96 h LC50, behavioural alterations and histopathological effects due to wastewater toxicity in a freshwater fish Channa punctatus

RESEARCH ARTICLE

96 h LC50, behavioural alterations and histopathologicaleffects due to wastewater toxicity in a freshwater fish Channapunctatus

Rajbir Kaur & Anish Dua

Received: 10 April 2014 /Accepted: 9 October 2014 /Published online: 24 October 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The aim of the study was to evaluate the toxicimpact of wastewater from sites 1 and 2 of Tung Dhab drainin the state of Punjab, India, on fish behaviour, morphologyand gill histopathological biomarkers in comparison to controlgroup. Static non-renewal tests were conducted for 96 h todetermine LC50 of the wastewater for both sites using fiveconcentrations (6.25–100 %). Fish were regularly noticed forany deviation in behaviour and external morphology. Physico-chemical analysis of wastewater was done using standardmethods recommended by APHA/AWWA/WEF (2005).Chronic toxicity tests were conducted for 15 and 30 days withsublethal concentrations of wastewater (50–90 % of LC50)and gill histopathology was assessed. Wastewater near a papermill was more toxic as observed from LC50 values of72.45 %. There was evident deterioration of water quality asthe recorded values of some parameters were higher than thestandard discharge limits. The test fish exhibited increased airgulping and surfacing, erratic movements initially and de-creased opercular movements as the exposure period in-creased. Morphological observations include increased bodycolouration, mucus secretion, scale loss and haemorrhages onthe skin and lower lip. Alterations in the gill histology such ascomplete lamellar fusion, epithelial lifting and intraepithelialoedema, haemorrhages, lamellar necrosis and aneurysm werenoted in the test fish. Results demonstrate that the fish exposedto wastewater from both sites showed significantly greaterchange in gill organ index (IG) as compared to control fishfor 15 and 30 days.

Keywords Municipal wastewater . Paper mill effluent .

Physicochemical parameters .Channa punctatus . Acutetoxicity . Behaviour . Morphology . Gill histopathology

Introduction

In an annual report, the CPCB (2010) stated that depletion ofavailable freshwater resources, falling ground water levels anddeteriorating water quality are all posing a variety of chal-lenges in managing India’s water resources. The impropertreatment and disposal of inadequately treated wastewaterfrom industries have become the main cause of water pollu-tion. According to Khapekar et al. (2008), about 70 % of theavailable water is polluted in India, out of which, 8–16 % ispolluted by industrial pollution and 84–92 % by sewagepollution. The pollution of surface and ground water due tothe ever increasing industrial, agricultural and other humanactivities often places severe physiological stress on manyaquatic ecosystems. These pollutants can cause damage toaquatic habitats (Chaplen et al. 2007). As per AniladeviKunjamma et al. (2008), chemical additions have introducedor increased environmental stress on aquatic organisms and onfishes in particular. Fish are on top of the food chain, andthrough respiration or by ingestion of smaller species, theyreadily bioaccumulate and biomagnify a variety of contami-nants (Al-Sabti and Metcalfe 1995; Koca et al. 2008).

Acute toxicity tests give firsthand information on the ef-fects of such discharges on organisms and the ecosystem as awhole. These tests are valuable in creating awareness regard-ing potential harmful effects of such industrial discharges tothe environment (Adedeji et al. 2008; Onyedineke et al. 2010).Yadav et al. (2007) concluded that the changes of physical,chemical and biological parameters of water alter the behav-iour of fishes besides causing mortality. Different studiesreported that fish exposed to wide range of pollutants

Responsible editor: Philippe Garrigues

R. KaurAquatic Biology Laboratory, Department of Zoology, Guru NanakDev University, Amritsar 143005, Punjab, India

A. Dua (*)C-50, Guru Nanak Dev University Campus, Amritsar, Indiae-mail: [email protected]

Environ Sci Pollut Res (2015) 22:5100–5110DOI 10.1007/s11356-014-3710-1

exhibited abnormal behavioural and morphological alter-ations. These were erratic swimming, increased surfacing,decreased rate of opercular movement, reduced agility andinability to maintain normal posture and balance with increas-ing exposure time (Adedeji et al. 2008; Kasherwani et al.2009; Gupta and Dua 2011; Bhat et al. 2012).

Polluted freshwaters induce changes in the structural andphysiological aspects of the inhabiting biota, particularly fish-es. Gills are multifunctional and highly characteristic structur-al feature of fishes that serve a range of vital functions includ-ing osmotic and ionic regulation, acid-base regulation, gasexchange, nitrogen excretion and detoxification in fishes(Evans 1987; Roy and Datta-Munshi 1995; Evans et al.2005; Koca et al. 2005). Fish gills are primary site of toxicaction of many waterborne pollutants and particularly sensi-tive to adverse environmental conditions. This is due to theirdirect and continuous contact with the environment, largesurface area and the small diffusion distance between thewater and blood (Evans 1987; Dua and Johal 1994;Bhagwant and Elahee 2002; Olson 2002). Jiraungkoorskulet al. (2003) concluded that changes in gill epithelia can beconsidered as good indicators of xenobiotics on fish. Therefore,histopathological studies are recommended as powerful anduseful biomarkers for evaluation of fish health. These are potentindicators of biochemical and physiological responses in ani-mals exposed to a variety of environmental stressors (Santoset al. 2011; Dhevakrishnan and Zaman 2012).

Various studies have been conducted on Tung Dhab drainfrom time to time, including DNA damage in peripheral bloodlymphocytes on human population, high frequency ofmicronuclei in buccal mucosa of women residing along thedrain (Gandhi and Kumar 2004; Sambyal et al. 2004) and fishscales as indicators of wastewater toxicity (Kaur and Dua2012). Until date, little work has been done regarding theeffects of drain water upon the health of fish fauna. Uponconsidering this, the study was conducted to determine theacute toxicity along with behavioural, morphological and gillhistopathological studies of effluent collected from TungDhab drain near a paper mill outlet (site 1) and near villageMahal (site 2).

Materials and methods

Sampling sites

The two sites, first (site 1) near a paper mill (31° 67′ 093″ Nand 74° 88′ 411″ E) and second (site 2) near village Mahal ofTung Dhab drain (31° 67′ 612″ N and 74° 74′ 280″ E), werechosen for the present study (Fig. 1). Tung Dhab drain is20 km long, has a catchment area of 208.83 km2, capacity of53m3/min and bed width of 13.72m (at outfall) and 1.22m (atstarting point). The drain is a natural storm water drain

designed to drain excess rain and ground water but is beingused by industries to dump their untreated waste. The drainreceives effluents from the Gumtala drain (carrying paper milland textile processing mill effluents) and the Verka drain(carrying milk plant, iron foundries and woollen dyeing milleffluents) and also receives sewage water of the Amritsar city.The level of pollution in the drain is extremely high, as notreatment plant is yet installed along the drain. At present,sewage (91.98 MLD) of the Amritsar North zone is beingpumped out through temporary disposal works into the TungDhab drain, as mentioned in the report of PWSSB (2006).Along its course, it covers many areas/villages such asFatehgarh Shukarchak, Verka, Othian, Nashera, Gumtala,Mahal, Wadala Bhitewadh, etc., and after that, it joins withHudiara drain near Khiala Khurd and further enters the riverRavi near the international border (PUDA 2010).

Sampling

Samples were collected from both sites separately for settingthe experiments and for physicochemical analysis every2 weeks during the months of February to May 2012. Bothgrab and composite samples were collected in pre-treated andproperly labelled plastic and glass bottles as recommended bythe manual of APHA/AWWA/WEF (2005). Samples wereimmediately preserved in ice packs and brought to the labo-ratory without any delay. Samples were then refrigerated at4 °C tominimize the biodegradation of samples. Grab sampleswere collected in tank of 120 l capacity from both sites forsetting the experiments.

Physico-chemical analysis of wastewater

A portable water analysis kit (WTW Multy 340i/ SET) wasused for measuring four parameters, i.e. pH, temperature(Temp), dissolved oxygen (DO) and electrical conductivity(EC) at the sampling sites itself. Electrodes in the kit werecalibrated prior to every sampling event by following theinstructions supplied with the equipment. Biological oxygendemand (BOD) was calculated using Oxitop measuring sys-tem for 5 days at 20 °C in a thermostat (TS 606-G/2-i). Acidity(AD), alkalinity (AK), total hardness (TH), calcium (Ca),magnesium (Mg), total solids (TS), total dissolved solids(TDS) and total suspended solids (TSS) were calculated usingstandard methods recommended by APHA/AWWA/WEF(2005). Chemical oxygen demand (COD), ammonium as N(NH4

+–N), nitrate as N (NO3–N), nitrogen (N), total phos-phates (∑P), potassium (K), chlorides (Cl−) and heavy metalanalysis of lead (Pb), manganese (Mn), nickel (Ni), chromium(Cr), cadmium (Cd), iron (Fe) and copper (Cu) was done byusing Merck cell test kits and heavy metal testing kits, andtheir concentrations were measured using the UV/VIS spec-t rophotometer (Spec t roquant® Pharo 300) . For

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physicochemical parameters of wastewater, mean±SE valueswere obtained from data using Minitab statistical software.

Student’s t test was applied to check significant differencesbetween waste samples collected from two different sites.

Fig. 1 Map showing Tung Dhab Drain and Hudiara Drain. a Thesampling sites are marked by stars: ( ) in site 1 and ( ) in site 2; originof drains is shown by ; confluence of Tung Dhab drain and Hudiaradrain is marked by . b Map showing main industries and sewer outfalls

along Tung Dhab drain. is indicating sewer outfalls; ( ) metalfoundries, ( ) paper mill, ( ) food, ( ) leather and ( ) chemicalindustries (source adapted from Google earth maps(R), accessed August,2014)

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Test fish

The test fish Channa punctatus (19.01±1.48 g in weight,11.97±0.98 cm in length, Family Channidae; OrderChanniformes) were procured from the local fish market(Hall Bazaar) of Amritsar City and carefully brought to thelaboratory avoiding any injury to fish during transport. Thefish were disinfected with 0.1 %KMnO4 solution for 2–3min.Fish were then transferred for acclimatization to plastic tankscontaining laboratory tap water (Temp 26.2±0.80 °C, pH 7.1±0.04, DO 6.85±0.54 mg/l, EC 474.2±4.38 μS/cm and TDS120±6.20 mg/l). Normal photoperiod was maintained duringacclimatization period and during experimentation. Fish werefed ad libitum on boiled chicken eggs during the acclimationperiod. Every effort was made to provide optimal conditionsfor fish, and there was no fishmortality during this period. Theair-breathing fish (C. punctatus) have several characteristicsrequired in a sentinel species, such as wide geographic distri-bution, great abundance, salinity and temperature toleranceand common occurrence in polluted waters.

Acute and chronic toxicity tests

The range finding tests were conducted by exposing the testfish to a wide range of wastewater concentrations at whichmaximum and minimum number of fish died. Thereafter, twoseparate definitive, static non-renewal, acute toxicity bioas-says were conducted by exposing fish to 100, 50, 25, 12.5 and6.25 % (v/v) concentrations of sample collected from site 1and site 2. The wastewater was thoroughly mixed, and con-centrations of the quantity 80 L (v/v) were prepared using tapwater for dilutions (Temp 25.8±0.7, pH 7.02±0.04, DO 6.78±0.35, AK 194±14.0, TH 134±11.0, TDS 123±7.6 and EC469±8.37). Static non-renewal tests were conducted undernatural photoperiod for 96 h in 100 L colourless plastic tanks.Ten fish were exposed in each tank, and each experiment wasconducted in triplicate (n=10; total 360). A control was si-multaneously run using tap water with similar number of fish.All the toxicity tests were conducted in accordance withstandard methods given in the manual of APHA/AWWA/WEF (2005) and EPA (2002). Feeding was suspended a daybefore exposure experiment; fish were not given any foodduring the tests and dead fish were immediately removed fromthe experimental tanks. The fish mortality was recorded at 24,48, 72 and 96 h for each concentration and subjected to EPAcomputer probit analysis program (version 1.5) based onFinney’s (1971) method.

Behavioural and morphological studies

During days of the experiment, fish were regularlynoticed for any deviation in their external morphologyand behaviour. Fish were examined for different

behavioural observations such as swimming movements,general activity and equilibrium every 24 h until 96 h.Opercular movements were noted per minute and airgulps for a duration of 15 min during the 96-h staticbioassay. Student’s t test was applied to data so as tofind the significance of the difference between themeans of control and test fish.

Gill histopathology studies

For histological studies, fish were divided into six groups (n=10; total 120). One group served as control and the other fiveas exposed groups for site 1, and similar number of groupswere used for site 2. Static chronic toxicity tests were con-ducted for exposure duration of 15 and 30 days with sublethalconcentrations of wastewater (50, 60, 70, 80 and 90 % ofLC50) that is 36.2, 43.4, 50.7, 57.9 and 65.2 % for site 1 and41.6, 49.9, 58.2, 66.5 and 74.8 % for site 2. All the testexposures were carried out in duplicates. Control and treatedfish (both sites) were dissected after giving a stunning blow tothe head on completion of exposure durations of 15 and30 days. Gills were skillfully removed by cutting the upperand lower attachments of the bony arch. The third pair of gillarches was selected to study histology since it has large gillrakers and gill filaments, providing a larger surface area. Gillsof control fish not exposed to the toxicant were also simulta-neously examined. The third gill arches were excised andimmediately fixed in 10 % formalin, dehydrated in acetone,cleared in xylene, embedded in paraffin wax and sectioned at3 μ with the help of rotary microtome. Slides were stainedwith Harris haematoxylin stain and counterstained with eosin,dipped in xylene and mounted in DPX. Three sections wereprepared from each fish gill, and histological alterations wereexamined under light microscope (Olympus C3040-ADU)and were photographed at different magnifications (×10,×40 and ×100) by using a camera (Olympus CX31). Thehistological changes in the treated gills were classifiedaccording to the standardized assessment tool proposedby Bernet et al. (1999). Three histological sections (fiveimages/section) taken randomly per animal were studiedfor pathological changes. These were classified into fivereaction patterns including circulatory disturbances, re-gressive changes, progressive changes, inflammation andtumours. Each alteration was given an importance factorranging from 1 to 3 and score value ranging from 0 to6 according to the pathological importance of lesion anddegree and extent of damage. Gill organ index (Iorg orIG) was calculated using importance factors and scorevalues from the below given formula:

Iorg ¼X

rp

X

alt

aorg rp alt � worg rp alt

� �

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where org is the organ (constant); rp is the reaction pattern, altis the alteration, a is the score value and w is the importancefactor. This index represents the degree of damage to an organ.A high index indicates a high degree of damage of the sameorgan in different individuals. The calculated mean Gill organindex values obtained for all the experimental groups weresubjected to one-way ANOVA using MINITAB (version 14)software to compare significant differences (p≤0.01) betweencontrol-treatment groups from both sites. Gill organ index datawas further subjected to linear regression analysis usingMicrosoft Excel 2007 for Windows.

Results

Analytical results

It is important to assess the physical and chemical parametersof wastewater to relate the behavioural, morphological andhistopathological alterations to the toxicants present in waste-water. The limit, mean±SE along with t values of physical andchemical parameters of wastewater collected from site 1 andsite 2 are listed in Table 1. Significant differences were notedfor parameters such as Temp, Mg, TS, Cl− and BOD at 5 %and pH, TSS at 1 % level of significance for samples collectedfrom the two sites. There was evident deterioration of waterquality as the recorded values of some parameters were higherthan the standard discharge limits stated under EnvironmentProtection Amendment Rules (EPAR 2012) for TSS, NO3–N,BOD, Cr, Mn, Pb and As for both sites and COD and Ni forsite 1.

Acute toxicity

96 h LC50

The calculated 96 h LC50 values along with 95 % confidencelimits were found to be 72.45 % (61.11–89.35) for site 1 and83.20 % (69.11–108.92) for site 2 (obtained from the probitanalysis software). The regression equation of the expectedprobit (Y) and log concentration X is Y=a+bX that is −2.83+4.07X for site 1 and −3.21+4.41X for site 2.

Behavioural studies

Control fish showed normal behaviour, with active feedingand well-coordinated body movements and were very atten-tive towards slight stimulus. Therefore, control fish behaviourwas taken as standard to study altered fish behaviour in treatedfish. Mean±SE values of behavioural response observationsof test fish exposed to wastewater concentrations (6.25–100 %) from both sites are listed in Table 2. Increased air

gulping and surfacing was recorded immediately after expo-sure of fish to wastewater concentrations. The number of airgulps (per 15 min, 24–96 h) increased from 8.30±0.03 in thecontrol group to 21.0±0.58 in the group exposed to 100 % ofwastewater collected from site 1. The number of gulps wasincreased to 19.6±1.20 in group exposed to 100 % concen-tration of wastewater collected from site 2 when compared tothe control group (8.00±0.33). A clear decrease in number ofopercular movements (for 1 min, 24–96 h) was recorded ingroups exposed to wastewater collected from both sites ascompared to control. The recorded values ranged from 46.6±1.45 in control group to 30.3±0.66 in exposed groups andfrom 46.3±0.66 in control group to 31.0±1.00 in exposedgroup for sites 1 and 2, respectively. Erratic and speedymovements were found in exposed (12.5–50 %) fishes incomparison to uniform movements in control and also in thelowest concentration for 96 h. Swimming movements of thecontrol fish remained normal throughout the experiment peri-od. The test fish at 100 % concentration showed exodusmovements and tried to move out of the tank to avoid thetoxicant (from both sites). Fish showed rapid movementsinitially and, later on, spent most of its time at the bottomwith little movement. Equilibrium was normal in control fishand was lost in treated groups (50 and 100 %). Some speci-mens at 50 and 100 % remained in vertical position withmouth pointed towards the surface prior to death. Fish wereviolent at highest concentrations (50 and 100 %), hyperactiveat 25 % and normal at 6.25–12.5 % concentrations whensubjected to wastewater from both sites. Results indicatedapparent changes in the external morphology of the test fishwhen exposed to wastewater concentrations collected fromboth sites. Sinking of eyeballs, copious amounts of mucussecretion and an increased body colouration along with ex-cessive mucus secretion were recorded in exposed fish.Simultaneously, loosening of scales and complete loss ofscales from the head region were also observed. Intensity ofeffect increased with the increased concentration of wastewa-ter. Scale loss wasmore intense in fish subjected to wastewatercollected from site 1. Additionally, haemorrhages on skin andlower lip were recorded and were seen even at the lowestconcentration in specimens subjected to toxicant of site 1.

Chronic toxicity

Gill histopathology

C. punctatus that served as control showed normal, straightand evenly spaced gill lamellae. Each gill consisted of tworows of primary gill filaments that bear a series of alternatelyarranged secondary lamellae (respiratory lamellae) on bothsides (Fig. 2a, a1). The secondary gill lamellae are composed

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of a single layer of epithelial cells supported by pillar cells Thesurface of gill filament and secondary lamellae is covered withsquamous epithelial cells, mucous cells in the interlamellarepithelial lining as well as on the distal tip of the gill filamentand chloride cells were located at different locations on thegills. No significant histopathological lesions were recordedfor control fish, although slight changes like fusion of fewsecondary lamellae and fusion of few secondary lamellae inbetween the primary lamellae were observed (Fig. 2a) andalthough the calculated mean Gill organ index values (1.5±0.16; 1.9±0.27) for site 1 and (1.3±0.15; 1.8±0.24) for site 2remained within the range expected for gills with normalfunction for 15 and 30 days (Fig. 3a, b).

Circulatory disturbances, regressive and progressivechanges in lamellar epithelia and supporting tissue were ob-served in all tested specimens exposed to wastewater. Theintensity of these reaction patterns varied and became moresevere with increase in exposure time and type of toxicant.Fish exposed to toxicant collected from the site 1 and site 2 for15 and 30 days revealed circulatory disturbances such asepithelial lifting and intraepithelial oedema (Fig. 2b, d, e andh–k), haemorrhages and hyperemia at primary lamellae(Fig. 2d, g and j) and aneurysm in secondary lamellae andrupture of pillar cell system (Fig. 2b, c, d, h, i and k). Inaddition, loss of secondary lamellae and necrosis (Fig. 2b, c,e and k), severely damaged and lost secondary lamellae

Table 1 Physico-chemical parameters of paper mill effluent collected from site 1 and municipal wastewater collected from site 2 of Tung Dhab drain

Parameters Site 1 Site 2 t value EPAR

Limit Mean±SE Limit Mean±SE MPL

Temp 27.9–30.3 29.3±0.72 26.4–27.2 26.8±0.23 3.301a 20–35

pH 7.34–7.56 7.42±0.06 7.01–7.08 7.05±0.02 5.250b 5.5–9

DO 0.26–0.42 0.34±0.04 0.39–0.55 0.48±0.04 2.15 NS –

EC 461–488 975.6±7.88 952–968 960.3±4.63 1.677 NS –

TD 132–157 146.3±7.45 128–145 135.7±4.98 1.190 NS 300

AD 76.2–81.9 79.3±1.51 72.4–77.3 75.1±1.44 1.910 NS –

AK 563–582 574.7±5.90 540–552 546.6±3.53 4.074a –

TH 218.9–312.4 277.1±29.3 209.7–298.4 261.4±26.6 0.397 NS –

Ca 79.4–94.7 85.1±4.83 72.3–86.4 77.83±4.34 1.118 NS

Mg 46.6–54.3 51.2±2.35 35.9–42.5 38.53±2.02 4.092a

TS 935–988 959.3±15.5 832–897 858.0±19.9 4.027a –

TDS 728–784 753.7±16.3 699–746 721.0±13.7 1.534 NS –

TSS 204–207 205.6±0.88 127–151 137±7.21 9.541b 100

Cl− 78.0–89.0 83.1±3.20 53.8–68.2 62.2±4.34 3.871a 1000

N 25.4–32.4 29.1±2.03 21.8–28.2 24.8±1.85 1.550 NS –

K 19.7–23.6 21.97±1.17 17.4–19.8 18.64±0.69 2.451 NS –

∑P 3.24–4.54 4.0±0.39 2.98–4.25 3.63±0.36 0.688 NS 10

NH4-N 17.3–32.4 26.5±4.66 13.4–16.8 15.3±1.01 2.341 NS 50

NO3-N 14.3–26.6 21.90±3.84 9.9–12.6 11.46±0.81 2.662 NS 10

BOD 265.3–302.5 283.5±10.7 232.3–248.9 241.4±4.86 3.572a 30

COD 213.5–295.3 262.3±24.9 198.0–273.0 246.0±24.1 0.469 NS 250

Cd 0.21–0.34 0.28±0.04 0.16–0.23 0.19±0.02 1.994 NS 2.0

Cr-VI 0.34–0.45 0.40±0.03 0.23–0.38 0.32±0.04 1.394 NS 0.1

Mn 2.03–2.25 2.14±0.06 1.86–2.19 2.01±0.09 1.178 NS 2.0

Ni 2.67–3.43 3.14±0.23 1.83–3.24 2.68±0.43 0.924 NS 3.0

Pb 2.02–2.98 2.45±0.30 1.92–2.43 2.15±0.15 0.909 NS 0.1

Cu 1.08–1.15 1.12±0.02 1.02–1.09 1.05±0.02 2.208 NS 3.0

Fe 0.97–1.24 1.14±0.08 0.88–1.02 0.96±0.04 1.903 NS 3.0

As 0.30–0.60 0.43±0.08 0.10–0.30 0.20±0.05 2.213 NS 0.2

All the values except pH, Temp (°C), TD (NTU) and EC (μS/cm) are reported in milligrams per litre. Values represent mean±SE; n=6

EPAR Environment Protection Amendment Rules, MPL maximum permissible limits, NS non-significant by Student’s t testa A significant difference at 5 % levelb A significant difference at 1 % level

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(Fig. 2f), destruction of cartilage in primary lamellae (Fig. 2f)and decreased mean length of primary and secondary lamellae(Fig. 2g, k) were the observed regressive changes. Progressivechanges included fusion of secondary lamellae between pri-mary lamellae (Fig. 2d, g, i and j), gill lamellar hyperplasiaand hypertrophy which led to complete lamellar fusion(Fig. 2b, k), curving of primary and secondary lamellae(Fig. 2d, f), ballooning dilatations of secondary lamellae(Fig. 2e, h), proliferation of mucous and chloride cells in baseof secondary lamella and blood congestion (Fig. 2h, i), inten-sive vasodilatation of the lamellar vascular axis (Fig. 2f),proliferation of cartilage cells (Fig. 2e, h) and predominanceof muscular tissue (Fig. 2f).

The number and severity of circulatory disturbances suchas aneurysm, intercellular oedema and progressive changeslike hypertrophy and hyperplasia of epithelium and infiltrationof cells in supporting tissues of gills increased as the concen-tration and exposure duration increased. Gill organ index(11.08±0.42) at lowest concentration of 50 % showed slightdamage to the organ in fish exposed to effluent from site 1 incomparison to fish treated with wastewater from site 2 thatshowed normal gill structure (7.0±0.31) with alterations onlyin few studied specimens. Moderate damage (25.09±0.43,37.96±0.89, 28.77±0.22) was noted in case of 60 and 70 %(site 1) and 70 % (site 2), severe lesions (80.56±0.47, 53.49±0.87, 89.11±0.65) were seen in 80 % (site 1) and 80 and 90 %(site 2) and irreparable lesions (144.99±0.38) were observedin fish subjected to highest concentration of 90 % (site 1) for15 days (Fig. 3a).

Degree of damage increased as the exposure duration in-creased from 15 to 30 days that clearly indicated the increasedcalculated values of histological organ index from both sites.Moderate damage (24.46±0.55, 42.69±0.54, 35.29±0.34) inconcentrations of 50 and 60% (site 1) and 60% (site 2) severe

lesions (65.55±0.48, 57.31±0.48, 92.10±0.48) in 70 % (site1) and 70 and 80 % (site 2) irreparable lesions (131.95±1.42,173.65±0.55, 153.32±0.36) in case of two highest concentra-tions of 80 and 90% (site 1) and 90% (site 2) were reported intest fish at the end of 30 days (Fig. 3b). Results demonstratethat the fish exposed to effluent from site 1 showed signifi-cantly greater change in Gill organ index (IG) for 15 and30 days as compared to fish exposed to effluent from site 2.Values of IG varied from 11.08±0.42 to 144.99±0.38(15 days) and 24.46±0.55 to 173.65±0.55 (30 days) for site1 in comparison to 7.0±0.31 to 89.11±0.65 (15 days) and16.57±0.50 to 153.22±0.36 (30 days) for site 2. The calcu-lated Gill organ index for treated fish from both sites wassignificantly different from control (p≤0.01) (Fig. 3a, b). Therecorded damage in fish exposed to wastewater from sites 1and 2 was 91.39 and 85.12 fold more in comparison to controlat the highest concentrations and exposure durations. The gilldamage in fish exposed to wastewater collected from site 1was 94.1 and 92.5 % from site 2 for a duration of 30 days(Fig. 3b).

Discussion

The physicochemical results of this study suggested the dete-rioration of water quality due to discharge of harmful effluentsfrom various industries and domestic sewage from AmritsarCity as indicated by the lower values of DO and higher valuesof BOD, COD, TSS and heavy metals. Fish mortality rate wasincreased proportionally with an increase in the concentrationof toxicant as 100 % mortality was recorded at 100 % con-centration for both sites. Wastewater from site 1 was moretoxic to C. punctatus as compared to site 2. The LC50 values

Table 2 Effect of wastewater on different behavioural parameters of C. punctatus up to 96 h

Parameter Site Control Wastewater concentration (v/v)

6.25 % 12.5 % 25 % 50 % 100 %

Air gulps(per 15 min)

Site 1 8.30±0.33 10.0±0.57NS 11.3±0.88* 14.0±0.57** 17.3±1.20** 21.0±0.58**

Site 2 8.00±0.57 10.6±1.20NS 11.0±0.57* 12.0±1.00* 15.0±1.15** 19.6±1.20**

Opercular movement(per min)

Site 1 46.6±1.45 42.3±1.45 NS 40.6±0.67* 38.0±0.57** 34.3±1.20** 30.3±0.66**

Site 2 46.3±0.66 43.0±1.15 NS 40.0±0.57** 39.0±0.58** 35.6±0.88** 31.0±1.00**

Swimming movements(24–96 h)

Sites 1 and 2 U U ES ES ES EX

Equilibrium(24–96 h)

Sites 1 and 2 N N N N EL EL

General activity(24–96 h)

Sites 1 and 2 N N N H V V

Values represent mean±SE; n=10

NS non-significant by Student’s t test (one tailed),U uniform, ExExodus trials,ES erratic and speedymovements,H hyperactive,N normal,V violent, ELequilibrium lost

*p≤0.05 (a significant difference at 5 % level); **p≤0.01 (a significant difference at 1 % level)

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reported in the present study for wastewater are in goodagreement with the values reported by different workers tothe same fish when exposed to wastewater of different origins.Yadav et al. (2007) reported 96 h LC50 value of 70.0 % toC. punctatus when exposed to industrial wastewater. Mishraand Poddar (2013) calculated 70 % 96 h LC50 on coke oveneffluent exposure to the same fish.

The behavioural and histopathological alterations revealedtoxic nature of contaminants present in Tung Dhab drain. Thebehavioural alterations are strongly correlated with the gillhistopathological changes observed in the present study.Alterations such as erratic swimming, surfacing, restlessnessand decreased rate of opercular movements are the avoidancereactions of fish to the toxicant. This is substantiated furtherthrough gill lamellar fusion, epithelial lifting, hypertrophy andhyperplasia of epithelial cells that obstruct the water spaces inbetween the secondary lamellae. The copious amount of mu-cus secretion also decreases the respiratory surface area and

increases the toxicant-blood diffusion distance. This is a de-fensive mechanism that provides a barrier to the entry oftoxicant, although this phenomenon reduces the total surfacearea for gas exchange leading to suffocation of fish. To com-pensate this loss, fish meets its oxygen needs via increased airgulping.

Behaviour is an important animal response that may intro-duce a pollution marker more sensitive and more relevant thanphysiological and anatomical changes (Biuki et al. 2010). Theloss of equilibrium might be due to the inactivation of centralnervous system and failure of latero-acoustic system due tothe inactivation of acetylcholine esterase activity, as suggestedin earlier studies (Verma et al. 1978; Pathan et al. 2009; Bhatet al. 2012). Decreased opercular movement possibly helps inreducing absorption of toxicants through gills as reported byBhat et al. (2012) in Labeo rohita when exposed to NEEM-ON biopesticide. Behavioural alterations like surfacing, errat-ic swimming and restlessness directly point towards the

Fig. 2 Light micrographs of gills of C. punctatus. b, c, d, e, f Fishexposed to wastewater collected from site 1 for a duration of 15 days(60 and 90 % of LC50) and 30 days (50, 70 and 90% of LC50). g, h, i, i, kFish exposed to wastewater collected from site 2 for duration of 15 days(60 and 80 % of LC50) and 30 days (70, 80 and 100 % of LC50).indicates epithelial lifting and intraepithelial oedema, for haemorrhagesand hyperaemia at primary lamellae, for aneurysm in secondary lamel-lae, for loss of secondary lamellae and necrosis, for fusion of

secondary lamellae between primary lamellae, for proliferation anddestruction of cartilage in primary lamellae, for hypertrophy and hyper-plasia of epithelial cells leading to lamellar fusion, for ballooningdilations at the tips of secondary lamellae, for severely damaged andlost secondary lamellae, for intensive vasodilation of the lamellarvascular axis, for predominance of muscular tissue, for proliferationof cartilage cells, for hypertrophy of chloride and pillar cell systems. a–k Haematoxylin and Eosin; a, d, f ×10 and the rest is ×40

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avoidance behaviour as observed by various workers (PatilandDavid 2008; Kasherwani et al. 2009). The phenomenon ofincreased air gulping reflects an attempt by the fish to extractmore oxygen to meet the increased energy demand, and thisaction may also be correlated to the formation of a hypoxiccondition due to the interference in gaseous exchange causedby the accumulation of mucous on the gill epithelium asreported by Kasherwani et al. (2009) while working withcadmium-treated Heteropneustes fossilis. Body surface dark-ening along with increased mucous secretion on body andgills was thought to be a reaction of fish to the toxicant thatminimizes the irritation by forming a barrier between the fishbody and toxic media as reported previously by Caglan et al.(2005), Adedeji et al. (2008) and Kaur and Dua (2014) inOreochromis niloticus, Clarias gariepinus and Labeo rohitawhen exposed to ammonia, diazinon and municipal wastewa-ter, respectively. Scale loss due to lepidontal damage andhaemorrhages near the mouth and caudal fin were reportedin C. punctatus due to zinc and municipal wastewater toxicityand in Cirrhinus mrigala exposed to dyeing industry effluent(Khangarot 2006; Kaur and Dua 2012; Kaur et al. 2013).

The histopathological changes revealed structural damage(slight, moderate, severe and irreparable) to the gills ofC. punctatus due to contaminants present in Tung Dhab drain.Epithelial lifting, proliferation of pavement and chloride cells,necrosis, lamellar fusion, epithelial hypertrophy and hyperpla-sia, proliferation of filamentary epithelium and aneurysm havealso been described by several authors for fish exposed toxenobiotics (Dua and Johal 1994; Escher et al. 1999; Erkmenand Kolankaya 2000; Gupta and Dua 2002; Alberto et al.2005; Elahee and Bhagwant 2007; Fontainhas-Fernandeset al. 2008). Epithelial lifting and intraepithelial oedema andlamellar fusion were observed to be the most important signsof ecological degradation of the ambient water as suggestedby Ojha (1993). The lamellar fusion reduces the total gillrespiratory area and reduces the oxygen uptake capacity ofthe gills (Mallatt 1985). The reported epithelial hypertrophyand hyperplasia, proliferation of filamentary epithelium and

aneurysm in the secondary lamellae may drastically increasethe toxicant-blood diffusion distance; hence, the reduction ofthe oxygen uptake and can lead to the rupture of blood vesselswith small hemorrhagic foci (Jiraungkoorskul et al. 2003;Koca et al. 2005; Abdel-Moneim et al. 2008). The presenceof ballooning dilatations observed in the secondary gill fila-ments may be considered as an ion trap to concentrate tracesof metals from water favouring cell adhesion betweenneighbouring secondary lamellae (Bhagwant and Elahee2002; Koca et al. 2008). The leukocyte infiltration along withhaemorrhages and hyperemia at primary lamellae markedlyshowed an immunological response to environmental contam-inants clearly indicates an inflammatory reaction and cellularinfiltration (Tao et al. 2000; Bhagwant and Elahee 2002; Kocaet al. 2008). Observations such as aneurysm, haemorrhages atprimary lamellae and sloughing of epithelium were recordedbyMishra and Mohanty (2008) in C. punctatus when subject-ed to chromium concentrations and by Alberto et al. (2005) infish treated with domestic sewage. According to them, thedelicate epithelium of the gill tissue is affected byecodegradation of the water in which fish live. Lamellar bloodcongestion can harm the gas exchange function of the gillstructure causing rupture of the pillar cell system, with loss oftheir support capacity and consequently structural disorder ofthe lamellae (Nascimento et al. 2012).

Conclusions

The current study suggests that the various physico-chemicalparameters of wastewater in which the experimental fish wereexposed to toxicological studies were above the maximumpermissible discharge limits. The calculated LC50 valuesclearly demonstrate the higher toxicity of site 1 to fingerlingsof C. punctatus. Apparent changes were observed in fishbehaviour and morphology to cope up with the stress ofdeteriorated water quality. The observed histopathologicalalterations in gills of C. punctatus clearly indicate the damage

Fig. 3 Effect of wastewater from sites 1 and 2 in fish C. punctatus exposed for durations of 15 (a) and 30 days (b). Bars indicating mean±SE values ofgill index; R2 coefficient of determination. Double asteriks significant at 1 % level

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at the cellular and organ levels, sensitivity and respiratorydistress of fish towards effluents. The studied parameters aredirect indicators of fish health and further validate a useful aswell as rapid method for evaluating water quality. The studyalso recommendsC. punctatus to be used as sentinels in waterquality monitoring programmes. The present study stronglyrecommends continuous monitoring of water bodies, and thegovernment agencies must take an action in response to anyviolation regarding discharge of pollutants beyond specificacceptable levels to maintain the health of aquatic ecosystemsand biodiversity inhabiting therein.

Acknowledgments The authors would like to thank the Department ofZoology, Guru Nanak Dev University for providing necessary infrastruc-tural facilities for the execution of this work. Further, the authors assurethat the studies involving experimental animals were conducted in accor-dance with national and institutional guidelines for the protection ofhuman subjects and animal welfare.

Conflict of interest The authors declare that they have no conflict ofinterest.

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