INTRODUCTION - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/33884/6/chapter4.pdf · INTRODUCTION Contaminants occur in the environment as complex mixtures with interactive

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INTRODUCTION

Contaminants occur in the environment as complex mixtures with interactive

effects, making the use of biomarkers particularly relevant for environmental health

assessment (Orbea et al., 2005). In order to properly evaluate the effects of

contaminants in organisms, the complementation of bioaccumulation with effect

biomarkers is strongly recommended (Fernandes et al., 2008). The term

bioaccumulation refers to the wastes which have been reconcentrated in organisms

often having undergone initial dilution in environment producing toxic effects in

fishes (Dallinger et al., 1987). Yazdandoost and Katdare (1999) reported that

metalloids and high concentrations of transitional metals tend to accumulate in

different tissues of body and hence become bioaccumulated. Bioaccumulation through

food chain leads to biomagnifications, which causes severe physiological

abnormalities. The level of pollutants detected in tissues of organisms is the only

direct measure of the proportion of total toxicant delivery to biota and therefore,

indicates the fraction that is likely to enter and affect aquatic ecosystem (Phillips,

1978; Yazdandoost and Katdare, 1999).

Heavy metals from natural sources and anthropogenic activities are

continually released into aquatic systems, causing serious threat because of their

toxicity, bioaccumulation, long persistence and bio-magnification in the food chain as

well as persistence in the natural environment (Miller et al., 2002). Availability of

heavy metals in the aquatic ecosystem and its impact on the flora and fauna had been

reported by many investigators (Shrinivas and Balaparamaeswaran, 1999). The fact

that they increase in values by passing from lower to higher organisms, is even more

disturbing (Hammond, 1971). Under certain environmental conditions, heavy metals

may be accumulated to a toxic concentration (Güven et al., 1999), and cause

ecological damage (Freedman et al., 1989). In fact, all water bodies are polluted by

heavy metals (HM). Many of these pollutants possess biological activity and, as

opposite to organic compounds, do not undergo transformation in the organisms of

aquatic animals. As a result HM leave biological cycles very slowly.

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Metals are present in very low concentrations in natural aquatic ecosystems

(Nussey, 1998), usually at the nanogram to microgram per liter level. The problem of

appearance of toxic materials in water ecosystem is presently closely connected with

increased concentration of different types of pollutants, which enter water bodies with

industrial and communal waste waters or from non point sources. Measurements of

total metal concentration provide useful information concerning the pollution status of

the environment by these chemicals; they do not show more direct evidence of

bioavailability, bioaccumulation, or toxicity of metals to aquatic organisms (Glynn,

2001). Such studies can provide critical information for the environmental risk

assessment of metals in aquatic environments (Wang and Rainbow, 2008). Due to the

deleterious effects of metals on aquatic ecosystems, it is necessary to monitor their

bioaccumulation in key species, because this will give an indication of the temporal

and spatial extent of the process, as well as an assessment of the potential impact on

organism health (Kotze et al., 1999).

In the last few years there has been interest in developing predictive models

for acute toxicity based on the short-term binding of waterborne metals to fish gills

(‘biotic ligand model’; Playle, 1998). Recently, extension of this approach to

predictive models for chronic toxicity based on longer term tissue-specific metal

residues has been advised (Bergman and Dorward-King, 1997). Tissue specific

accumulation of metal has been proposed as a key indicator of chronic exposure

(Bergman and Dorward-King, 1997) and an understanding of the toxicokinetics of

accumulation of a metal during chronic sublethal exposure is a critical element in

establishing links between toxicity and exposure in risk assessment (McCarty and

MacKay, 1993). Overall this approach allows assessment of the relative mobility and

accumulation of metals among intracellular compartments with quantification of the

toxicologically relevant metal pool. However, accumulated metals are exchangeable

between the toxicologically relevant and detoxified pools, as well as being present in

free form, although cellular free metal concentrations are extremely low even for

essential metals (O’Halloran and Culotta, 2000). It is believed that the pattern of

intracellular metal binding determines the functional significance of the metal and

may reveal potential mechanisms of toxicity and fate of accumulated metal. Aquatic

organisms are able to accumulate heavy metals up to concentrations that are tenths

and even thousands of times higher than their concentrations in the environment


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(Perevoznikov and Bogdanov, 1999; Podgurskaya et al., 2004; Gremyachikh et al.,

2006).

Heavy metals accumulate in the tissues of aquatic animals and may become

toxic when accumulation reaches substantially high levels (Dural et al., 2006). Some

metals have become a matter of concern because of their toxicity and tendency to

accumulate in food chains (Parlak et al., 1999; Al-Yousuf et al., 2000). Accumulation

of heavy metals in fish tissues are of particular interest can affect not only natural

populations, but also their consumers lead to potential risk (Abel, 1998; Schmitt et al.,

2006). The heavy metal levels of fish and other fishery products are widely

documented in the literature (Adekunle and Akinyemi, 2004; Dalman et al., 2006;

Tuzen and Soylak, 2007). Fish absorb dissolved or available metals and can therefore

serve as a reliable indication of metal pollution in an aquatic ecosystem (Nussay et al.,

1999). The significance of experimental exposure of fish to heavy metal

concentrations for predicting potential damage to aquatic ecology has been advocated

(Kargin and Cogun, 1999).

In natural waters observed that some non-essential trace metals such as

mercury, lead and cadmium are toxic at concentrations (McKim, 1985, Javed and

Mahmood, 2001). The metal causes both acute and chronic effects that usually result

from its accumulation in the body over a certain period (WHO, 1995). Elements such

as Hg, Cd, Cu, and Zn are considered most dangerous in the ecotoxicological aspect

(Spry and Wiener, 1991). The pattern of bioaccumulation of metals in animals differs

from metal to metal and organ to organ during their functional status. Most of the

investigations pertaining to heavy metals contaminants in aquatic systems are dealt

either with toxicity or with accumulation. Pollutants rarely distribute uniformly within

the body tissues of fish, but are accumulated by particular target organs (Cinier et al.,

1999).

The majority of metal contamination studies on fish focused on accumulation

in soft tissues, such as liver, kidney, gill and/or muscle. Most often highest

concentrations of heavy metals are found in fish liver, kidney, gills (Perevoznikov and

Bogdanov, 1999; Farkas et al., 2003), and in some cases in the gut (Farag et al., 1995;

Sobolev, 2005). The order in which organs are sensitive to heavy metals effects may


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differ in cases of acute and chronic exposures (Brown et al., 1990). Liver, kidney and

gill have efficient mechanisms for the elimination of potentially toxic compounds

(Olsson et al., 1998) and for the regeneration of damaged tissues (Tsonis, 2000).

Kidney and gills are among the organs most heavily involved in the sublethal

chronic toxicity processes of various toxic agents (Gargiulo et al., 1996). Kidney

and liver, which are considered to be critical target organs (Yamano et al., 1999; Ryan

et al., 2000) and result in their damage (WHO 1992a).

The fish gill is a multifunctional organ performing vital functions such as

respiration, osmoregulation, acid–base balance and nitrogenous waste excretion

(Evans et al., 2005). Gills are the first organs which come in contact with

environmental pollutants. Paradoxically, they are highly vulnerable to toxic chemicals

because firstly, their large surface area facilitates greater toxicant interaction and

absorption and secondly, their detoxification system is not as robust as that of liver

(Evans, 1987). Additionally, absorption of toxic chemicals through gills is rapid and

therefore toxic response in gills is also rapid (Evans, 1987). Gills have frequently

been used in the assessment of impact of aquatic pollutants in marine as well as

freshwater habitats (Athikesavan et al., 2006; Craig et al., 2007). Metal absorption in

fish takes place primarily through gills. Accordingly, gills are the primary targets of

toxicity (Evans et al., 1987; Jiraungkoorskul et al., 2007). In fact, pollutants not only

enter the organism through the gills, but also exert their primary toxic effects on the

branchial epithelium (Playle et al., 1992) which in turn may influence general gill

functions. Metal concentrations measured in gills of fishes corresponded to the

background study of many authors. The concentration and distribution of Cr, Cu, Pb

and Zn were studied in Clarias gariepinus exposed to combined (composite) tannery

effluent (Gbem et al., 2001). Similarly, copper, zinc and cadmium concentrations

in Perca flavescens (Kraemer et al., 2005) and Cu in Oreochromis niloticus (Monteiro

et al., 2005) were observed. Likewise, Cu, Zn, Mn, Fe, Mg, Ni, Cr, Co and

B accumulation were studied in Lithognathus mormyrus, Liza aurata, Chelon labrasu,

Mugil cephalus, Sparus aurata and Liza ramada (Uysal et al., 2008).

The liver and kidneys play a crucial role in detoxification and excretion

of toxicants mainly through the induction of metal-binding proteins such as

metallothioneins (MTs) (Klaverkamp et al., 1984; Cousins, 1985). Studies carried


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out in various fish species have shown that liver and kidney are the main sites of

accumulation and storage (Capelli and Minganti, 1987). Liver is the major site of

metal storage and excretion in fish and as a result of its major role in metabolism and

its sensitivity to metals in the environment; particular attention has been given to liver

in toxicological investigations (Parvez et al., 2006). Liver plays an important role in

vital functions in basic metabolism and it is the major organ of accumulation,

biotransformation, and excretion of contaminants in fish (Triebskorn et al., 1997;

Figueiredo-Fernandes et al., 2006).

The liver is the main organ used for metal homeostasis and has the ability to

reduce metal toxicity and cellular damage by metals binding to nuclear proteins, such

as metallothioneins (Heath, 1995). Liver tissues contain a large number of

parenchymal cells (hepatocytes) lined with secretory and biosynthetic structures used

to sequester, transport and/or excrete metals and other contaminants (Heath, 1995).

The liver is involved in the main metabolic activities, such as detoxification; thus, it is

probable that metal is transported from other tissues to this organ for its subsequent

elimination through induction of metal-binding proteins or binding to insoluble

fractions (Klavercamp et al., 1984). The liver tissues in fish are more often

recommended as an environmental indicator of water pollution than any other organs.

Heavy metals have been shown to be concentrated in the liver of various fishes by

numerous investigators (Sorensen, 1991; Thiruvalluvan et al., 1997). Arsenic in

Tribolodon hakonensis (Takatsu et al., 1999), Zn, Cu and Mn in Lethrinus lentjan

(Al-Yousuf et al., 2000), lead, copper, zinc and antimony in Salmo trutta (Heier et al.,

2009), cadmium in Sparus aurata and Solea senegalensis (Kalman et al., 2010) and

zinc in Synechogobius hasta (Zheng et al., 2011) has been reported.

In fish, as in higher vertebrates, the kidney performs an important function

related to electrolyte and water balance and the maintenance of a stable internal

environment. The kidney excretes nitrogen-containing waste products from the

metabolism such as ammonia, urea and creatinine. The key roles of fish kidney in the

maintenance of homeostasis, hematopoietic, immune and endocrine functions and,

mainly, in the elimination and rapid clearance of xenobiotics, make it prone to

damage (Pereira et al., 2010). Despite its relevance on fish physiology, kidney has

been under employed in environmental health assessment and ecotoxicology studies


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(Filipovic and Raspor, 2003). Only few studies are available on metal levels in fish

kidney (Pereira et al., 2010). Several biological studies have associated with metals in

kidney tissues of different fish species. Cd exposed to Oncorhynchus mykiss (Zohouri

et al., 2001), Cyprinus carpio (Reynders et al., 2006), Pb, Co, Cu, Ni, Zn, Mn and Cd

in Scarus ghobban (Ashraf and Nazeer, 2010) and cadmium, copper and zinc in

rainbow trout, Oncorhynchus mykiss (Kamunde and MacPhail, 2011).

Studies dealing with degree of accumulation among the tissues of fish under

pollutant exposure are reviewed by many authors. Cd and Pb were determined in

different tissues (muscle, gill, stomach, intestine, liver, vertebral column and scales)

of Tilapia nilotica from lake water pollution (Rashed, 2001). Channa punctatus were

exposed to Zn, Cd and Cu and their levels were observed in gill, liver, kidney, blood

and muscle (Shukla et al., 2007). The Cr, Zn, Cu, Cd, Pb contents were determined in

muscles, liver, gonads, skin, encephalon of Cyprinus carpio, Carassius auratus,

Hypophthalmichthys molitrix and Aristichthys nobilis from lead–zinc mining areas

(Chi and Zhu et al., 2007). The concentrations of Ag, Al, As, B, Ba, Cd, Co, Cr, Cu,

Fe, Mn, Mo, Ni, Pb, Se, Sr, Zn and Li in the muscle, gills, liver and intestine of the

starlet Acipenser ruthenus from the Danube River have been assessed (Jarić et al.,

2011).

Cadmium is a problem of magnitude and of ecological significance due to its

high toxicity and its ability to be accumulated in living organisms. The studies in

literature generally dwell upon the accumulation of cadmium metal in tissues (Duncan

and Klaverkamp, 1983; Suresh et al., 1993). Eisler (1985) carried out a very

comprehensive study on Cd accumulation in fish and invertebrates. Cd has been

shown to alter the structure and to cause morphopathological changes of varying

severity in various organs of fish (Battaglini et al., 1993). For example the binding of

Cd to mitochondria impairs oxidative phosphorylation with reduction in ATP

production (Sokolova et al., 2005) while binding to cytosolic thionein ligands reduces

toxicity and imparts tolerance. Cd concentration in organs of fish associated with

osmoregulation (gills), metal detoxification (liver, kidney), digestion (intestine),

neuro-endocrine regulation (brain, head kidney), locomotion (muscle) and

reproduction (gonads) (Pelgrom et al., 1995). Available reports indicate that the gill,

liver and kidney are the critical targets for cadmium in fishes (Tjalve et al., 1986), in


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which they have been reported to cause significant metabolic, biochemical and

physiological effects (WHO, 1992b).

Tissue specific accumulation of metal has been proposed as a key indicator of

chronic exposure (Bergman and Dorward-King, 1997) and an understanding of the

toxicokinetics of accumulation of a metal during chronic sublethal exposure is a

critical element in establishing links between toxicity and exposure in risk assessment

(McCarty and MacKay, 1993). Some progress has been made in understanding

accumulation - response relationships on a short term basis in fish chronically exposed

to sublethal metals (Hollis et al., 1999; Taylor et al., 2000). However, much less is

known about the pattern of metal accumulation over the longer term during chronic

sublethal exposure, and its possible association with chronic physiological

disturbances. The relationships among tissue metal burdens and overall toxic response

during chronic exposure is complicated by the fact that metals accumulate

differentially in tissues.

Therefore, the present study is focused on monitoring the accumulation levels

of cadmium in Cirrhinus mrigala, as well as to analyze their distribution among

different tissues which have been frequently used as means of evaluating heavy metal

pollution in fresh waters. Cirrhinus mrigala is one of the most popular fish in India

has a commercial importance, valuable, cheap food item and a source of protein in the

human diet. Thus, it is important to identify the extent of heavy metal concentration in

fish samples and consider its potential impacts on the food chain and its human health

risks.


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MATERIAL AND METHODS

EXPERIMENTAL MANIPULATIONS

A static acute and sublethal toxicity studies were used in the test series. In

each study five replicates of control and treatment groups were maintained.

ACUTE STUDIES

Fish were submitted to acute (24 h) static toxicity tests, performed in aerated

glass tank of 50 L (Five replicates per treatment) filled with 30 L of tap water. Then,

LC 50 (24 h) concentration of cadmium (35.97 mg L-l) was added to the respective

experimental aquaria and 10 healthy fishes were introduced into each tank after

removal of the same quantity of water. Then, a common control was also maintained

simultaneously (free from toxicant). During the tests, water was continuously

monitored for physico-chemical parameters. After 24 h, fish from control and Cd

treated groups were selected randomly and sacrificed for accumulation (Chapter II),

hormonal (Chapter III), enzymological (Chapter IV), biochemical (Chapter V), and

histological (Chapter VI) analysis.

SUBLETHAL STUDIES

For sublethal studies, two glass tanks of 500 L capacity were taken and filled

with 400 L of tap water and marked as Treatment I and II. Then 200 healthy fish from

the stock were randomly collected from the stock and transferred to each tank. After

introduction of the fish, cadmium concentrations viz., 1/10th (3.59 mg L-1; Treatment

I) and 1/5th (7.19 mg L-1; Treatment II) of 24 h LC50 (Sprague, 1971) added and they

were continuously exposed for 35 days and the toxicant water was renewed every day.

Five similar replicates were maintained for each treatment group. For each treatment

group a common control was also maintained (toxicant free). During the study period

fish were fed with ad libitum with rice bran and groundnut oil cake (2:1 ratio), excess

food and fecal matter were removed from the aquaria to reduce water quality


36

deterioration. The pH of the water was maintained at 7.1 ± 0.08. Sampling was done

at the end of 7, 14, 21, 28 and 35th day for further investigation.

SAMPLING FREQUENCY

A minimum of 20 fish per treatment, 5 replicates per treatment and 20 fish per

replicate were used for both acute and sublethal studies. After removal of fish at

various intervals of time, the volume of the experimental and control media were

adjusted to maintain a constant density of fish per unit volume of water. Care was

taken to avoid stress during sampling. No mortality was observed during the

experimental period.

PREPARATION OF SAMPLES

Fish from control and cadmium treated groups were sacrificed and blood was

drawn by cardiac puncture using plastic disposable syringe fitted with 26 gauge

needle. The syringe and needle were prechilled and moistured with heparin

(BeparineR heparin sodium, IP 1000 IU ml-1 derived from beef intestinal mucosa

containing 0.15% w/v chlorocresol IP preservative), an anticoagulant manufactured

by Biological E Limited, Hyderabad, India. It was transferred into small vials, which

is previously rinsed with heparin. The whole blood was used for the estimation of

reduced glutathione (GSH), glutathione S-transferase (GST), glutathione peroxidase

(GPx) and lipid peroxidation (LPO). The remainder of the blood sample was

centrifuged at 10,000 rpm for 20 min. to separate the plasma and transferred into

clean vials for the analysis viz., thyroxine (T4), triiodothyronine (T3), glucose,

protein, total bilirubin, acid phospahatase (ACP), and alkaline phosphatase (ALP).

TISSUE PREPARATIONS

After blood collection, gill, liver, and kidney tissues were removed

immediately from control and Cd treated for accumulation studies (Chapter II) to

overcome autolysis; then they were stored in respective plastic vials for further study.


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For histopathological studies (Chapter VI), gill, liver and kidney were

dissected out from control and experimental fish and fixed in 10% formalin after that

stored in respective plastic vials for further investigation.

ACCUMULATION STUDY

Accumulation of cadmium metal in gill, liver and kidney were measured as

described by Topping (1973).

SAMPLE PREPARATION AND ANALYSIS

0.5 g of gill tissue was wet weighed (ww) from control and cadmium exposed

groups and transferred to a 100 ml beaker. Then, analytical grade concentrated nitric

acid and perchloric acid was added in 3:1 ratio. For digestion, the acid solution was

gently heated on a hot plate, until the tissue completely dissolved. After the solution

reduced in volume of about 1 to 2 ml it was transferred to a 25 ml graduated flask and

made up to the mark with deionized water. Finally, the metal concentrations were

estimated with the help of Atomic Absorption Spectrophotometer (AAS), Shimadzu

model 7000 and are reported as µg/g wet weight (ww). Liver and kidney samples

were also digested in the same manner as like the gill. All the samples were measured

using Atomic Absorption Spectrophotometer.

STATISTICAL PROCEDURES

The results are mean ± standard error of mean of five individual observations

for each parameter. Analysis of data concerning differences between control and

cadmium treated groups was made using the Students‘t’ test and significance level for

the tests performed for acute studies. The statistical analysis of the data of variance

included one-way and two-way ANOVA followed by Duncan Multiple Range Test

(DMRT) were made for sublethal studies. The data were tabulated as well as bar

diagrams placed in the text of all the chapters.


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RESULTS

Accumulation of cadmium in tissues of fish Cirrhinus mrigala after acute

exposure is given in Table. 4 and Fig. 3a, b & c. The mean concentrations of cadmium

were found to be increased significantly (P<0.05) in gill, liver and kidney of

cadmium treated fish compared to control groups. There is maximum accumulation

found in liver (11.53 ± 0.03 µg/g) followed by substantial accumulation of cadmium

in kidney (10.55 ± 0.08 µg/g), while minimum accumulation is seen in gill (4.69 ±

0.13 µg/g). The accumulation of cadmium was found to be in the order: Liver <

Kidney <Gill.

The levels of cadmium accumulation in gill tissues of Cirrhinus mrigala

exposed to Treatment I and II are shown in Table 5 and Fig. 4. Mean percentage of

gill generally increased due to increase in concentration and duration of both

Treatments (I & II). Moreover, in extreme sublethal exposure the initial frequency of

3.10 ± 0.31 µg/g could increase to a maximum of 6.42 ± 0.16 µg/g for Treatment I

and then, 3.55 ± 0.19 µg/g to 7.43 ± 0.26 µg/g for Treatment II, respectively. In

comparison, Treatment II showed highest accumulation in gill. There were significant

(P<0.01) variation among the treatments (F2, 60 = 322.20; P<0.01), significant among

the periods (F4, 60 = 85.47; P < 0.01) and their interactions (F8, 60 = 23.78; P < 0.01).

Table 6 and Fig. 5 show the accumulation of cadmium level in liver of treated

fish at sublethal concentrations for 35 days. Liver exhibited a maximum percent

increase of 124.54 and 179.36 for Treatment I and II at the end of 35 days of exposure

compared to control groups, respectively. However, Treatment II showed a maximum

percent increase in relation to Treatment I. There were significant (P <0.01) variation

among the treatments (F2, 60 = 5452.40; P < 0.01), significant among the periods (F4, 60

= 1553.10; P < 0.01) and their interactions (F8, 60 = 411.51; P < 0.01).

Fish exhibited an increase in cadmium levels in kidney tissue at both sublethal

concentrations (3.59 and 7.19 mg L-1), for different periods of exposure (Table 7 and

Fig. 6). Accumulation in the kidney tissue of exposed fish increased throughout the

study in associated to the unexposed control groups. A maximum percent increase


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was observed at the end of 35th day in both the Treatments (91.17 and 113.26),

respectively. Among treatments, Treatment II showed maximum accumulation pattern

in the kidney of fish. There were significant (P<0.01) variation among the treatments

(F2, 60 = 4694.31; P<0.01), significant among the periods (F4, 60 = 877.83; P<0.01) and

their interactions (F8, 60 = 247.29; P < 0.01).


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DISCUSSION

According to Szefer et al. (1990) knowledge of the distribution of metals in

tissues is useful in identifying particular organs that are sensitive and selective to

heavy metal accumulation Under conditions of acute, high-dose metal exposure, the

maintenance of branchial osmoregulation and gas exchange is of prime importance

for the survival of the fish and gills could be important as a site of direct metal uptake

from water (Storelli et al., 2006), whereas under conditions of sublethal, chronic

metal intoxication the adaptive capacity of internal, metal-accumulating organs such

as the liver and kidney may gain importance (Stubblefield et al., 1999; Goyer and

Clarsksom, 2001). In the latter case, the key factors determining metal toxicity are

tissue distribution of accumulated metal ions, the intracellular metal sequestration,

and the relation between tissue metal dose and toxic response.

Sindayigaya et al. (1994) reported that there are four possible routes for metals

to enter a fish: the food ingested; simple diffusion of the metallic ions through gill

pores; through drinking water; and by skin adsorption. Heavy metals in dissolved

form are easily taken up by aquatic organisms where they are strongly bound with

sulfhydryl groups of proteins and accumulate in various tissues and organs (Hadson,

1988; Kargin, 1996). Fish have a tendency to accumulate heavy metals in higher rates

as shown for many species of fish in different areas (Kargin, 1996; Yilmaz, 2003).

Each fish species has a particular way to accumulate (and/or to eliminate) metal when

exposed to contaminants (Terra et al., 2008). Terra et al. (2008) suggested that

pollutants rarely distribute uniformly with the body tissues of fish, but are

accumulated by particular target organs, such as liver, gonads, kidney and gills. These

are metabolically active tissues and accumulate heavy metals of higher levels, as

observed in experimental and field studies (Karadede and Unlu, 2000). Rao et al.

(1998) have found a steady accumulation of heavy metal in the liver and kidney of

most of the fishes. Highest metal accumulation observed in kidney and liver (Hamza-

Chaffai et al., 1993). The highest metal concentrations were found in the liver, kidney

and gill, while the muscle tended to accumulate less metal (Gbem et al., 2001;

Erdogrul and Ates, 2006).


41

Cadmium (Cd) is bio-accumulative, that means it is persistent and increase

with time since it is non-biodegradable. Numerous scientific papers cite a high

degree of Cd accumulation in live organisms, which is indicative of the pollution of

both the investigated water body and the whole region with this metal. There are

various reasons explaining the overall increase in the concentration of Cd in the

environment: transport, burning of fuel, industrial contamination, transboundary

pollutant transport, urbanization, etc. Cd is toxic, especially in combination with other

elements. Known world-wide threshold values of Cd toxic effect on aquatic

organisms is 0.15 µg/l (Lithner, 1989) and 0.08 µg/l (Crommentuijn et al., 2000). The

toxicological properties of this metal largely depend on the concentration of CaCO3 in

the water. In the aqueous medium in which the cadmium salt was dissolved there are

two major routes through which cadmium can be taken up by the fish. The principal

one is the oral route with subsequent intestinal absorption, while the other is the gill.

The gills are the main targets of a direct contact with cadmium (Evans et al., 1987;

Jiraungkoorskul et al., 2007) due to its highly branched structural and vascular nature,

with the resultant highly increased surface area through which large volumes of water

pass through the gill surface, amongst others (Jayakumar and Paul, 2006).

Heath (1987) reported that metal concentration in the gills could be due to the

element complexing with the mucus, which is impossible to remove completely from

between the lamellae before tissue is prepared for analysis. Moreover, the amount of

mucus on the gill surface increases during metal exposure (Handy and Eddy, 1991),

which may contribute to higher metal concentrations at the gill surface (Reid and

McDonald, 1991). The comparatively higher Cd concentration in gills is a result of

the direct contact with the water. Failure of the gill to function during acute exposure

to cadmium can lead to the death of fish. In the present study also the accumulation of

cadmium in gill may be due to the element complexing with the mucus, which is

impossible to remove completely from between the lamellae.

The concentrations of metals in the liver represent the storage of metals from

the water where the fish species live (Karadede et al., 2004). The higher levels of

trace elements such as lead and chromium in liver relative to other tissues may be

attributed to the affinity or strong coordination of metallothionein protein with these

elements (Ikem et al., 2003). Al-Yousuf et al. (2000) reported that the accumulation


42

of metals in the liver could be due to the greater tendency of the elements to react

with the oxygen carboxylate, amino group, nitrogen and/or sulphur of the mercapto

group in the metallothionein protein, whose concentration is highest in the liver.

Further the liver exhibited higher concentration of metal accumulation than any other

organ/ tissue (Coombs, 1979). Arsenic accumulation in trout liver may reflects the

role of liver in detoxification mechanisms (Sorensen, 1991).

Nemcsok et al. (1987) reported high amounts of Zn in fish liver. Annune and

Iyaniwura (1993) also reported the liver of O. niloticus and C. gariepinus to have

accumulated Zn and Cd more than other tissues. Lanno et al. (1985) found increased

Cu concentrations in the liver of rainbow trout exposed to Cu. Levels of heavy metals

including mercury, lead and cadmium, in fish, have been widely reported (Voegborlo

et al., 1999; Canli and Atli, 2003). The highest Cd, Pb, and Zn concentrations were

found in liver, which supports the idea of metal accumulation in metabolically active

tissues that are rich in metallothionein (Kargin 1998). Heavy metals mainly

accumulate in metabolically active tissues (Thomas et al., 1985) by producing

metallothionein, particularly liver (Heath, 1987).

Cadmium has the potential to bioaccumulate in liver (Al-Yousuf et al., 2000)

and be retained in the liver (Rie et al., 2001). Liver Cd is considered a good

indicator of exposure, together with induction of liver metallothioneins (Olsson

and Haux, 1986). High values of cadmium accumulation in liver were reported by

many authors (Hilmy et al., 1985; Farkas et al., 2003). Cadmium is redistributed by

circulation to the liver and kidney following uptake through the gills (Olsson and

Hogstrand, 1987; Glynn et al., 1992). In these organs, it is mainly found to be bound

to MT which may explain the long half-life of cadmium (more than one year) in fish

(Haux and Larsson, 1984). It has been reported that fish liver accumulated substantial

amounts of Cd after both acute and chronic exposures. With regard to the liver, it is

possible that the slow rate of accumulation in the early stages of the exposure allowed

synthesis of metal-binding ligands at a rate that matched the Cd influx (Haux and

Larsson, 1984).

In the present study the accumulation of cadmium during acute and sublethal

treatment in the liver may be attributed to the affinity or strong coordination of


43

metallothionein protein with cadmium. However, the key factors determining metal

toxicity are tissue distribution of accumulated metal ions, the intracellular metal

sequestration, and the relation between tissue metal dose and toxic response.

Kidney is one of those organs where metal tends to accumulate in large

quantities at increased exposure times. The highest cadmium accumulation in

kidney was obtained by Asagba et al. (2008) in Clarias gariepinus. Kidney is one of

those organs where cadmium metal tends to accumulate in large quantities at

increased exposure times. Kidney is one of the main targets for cadmium

accumulation even if its concentration in the water is very low (Cinier et al., 1997).

Olsson et al. (1996) reported maximum values of Cd concentration (up to 5.66 µg per

one gram of fish dry weight) in kidneys. Kidneys accumulated the greatest

concentration of Cd followed by gills, liver, whole body (and carcass) (Hollis et al.,

2001). Drastichova et al. (2004) observed the highest accumulation of cadmium in the

kidney followed by liver and muscle. Woo et al. (1993) observed the highest

cadmium accumulation in the blue tilapia Oreochromis aureus after chronic exposure

in the kidney, followed by liver, brain, gill filaments and muscles.

Several other studies have demonstrated equal or higher concentrations of Cd

(relative to gills or liver) in kidneys of trout chronically exposed to waterborne Cd,

depending on uptake route. Harrison and Klaverkamp (1989) observed that kidney Cd

concentrations remained high even after the fish were returned to Cd-free water,

indicating the importance of the kidney as an accumulator of Cd. The kidney is thus

the final destination of all the cadmium from various tissues as it has also been shown

that Cd-MT is filtered through the glomerulus, and is reabsorbed by the proximal

tubular cells, possibly by endocytosis (Timbrell, 1991). Within these cells the

complex is taken up by lysosomes and degraded by proteases to release Cd, which

may result in renal accumulation of the metal. Thus, this may have accounted for the

raised level of the heavy metal in the kidney.

Many studies have investigated Cd accumulation and distribution among

organs. Karlsson-Norrgren and Runn (1985) noticed high activity of Cd in the gills,

liver and kidneys of zebra fish exposed to cadmium for 10 days. Also high

concentrations of cadmium were found in the gills, liver and kidneys of Girella


44

punctata exposed to different concentrations of waterborne cadmium for 24 weeks

(Kuroshima, 1987). In rainbow trout a progressive and dose-dependent accumulation

of cadmium was found in the liver during the 30 weeks exposure period (Haux and

Larsson, 1984). Cinier et al. (1999) commented that cadmium accumulation in carp’s

kidney was fourfold higher than in liver and 50-fold higher than in muscle, and that

toxic concentration increased as the concentration of pollutant in water increased.

However, the distribution of accumulated Cd in organs differs among studies (Cattani

et al., 1996). Various studies on fish exposed to cadmium have shown that, although

the highest metal concentration was found in the gills, liver and kidneys, the pattern of

accumulation tended to vary between different species (Asagba et al., 2008). Highest

cadmium concentrations were measured in kidney followed by gills, intestine and

liver and reflected the importance of these organs as target sites for cadmium toxicity

(Sörensen, 1991).

The accumulation of cadmium in the liver and kidney could be due to the

involvement of these organs in the detoxification and removal of toxic substances

circulating in the blood stream. Moreover, since these organs are the major organs of

metabolic activities including detoxification cadmium might also be transported into

these organs from other tissues like the muscle and gills, for the purpose of

subsequent elimination. Elevated cadmium concentrations in liver and kidney on the

other hand pointed towards their vital function in the chronic accumulation,

detoxification and elimination of metals through the induction of metal-binding

proteins such as metallothioneins (MTs) (De Conto Cinier et al., 1998; De Smet et al.,

2001). In an attempt to detoxify cadmium, the liver produces the protein MT

(Klavercamp et al., 1984). It is assumed that Cd-MT released from the liver cell is

then gradually redistributed to the kidney, which is the main target organ for chronic

cadmium toxicity (WHO, 1992a). The gill and the liver, along with the kidney, are the

main sites of metallothionein (MT) production and metal retention (Klavercamp et al.,

1984). One of the main reasons for the increased presence of cadmium in these organs

is their capacity to accumulate cadmium by induction of the metal binding protein,

MT, which is believed to influence the uptake, distribution and toxicity of cadmium

by binding to it (Wimmer et al., 2005).


45

Fish possess different defensive mechanisms to counteract the impact of

toxicants. One is the immune system, where the kidney is the one of the major organs

of the fish immune system (Jiraungkoorskul et al., 2007). It is known that kidney is

preferentially targeted by chemicals when taken up through the gills (Pritchard and

Bend, 1984). Fish kidney can present toxicant metabolizing rates (Ortiz-Delgado et al.,

2008). Exposure of fish to elevated levels of heavy metals induces the synthesis of

metallothioneine proteins (MT), which are metal binding proteins (Noel-Lambot et al.,

1978; Phillips and Rainbow, 1989). Fishes are known to posses the MT (Friberg et al.,

1971). MT has high affinities for heavy metals and in doing so, concentrate and

regulate these metals in the liver. MT binds and detoxifies the metal ion. It has been

reported that when MT becomes saturated with cadmium, it may lead to tubular

epithelial cell necrosis (Chan and Rennert, 1981). Freshwater teleosts incorporate

dissolved Cd through the gills and accumulate it mainly in the kidney (Hawkins et al.,

1980). In the present study the accumulation of cadmium in kidney may be associated

with the excretory/ionoregulatory function of the kidney in fish. Cinier et al. (1999)

suggested that the unexpected increase may occur due to a redistribution of cadmium

among the tissues before its ultimate excretion.

The accumulation of Cd is dependent on the presence of other metals as well

as the concentration and length of exposure (Lange et al., 2002). The highest

cadmium accumulation was found in the kidneys and relatively low concentration in

the liver and the gills suggesting that some cadmium was probably bound to the

mucus on the apical side of the gills and subsequently released to the water, while

some cadmium that may have entered the gills was transported via the blood to the

kidneys or the liver (Pratap and Wendelaar Bonga, 2004). The inconsistencies among

previous studies may be ascribed to differences in doses and exposure times of Cd

(Wu et al., 1999). The ATSDR (2002) review of the literature indicates that Cd may

bioaccumulate in all levels of aquatic and terrestrial food chains. It accumulates

largely in the liver and kidneys of vertebrates and not in muscle tissue (Vos et al.,

1990).

Studies have shown that fish are able to accumulate and retain heavy metals

from their environment and it has been shown that accumulation of metals in tissues

of fish is dependent upon exposure concentration and duration, as well as other factors


46

such as salinity, temperature, hardness and metabolism of the animals and biological

half-life of the metal (Nielsen and Andersen, 1996). Studies from the field and

laboratory experiments showed that accumulation of heavy metals in a tissue is

mainly dependent upon water concentrations of metals and exposure period; although

some other environmental factors such as salinity, pH, hardness and temperature play

significant roles in metal accumulation (Has-Schön et al., 2006). Metal accumulation

in tissues and whole fish was seen to be directly related to concentration and duration

of exposure. This agrees with the findings of Hodson et al. (1978), and Oladimeji and

Offem (1989). These authors exposed various fish to Pb and found concentration in

tissues to be directly related to concentration in water. Radhakrishnainah (1988) found

the accumulation of Cu in the organs of O. niloticus to be dose and time-dependent.

Annune and Iyaniwura (1993) exposed O. niloticus and C. gariepinus to Zn and Cd

and observed tissue accumulation in both fish to be dose-and time-dependent.

Bioaccumulation of Cd is known to be influenced by physico-chemical

parameters of water. Moore (1991) pointed out that uptake of Cd increased with low

DO levels, owing to increased gill ventilation and availability of free Cd2+. Uptake of

Cd is also known to be inversely related with alkalinity and hardness of water

(McCarty et al., 1978). Cadmium toxicity is also closely correlated with temperature,

simply because increased metabolic activity leads to high respiratory water flow

across the gills (Taylor, 1983). Not surprisingly, waterborne Ca2+ has a marked

protective effect against waterborne Cd toxicity to brook trout (Carroll et al., 1979),

tilapia (Pratap and Wendelaar Bonga, 1993), rainbow trout (Hansen et al., 2002), and

zebrafish embryos (Meinelt et al., 2001). Higher water Ca2+ levels reduce the amount

of Cd binding to gills (Playle et al., 1993) and reduce branchial Cd uptake rates,

resulting in lower accumulation in the kidney and liver (Hollis et al., 2000a).

Accumulation of Cd2+ can lead to severe tissue dysfunction and organ damage (Jones

et al., 2001). In fish, cadmium has adverse effects on growth (Lemaire and Lemaire,

1992), damage gills (Voyer et al., 1975) result in skeletal deformities (Muramoto,

1981), and disturb calcium balance (Wicklund-Glynn et al., 1994), inhibits calcium

uptake in gills (Verbost et al., 1987) and alters liver function (Soengas et al., 1996).

The fate of each trace element (species) is different, in terms of distributions

into different organs and tissues as well as possible biotransformation and, eventually,


47

elimination. Some tissues can act as accumulation sites; others are primarily involved

in the possible biotransformation of foreign compounds (e.g., liver) or their

elimination (e.g., kidney). In general terms, it can be speculated that trace elements

accumulate in multiple compartments in fish, each with a different elimination kinetic

(Reinfelder et al., 1998). Ecological needs, size and age of individuals (Newman and

Doubet, 1989), their life cycle and life history, feeding habits (Canpolat and Çalta,

2003) and the season of capture were also found to affect experimental results from

the tissues.

Further, many authors reported that heavy metals can accumulate in their

tissues in different amounts depending on the size and age of fish (Tuzen and Soylak,

2007). Accumulation levels vary considerably among metals and species (Heath,

1987). The accumulation of heavy metals of fish was size specific, with higher

concentrations of metals generally found in smaller fish (Yildirim et al., 2009). The

uptake of trace elements in fish occurs through food ingestion and water via the gills.

For a given trace element, accumulation sites within fish may vary with route of

uptake (i.e., food vs water) and also with the intensity and duration of exposure

(Reinfelder et al., 1998). Heavy metal concentrations in the tissue of freshwater fish

varies considerably among different studies, possibly due to differences in metal

concentrations and chemical characteristics of water from which fish were sampled,

ecological needs, metabolism and feeding patterns of fish and also the season in which

studies were carried out (Erdogrul and Ates, 2006). Different tissues of the fish

showed significant difference for heavy metal accumulation. Normally, the kidney

and liver showed higher enrichment coefficients than gill, muscle and swim bladder

(Liu et al., 2001).

A chronic sublethal exposure to waterborne Cd leads to accumulation of Cd

metal mainly in fish kidneys, liver, and gills (Hollis et al., 1999; McGeer et al., 2000).

The highest levels of Cd are detected in the kidney, liver and gills of fish (Kay et al.,

1986; Olsson et al., 1996). A large amount of Cd may be accumulated rapidly but lost

very slowly (Thophon et al., 2003). The main target organs are the kidney and the

liver. Cd poisoning in fish is produced by its bioaccumulation potential (Health, 1987)

and hence the important need to monitor pollution levels of this metal in aquatic


48

systems such that appropriate measures may be put in place in conditions of excessive

pollution. As a non-degradable cumulative pollutant, cadmium is a large and

ecologically significant problem due to its ability to be accumulated in living

organisms (Alazemi et al., 1996). A biological function of Cd is unknown to date.

However, even biogenic elements, if present in excessive amounts, are toxic for

organisms. Background concentrations of this element in ambient waters are usually

within a range of ≤ 0.01 to 0.02 mg/l (Alabaster and Lloyd, 1980).

The results of the present study exhibited that fish liver showed higher metal

concentrations having tendency to accumulate more cadmium, whereas the

accumulation of Cd was minimum in fish gills and kidney. The accumulation of Cd in

tissues of Cirrihinus mrigala was in the following order: gill > kidney > liver. The

increased concentrations of Cd in the present study may be due to the induction of

metallothionein protein or the fish might have undergone detoxification mechanism to

eliminate Cd and also indicate the importance of these organs as target sites for

cadmium toxicity. The present study makes an evident that Cirrihinus mrigala was

found to be a good indicator for assessing Cd accumulation.


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INTRODUCTION - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/33884/6/chapter4.pdf · INTRODUCTION Contaminants occur in the environment as complex mixtures with interactive