Metal Levels in the Liver, Muscle, Gill, Intestine, and Gonadof Lake Van Fish (Chalcalburnus tarichi) with Abnormal Gonad

Ahmet R. Oğuz & Aslı Yeltekin

Received: 25 March 2014 /Accepted: 10 April 2014 /Published online: 26 April 2014# Springer Science+Business Media New York 2014

Abstract Recently, an increasing number of studies haveshown that Lake Van—the biggest soda lake in the world—is polluted due to an increasing population. Studies haveshown abnormalities in the Lake Van fish (Chalcalburnustarichi), the sole fish species that inhabits the lake. Unlikethe vitellogenic and mature oocytes in normal gonads, abnor-mal gonads show large amounts of connective tissue andyoung oocytes. In this study, metal levels (nickel [Ni], copper[Cu], cobalt [Co], iron [Fe], zinc [Zn], cadmium [Cd], lead[Pb], and manganese [Mn]) in the muscle, liver, gill, intestine,and gonad of Lake Van fish with normal and abnormal gonadswere assessed. Further, the metal contents in the wastewaterfrom the wastewater treatment facility situated near Lake Vanin Van City were assessed. All the metal levels, except that ofZn, were high in the Lake Van environment (P<0.05). Thehighest metal content in the tissues was for Fe, while thelowest level was for Co. The Pb level was found to be veryhigh in both fish groups. Cd was not found in the tissues ofboth fish groups. The levels of Fe, Cu, Pb, and Mn were notsignificant in the tissues of both normal and abnormal fishgroups. Zn level was significantly high in the livers andgonads of fish with abnormal gonads, and Co level wassignificantly high only in the livers (P<0.05). Consequently,high levels of Zn in the liver and gonads and high levels of Coin the liver may be factors causing the abnormal gonads in theLake Van fish.

Keywords Heavymetal . Abnormal gonad . LakeVan fish .

Chalcalburnus tarichi . LakeVan

Introduction

The physiological functions of metals are widespread andnecessary in biological systems. Zinc (Zn), copper (Cu), co-balt (Co), iron (Fe), manganese (Mn), magnesium (Mg), mo-lybdenum (Mo), selenium (Se), and nickel (Ni) are essentialmetals, while cadmium (Cd), mercury (Hg), lead (Pb), andarsenic (As) are nonessential elements. Metals play importantroles in oxygen transport [1], DNA and protein syntheses [2],prevention of apoptosis [3], and the structure of enzymes andbone tissue [4]. Additionally, metals ensure stabilization ofcell and organelle membranes and macromolecules such asDNA, RNA, and enzymes. Excessive metal levels have toxiceffects and cause disorders in the histologic, metabolic, andendocrine functions in animals. Further, excessive metallevels cause oxidative stress [5], which adversely affects thereproductive cells [6].

Lake Van is the biggest lake in Turkey and the fourthbiggest lake in the world. Because of the high pH and alka-linity levels, to a great extent, life forms are restricted in thelake [7]. The only fish species to adapt to the conditions in thelake is the Lake Van fish (Chalcalburnus tarichi, Pallas 1811).Although the Lake Van fish is the only species, it constitutesone third of the domestic fish production (10.000 t/year) ofTurkey. The fish offers a great and cheap protein resource forthe local community.

In recent years, abnormalities in the Lake Van fish causedby pollution in Lake Van have been observed. Studies haveshown that abnormalities in the gonad might be caused byendocrine-disrupting chemicals [8]. In the preliminary studiesby Oğuz and Kankaya [9], some localities of the lake wereshown to be contaminated by endocrine-disrupting chemicals.

A. R. Oğuz (*)Department of Biology, Faculty of Science, Yüzüncü Yıl University,65080 Van, Turkeye-mail: ahmetoguz@yyu.edu.tr

A. YeltekinDepartment of Chemistry, Faculty of Science, Yüzüncü YılUniversity, 65080 Van, Turkey

Biol Trace Elem Res (2014) 159:219–223DOI 10.1007/s12011-014-9980-0

Endocrine-disrupting chemicals cause endocrine system dis-orders by displaying agonist or antagonist functions in thehormones of animals and human beings. Such endocrine-disrupting chemicals are found in insecticides, herbicides,detergents, resins, and plasticizers. In addition to thechemicals, some heavy metals show endocrine-disrupting-chemical characteristics and are defined as “endocrine-disrupting metals” [10, 11]. Such metals not only influencethe endocrine system pathways but also directly impart nega-tive effects on the gametes in the gonad [12, 13].

In this study, some somatic index values and the histologyof the Lake Van fish with normal and abnormal gonads wereevaluated. The levels of Ni, Cu, Co, Fe, Zn, Cd, Pb, andMn inthe surface water of Lake Van and wastewater discharged intothe lake from a wastewater treatment plant were assessed.Furthermore, the metal levels in different tissues of fish withnormal and abnormal gonads were determined, and the corre-lation between the metal levels and abnormal gonad wasexamined.

Material and Methods

Fish

Freshly caught fish (C. tarichi) were purchased from a localfisherman who captured them from Lake Van. The fish werethen immediately transported on ice to the laboratory. Thegonad tissue samples were collected and fixed for histologicaluse and gender identification. The fork lengths and total andgonad weights of the fish were measured. Tissue samples(muscle, gill, liver, intestine, and gonad) were collected andstored at −80 °C until analysis. Age determination was per-formed through the operculum samples belonging to the fish.All the procedures were carried out according to the NationalAnimal Care regulations.

The gonadosomatic index (GSI) was measured using thefollowing formula: GSI=gonad weight/total weight×100.

Histology

Pieces of gonad tissue were fixed in Bouin’s solution for 12 h.Afterward, the tissues were dehydrated through a gradedseries of ethanol, cleared in xylene, and embedded in paraffinwax. Histologic sections of the paraffin-embedded tissue werecut at a thickness of 5–7 μm by using a rotary microtome (HM325 Manual Microtome, MICROM International GmbH,Walldorf, Germany). Slide-mounted sections weredeparaffinized in xylene and rehydrated through graded seriesof methanol washes. Sections were then stained using hema-toxylin and eosin. All slides were mounted with coverslipsand examined under a light microscope (Leica Microsystems,Wetzlar, Germany) attached with a digital camera system.

Metal Analysis

For metal analysis, all the chemicals used were of analyticalgrade and obtained from Merck (Germany). Freeze-driedtissue samples (liver, muscle, gill, intestine, and gonad) wereweighed (approximately 0.5 g) and digested in nitric acid(65 %) at 120 °C in an oven. After the solution cooled toroom temperature, perchloric acid (65 %) was added. Afterdigestion, the residues were diluted with deionized water andfiltered using a filter paper. The residues were maintained at4 °C until analysis [14]. Metal levels (Ni, Cu, Co, Fe, Zn, Cd,Pb, and Mn) were determined by atomic absorption spectro-photometry (AAS; ICE3000 series, Thermo, USA). Metalswere determined at the following wavelengths (λ): Ni,232.0 nm; Cu, 324.8 nm; Co, 240.0 nm; Fe, 248.3 nm; Zn,213.9 nm; Cd, 228.8 nm; Pb, 217.0 nm; and Mn, 279.5 nm.All tissue samples were analyzed in duplicate. The heavymetal concentrations were expressed as micrograms per gramof wet tissue.

Statistical Analysis

Data were expressed as means±standard deviation (SD), andthe differences between sample groups were evaluated usingtheMann–WhitneyU test. Significant differences between thetwo fish groups were considered when P was ≤0.05.

Results

After the morphologic examination of the fish gonads,the abnormal gonads were noted to be smaller than thenormal gonads. Consequently, the total weight, gonadweight, and GSI of fish with abnormal gonads werelesser than those of normal fish (Table 1, P<0.05).After histologic examination of the tissues, normal fishgonads were observed to have vitellogenic oocytes,whereas the abnormal fish gonads had younger, imma-ture oocytes (Fig. 1). Additionally, the abnormal gonadshad remarkably dense connective tissue. Age determina-tion showed that all the fish were aged more than

Table 1 Morphological indexes in the Lake Van Fish having normal andabnormal gonads

Normal Abnormal

Fork length (cm) 19.20±1.55 18.76±0.72

Weight (g) 100.92±10.34 76.08±6.39*

Gonad (g) 6.97±0.87 3.07±0.63*

GSI (%) 6.93±0.70 4.09±1.07*

Fish sampled on March 2013. All values are means±SD, N=10

*P<0.001

220 Oğuz and Yeltekin

3 years (data not shown) and sufficiently mature toreproduce.

The mean metal concentrations of the Lake Van water andwastewater are shown in Table 2. Cd was not detected in the

wastewater. Further, all the measured metal levels, except thatof Zn, were high in the lake water. The highest level was notedfor Pb, and the lowest was for Cd.

Metal (Ni, Cu, Co, Fe, Zn, Cd, Pb, and Mn) levels in themuscle, liver, gill, intestine, and gonad tissues of fish withnormal and abnormal gonads are shown in Table 3 as micro-grams per gram wet tissue. The highest metal level in thetissues was noted for Fe, and the lowest was for Co. Cd wasnot detected in any of the tissue samples. The Zn and Co levelsin the liver and gonad tissues were found to significantly differ(P<0.05) between fish with normal and abnormal gonads.

Discussion

Fish are highly sensitive to aquatic pollution. Studies haveshown metal accumulation in the gonads of fish exposed tometals [15–17], which results in reductions in the productionand viability of gametes. Further, the fertilization of sperm andoocyte and embryonic development in fish are adverselyaffected by metal contamination [18].

The examination of the somatic indexes of the fish showedthat reductions in the total weight, gonad weight, and GSI offish with abnormal gonads were because of immature ovaries(Fig. 1). Previous studies of the abnormal gonad in fish haveshown a low plasma 17-β-estradiol level [8]. Hence, similar-ities are present in the symptoms of abnormal fish in Lake Vanand those of fish in aquatic areas contaminated with heavymetals. Hence, similarities are present in the symptoms ofabnormal fish in Lake Van and those of fish in aquatic areascontaminated with heavy metals, such as decrease in GSI and17-β-estradiol level and presence of immature and atreticfollicles in the ovaries [13, 19].

The wastewater discharged into the lakewas shown to havelow metal levels (Table 2). This could be because the waste-water that is treated at the treatment plant is domestic waste-water, since there are no developed industries situated near thelake. Therefore, the heavy metal concentration in the lake andconsequently accumulated in the Lake Van fish is caused bythe geochemical structure of the region and partially by thedomestic wastewater discharged into the lake. In addition, thedischarge of untreated effluents from nearby towns and agri-cultural activities may have contributed to the accumulation ofmetals in the lake.

The Pb level in the Lake Van fish was higher than that inseveral other fish species [20–23]. In a previous study relatedto the fish, the Pb level was shown to be high [24]. Afterexamination of the lake water metal levels (Table 2), the Pblevel was confirmed to be very high, which suggests that thePb accumulation in the tissues is because of the high Pb levelin the water. No significant differences were noted for the Ni,Cu, Fe, Cd, and Mn levels between the tissues of fish withnormal and abnormal gonads. Since the levels of essential

Fig. 1 Gonad tissues of normal (a) and abnormal (b) gonad samples ofLake Van fish (asterisks connective tissue, arrowheads vitellogenic oo-cytes, arrow young oocytes; scale bar 200 μm)

Table 2 Metal concentrations (mg/L) in Lake Van and the efflux of thewastewater treatment plant

Lake Van Wastewater

Ni 0.186±0.015 (9) 0.008±0.006 (4)**

Cu 0.042±0.014 (10) 0.0262±0.007 (9)**

Co 0.122±0.044 (10) 0.0150±0.013 (8)***

Fe 0.211±0.067 (10) 0.0664±0.024 (9)**

Zn 0.040±0.019 (10) 0.036±0.013 (9)

Cd 0.0033±0.002 (7) <LOD

Pb 0.214±0.050 (10) 0.0614±0.016 (9)***

Mn 0.043±0.005 (10) 0.055±0.009 (9)*

Data are expressed as mean±SD. N=10. Numbers in parentheses repre-sent the number of determinations

LOD limit of detection

*P<0.05; **P<0.01; ***P<0.001

Metal levels in the tissues of Lake Van Fish with abnormal gonad 221

metals, which are vital for living organisms, especially as theyplay important roles in several metabolic processes such aselectron transport, DNA synthesis, antioxidant defense sys-tem, and oxygen transport [1, 25], did not differ between fishwith normal and abnormal gonads, it can be confirmed that theabnormality did not occur because of the presence of theessential metals.

The hypothalamic–pituitary–gonadal axis plays a criticalrole in vertebrate oogenesis, including that in fish. Vitellogen-esis is an important stage in oogenesis where a specific pro-tein—vitellogenin—is secreted by the liver via estrogens andabsorbed by the growing oocyte. Several factors affect oocytedevelopment, and metals are essential for oocyte maturation[26, 27]. A previous study showed high Zn levels in zebrafishduring oocyte development [28]. In contrast, some other stud-ies have shown Zn as a potential endocrine disrupter [29, 30].Further, Zn has been shown to have specific endocrine toxicityin the interrenal tissue in rainbow trout [31], and Co and Znhave been shown to repress insulin-like growth factor (IGF)and growth hormone (GH) mRNA levels in rainbow trout[32]. In this study, Zn levels were high in the gonads andlivers and Co was high in the livers of fish with abnormalgonads (P<0.05). Thus, the abnormalities in the gonads of theLake Van fish might be because of the accumulation of themetals in the tissues. Further, the metals might have inhibitedthe synthesis and storage of vitellogenin in the liver andoocytes, respectively. Moreover, the combination of themetals with other chemicals (pesticides, herbicides, estrogen,and estrogen-mimicking chemicals) present in the aquaticenvironments might have led to the abnormalities. Furtherdetailed in vivo and in vitro studies are required to determinethe precise effects of Co and Zn on the Lake Van fish.

Acknowledgments The authors kindly thank Burcu Ergöz for thehistological process and Van WWTP personnel for providing wastewatersamples.

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ble3

Heavy

metalconcentrations

indifferenttissues

oftheLakeVan

fish

having

norm

alandabnorm

algonads.C

oncentratio

nsareexpressedas

microgram

spergram

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Normal

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Normal

Abnormal

Normal

Abnormal

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Ni

0.831±0.097

0.883±0.095

0.706±0.058

0.684±0.073

0.864±0.296

0.322±0.143

0.859±0.044

0.652±0.072

0.670±0.072

0.693±0.833

Cu

1.114±0.194

0.954±0.121

21.892

±6.996

13.632

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2.509±0.447

2.262±0.437

1.462±0.167

1.534±0.375

Co

0.664±0.127

0.657±0.156

0.273±0.067

0.616±0.087**

0.544±0.187

0.398±0.179

0.636±0.102

0.435±0.127

0.392±0.082

0.478±0.127

Fe12.984

±1.999

10.784

±2.274

397.222±38.168

405.210±54.420

34.424

±6.792

10.974

±4.907

60.00±15.565

42.838

±12.733

40.960

±5.081

29.751

±3.630

Zn

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±2.420

28.164

±3.496*

16.755

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17.590

±7.866

16.627

±2.602

20.479

±7.661

26.313

±5.765

47.016

±6.886*

Cd

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Pb2.419±0.208

2.255±0.240

1.801±0.273

2.130±0.202

1.523±0.272

1.465±0.655

2.034±0.245

1.931±0.368

2.485±0.202

1.508±0.181*

Mn

0.697±0.060

0.719±0.062

2.523±0.234

2.353±0.268

3.576±0.871

0.592±0.265

1.246±0.132

1.170±0.122

2.321±0.465

1.281±0.311

Dataareexpressedas

mean±SD

.N=10

LODlim

itof

detection

*P<0.05;*

*P<0.01

222 Oğuz and Yeltekin

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Metal levels in the tissues of Lake Van Fish with abnormal gonad 223