Chemico-Biological Interactions 207 (2014) 67–73

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journal homepage: www.elsevier .com/locate /chembioint

Hematological, biochemical and enzymological responses in an Indianmajor carp Labeo rohita induced by sublethal concentration of water-borne selenite exposure

0009-2797/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.cbi.2013.10.018

⇑ Corresponding author. Tel.: +91 422 2428493; fax: +91 422 2422387.E-mail address: poopalramakrishanan@ymail.com (R.K. Poopal).

Mathan Ramesh, Marimuthu Sankaran, Velusami Veera-Gowtham, Rama Krishnan Poopal ⇑Unit of Toxicology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641046, Tamil Nadu, India

a r t i c l e i n f o

Article history:Received 6 July 2013Received in revised form 28 September 2013Accepted 22 October 2013Available online 30 October 2013

Keywords:SeleniumSublethal toxicityHematologicalBiochemicalEnzymological parametersLabeo rohita

a b s t r a c t

Selenium (Se) pollution in aquatic ecosystem is an environmental issue throughout the world. Elevatedconcentrations of inorganic Se from agricultural and industrial processes may cause adverse biologicaleffects in aquatic organisms such as fish. In the present study, Labeo rohita an Indian major carp wereexposed to sublethal concentration of Se (sodium selenite) for 35 days and certain hematological, bio-chemical and enzymological parameters were estimated. The median lethal concentration of waterbornesodium selenite (Na2SeO3) to L. rohita was found to be 23.89 mg L�1 for 96 h. During sublethal(2.38 mg L�1) treatment, hematological and biochemical parameters such as hemoglobin (Hb) (except14th day), hematocrit (Hct), erythrocyte (RBC) count and protein levels were found to be decreased inSe treated fish whereas leucocyte (WBC) count and glucose level increased in Se treated fish throughoutthe study period. The enzymatic parameters such as glutamate oxaloacetate transaminase (GOT), gluta-mate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) activities were found to beincreased in liver of Se treated fish L. rohita. A biphasic response was observed in the value of mean cor-puscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin con-centration (MCHC). The alterations of these parameters can be used as suitable biomarkers inmonitoring of selenium in the aquatic environment and to protect aquatic life.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Metals and their components are considered to be a major eco-logical health concerns worldwide [1–3]. Selenium (Se) is a natu-rally occurring non metallic element and exists in theenvironment in different forms; they are selenide (Se [II]), elemen-tal selenite (Se [IV] or SeO2�

3 ) and selenate (Se [VI] or SeO2�4 ) [4–6].

The inorganic and/or organic selenium compounds are essential forthe development of the acquired immune system in many organ-isms [7]. Moreover, Se is an important element of many proteinmolecules with diverse physiological functions [8,9]. However,anthropogenic activities such as metal mining and smelting, coalcombustion, and agriculture may leads to large quantities of Sein aquatic environment [10,11]. High concentrations of Se were de-tected in surface water, sediments, and aquatic organisms in differ-ent parts of the world and are an environmental issue throughoutthe world [9]. In the dynamic aquatic ecosystem selenium can becycled back into the biota and continue to high levels for manyyears [12]. Furthermore, Se can be ingested by organisms or bindswith particulate matter, or be free without binding to any

substance [13]. The accumulation of selenium in aquatic organismsmay leads to hematological alterations, tissue pathology, reproduc-tive failure, teratogenic deformities and cytotoxicity [11,14,15].

In aquatic ecosystem, Se contamination is more challengingthan any other chemical contamination [16]. Moreover, all chemi-cal form of Se has different toxicological and biological properties[17,18]. Generally, fish model is widely used to study Se toxicitydue to their higher nutritional requirement of selenium than mam-mals [8]. Moreover, the biochemical pathways involved in sele-nium metabolism in fish are almost unknown. In toxicologicalassay, hematological and biochemical parameters are widely usedas health indicators, because they react before the toxicant entersinto the body of the organism [19,20]. Hematological parameterssuch as hematocrit (Hct), hemoglobin (Hb), red blood cells (RBCs),white blood cells (WBCs), are used to assess the functional status ofthe oxygen carrying capacity and also indicate secondary re-sponses of an organism to irritants such as metals [21,22]. Theerythrocyte blood indices such as MCV, MCH and MCHC are widelyused to diagnose anemia in animals under stress conditions [23].

Likewise, biochemical parameters such as protein and glucoseare highly sensitive to stress conditions and often used in detectionof status of stress condition [24]. Furthermore, enzymologicalparameters are frequently used to assess the impact of metals in

68 M. Ramesh et al. / Chemico-Biological Interactions 207 (2014) 67–73

aquatic organisms. Enzymes such as, carboxyl esterase (CE), lactatedehydrogenase (LDH), alkaline and acid phosphates (ALP, ACP),transaminases (ASAT or GOT and ALAT or GPT) are considered tobe useful biomarkers in biomonitoring of chemical pollutants inaquatic organisms [25,26]. The alterations of transaminasesactivity can be taken as a measure of compensatory mechanismto impaired metabolism [27]. Likewise, LDH activity is used as abiomarker in the assessment of stress condition in variousorganisms [28,29] and serves as a good diagnostic tool intoxicology.

In India during recent years aquaculture receives much atten-tion than the agriculture activity. The Indian major carps, such ascatla (Catla catla Ham.), rohu (Labeo rohita Ham.) and mrigal (Cir-rihinus mrigala Ham.) are cultivated almost in all Indian fresh wateraquaculture farms [30]. In many feed additives, selenium is addedas a vital element to preserve health and to maximize animal pro-ductivity [31,32]. To our knowledge the impact of selenium toxic-ity on Indian major carps are very limited. Hence, the present studyis aimed to evaluate the sublethal concentration of sodium seleniteon hematological, plasma biochemical and enzymological activi-ties of an Indian major carp, L. rohita. The fish L. rohita is a wide-spread species and cultured throughout India. They are widelyused in carp polyculture systems and have a higher consumer pref-erence and market demand. Moreover, the selected biomarkersendpoints can be used in environmental monitoring of seleniumcontamination in aquatic ecosystem.

2. Materials and methods

The Department of Zoology, School of Life Sciences, BharathiarUniversity, Coimbatore 46, Tamil Nadu, India, has been registeredwith the Committee for the Purpose of Control and Supervisionof Experiments on Animals (CPCSEA), Government of India. Theexperiments and the handling of the organisms were carried outas per the guidelines of CPCSEA.

2.1. Collection of fish and maintenance

Specimens of L. rohita (length 8.0 ± 0.6 cm and weight11.0 ± 1.5 g) were obtained from Tamil Nadu Fisheries Develop-ment Corporation Limited, Aliyar Fish Farm, Tamil Nadu, Indiaand safely brought to the laboratory. The fish were acclimatizedto laboratory conditions for couple of weeks in a large tank(1000 L capacity) and fed ad libitum with rice bran and groundnutoil cake in the form of dough once in daily. Water was renewed(one – third of the water) daily to avoid accumulation and contam-ination of excretory materials. Feeding was withheld for 24 h be-fore to the commencement of the experiment to keep thefingerlings more or less in the same metabolic state. Fish showingany abnormal behavior was removed from water tank as soon aspossible. Tap water free from chlorine was used with the followingphysicochemical characteristics [33]; temperature (27.3 ± 1.2 �C),pH (7.1 ± 0.05), dissolved oxygen (6.3 ± 0.02 mg L�1), total hard-ness (18.9 ± 0.3 mg L�1) and salinity (0.3 ± 0.05 ppt). Prior to thecommencement of experiment, healthy fingerlings were collectedrandomly and transferred into two glass aquaria (200 L capacity)which were continuously aerated. The study was conducted in12:12 light–dark cycle.

2.2. Toxicant

Sodium selenite (anhydrous, Na2SeO3) was obtained from M/SLoba chemic Pvt. Mumbai, India and used without further purifica-tion for the experiment.

2.3. Toxicity assessment and determination of 96 h LC50 of sodiumselenite

Preliminary toxicity tests were conducted for the determinationof 96 h median lethal concentration of Na2SeO3 to L. rohita. Sepa-rate glass tanks (50 L capacity) were taken and different concentra-tions of Na2SeO3 (5, 10, 15, 20 and 25 mg L�1) were added. Then, toeach tank, 10 healthy fish randomly collected from the stock wereintroduced. To each concentration three replicates were main-tained. Control groups (toxicant free) were also maintained simul-taneously with three replicates for each concentration. Themortality/survival of fish was recorded at the end of 96 h and theconcentration at which 50% mortality of fish occurred was takenas the median lethal concentration (LC50), which was23.89 mg L�1. The LC 50 concentration was calculated by probitanalysis method of Finney [34].

2.4. Sublethal toxicity studies (1/10th of 96h LC 50 of Na2SeO3)

To assess the sublethal toxicity of Na2SeO3, 300 healthy fishwere selected from the stock and divided into three groups (onecontrol and two experiments) and then introduced into three sep-arate aquarium tanks (100 fish in each tank). 1/10th of 96 h LC 50value of Na2SeO3 (2.38 mg L�1) was added directly into two exper-imental aquarium tanks after removal of the same volume ofwater. Experiment was conducted for a period of 35 days. The con-centration of Na2SeO3 (2.38 mg L�1) in experimental tanks were re-newed daily in order to maintain constant concentration of theNa2SeO3 after removal of the same volume of water. Fish werefed ad libitum every day. At the end of 7th, 14th, 21st, 28th, and35th day of exposure fish were randomly collected from controland experiment aquarium for the study of hematological, biochem-ical and enzymological assay. No mortality was observed duringthe exposure period.

2.5. Preparation of samples and analytical procedures

2.5.1. Blood samplesCardiac blood were collected in plastic disposable syringes fit-

ted with 26 gauge needle which was pre-chilled and coated withheparin and expelled into separate heparinised plastic vials andkept immediately on ice. The whole blood was used for the analysisof hematological parameters (Hb, Hct, RBCs, WBCs,) and theremaining of the blood samples were centrifuged at 93.9g, at 4 �Cfor 20 min to separate the plasma, which was used for the estima-tion of biochemical parameters (glucose and protein).

2.5.2. Organ samplesAfter drawing the blood, fish were washed thoroughly in double

distilled water and blotted dry with absorbent paper. 100 mg of li-ver tissues were separated from the control and Na2SeO3 exposedfish and homogenized with 1.0 ml of 0.1 M Tris–HCl buffer (pH 7.5)using a Teflon homogenizer in ice cold condition, and then centri-fuged at 93.9g at 4 �C for 15 min. The supernatant was used for theenzymological assay (GOT, GPT and LDH).

2.5.3. Hematological analysisHemoglobin content of the blood was estimated by cyanmethe-

moglobin method [35]. Hematocrit was estimated by microhemat-ocrit (capillary) method [36]. RBC and WBC were counted byhemocytometer method of Rusia and Sood [37]. Erythrocyte indi-ces like MCV, MCH and MCHC were also calculated according tostandard formulas.

MCVðcupic micraÞ ¼ Hctð%ÞRBCðmillions� cu mm� 106Þ

� 100 ð1Þ

M. Ramesh et al. / Chemico-Biological Interactions 207 (2014) 67–73 69

MCHðpicogramsÞ ¼ Hbðg=dlÞRBCðmillions� cu mm� 106Þ

� 100 ð2Þ

MCHCðg=dlÞ ¼ Hbðg=dlÞHctð%Þ � 100 ð3Þ

2.5.4. Biochemical assayPlasma glucose was estimated by the method of Cooper and

McDaniel [38]. To 0.1 mL of plasma, 5.0 mL of O-Toluidine color re-agent was added, mixed well and kept in boiling water for 10 min.After 10 min. the samples were cooled under running tap water for5 min and the optical density (OD) of the samples were measuredat 630 nm in a UV spectrophotometer within 30 min and expressedas mg/100 mL.

Plasma protein was estimated by the method of Lowry et al.[39]. To 0.10 mL of plasma, 0.90 mL of distilled water was addedand mixed well. The contents were treated with 5.0 mL of solutionC [50 mL of solution A (2.00 g of sodium carbonate was dissolved in100.00 mL of 0.1 N NaOH), was dissolved with 1 mL of solution B(500.00 mg of copper sulfate was dissolved in 100.00 mL of 1% so-dium potassium tartarate solution)] and allowed to stand at roomtemperature for 10 min. After 10 min 0.5 mL of Folin-phenol re-agent was added and kept at room temperature for 15 min andthe color intensity was read at 720 nm in a UV spectrophotometerand expressed as lg/mL.

2.5.5. Enzymological assayLiver GOT activity was estimated following the method of Reit-

men and Franckel [40]. To 50 mL of supernatant, 0.25 mL of buf-fered aspartate was added and incubated at 37 �C for 60 min.Then, 0.25 mL of 2,4-DNPH was added and allowed to stand for20 min at room temperature. To this 2.5 mL of 0.4 (N) NaOH wasadded, mixed well and allowed to stand for 10 min. The OD wasmeasured at 505 nm in a UV spectrophotometer. A standard curvewas also run simultaneously. The values were interpreted in thestandard curve and the enzyme activity was expressed as IU/L.

Liver GPT activity was estimated following the method of Reit-men and Franckel [40]. To 50 mL supernatant, 0.25 mL of bufferedL alanine was added and incubated at 37 �C for 30 min. To this,0.25 mL of 2,4-DNPH was added and allowed to stand for 20 minat room temperature. Then, 2.5 mL of 0.4 (N) NaOH was added,mixed well and allowed to stand for 10 min. The OD was measuredat 505 nm in a UV Spectrophotometer. A standard curve was alsorun simultaneously. The values were interpreted in the standardcurve and the enzyme activity was expressed as IU/L.

The LDH activity was measured following the method of Tietz[41]. To 10 ll of sample 1000 ll of buffer pyruvate was added.The contents were mixed well and the absorbance was noted for2 min at 37 �C at 340 nm. A standard curve was also run simulta-neously. The values were interpreted in the standard curve andthe enzyme activity was expressed as IU/L.

2.6. Statistical analysis

The results of the present studies were expressed as mean ± SE.The significance of sample mean between control and sodium sel-enite treated fish was tested using Student’s‘t’ test. Differenceswere considered significant at p < 0.05.

3. Results

Upon exposure to different concentration of Na2SeO3 the fishshowed behavioral changes such as excess mucus secretion, fastswimming, convulsions, jerky movement and static condition atbottom. The observed behavioral changes were severe when the

concentration of the Na2SeO3 was increased indicating that behav-ioral changes were dose dependent. Probit analysis methodshowed that the 96 h median lethal concentration of Na2SeO3 toL. rohita was 23.89 mg L�1. Based on Chi-square test, fish popula-tion used in the present investigation was found be homogeneous.In the present study, 1/10th of the 96 h LC50 value (2.38 mg L�1)was taken to assess the sublethal toxicity studies.

3.1. Hematological parameters

During sublethal treatment, Hb value was found be significantly(p < 0.05) decreased throughout the exposure period (except at theend of 14th day) (Table 1). The observed increase in Hb value at theend of 14th day was not significant when compared to controlgroups. Likewise, Hct value also decreased significantly (p < 0.05)throughout the study when compared to control groups (Table 1).The RBC count was decreased in sodium selenite treated fishthroughout the study showing a maximum percent decrease of91.40 at the end of 7th day (Table 1). WBC count was gradually in-creased from day 7 to 35 (p < 0.05) (Table 1). The MCV value wasincreased (except 7th and 21st day) significantly (p < 0.05) in so-dium selenite treated fish throughout the study period (Table 1).A biphasic response was observed in the value of MCH and MCHC(Table 1).

3.2. Biochemical parameters

Plasma glucose level was found to be increased significantly(p < 0.05) in sodium selenite treated fish throughout the study per-iod when compared to the control groups (Fig. 1). The increase inplasma glucose level was found to be time dependent showing amaximum percent increase of 90.53 at the end of 35th day. Plasmaprotein level in sodium selenite treated fish was found to be in-creased (p < 0.05) throughout the experimental period showing adirect relationship with the exposure period (Fig. 2).

3.3. Enzymological parameters

Liver GOT, GPT and LDH activities were increased throughoutthe study period (Figs. 3–5). The values obtained in liver GOT,GPT (except 7 day) and LDH activities were statistically significant(p < 0.05) in relation to the respective controls.

4. Discussion

Among the various techniques to assess the toxicity of environ-mental contaminants on living organisms, static bioassay has con-siderable attraction in ecotoxicological studies. In this study, the96 h LC 50 value of Na2SeO3 to the fish L. rohita was found to be23.89 mg L�1. In previous reports, the 96 h LC 50 value of Na2SeO3

was found to be 85.8 mg L�1 in Morone saxatilis [42], 1–35 mg L�1

in Danio rerio [43], 39.0 mg L�1 in Oncorhynchus kisutch [44],11.7 mg L�1 Juvenile walleye [45] and 70.0 mg L�1 in Pagrus major[5]. The differences in LC 50 value of Na2SeO3 to different fish spe-cies depends on many factors such as the life stage of the organism,water quality, sex, species, dose and the exposure period [10].Furthermore, the toxicity of Se also varies among the teleost fishspecies [46] and its chemical form. It has been reported that someof the seleno-compounds are found to be toxic at low levelwhereas the other compounds are relatively less toxic [8]. Forexample, Se [IV] is highly toxic than other forms which indicatethat different forms of Se have different chemical, biological andtoxicological properties [18]. Selenium can be present in four dif-ferent oxidation states (i.e., +6, +4, 0 and �2). Higher oxidationstates are mostly found in inorganic forms (i.e., selenate, +6;

Table 1Changes in the hematological values (Hb, HCT, RBC, WBC, MCV, MCH, and MCHC) of Labeo rohita exposed to sublethal concentration of sodium selenite for 35 days.

Hematological parameters Exposure period (d) Control Na2SeO3 exposed Percent change

Hb (g/dl) 7 3.598 ± 0.085 2.462 ± 0.224 (�31.55)*

14 2.292 ± 0.138 2.632 ± 0.522 (+14.19)21 3.908 ± 0.071 3.804 ± 0.154 (�2.66)*

28 2.155 ± 0.022 1.986 ± 0.015 (�7.82)*

35 3.873 ± 0.044 2.361 ± 0.043 (�39.02)*

Hct (%) 7 9.900 ± 0.189 5.220 ± 0.073 (�47.27)*

14 15.12 ± 0.037 6.360 ± 1.684 (�57.93)*

21 10.16 ± 0.024 9.140 ± 0.935 (�10.03)*

28 5.140 ± 0.024 5.060 ± 0.024 (�1.55)*

35 10.12 ± 0.037 5.140 ± 0.040 (�49.20)*

RBC (million/cu.mm) 7 2.140 ± 0.060 0.184 ± 0.116 (�91.40)*

14 2.480 ± 0.037 0.352 ± 0.096 (�85.80)*

21 2.840 ± 0.237 0.518 ± 0.128 (�81.76)*

28 2.440 ± 0.050 0.678 ± 0.242 (�72.21)*

35 2.320 ± 0.091 0.860 ± 1.415 (�62.93)

WBC (1000/cu.mm) 7 28.20 ± 8.511 51.30 ± 1.985 (+81.91)*

14 32.20 ± 0.583 62.04 ± 3.619 (+92.67)*

21 29.80 ± 1.529 64.80 ± 3.023 (+117.44)*

28 31.20 ± 0.583 78.54 ± 3.597 (+151.73)*

35 30.40 ± 0.600 81.44 ± 2.870 (+167.89)*

MCV (cubic micra) 7 464.57 ± 19.148 288.51 ± 19.212 (�37.89)*

14 206.62 ± 3.332 252.39 ± 48.267 (+22.14)*

21 367.22 ± 28.339 175.49 ± 16.055 (�52.21)*

28 186.57 ± 8.756 751.11 ± 29.758 (�302.52)*

35 438.41 ± 19.503 612.40 ± 47.545 (�39.68)*

MCH (picograms) 7 16.882 ± 0.689 19.899 ± 1.316 (+17.87)*

14 89.275 ± 2.772 61.377 ± 2.478 (�31.24)*

21 14.143 ± 1.164 75.558 ± 1.488 (+434.24)*

28 79.478 ± 3.936 32.028 ± 1.507 (�59.70)*

35 16.834 ± 0.876 46.019 ± 3.180 (+173.36)*

MCHC (g/dl) 7 36.401 ± 1.141 47.076 ± 3.654 (+29.29)*

14 42.504 ± 0.703 41.150 ± 4.472 (�3.18)*

21 38.462 ± 0.624 43.732 ± 5.340 (+13.70)*

28 42.044 ± 0.674 39.264 ± 0.467 (�6.61)*

35 38.270 ± 0.343 45.948 ± 0.779 (+20.06)*

Data represent mean ± SD (n = 5).* Significant, p < 0.05, (based on t test).

Fig. 1. Changes in the plasma glucose level of Labeo rohita exposed to sublethalconcentration of sodium selenite for 35 days. Data represent mean ± SD (n = 5),⁄Significant, p < 0.05, (based on t test).

Fig. 2. Changes in the plasma protein level of Labeo rohita exposed to sublethalconcentration of sodium selenite for 35 days. Data represent mean ± SD (n = 5),⁄Significant, p < 0.05, (based on t test).

70 M. Ramesh et al. / Chemico-Biological Interactions 207 (2014) 67–73

selenite +4). Elemental selenium with zero oxidation state (Se0) isusually considered to be unreactive. Selenium in organic seleniumcompounds (e.g., selenomethionine, selenocysteine) is predomi-nantly in �2 oxidation state [8].

The toxicity of selenium is ascribed to its similar chemical prop-erties of sulfur and its ability to substitute for that element duringthe assembly of proteins [18]. The excess amount of Se can be

substituted for sulfur resulting formation of a triselenium or a sele-notrisulfide linkage which may stop the formation of disulfidechemical bonds [11]. The prevention of formation of disulfidechemical bonds may leads to dysfunctional enzymes and proteinmolecules resulting impairment of normal cellular biochemistry[47,48]. In fish, waterborne selenium is taken up by the gill and

Fig. 3. Changes in the GOT activity of Labeo rohita exposed to sublethal concen-tration of sodium selenite for 35 days. Data represent mean ± SD (n = 5), ⁄Signifi-cant, p < 0.05, (based on t test).

Fig. 4. Changes in the GPT activity of Labeo rohita exposed to sublethal concen-tration of sodium selenite for 35 days. Data represent mean ± SD (n = 5), ⁄Signifi-cant, p < 0.05, (based on t test).

Fig. 5. Changes in the LDH activity of Labeo rohita exposed to sublethal concen-tration of sodium selenite for 35 days. Data represent mean ± SD (n = 5), ⁄Signifi-cant, p < 0.05, (based on t test).

M. Ramesh et al. / Chemico-Biological Interactions 207 (2014) 67–73 71

subsequently distributed to all the major tissues before reachingthe liver (receives blood via hepatic portal system) [49] and thecellular transport of selenite is mediated by multiple types of anio-nic transporters [8]. Recently, selenium-induced oxidative damagehas been suggested as another possible mechanism of toxicity [50].The metabolism of selenium leads to the generation of redox-active metabolites, which may result in generation of intracellularreactive oxygen species (ROS) [8,51]. Induction of enzymatic anti-oxidants such as superoxide dismutase (SOD), glutathione peroxi-dase (GPx) and hepatic glutathione (GSH) has been reported introut exposed to waterborne sodium selenite [49,52]. In thepresent study, selenium induced oxidative stress may be another

possible reason for the observed mortality of fish during acutestudy. Spallholz et al. [53] suggested three major mechanisms forSe toxicity: (1) substitution of Se for sulfur during the assemblyof proteins, (2) inhibition of Se methylation metabolism resultingin hydrogen selenide accumulation, and (3) membrane and proteindamage from a Se generated reactive oxygen species (ROS). Thepresent results indicate that Se is moderately toxic to L. rohitaand further studies are needed on other Indian major carps suchas Cirrhinus mrigala and C. catla to compare the tolerance of Indianmajor carps to selenium.

The accumulation of selenium at high concentration may affectthe physiology of aquatic organisms [10,49]. Clinical hematologicalparameters have been widely used as a potent bioindicators inaquatic toxicology [54]. In the present study, when L. rohita wereexposed to sublethal concentration of sodium selenite Hb, Hctand RBC levels were decreased. The significant decreases in hema-tological parameters usually indicate the anemic condition of fishwhich might have resulted from the hemolysis caused by the tox-icant. In the present study sodium selenite might have damagedthe gill leading to anemic condition of fish during the study period.A similar observation was also noticed in rainbow trout, Oncorhyn-chus mykiss exposed to sodium selenite [55]. The authors pointedout that the decrease in these parameters indicates the anemiccondition of the fish which results in inhibition of erythropoiesisin the hemopoietic organism. Release of oxygen radical broughtabout by sodium selenite may be another possible reason for theobserved decrease in Hb content. Generally, the Hct value dependson the oxygen carrying capacity of blood [56]. In the present studythe observed decrease in Hct value may be due to the less oxygencontent in blood of fish. Moreover, lower Hct values also indicateshrinkage of cell due to toxicant stress on erythropoietic tissue[57]. Failure of erythrocyte production, internal hemorrhages orimpaired osmoregulation during stress condition may leads to areduction in RBC count [24,58,59]. In the present study the ob-served reduction in RBCs count might have resulted from inhibi-tion of erythropoiesis due to toxic action of sodium selenite.Immunological activities and defense mechanisms are usuallymaintained by WBC cells [60]. In the present study, the observedincrease in WBC count indicates a generalized immune responseand a protective response to the toxicant.

In the present investigation the decrease in MCV and MCH valuemight have resulted from a compensation for impaired oxygen up-take due to gill damage caused by sodium selenite. Release ofimmature RBCs in circulation may be another possible reason forthe decrease in MCV value [24,61]. However, the observed increasein MCV and MCH value during sublethal treatment indicates themacrocytic anemia or increased RBC volume [62,63]. Moreover,high concentration of smaller immature erythrocytes in the circu-lation due to hyperplasia in the erythropoietic (erythrocyte form-ing) sites also leads to higher values of MCV [64]. The observedlow value of MCHC indicates a decrease in Hb synthesis. Se canbind to hemoglobin and prevent the carrying capacity of oxygenwhich may leads to metabolic stress and death of fish [65].Whereas the observed increase of MCHC value during sublethaltreatment may be due to congenital sphaerocytosis as suggestedby Sobecka [66]. In this study, we conclude that the alterationsof these hematological parameters may provide the general healthcondition of the fish under sodium selenite intoxication in fish.

The aquatic pollutants may influence the carbohydrate metabo-lism resulting alterations in the level of glucose, glycogen, lacticacid etc., [26]. Among these parameters, plasma glucose has beenwidely used as a sensitive indicator of environmental stress in fish[67]. In our study, plasma glucose level of fish L. rohita exposed tosodium selenite was gradually increased as the exposure periodextended. The elevation of blood glucose level might have resultedfrom high utilization of glucose to meet the metabolic demands

72 M. Ramesh et al. / Chemico-Biological Interactions 207 (2014) 67–73

caused by sodium selenite. Moreover, sodium selenite might haveaffected the glycogenesis and glycolytic pathway. Elevation of plas-ma glucose level is a common response of fish under stress condi-tion and help to provide energy substrates to various organs/tissueto cope with the increased energy demand [68]. Likewise, proteinserves as an immediate source of energy during stress conditionin many organisms. The observed reduction in plasma protein levelmay be due to impaired protein synthesis or their possible utiliza-tion for metabolic demands. Accumulation of toxicants in organssuch as liver and kidney may leads to impaired protein synthesis[65,32].

Fish react to xenobiotics by changing and adapting their meta-bolic functions and their enzymatic systems appear to be very sim-ilar as that of the mammalian system [69,70]. Like mammals, fishexhibit a characteristic response to many stressors and this re-sponse may be measured through a variety of enzyme activitiesin blood, liver, and muscle [71]. Enzymes such as GOT and GPT usu-ally present within cell membranes, cytoplasm and mitochondriaand play a very important role in protein and carbohydrate metab-olism [67,70]. In the present investigation, the significant increasein GOT and GPT activity in liver might have resulted from the dam-age of the liver due to sodium selenite accumulation and toxicity. Ithas been reported that liver is the major site of selenium accumu-lation in fish [72], and structural degeneration of liver tissues havebeen observed in fish following selenium exposure [73]. Toxicantsinduced vascular degeneration and liver cell necrosis may leads tosignificant increase in GOT and GPT activities in liver and muscle[70]. A change in protein and carbohydrate metabolism duringstress condition may also leads to significant changes in GOT andGPT activity [30,74]. Likewise, LDH is also involved in energy pro-duction through the anaerobic metabolism and play a role duringenergy demand in fish exposed to chemical stress [75,76]. The en-zyme LDH is present in all tissues and mostly involved in carbohy-drate metabolism [73,77]. In the present study, the significantincrease in LDH activity may be due to metabolic changes in livercaused by sodium selenite. The activity of LDH has been used fordemonstrating tissue damage and also an indicative criterion ofexposure due to chemical stress in fish [78,79].

5. Conclusion

The present study concludes that exposure of fish L. rohita tosublethal concentration of sodium selenite alters the hematologi-cal, biochemical and enzymological parameters. The alterationsof these parameters can be used as suitable biomarkers or offeras a sensitive tool to determine the toxicity of chemicals at suble-thal level in aquatic environment. The findings of the present studyalso provide a better understanding of the toxicological endpoint ofaquatic pollutants and to ascertain a safer level of these chemicalsin the aquatic environment in order to protect aquatic organisms.

Conflict of interest statement

None declared.

References

[1] S.S. Soares, H. Martins, C. Gutiérrez-Merino, M. Aureliano, Vanadium andcadmium in vivo effects in teleost cardiac muscle: metal accumulation andoxidative stress markers, Comp. Biochem. Physiol. 147C (2) (2008) 168–178.

[2] P.L.R.M. Palaniappan, T. Nishanth, V.B. Renju, Bioconcentration of zinc and itseffect on the biochemical constituents of the gill tissues of Labeo rohita: an FT-IR study, Infrared Phys. Technol. 53 (2) (2010) 103–111.

[3] R.K. Poopal, M. Ramesh, K.P.B. Dinesh, Short-term mercury exposure on Na+/K+-ATPase activity and ionoregulation in gill and brain of an Indian major carp,Cirrhinus mrigala, J. Trace Elem. Med. Biol. 27 (2013) 70–75.

[4] D.G. Barceloux, Selenium, J. Toxicol. Clin. Toxicol. 37 (1999) 145–172.

[5] K. Takayanagi, Acute toxicity of waterborne Se (IV), Se (VI), Sb (III), and Sb (V)on red seabream (Pargus major), Bull. Environ. Contam. Toxicol. 66 (6) (2001)808–813.

[6] S. Mechora, V. Stibilj, M. Germ, The uptake and distribution of selenium inthree aquatic plants grown in Se (IV) solution, Aquat. Toxicol. 128–129 (2013)53–59.

[7] D.D. Mruk, B. Silvestrini, M. Mengyun, Y.C. Cheng, Antioxidant superoxidedismutase-a review: its function, regulation in the testis, and role in malefertility, Contraception 65 (2002) 305–311.

[8] S. Misra, Cellular transport, metabolism and toxicity of selenium in rainbowtrout (Oncorhynchus mykiss), A Thesis Submitted to the College of GraduateStudies and Research In Partial Fulfillment of the Requirements For the Degreeof Doctor of Philosophy In the Department of Biology University ofSaskatchewan Saskatoon, 2011.

[9] M.M. Patterson, G.B. Paige, K.J. Reddy, Selenium in surface and irrigation waterin the Kendrick irrigation district, Wyoming, Environ. Monit. Assess. 171(2010) 267–280.

[10] T. Pieterek, M. Pietrock, Comparative selenium toxicity to laboratory-rearedand field-collected Hyalella azteca (Amphipoda, Hyalellidae), Water Air SoilPollut. 223 (2012) 4245–4252.

[11] A.D. Lemly, Symptoms and implications of selenium toxicity in fish: theBelews Lake case example, Aquat. Toxicol. 57 (2002) 39–49.

[12] A.D. Lemly, A teratogenic deformity index for evaluating impacts of seleniumon fish populations, Ecotoxicol. Environ. Saf. 37 (1997) 259–266.

[13] A.D. Lemly, R.J.F. Smith, Effects of chronic exposure to acidified water onchemoreception of feeding stimuli by fathead minnows, Pimephales promelas:mechanisms and ecological implications, Environ. Toxicol. Chem. 6 (1987)225–238.

[14] E.M.B. Sorensen, The effects of selenium on freshwater teleosts, in: E. Hodgson(Ed.), Reviews in Environmental Toxicology 2, Elsevier, New York, NY, USA,1986, pp. 59–116.

[15] T.W. Lohner, R.J. Reash, V.E. Willet, L.A. Rose, Assessment of tolerant sunfishpopulations (Lepomis sp.) inhabiting selenium-laden coal ash effluents-1.Hematological and population level assessment, Ecotoxicol. Environ. Saf. 50(3) (2001) 203–216.

[16] S.N. Luoma, T.S. Presser, Emerging opportunities in management of seleniumcontamination, Environ. Sci. Technol. 43 (2009) 8483–8487.

[17] S. Wiseman, J.K. Thomas, L. McPhee, O. Hursky, J.C. Raine, M. Pietrock, J.P.Giesya, M. Hecker, D.M. Janz, Attenuation of the cortisol response to stress infemale rainbow trout chronically exposed to dietary selenomethionine, Aquat.Toxicol. 105 (3–4) (2011) 643–651.

[18] K.J. Maier, A.W. Knight, Ecotoxicology of selenium in freshwater systems, Rev.Environ. Contam. Toxicol. 134 (1994) 31–48.

[19] J.V. Rao, Biochemical alterations in euryhaline fish, Oreochromis mossambicusexposed to sub-lethal concentrations of an organophosphorus insecticide,monocrotophos, Chemosphere 65 (10) (2006) 1814–1820.

[20] C.T. Pimpao, A.R. Zampronio, H.C.S. de Assis, Effects of deltamethrin onhematological parameters and enzymatic activity in Ancistrus multispinis(Pisces, Teleostei), Pestic. Biochem. Physiol. 88 (2007) 122–127.

[21] G. Nussey, J.H.J. Van Vuren, H.H.D. Preez, Effect of copper on the haematologyand osmoregulation of the Mozambique tilapia, Oreochromis mossambicus(Cichlidae), Comp. Biochem. Physiol. 11 (3) (1995) 369–380.

[22] I.A. Ololade, O. Ogini, Behavioural and hematological effects of zinc on Africancatfish, Clarias gariepinus, Int. J. Fish. Aquacult. 1 (2) (2009) 22–27.

[23] E.H. Coles, Veterinary Clinical Pathology, fourth ed., W.B. Saunders Co.,Philadelphia, 1986 (pp. 267–278).

[24] C. Kavitha, A. Malarvizhi, S. Senthil Kumaran, M. Ramesh, Toxicological effectsof arsenate exposure on hematological, biochemical and liver transaminasesactivity in an Indian major carp, Catla catla, Food Chem. Toxicol. 48 (2010)2848–2854.

[25] B. Asztalos, J. Nemcsok, I. Benedeczky, R. Gabriel, A. Szabo, O.J. Refaie, Theeffects of pesticides on some biochemical parameters of common carp(Cyprinus carpio), Arch. Environ. Contam. Toxicol. 19 (1990) 275–282.

[26] V. Sathya, M. Ramesh, R.K. Poopal, B. Dinesh, Acute and sublethal effects in anIndian major carp Cirrhinus mrigala exposed to silver nitrate: gill Na+/K+-ATPase, plasma electrolytes and biochemical alterations, Fish ShellfishImmunol. 32 (5) (2012) 862–868.

[27] S.L.N. Reddy, N.B.R.K. Venugopal, In vivo effects of cadmium chloride oncertain aspects of protein metabolism in tissues of a freshwater field crab,Barytelphusa guerini, Bull. Environ. Contam. Toxicol. 46 (1991) 583–590.

[28] A. Cohen, D. Nugegoda, L.M. Gagnon, Metabolic responses of fish followingexposure to two different oil spill remediation techniques, Ecotoxicol. Environ.Saf. 48 (2001) 306–310.

[29] S. Lavanya, M. Ramesh, C. Kavitha, A. Malarvizhi, Hematological, biochemicaland ionoregulatory responses of Indian major carp Catla catla during chronicsub-lethal exposure to inorganic arsenic, Chemosphere 82 (2011) 977–985.

[30] P.C. Das, S. Ayyappan, J.K. Jena, B.K. Das, Acute toxicity of ammonia and its sub-lethal effects on selected hematological and enzymatic parameter of mrigal,Cirrhinus mrigala. (Hamilton), Aquacult. Res. 35 (2004) 134–143.

[31] T. Watanabe, V. Kiron, S. Satoh, Trace minerals in fish nutrition, Aquaculture151 (1–4) (1997) 185–207.

[32] A.C. Elia, M. Prearo, N. Pacini, A.J.M. Dorr, M.C. Abete, Effects of selenium dietson growth, accumulation and antioxidant response in juvenile carp,Ecotoxicol. Environ. Saf. 74 (2011) 166–173.

[33] APHA, 20th ed., in: A.D. Eaton, L.S. Clesceri, A.E. Greenberg, M.A.H. Franson(Eds.), Standard Methods for the Examination of Water and Wastewater,

M. Ramesh et al. / Chemico-Biological Interactions 207 (2014) 67–73 73

American Public Health Association, American Water Works Association,Water Environment Federation, Washington, DC.

[34] D.J. Finney, third ed., Statistical Methods in Biological Assay, Griffin Press,London, UK, 1978, p. 508.

[35] D.L. Drabkin, Spectrometric studies, XIV-the crystallographic and opticalproperties of the hemoglobin of man in comparison with those of otherspecies, J. Biol. Chem. 164 (1946) 703–723.

[36] D.A. Nelson, M.W. Morris, Basic methodology: hematology and coagulation,Chap. 27, part IV, 17th ed., in: D.A. Nelson, J.B. Henry (Eds.), Clinical Diagnosis,Management by Laboratory Methods, W.B. Saunders Company, Philadelphia,USA, 1989, pp. 578–625.

[37] V. Rusia, S.K. Sood, Routine hematological tests, in: L. Kanai, I. Mukerjee (Eds.),Medical Laboratory Technology, Vol. I, Tata McGraw Hill, New Delhi, 1992, pp.252–258.

[38] G.R. Cooper, V. McDaniel, The determination of the glucose by theOrthotoluidine method, in: Standard Methods of Clinical Chemistry, Vol. 6,Academic Press, New York, 1970, pp. 159–170.

[39] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.I. Randall, Protein measurement withFolin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.

[40] S. Reitmen, S. Frankel, A colorimetric method for the determination of serumglutamic oxalacetic and glutamic pyruvic transaminases, Am. J. Clin. Pathol. 28(1) (1957) 56–63.

[41] N.W. Tietz, Fundamentals of Clinical Chemistry, Saunders, W.B., Co.,Philadelphia, PA, 1976 (p. 652).

[42] USEPA, Ambient Water Quality Criteria for Selenium, EPA 440587006,Washington, DC, 1987.

[43] A.J. Niimi, Q.N. Laham, Relative toxicity of organic and inorganic compounds ofselenium to newly hatched zebrafish (Brachydanio rerio), Can. J. Zool. 54 (1976)501–509.

[44] M.A. Hamilton, K.J. Buhl, Acute toxicity of boron, molybdenum and selenium tofry of chinook salmon and coho salmon, Arch. Environ. Contam. Toxicol. 19(1990) 366–373.

[45] R.J. Mauk, M.L. Brown, Acute toxicity of sodium selenite to Juvenile walleye,Bull. Environ. Contam. Toxicol. 63 (1999) 188–194.

[46] L.L. Miller, F. Wang, V.P. Palace, A. Hontela, Effects of acute and subchronicexposures to waterborne selenite on the physiological stress response andoxidative stress indicators in juvenile rainbow trout, Aquat. Toxicol. 83 (2007)263–271.

[47] A.T. Diplock, W.G. Hoekstra, Metabolic aspects of selenium action and toxicity,CRC Crit. Rev. Toxicol. 5 (1976) 271–329.

[48] J.E. Spallholz, D.J. Hoffman, Selenium toxicity: cause and effects in aquaticbirds, Aquat. Toxicol. 57 (1–2) (2002) 27–37.

[49] P.V. Hodson, J.W. Hilton, The nutritional requirements and toxicity to fish ofdietary and waterborne selenium, Ecol. Bull. (1983) 335–340.

[50] L.L. Miller, A. Hontela, Species-specific sensitivity to selenium-inducedimpairment of cortisol secretion in adrenocortical cells of rainbow trout(Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis), Toxicol. Appl.Pharmacol. 253 (2011) 137–144.

[51] V.P. Palace, J.E. Spallholz, J. Holm, K. Wautier, R.E. Evans, C.L. Baron,Metabolism of selenomethionine by rainbow trout (Oncorhynchus mykiss)embryos can generate oxidative stress, Ecotoxicol. Environ. Saf. 58 (2004) 17–21.

[52] I. Orun, B. Ates, Z. Selamoglu, H. Yazlak, E. Ozturk, I. Yilmaz, Effects of varioussodium selenite concentrations on some biochemical and hematologicalparameters of rainbow trout (Oncorhynchus mykiss), Fresenius Environ. Bull.14 (2005) 18–22.

[53] J.E. Spallholz, V.P. Palace, T.W. Reid, Methioninase and selenomethionine butnot Se-methylselenocysteine generates methylselenol and superoxide in anin vitro chemiluminescent assay: implications for the nutritional carcinostaticactivity of selenoamino acids, Biochem. Pharmacol. 67 (2004) 547–554.

[54] N.N. Singh, A.K. Srivastava, Hematological parameters as bioindicators ofinsecticide exposure in teleosts, Ecotoxicology 19 (2010) 838–854.

[55] B. Ates, I. Orun, Z.S. Talas, G. Durmaz, I. Yilmaz, Effects of sodium selenite onsome biochemical and hematological parameters of rainbow trout(Oncorhynchus mykiss Walbaum 1792) exposed to Pb2+ and Cu2+, FishPhysiol. Biochem. 34 (2008) 53–59.

[56] A. Larsson, C. Haux, M. Sjobeck, Fish physiology and metal pollution: resultsand experiences from laboratory and field studies, Ecotoxicol. Environ. Saf. 9(3) (1985) 250–281.

[57] M. Saravanan, K. Prabhu Kumar, M. Ramesh, Hematological and biochemicalresponses of freshwater teleost fish Cyprinus carpio (Actinopterygii:

Cypriniformes) during acute and chronic sublethal exposure to lindane,Pestic. Biochem. Physiol. 100 (2011) 206–211.

[58] P.K. Joshi, M. Bose, D. Harish, Changes in certain hematological parameters in asiluroid catfish, Clarias batrachus (L.) exposed to cadmium chloride, Pollut. Res.21 (2) (2002) 119–131.

[59] O.B. Adedeji, O.A. Adedeji, O.K. Adeyemo, S.A. Agbeda, Acute effects of diazinonon blood parameters in the African catfish Clarias gariepinus, Internet J.Haematol. 5 (2) (2009) 708–715.

[60] B.D. Abhijith, M. Ramesh, R.K. Poopal, Sublethal toxicological evaluation ofmethyl parathion on some hematological and biochemical parameters in anIndian major carp Catla catla, Comp. Clin. Pathol. 21 (2012) 55–61.

[61] S.S. Vutukuru, Acute effects of hexavalent chromium on survival, oxygenconsumption, hematological parameters and some biochemical profiles of theIndian major carp, Labeo rohita, Int. J. Environ. Res. Public Health 2 (3) (2005)456–462.

[62] M. Saravanan, S. Karthika, A. Malarvizhi, M. Ramesh, Ecotoxicological impactsof clofibric acid and diclofenac in common carp (Cyprinus carpio) fingerlings:Hematological, biochemical, ionoregulatory and enzymological responses, J.Hazard. Mater. 195 (2011) 188–194.

[63] E. Brucka-Jastrzebska, M. Protasowicki, Effects of cadmium and nickelexposure on hematological parameters of common carp, Cyprinus carpio L,Acta Ichthyol. Piscat. 35 (1) (2005) 29–38.

[64] M.D. Ferrando, E. Andreu-Moliner, Changes in selected biochemicalparameters in the brain of the fish Anguilla anguilla (L), exposed to lindane,Bull. Environ. Contam. Toxicol. 47 (1991) 459–464.

[65] A.D. Lemly, Teratogenic effects of selenium in natural populations offreshwater fish, Ecotoxicol. Environ. Saf. 26 (1993) 181–204.

[66] E. Sobecka, Changes in the iron leveling the organs and tissues of wels catfish,Silurus glanis L. caused by nickel, Acta Ichthyol. Piscat. 31 (2) (2001) 127–143.

[67] J. Nemcsok, L. Boross, Comparative studies on the sensitivity of different fishspecies to metal pollution, Acta Biol. Acad. Sci. Hung. 33 (1) (1982) 23–27.

[68] B.A. Barton, Stress in fishes a diversity of responses with particular reference tochanges in circulating corticosteroids, Integr. Comp. Biol. 42 (2002) 517–525.

[69] D.B. Huggett, J.C. Cook, J.F. Ericson, R.T. Williams, A theoretical model forutilizing mammalian pharmacology and safety data to prioritize potentialimpacts of human pharmaceuticals to fish, Hum. Ecol. Risk Assess. 9 (2003)1789–1799.

[70] A. Malarvizhi, C. Kavitha, M. Saravanan, M. Ramesh, Carbamazepine (CBZ)induced enzymatic stress in gill, liver and muscle of a common carp, Cyprinuscarpio, J. King Saud Univ. Sci. 24 (2012) 179–186.

[71] M.B. da Fonseca, L. Glusczak, B.S. Morales, C.C. de Menezes, A. Pretto, M.A.Tierno, R. Zanella, F.F. Goncalves, V.L. Loro, The 2,4-D herbicide effects onacetylcholinesterase activity and metabolic parameters of piava freshwaterfish (Leporinus obtusidens), Ecotoxicol. Environ. Saf. 69 (2008) 416–420.

[72] P.V. Hodson, D.J. Spry, B.R. Blunt, Effects on rainbow trout (Salmo gairdneri) of achronic exposure to waterborne selenium, Can. J. Fish. Aquat. Sci. 37 (1980)233–240.

[73] E.M.B. Sorensen, M. Cumbie, T.L. Bauer, H.S. Bell, C.W. Harlan,Histopathological, hematological, condition-factor and organ weight changesassociated with selenium accumulation in ‘‘fish from Belews Lake, NorthCarolina, Arch. Environ. Contam. Toxicol. 13 (1984) 153–162.

[74] P. Subha Rao, K. Rajender, Kinetics of aspartate amino transferase inhibition bycertain insecticides in haemolymph of Periplaneta americana, J. Environ. Biol.11 (4) (1990) 383–387.

[75] E. Sancho, C. Fernandez-Vega, M.J. Villarroel, E. Andreu-Moliner, M.D.Ferrando, Physiological effects of tricyclazole on zebrafish (Danio rerio) andpost-exposure recovery, Comp. Biochem. Physiol. 150 (2009) 25–32.

[76] J.R. Almeida, C. Oliveira, C. Gravato, L. Guilhermino, Linking behaviouralalterations with biomarkers responses in the European seabass Dicentrarchuslabrax L. exposed to the organophosphate pesticide fenitrothion, Ecotoxicology19 (8) (2010) 1369–1381.

[77] M.M. Gagnon, Metabolic enzymes as biochemical markers of effect followingexposure of fish to sodium pentachlorophenate (Na-PCP), Bull. Environ.Contam. Toxicol. 69 (2002) 570–575.

[78] T.C. Diamantino, E. Almeida, A.M.V.M. Soares, L. Guilhermino, Lactatedehydrogenase activity as an effect criterion in toxicity tests with Daphniamagna straus, Chemosphere 45 (2001) 553–560.

[79] J. Rendon-von Osten, A. Ortız-Arana, L. Guilhermino, A.M. Soares, In vivoevaluation of three biomarkers in the mosquito fish, Gambusia yucatanaexposed to pesticides, Chemosphere 58 (5) (2005) 627–636.