion is the dominant form in
natural waters at pH range 5–9. In aquatic ecosystems, Ni occurs as soluble salts adsorbed onto or associated with clay particles, organic matter and other substances.
Metallurgical plants, silver refineries, industries concerned Fig. 1 Overall health effects on fish and fish consumers
primarily with copper or brass plating, storage batteries, printing fabrics, automobile plants, electrodes, paints, pigments, combustion of oil, pulp and paper board mills, fertilizers and burning of fossil fuels cause an increase in nickel concentration in aquatic systems.
Copper (29Cu63.5)
It exists in ?1 (cuprous) and ?2 (cupric) oxidation states. Natural concentrations in water are at B5 lg/L
[25]. Cu reaches into the aquatic systems through anthropogenic sources such as industrial, coolant water discharge, corrosion of pipe lines, municipal drainage/
sewage, combustion of coal, fly ash, mining, plating operations, antifouling paints, pulp and paper board mills, fertilizers, petroleum refining, steel works, foun- dries, copper fungicides, use of copper salts in control- ling aquatic vegetation and influx of Cu-containing fertilizers [26].
Table 1 Heavy metal content and pollution source of different water bodies of the world
Site Effluent Heavy metals (ppm or mg/L)
Cr Mn Fe Co Ni Cu Zn References
River Olifants, South Africa
Cu mining 0.003 0.618 7.9 – – 0.004 – [8]
River Khan, India Dyes from currency printing, leather effluents – – 2800 – – 50 220 [9]
River Kshipra, India
Untreated sewage, textile effluent, agricultural runoff
– – 1570 – – 50 200 [9]
Cine stream, Turkey
Feldspar thermal power plant – – – 0.0246 0.031 0.288 – [10]
River Buyuk Menderes, Turkey
Domestic, garbage, fertilizer, textile, cement, sugar mill
– – – 0.01 0.01 0.11 0.054 [11]
Cine stream, Turkey
Feldspar thermal power plant – – – 0.067 0.081 0.009 0.023 [11]
River Orontes, Turkey
Leather tanning, oil plants, sewage, domestic waste, agricultural runoff
15.3 – 98.8 – 22.5 40.3 39 [11]
Madiwala Lake, India
Untreated municipal sewage, domestic sewage, storm water
2.5 – – – 5.6 – – [12]
Upper Lake, India Sewage, hospital waste, religious activities 0.043 – – – 0.188 0.013 0.302 [13]
River Gediz, Turkey
Agricultural, industrial, residential waste – 0.13 0.232 0.03 0.02 0.008 0.05 [14]
River Gwebi, Zimbabwe
Urban waste, agricultural runoff 0.018 – 21 – 3.7 46 650 [15]
River Manyame, Zimbabwe
Mining, metallurgical, urban waste, agricultural runoff, manufacturing industries
42 – 46 – 3.4 17 542 [15]
River Mukuvisi, Zimbabwe
Residential, industrial, agricultural runoff 14 – 32 – 4.4 73 586 [15]
River Ganga (Garhwal Region), India
Industrial, sewage, dairy 29 – 0.27 – – 0.24 0.09 [16]
River Yamuna (Garhwal Region), India
Industrial, sewage, dairy – – 0.2 – – 0.42 – [16]
River Ram Ganga, India
Sewage, pulp, paper, sewage – – – – – – – [16]
Naini Lake Sewage, industrial – – 0.7 – – 0.2 0.08 [16]
River Gomti, India Organic matter, urea, pharmaceuticals, drugs, antibiotics, disinfectants
– – – – – – – [17]
Sewage-fed pond Dairy factory, sugar mill, cold store waste, agricultural runoff, domestic waste, mechanical shops
0.07 2.32 8.08 0.24 0.08 0.07 0.45 [18,19]
Satha canal Sugar mill, agricultural runoff 0.21 2.13 8.69 0.08 0.24 0.12 0.6 [20,21]
Kasimpur canal Thermal power plant effluent 0.10 0.21 8.71 0.11 0.12 0.86 0.3 [22–24]
Zinc (30Zn65.4)
It exists only in most stable ?2 oxidation state. Major sources of zinc in water are galvanized materials, and domestic products containing zinc are batteries, pigments and paints, electroplating, fly ash, combustion of coal, petrochemicals, organic chemicals, fertilizers, steel work foundries, zinc, lead and copper smelting industries, brass alloy manufacturing, galvanizing iron and steel, dry bat- teries, municipal waste, pesticides, automobiles, fungi- cides, pigments and printing.
All heavy metals exist in surface waters in colloidal, particulate and dissolved phases, although dissolved con- centrations are generally low [27]. The solubility of heavy metals in surface waters is predominately controlled by the water pH, the type and concentration of ligands on which the metal could adsorb, and the oxidation state of the mineral components and the redox environment of the system [28].
Adverse Effects of Heavy Metals on Fish
The toxic effects of heavy metals on fish are multidirec- tional. They are manifested by numerous changes in the physiological and chemical process of their body system.
Accumulation of metals in various organs of fish may cause structural lesions and functional disturbances in them. A survey of heavy metals toxicity shows that the presence of heavy metals, in general, causes alterations in condition indices (condition factor and hepatosomatic index), biochemical disorders including oxidative stress and associated genotoxicity, and histopathology on aquatic organisms.
Condition Indices
Some physical indices of fish have been routinely used in monitoring the changes in fish health. The most common morphometric index is the condition factor (K) and in addition to this is the organosomatic index in particular hepatosomatic index (HSI) relates mass of liver to the body mass. They serve as very simple tool to monitor fish health in field studies since these indices like many other biomarkers are also affected in polluted environments.
Condition Factor (K)
Kis expressed as weight (g)/length3(cm). It provides only the general well-being of the fish or used to assess the growth conditions of the fish. A high value ofK reflects good environmental quality, while a low value ofKreflects poor environmental quality. Polluted environment often
decreases K, which could be interpreted as increase in metabolic rate, enhancement of fat metabolism as part of toxic action and decline in stored glycogen in liver.
Hepatosomatic Index (HSI)
HSI is the ratio of the liver weight/the fish body weight9100. It provides more specific information relating to the function of liver in response to the pollution.
In a poor environment, fish usually have a smaller liver (with less energy reserved in the liver). It is more advan- tageous overKsince liver plays a central role in detoxifi- cation of contaminants. Goede and Barton [29], however, pointed out that HSI may decrease in response to starvation (glycogen depletion), but liver weight may increase due to pathological changes (hyperplasia). The resultant effect could, in theory, be not net change in HSI even if resultant histological examination of the tissue reveals toxic responses [30], in spite of the fact that HSI proves useful in field and laboratory studies.
Very few fish toxicologists had used the condition factor (K) and hepatosomatic index (HSI) as a biomarker of fish health in relation to pollution in general and heavy metals in particular. Eastwood [31] investigated the effect of Ni, Al, Cu and Zn on theK(6 g cm-3) and HSI (1.42%) of fish Perca flavescensinhabiting lake in Ontario and found that Ni and Al did not appreciably bioaccumulate and so showed no relationship toKand HSI. However, Cu and Zn concentrations of liver were negatively correlated with these indices. Lowered K (2 g cm-3) and HSI (0.63%) were also reported in fishOreochromis niloticusinhabiting pond received irrigation water, containing heavy metals Fe, Zn, Cu, Cd and Pb [32]. El-Serafy et al. [33] reported that K (-7.93%) and HSI (-22.79%) exhibited marked reduction in fishOreochromis niloticuson exposure to Cu and Cd. Saeed [34] recorded variation inKand HSI values in three fishes inhabiting Lake Edku, Egypt, polluted by drain water and agricultural runoff. The trend ofKwasO.
niloticus(1.72 g cm-3)[Tilapia zilli(1.68 g cm-3)[O.
aureus (1.53 g cm-3); however, trend of HSI was O.
aureus(1.87%)[T. zilli(1.59%)[O. niloticus(1.42%).
Bervoets et al. [35] investigated the effect of Cd, Cu and Zn in fishesGobio gobio, Rutilus rutilus andPerca fluviatilis inhabiting Scheldt River, Belgium, that despite appreciable accumulation in fish tissues, very weak or no changes were observed inKand HSI of each fish.
K and HSI are not only responsive to pollution; how- ever, they can also be affected by other factors such as temperature and food availability. It might therefore be preferable to choose an index which is more closely related to the target organs of the pollutant of concern. Hence, tissue contaminant analysis becomes a necessary step for pollution monitoring studies.
Bioaccumulation
Bioaccumulation can be defined as the process by which certain toxic substances (such as heavy metals or other compounds) occurring in the environment accumulate in the living organisms. Their uptake is considered to be passive and involves diffusion gradients created by adsorption or binding of the metal to the tissue and cell surfaces [36].
The metal concentration in any organism is exhibited by the cumulative effect of a number of processes such as their uptake, elimination, storage and transformation. It varies among the metals, the species and even the indi- viduals because of differences in their permeability, metabolic rates as well as on the quantities and the types of metal binding ligands present at the surface of the organ- ism. Although it may not be essential to quantify all the processes precisely, some idea of their relative contribution to the overall pattern of metal turnover is often the only way of interpreting tissue residue data. The incorporation of metals into cells from solution is common to all aquatic animals. It may be at the surface (from surrounding water) or in the alimentary tract following intestinal digestion of food. Consequently, the kinetic studies of the dissolved metals have received the maximum attention vis-a`-vis.
their uptake and loss. Net accumulation usually follows either exponential or linear patterns. Exponential accumu- lation arises when uptake is initially rapid relative to the loss. This signifies that a preliminary adsorption phase is dependent on the surface characteristics of the organism.
Once the external adsorption sites become filled, the net accumulation slows (limited by the rate of inward diffusion and provision of internal ligand systems) and loss of metal becomes increasingly significant. Eventually, the uptake and the loss rates may cancel each other out, and the organisms body approaches steady state with respect to ambient concentrations (often proportionality is observed).
Steady-state concentrations in tissues are usually charac- terized in this scenario by simple first-order equation as follows:
Ct¼Cssð1ectÞ
whereCtandCssare concentration in the organism at time t and at steady state*, respectively; c(elimination coeffi- cient)=0.693/t1/2whilet1/2being the biological half-life) [37].
Linear accumulation over a time arises when the excretion of metal is negligible or slow in relation to uptake, and therefore, body burdens continue to increase during exposure, provided that metal binding ligands do not become saturated, or the rate of organism growth does not outstrip that of metal assimilation. Steady state* may take a considerable time if it occurs at all [37].
If there is excess in the body, the metal content in the organism can be regulated by homeostasis [38]. However, if the heavy metal concentration at the source of supply (e.g., water and food) is too high, the homeostatic mech- anism ceases to function and the essential heavy metals act in either acutely or chronically toxic manner. Thus, in the event of a resulting extended bioaccumulation of heavy metals, the organism may be damaged.
Among the aquatic animals fishes are the inhabitants that cannot escape the detrimental effects of heavy metals [39]. Moreover, they are at the top of aquatic food chain.
The effect of heavy metals on fish is multidirectional that they bioaccumulate in the different tissues and can knock down respiratory, digestive, excretory, immune, reproduc- tive, nervous, endocrine system. Level of trace metals in different organs of fish is used as an index of metal pol- lution in an ecosystem. This is considered as an important tool to highlight the role of elevated level of metals in aquatic organisms [40].
Generally, heavy metal accumulates in all the vital organs of the fish. Most often, the highest concentrations of heavy metals are found in fish liver, kidney, gills [21,41–43] and in some cases in the gut [44,45]. However, little is known about the uptake of heavy metals through the integument. Javed et al. [24] in their study onChanna punctatus inhabiting heavy metal-polluted canal reported high accumulation of Ni in integument. Other workers also reported highest accumulation of metals in integument of fish Labeo dyocheilus [46] and Clarias gariepinus [43].
The exact cause is unknown and needs to be worked out.
Several studies have been conducted to observe the influence of contamination of water with heavy metal. The accumulation of Fe, Cu, and Zn was reported in gills, plasma and liver of Tilapia sparmanii; here gill was reported to be highly influenced organ [47]. Coetzee et al.
[48] estimated heavy metals in river Olifants, South Africa, and in gill, liver, muscle and skin of fishes Clarias gariepinus and Labeo umbratus. In both the species, gill and liver were found to contain higher concentrations of Zn, Mn, Fe, Ni, Al, Cr and Pb. Ahmad et al. [49] had reported bioaccumulation of heavy metals (Cu, Cr, Pb and Hg) in the target organs of O. niloticus and Poecilia latipinna of Wadi Namar, Saudi Arabia. In both the spe- cies, liver and kidney were more affected than gills.
Bioaccumulation of metals (Cu, Cd, Co, Ni, Pb, B and Zn) in gill, liver and muscle of freshwater fish L. gibbosus inhabiting Cine stream, Turkey, was estimated. Zn was the most accumulated metal and maximum accumulation occurred in both gill and liver [10]. In another study on Barbus capito pectoralis andChondrostoma nasus inhab- iting Buyuk Menderes River, Turkey, maximum accumu- lation of metals (Cu, Zn, Cd, Co and Pb) was found in liver of both species [11]. Mohamed [50] investigated
accumulation of Fe, Cu, Zn, Pb, Cd and Co in tissues (liver, gills, muscle, intestine, heart and testis) of Oreochromis niloticusandLates niloticus. In both species, accumulation was high in both gills and liver. Al-Weher [51] determined high heavy metal levels (Cu, Zn, Cd) in gills of three species (O. aureus, C. carpioandClarias lazera)collected from Northern Jordan Valley. Among the different tissues (gill, muscle, liver and skin) ofClarias gariepinusdwelling in river Vaal, South Africa, high accumulation was depicted in gills, followed by muscle, liver and then skin [52]. The maximum heavy metal concentration was reported in kidney and liver ofHypophthalmichthys moli- trix, Channa marulius,Catla catla,Cyprinus carpio,Cir- rhinus mrigala, Nandus nandus dwelling in Madiwala Lake, Bangalore, India [12]. Yildiz et al. [14] determined accumulation of Fe, Mn, Co, Zn, Cr, Cu and Ni in gill, liver and muscle of Anguilla anguilla, and accumulation was observed in the order gill[liver[muscle [14]. Baki et al.
[53] measured concentrations of Cu and Cr in gill, liver, kidney and muscle of Tilapia nilotica. These metals accumulated in the order: liver[kidney[gill[muscle.
InCyprinus carpio; also gills were the most affected organ [54]. Some other studies on exposed Channa punctatus also revealed that gills, liver, and kidney accumulated high levels of heavy metals [18, 24, 43, 55]. Chrysichthys nigrodigitatus and Clarias anguillaris also accumulated heavy metals (Mn, Pb, Cd, Cr, Ni and Cr) in gills, liver and muscle [56]. Mastacembelus armatus exposed to thermal power plant effluent also reported high accumulation in gills, liver and kidney [23].
Among the above named organs, the most affected organs were gills and liver. Gills were found to be the most influenced because they always remain in direct contact with the ambient environment, whereas liver is the main metabolic organ. Moreover, only few studies reported bioaccumulation in kidney.
Biochemical Parameters
The principal components of body like carbohydrates, proteins and lipids, which play a significant role in body construction and energy production, are also affected by heavy metal pollution [57]. The analysis of biochemical levels in serum and tissue is of considerable importance to assess the toxicity in environmental toxicology.
Protein
The heavy metal stress causing change in the glucose content indicates a change in the energy requirement and expenditure because anoxic or hypoxic condition increases carbohydrate consumption. When the glycogen reserves
deplete, the tissue protein supplies keto acids by the pro- cess of deamination of amino acids. So a study of the protein content of serum is necessary to understand the changing demands and expenditure of energy during metal stress. Estimation of total protein, albumin and globulin in serum is of considerable diagnostic value in fish as it relates to general nutritional status [58]. The albumin:
globulin ratio serves as a useful index to track relative changes in the composition of protein in serum. Moreover, the necrosis of the hepatic tissue may result in decrease in protein synthesis [59].
Gopal et al. [60] exposed C. carpioto lethal and sub- lethal concentrations of Cu (8–0.8 ppm, respectively) and Ni (10–1.0 ppm, respectively) for 72 h. Serum protein and globulin level showed an initial sharp increase from 2 to 20 h and then decline by 72 h; however, serum albumin showed an initial decrease from 2 to 4 h and increased over a period of 72 h. In C. gariepinus, serum total protein, albumin and globulin increased when intoxicated with Cu (0.1 g/kg) [61].L. rohita when reared in sewage-polluted (Hebbal Lake) and highly polluted (Chowkalli Lake) lakes of Bangalore showed gradual reduction in serum total protein [62]. Isani et al. [63] reported the amount of serum total protein and albumin 3.1±0.5 and 0.7±0.2 g/dL, respectively, on day (0) of exposure; however, no signifi- cant change was observed in these parameters on subse- quent exposure periods (14, 28 and 56 days) after Cu toxicosis in fish Sparus aurata. Barbus luteus inhabiting polluted sites of Al-Hammar marshes (Cu, Ni, Fe, Co and Mn) showed fall in total protein and albumin, whereas an increase in globulin [64]. When C. carpiowas exposed to sublethal concentration (1/10th of LC50) for 28 days, it showed significant (p\0.05) decline in serum total pro- tein [65]. Shaheen and Akhtar [66] exposed C. carpio to sublethal concentrations of Cr (VI) (0, 25, 50, 75, 100, 125, and 150 mg/L) for 6 months, and then, they observed that the total serum protein decreases significantly (p\0.05) with increasing toxicant concentration. In another similar study onC. carpio, when exposed to sublethal concentra- tion (2 mg/L) of trivalent Cr for 28 days, it showed no significant change in serum total protein; however, serum albumin revealed significant (p\0.05) increase in the examined fish as compared to the control [67].
Lipid
Biomembranes consist of lipid bilayers with various types of protein embedded in or associated with them. Phospholipids are the major lipids present in all biomembranes. Mainte- nance of the correct composition of the lipid component is critical for the functioning of the biomembrane. During stress, the biomolecules that help the fish to cope up are stored carbohydrates, lipids and proteins. In acute stress or
toxicity conditions, it is the stored glycogen which relieves the fish but on the continuous exposure to heavy metals, intensity of stress is strong then lipids and proteins start playing their role. Lipids may be mobilized to meet the energy demand either through the oxidation process or by gradual in saturation [68]. Phospholipids and cholesterol help to maintain integrity of cell membrane. Cholesterol present in the body in the form of low-density lipoproteins (LDL) [bad cholesterol], high-density lipoproteins (HDL) [good cholesterol] helps the body to get rid of bad cholesterol in the blood. The higher the level of HDL cholesterol, the better it is. Very-low-density lipoprotein (VLDL) is similar to LDL cholesterol in that it contains mostly the fats and not much protein. According to Perrier et al. [69], HDL is the main serum lipoprotein followed by LDL and VLDL in the fish rainbow trout. Triglycerides/triacylglycerols are the prominent form of reserve lipids, which are always mobi- lized before phospholipids during starvation. Heavy metals are known to induce changes in the lipid levels. Hence, the study of lipid profile also serves as a biomarker of fish health.
InC. gariepinus, serum total lipids increased significantly (p\0.05) from 0.95 to 2.27 g/L after Cu intoxication (0.1 g/kg) [61]. C. carpio when exposed to a mixture of Cr?Ni?Cd?Pb (1.25 mg/L of each metal ion) for a period of 32 days the concentrations of total cholesterol elevated [70]. In the control fish L. rohita, the serum cholesterol content was 239.23 mg/L; however, it showed a sudden decrease when reared in sewage-polluted (Hebbal Lake) and highly polluted (Chowkalli Lake) lakes of Ban- galore [62]. Salman [64] reported decrease in cholesterol level in fishBarbus luteus inhabiting polluted sites of Al- Hammar marshes (Cu, Ni, Fe, Co and Mn). WhenC. carpio was exposed to sublethal concentration (1/10th of LC50) for 28 days, it showed significant (p\0.05) increase in serum total cholesterol [65]. Shaheen and Akhtar [66] exposedC.
carpioto sublethal concentrations of Cr (VI) (0, 25, 50, 75, 100, 125, and 150 mg/L) for 6 months; then, they observed that the serum cholesterol decreases significantly (p\0.05) with increasing toxicant concentration. In serum of fishC.
gariepinuscollected from polluted Taiga Dam, Nigeria, the triglyceride levels fall significantly (p\0.05) as compared to the fish from unpolluted site [71].C. gariepinuscaught from heavy metal-polluted (Cu, Fe, Pb, Cd, Mn and Zn) EL- Rahawy drain, Egypt, revealed significant increase in serum total lipids, cholesterol and triglyceride levels as compared to the fish from reference site [72].
Tissue Damage Marker Enzyme Activity
A number of enzymes have also been shown as possible indicators of pollutant contamination. Estimation of enzyme levels has diagnostic value since under normal conditions very
low active amounts of intracellular enzymes are released in the serum and the comparison of changes in their activities pro- vides an indication of pathology. A sensitive analysis would give insight into the pathological changes caused due to heavy metal exposure. When tissue damage occurs, the intracellular enzymes are leaked into the blood depending upon the extent of tissue damage. Therefore, studies of the intracellular enzymes like alkaline phosphatase (ALP), aspartate transam- inase (AST) and alanine transaminase (ALT) serve as a useful index of the extent of pollution imposed.
Enzyme activities of the fish were also affected by the heavy metals. Seham and Soad [73] reported an increase in serum AST and ALT activities inTilapia zilliias well as in Clarias gariepinus and Mugil cephalus inhabiting polluted (Cu, Mn, Fe, Zn, Cd, Pb) River Nile. InC. gariepinus, the levels of serum AST and ALT increased significantly (p\0.05) when intoxicated with Cu (0.1 g/kg) [61]. Infected Oncorhynchus mykiss fry rearing in polluted fish farms revealed decline in serum AST activity; however, the serum ALT and ALP activity elevated [74]. Salman [64] reported an increase in AST, ALT and ALP enzyme levels in serum of fish Barbus luteusinhabiting polluted sites of Al-Hammar mar- shes (Cu, Ni, Fe, Co, Mn). WhenC. carpiowas exposed to sublethal concentration (1/10th of LC50) for 28 days, it showed significant (p\0.05) rise in serum AST and ALT [65]. Shaheen and Akhtar [66] exposedC. carpioto sublethal concentrations of Cr(VI) (0, 25, 50, 75, 100, 125, and 150 mg/
L) for 6 months; then, they observed that the serum AST, ALT and ALP activities increased significantly (p\0.05) with increasing toxicant concentration. In serum of fish C.
gariepinuscollected from polluted Taiga Dam, Nigeria, the marker enzymes AST, ALT and ALP showed significant rise as compared to the fish from unpolluted site [71].
Oxidative Stress
Lipid Peroxidation (LPO)
Biomembranes are made up of lipid bilayer. The polyun- saturated fatty acid (PUFA) present in them are highly susceptible to the oxidative deterioration. The oxidative degeneration of lipids by the ROS/[free radicals] generated as a result of exposure to toxicant is known as lipid per- oxidation. LPO is the basic deteriorative mechanism, which leads to the damage of membranes, enzymes and nucleic acids [75]. In normal cellular metabolism, the free radicals arise within the cells, which are usually metabo- lites of oxygen. However, when they are over produced or the levels of antioxidants become depleted, they become severely harmful causing oxidative stress. It is initiated when the double bonds in unsaturated fatty acids of
membrane lipids are attacked by oxygen-derived free rad- icals. The free radicals are generated in a series of reaction:
Initiation
Involves the conversion of fatty acid to free radicals
ðPolyunsaturated lipidÞLH ! LþH
ðFree radicalsÞ
Propagation
The carbon-centered lipid radical (L°) produced during initiation step is unstable and reactive species and therefore readily reacts with oxygen, resulting in the production of peroxyl radical (LOO°). This peroxyl radical, then, abstracts a hydrogen atom from another fatty acid, forming lipid hydroperoxide (LOOH) and another lipid radical (L°).
This radical will react with another molecule of oxygen, and thus, chain reaction is propagated.
LþO2! LOO
ðPeroxyl radicalÞ
LOOþLH!LOOHþL
ðHydroperoxideÞ
Termination
Oxidation once initiated proceeds in an accelerating man- ner due to the introduction of fresh radicals from the decomposition of lipid hydroperoxides to alkoxy and hydroxyl radicals. Termination of this chain reaction occurs when two radicals interact to form a non-radical product or when hydrogen atom is donated from an antioxidant compound to the peroxy radical.
Antioxidant Parameters
To guard against oxidative degeneration of PUFA, the animals have developed several enzymatic and non-enzy- matic parameters for the removal of ROS/[free radicals]
within cells. Enzymatic parameters involve catalase (CAT), superoxide dismutase (SOD) and glutathione S- transferase (GST), while non-enzymatic defense includes reduced glutathione (GSH).
SOD exists in both mitochondria and cytosol of the cell and carried out the dismutation of superoxide radical (O2 ) by converting it to H2O2by the following pathway:
O2 þHþ!HO2
HO2þO2þHþ!H2O2þO2
CAT is located in peroxisome and removes the H2O2by converting it to H2O and O2. This H2O2can also serve as the potential source of hydroxyl radicals (°OH) by the Fe-
catalyzed reaction (Fenton reaction) to convert its oxidation state, and the reaction is as follows:
Fe3þþH2O2!Fe2þþOHþOH
GST was previously called as ligandins. It comprises a family of eukaryotic and prokaryotic phase II metabolic isozymes, which are best known for their ability to catalyze the conjugation of the reduced form of glutathione (GSH) to xenobiotics for the purpose of detoxification.
GSH is a low molecular weight thiol in which major part (85–90%) of it is present in cytosol and remaining in the mitochondria, nuclear matrix and peroxisomes. GSH is synthesized from glutamate, cysteine and glycine, and the reaction is catalyzed sequentially by two cytosolic enzymes, namely c-glutamyl cysteine synthetase (GCS) and GSH synthetase. The cysteinyl part of this molecule has a sulfhydryl (SH) group, which accounts for its strong electron-donating character. Hence, GSH is readily oxi- dized non-enzymatically to glutathione disulfide (GSSG) by electrophilic substances (free radicals and ROS).
Moreover, GSH is an extremely important cell protectant since it directly quenches reactive°OH radicals, hydrogen and lipid peroxides, and radical centers on DNA and other biomolecules. Cellular GSH concentrations are reduced markedly in response to protein malnutrition, oxidative stress and many pathological conditions [76,77].
The effects of heavy metals on LPO and antioxidant defense system (enzymatic and non-enzymatic) of fish had also been investigated by many workers. When Thalas- sophryne maculosa was exposed to 25% oil–water-con- taminated fraction, then GST activity was increased in both gills and liver, while CAT activity was decreased in gills and, however, increased in liver [78]. In the River Ogun, Nigeria, Zn, Cu, Cd, As and Pb were detected due to which CAT activity reduced in liver, kidney and gills ofClarias gariepinus, while SOD, GST and GSH activities in liver and kidney were increased and fall in gills. Also, LPO levels also showed elevation in all these organs [79].
Almroth et al. [80] reported increase in antioxidant activ- ities of liver GST and GSH, whereas CAT and SOD decrease in female Symphodus melpos, inhabiting heavy metal-polluted site Visnes, near Norway. The activity of the antioxidant enzymes SOD, CAT and GST inCyprinus carpiowas increased after exposure to heavy metal Ni, Cr, Cd and Pb solution [70]. Velma and Tchounwou [81]
performed antioxidant enzyme assay in liver and kidney tissues ofC. auratus, exposed to different concentrations of Cr (VI) (LC12.5, LC25 and LC50). In both tissues, CAT activity was decreased, whereas SOD activity and lipid hydroperoxide levels were increased. Lopez et al. [82]
investigated the effect of Lake Yuriria water; received domestic sewage, industrial effluents, and municipal wastewaters contaminants in them are characterized as
heavy metals on the liver of fishGoodea atripinnis. Results of the investigation showed significant increase in LPO and CAT activity, whereas SOD activity decreased signifi- cantly. In another study on polluted River Vizela, Portugal, on fishBarbus bocagei, the elevation was reported in the hepatic LPO, SOD, CAT and GST activities and, however, decrease in the activity of GSH [83]. When Carassius auratus was exposed to 10, 25 and 50 mg/L Ni concen- trations, it showed initial decrease and then increase in LPO, decrease in SOD and increase in CAT at all con- centrations, while initial increase and then decline in GST activity in kidney [84]. In another study, Co-exposed C.
auratus exhibited elevated levels of LPO, SOD, GST activity in gills [85]. Sivakumar et al. [86] worked on the impact of heavy metals on antioxidant activity inChanos chanos, inhabiting polluted Kaattuppalli Island. Results revealed the significant increase in LPO, SOD, CAT, GST and GSH in different tissues. Similar results have shown in gill, liver and kidney ofC. punctatusfollowing exposure to thermal power plant effluent [24,112]. Cd and Cu (1 mg/
L) also showed the adverse effect on the hepatic antioxi- dant system of freshwater fish Oreochromis niloticus, whereas CAT and GSH showed an increasing trend while a decline was observed for SOD [87].
Genotoxicity
DNA is another molecule that is also susceptible to oxidative damage by ROS [88]. The polyanionic nature of DNA provides a useful substrate for adherence of metal cations, thus facilitating the formation of HO° radicals adjacent to these critical biological targets [89]. In addition to this, the heterogeneity of DNA allows for HO°attacks including the nucleobases and sugar–phosphate backbone [90]. When the attack of HO° directed toward the sugar–
phosphate backbone, cause lesions like fragmentation of deoxyribose with single-strand breaks and oxidation of sugar moiety [91].
Among the variety of methods developed for assessment of genotoxicity such as micronuclei test, chromosomal aberration test, DNA ladder assay and comet assay or alkaline single cell gel electrophoresis (SCGE) proves useful and is recommended by several workers. Comet assay is highly sensitive, easy and reliable technique to detect the single breaks in DNA molecule [92]. Moreover, in this assay the tail length (migration of DNA from the nucleus in micrometer), tail moment and tail intensity are used as an index of DNA breakage. However, most com- monly used is the tail length.
Furthermore, these heavy metals (24Cr, 25Mn, 26Fe,
27Co,28Ni,29Cu and 30Zn) are also known to cause DNA strand breaks, which may be double-strand or single-strand breaks. Ahmad et al. [93] reported that DNA integrity lost
at 1 Mm Cr concentrations, while at 100lM no change was observed in gills ofAnguilla anguilla. Mai et al. [94]
evaluated genotoxicity in larvae of oyster (Crassostrea gigas) and observed DNA strand breaks at 0.1lg/L Cu concentrations. Barsiene et al. [95] assessed environmental genotoxicity in blood erythrocytes of fishes, namely dab (Limanda limanda) and haddock (Melanogrammus aeglefinus) collected from North Sea and Atlantic Coastal waters. Results of the investigation reported that fishes of North Sea showed significantly higher level of genotoxicity than Atlantic waters. Ameur et al. [96] also reported higher DNA damage in liver of fishes Mugil cephalus and Di- centrarchus labrax inhabiting polluted waters of Bizerte Lagoon, Tunisia.
Histopathology
Histological examination of tissues is a useful method to determine the effects of environmental pollutants. Since majority of metals accumulate in gills, major damage has been reported in this organ by most of the workers. Liver is the main metabolic organ where detoxification occurs;
therefore, it would also be susceptible to damage by the toxicants. Heavy metals toxicity is mediated by producing ROS and free radicals. They produce lesions and damage the tissues.
In gills of fishes, the mean lengths of primary and sec- ondary lamellae decreased, curving of primary lamellae, separation of respiratory epithelium and lamellar fusion in secondary lamellae, loss of secondary lamellae, ballooning dilation and clavate lamellae formation at the tip of sec- ondary lamellae, cystic structures within secondary lamellae epithelium were observed in Barbus capito pec- toralis, Chondrostoma nasus [11]. Epithelial separation and lifting base of secondary lamellae showing edematous condition, alterations in chloride and mucous cells, prolif- eration of cartilage in primary lamellae, pyknotic nuclei, hyperplasia, atrophy, hypertrophy, necrosis, erythrocyte were observed in Tilapia mossambica [97], Channa punctatus [57], Cyprinus carpio [54], Anguilla anguilla [14], Mastacembelus armatus [23]. According to Mallatt [98], the edema of the gill epithelia is the main structural change caused by the exposure to heavy metals and sometimes referred as a first sign of pathology. Whereas the lifting of epithelia, hyperplasia were considered as the defense response of fish against heavy metal toxicity since they increase the diffusion distance of toxicant.
Mammalian and fish liver have some differences; most significant feature being hexagonal, hepatic lobule with distinct portal triad is not easily recognizable [99].
According to Gingerich [100], this apparent deficiency of feature could be attributed to the almost random pattern of
vascular branching that occurs during fish liver develop- ment. Generally, various histological lesions observed in exposed fish liver are distorted hepatocytes, vacuolization, necrosis of parenchyma, pyknosis and infiltration of leukocytes, damage to hepatopancreas, acute swelling, hemorrhage and development of large adipocytes. Such damages had been reported in liver ofClarias gariepinus exposed to fuel oil for 14 days [101], Channa punctatus exposed to hexavalent chromium and sugar mill effluents [102, 103], Clarias batrachus to ZnSO4 [104], Cyprinus carpioto lethal concentrations of Cr [65],Tilapia zillito Al [99], Clarias gariepinus to sewage/domestic wastewater containing Cu, Fe, Pb, Cd, Mn and Zn [105]. It has been noticed that only toxicant exposed liver shows vacuolation and pyknosis (Karyomegaly). Degeneration of liver tissue and necrosis could be due to the infiltration of leukocytes, and according to Hughes et al. [106] necrosis is the direct toxic effect of the pollutant and therefore most commonly reported. According to Hinton and Lauren [107], vacuo- lation in liver cells was the result of inhibition of protein synthesis, depletion of energy reserves or changes in sub- strate utilization. Lipidosis appears when the hepatocyte membrane disintegrates; consequently, the fat globules fuse with neighboring hepatocytes and it is irreversible [108]. Hepatic lipidosis may be correlated with lipid per- oxidation and suppression on non-enzymatic antioxidant like vitamin E [109]. The livers of some fishes also contain exocrine pancreatic tissue, which is termed as hepatopan- creas, aligned along the blood vessels in interstitial areas.
The negative aspect of this arrangement is that it could lead to the spread of inflammatory and non-inflammatory dis- eases of pancreas into the adjacent hepatic parenchyma. On the other hand, the operative advantage of this arrangement has yet not been explored [108].
Human Health
It is unfortunate that there is no water body, which does not have finger prints of human beings. Every water body receives the effluents containing heavy metals either from point or from nonpoint sources. Worst thing about heavy metals is their persistence in environment due to their unbiodegradable nature. It is the reason that aquatic fauna particularly fish bioaccumulate them, and thus, they remain in the tissues of the fish for long time. Fishes are the important source of protein and PUFA; therefore, Ameri- can Heart Association (AHA) recommended fish twice a week to the human adults. Unfortunately, fishes are now becoming the major source of heavy metals due to the pollution caused by industries. According to United States Environmental Protection Agency (USEPA) [110], these metals generally cause two types of health effects. One is
carcinogenic and other is non-carcinogenic effects. Both these effects can be measured in terms of target hazard quotients (THQ) and target cancer risk (TR) [110], and they worked on the amount and frequency of fish con- sumed. THQ should not exceed 1, if it is exceeding the limit then there are chances that one may have non-car- cinogenic risk sometimes in his/her lifetime. According to New York State Department of Health (NYSDOH) [111], the TR categories are described as: TR B10-6=low;
10-4to 10-3=moderate; 10-3to 10-1=high;C10-1-
=very high. Pregnant women, lactating mother and chil- dren are more prone to heavy metal health hazards. In one of the recent study on risk assessment via consumption of M. armatus, it has been reported that accumulation of Co (9.06 mg/kg dry weight) and Ni (58.98 mg/kg dry weight) pose non-carcinogenic risk to adult male and female indi- viduals, whereas carcinogenic risk posed by this fish was in the range 3.43910-3and 3.91910-3, respectively, for Ni [113].
Conclusion
Based on the current review reports and opinion of other investigators from the aquatic toxicology field, it is believed that for biomonitoring studies only single parameter is insufficient, rather there is a need of battery of biomarkers. Hence, biomarkers such as bioaccumulation, lipid and protein profiles, pathological marker enzyme activities, enzymatic and non-enzymatic antioxidants, lipid peroxidation, DNA damage and tissue damage could serve as useful tools to monitor the health of aquatic fauna. These heavy metals will enter the food web through water and food, to cause the adverse health effects like that in indi- cator organisms. There is no denying that industries are necessary for development, but on the other hand they are also creating heavy loss to the livelihood of humans.
Acknowledgements The authors wish to acknowledge Chairman, Department of Zoology for providing necessary facilities. First author is grateful to UGC for providing the research fellowship.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict of interest.
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