(ISSN: 0189 - 8779;

JOURNAL

OF

AQUATIC

SCIENCES

PUBLISHED BY

NIGERIAN ASSOCIATION OF AQUATIC SCIENCES

Volume 14 Apri/1999

Journal of Aquatic Sciences 14:.37 - 42 (1999).

HAEMATOLOGICAL CHANGES IN THE MUD FISH, CLARIAS GARIEPINUS(BURCHELL)EXPOSED TO MALACHITE GREEN

·S. O. MUSA AND E. OMOREGIEFisheries Research Unit, Department of Zoology, University of Jos, Nigeria.

ABSTRACTThe effect of sublethal concentrations of malachite green on blood compositions of the mudfish, Clarias

gariepinus (Burchell) was investigated under static bioassays during a 10 week exposure period. Concentrationsof malachite green used were 0.00 (control), 5.00, 10.00, 15.00, 20.00 and 25.00 ug.I". The exposure led toanaemic response in the fish, this response was observed to be time and dose dependent. There was significantreduction in the neutrophils of the fish exposed to 15.00, 20.00 and 25.00 f.lg.ri of the toxicant. The implicationsof these results were discussed with a suggestion ofthe total ban on the use of malachite green in aquaculture.

Key words: Haematological.mudfish, Clarias gariepinus, malachite green.

INTRODUCTIONThe use of haematological techniques in fish

culture is growing in importance for toxicologicalresearch, environmental monitoring and fish healthconditions as reported by Mulcahy (1975). Fish are sointimately associated with the aqueous environment,often physical and chemical changes in theenvironment are rapidly reflected as measurablephysiological changes in the fish. Sampath et al.(1993) noted that studies in fish blood lies in thepossibility that the blood will reveal conditions withinthe body of the fish long before there is any outwardmanifestation of disease. A thin epithelial membraneseparates fish blood from the water and anyunfavourable change in the water is reflected in theblood.

Many researchers have over the years studied theeffects of toxicants on haematological changes infish, also some researches have been carried out usingmalachite green as the toxicant. Malachite green haslong been used in fish culture since 1933 (Foster andWoodbury, 1936). Nelson (1974) noted thatmalachite green is a cornmon hatchery therapeutantused in fish culture.

Glagoleva and Malikova (1968) observed thatyoung Baltic salmon, Salmo salar exposed to 1.33ug.l" of malachite green for 20 minutes hadleucopaenia, slight erythropaenia and increasedmonocyte count. Increased in either theconcentrations of malachite green or length ofexposure, increase in the amount of changes in theblood cells were observed. Grizzle (1977) reportedthat neutrophilia was the most drastic haematologicalchange observed the channel catfish, Ictaluruspunctatus was exposed to 0.1 mg.I" of malachitegreen. Exposure of the Nile tilapia, Oreochromisniloticus to sublethal concentrations of malachitegreen Was reported by Omoregie et al. (1992) to leadto severe physiological impairment in the fish after a10 week exposure period.

The aim of this present study is to report on thehaematological changes in the mudfish, C. gariepinusexposed to sublethal concentrations of malachitegreen under laboratory conditions. This fish is

commonly cultured in several tropical countries ofthe world.

MATERIALS AND METHODSLive fingerlings of C. gariepinus used for this

investigation were collected from Rockwater FishFarm, Jos, Nigeria. The fish which were all of thesame brood stock were transported to the FisheriesResearch laboratory of the University ofJos, Nigeria.The fish had a mean weight of 15.63 ± 0.71 g at thestart of the exposure period. The fish were acclimatedto laboratory conditions for a period of 2 weeks.During the exposure period, the fish were fed oncommercial fish diet once daily (12.00 h) at 3% oftheir body weight.

After the period of acclimation, the fish wererandomly distributed into twelve class aquaria (56 x

28 x 28 em) with 50 litres of water at 12 fish peraquarium ..Each of the aquaria contained each of thefollowing concentrations of malachite green; 5.00,10.00, 15.00, 20.00 and 25.00 ug.l". The controlgroup of fish were kept in aquaria devoid of thetoxicant (0.00 Ilg.r1

). Each of these treatments had areplicate each. During the exposure period whichlasted for 10 weeks, the temperature, pH, dissolvedoxygen, free carbon dioxide and total alkalinity of theexperimental media were measured using standardlaboratory methods as described by APHA et al.(1989) on a weekly basis. Fresh preparations of thetest solution were introduced into the experimentalmedia on daily basis (08.00 h) during which time theset-up was thoroughly cleaned.

Two fish from each of the experimental mediawere sacrificed at bi-weekly. intervals, and blooddrawn into several heparinized micro-haematocrittubes from severed penduncle of the fish.Haematocrit values, haemoglobin, erythrocyte count,leucocyte count, neutrophils, lymphocytes andthrombocytes were ana1ysed using variousappropriate laboratory methods as described byBlaxhall and Daisley (1973). The mean values of thetwo fish taken from each of the experimental mediawere recorded for statistical analysis using two-wayanalysis of variance (ANOV A) at 0.05 level of

© Nigerian Association for Aquatic Sciences (ISSN: 0189 - 8779).

38

probability. Calculations were aided usingMicrosoft® Excel 97.

RESULTSThe water quality parameters measured during

the exposure period is presented in Table I. Thevarious parameters were not significantly different (P> 0.05) from each other, however, slight fluctuationswere observed. Results of the haematocrit andhaemoglobin values (Table 2) and erythrocyte andleucocyte counts (Table 3) of the fish exposed to15.00, 20.00 and 25.00 ug.l" decreased significantly(P <: 0.05) compared toiliose of the control fish.Though there was decrease in the values of fishexposed to 5.00 and 10.00 ug.l", this decrease invalues were however not significant (P > 0.05)compared to those of the control groups. Thedecrease in these values were also observed to beboth a factor of time and concentrations of thetoxicant.

The results of the lymphocytes, and neutrophilsof the exposed fish are presented in Table 4, while theresults of the thrombocytes are presented in Table 5.The percentage lymphocyte values of the exposedfish were not significantly (P > 0.05) changed at theend of. the exposure period, however, slightfluctuations occurred. The percentage neutrophils ofthe groups of fish exposed to 15.00, 20.00 and 25.00ug.l" reduced significantly (P < 0.05) at the end ofthe exposure period compared to the values recordedin the control groups of fish. Though the neutrophilsof the groups offish exposed to 5.00 and 10.00 ug.l"decreased, this decrease was however notsignificantly (P > 0.05) compared to the valuesrecorded for the control groups of fish. There wassignificant (P -c 0.05) increase in the values of thethrombocytes in fish exposed to 20.00 and 25.00 ug.l". while the values in the other groups of fish were

significantly (P >0.05) the same.

DISCUSSIONSublethal concentrations of toxicants in: the

aquatic environment will not necessarily result inoutright mortality of aquatic organisms. Omoregie etal. (1990) reported that they have significant effectswhich can result in several physiological dysfunctionin the fish. The non-significant change in the waterparameters of the various experimental mediareported in this investigation indicated that thesublethaleoncentrations of malachite green did notadversely lead to reduction in water quality. Whereslight changes were observed, the values were allwithin tolerance range.

Exposure of C. ganepmus to sublethalconcentrations of malachite green caused a significantdecrease in haematocrit, haemaglobin, erythrocytesand leucocytes of the fish. Similar reduction havebeen reported by Sampath et al. (1993) and Omoregie

et al. (1994) when they exposed fish to pollutedenvironment under laboratory conditions. Thesignificant reduction in these parameters is anindication of severe anaemia caused by malachitegreen on the exposed fish. The anaemic responsecould be as a result of destruction of erythrocytes orthe inhibition of erythrocyte production (Wintrobe,1978 and Omoregie, 1995), haemodilution(Johansson and Larsson, 1978; Omoregie et al., 1990and Sampath et al., 1993) and it could be as a resultof the destruction of intestinal cells (Gardner andYevich,I970).

In this investigation, apart from slightfluctuations, non-significant changes were observedin the lymphocytes of the exposed fish. However,Sampath et al. (I 993) had reported increase in thelymphocytes of the Nile tilapia, Oreochromisniloticus exposed to toxic environment, this theyattributed to the stimulation of the immunemechanism of the fish to eliminate the effects of thepollutants. The non-significant changes in thelymphocytes in this investigation is attributed tosublethal concentrations of malachite green usedwhich were within the recommended doses used inthe treatment of ectoparasites on fish (Roberts, 1978).The significant increase in the percentagethrombocytes reported in the exposed fish issupported by an earlier study documented by Rai andQayyan (1984).

The reduction in the neutrophils reported here isin agreement with the findings of Sharma and Gupta(1984) when juveniles of the mudfisli, Clariasbatrachus were exposed to carbon-tetrachloride.However, Storozhuk and Guleva (1983) reportedincreased neutrophils in the salmon, Oncorhynchuskisutch after exposure to mercury, this they attributedto tissue damage.

The use of malachite green in aquaculture iscurrently being discouraged in several developedcountries due to its toxicity. The report by Wright(1976) documented that this chemical is extremelytoxic at doses higher than the recommended levels.Result from this current study seems to agree withWright's report. Despite this however, malachitegreen is still being used in several third worldcountries where there are difficulties in enforcinglegislatures regarding chemical used in aquaculture.Finding from this investigation reveal deleteriousconsequences on the health ·of fish subjected tonominal chronic exposure of malachite green, andtherefore should not be used for any purpose inaquaculture.

ACKNO~EDGEMENTThe authors are grateful to the Departments of

Zoology and Health Services of the University of Jos,Nigeria for assistance given during this research.

Table 1: Water quality parameters* obtained during exposure of the mudfish, Clarias gariepinus to sublethalconcentrations of malachite green for 10 weeks.

Parameters Malachite green concentrations (Ilg.rl)0.00 5.00 10.00 15.00 20.00 25.00

Temperature (0C) 23.08(1.48)

23.18 23.39 22.94 23.11(1.40) (1.19) (1.25) (1.32)

23.04(1.36)

Dissolved oxygen (rng.I'") 7.44 7.40 7.18 7.13 7.14 6.90(0.48) (0.54) (0.55) (0.62) (0.69) (0.37)

Free carbon-dioxide (mg.l") 3.97 4.05 3.99 4.03 4.05 4.00(0.17) (0.36) (0.32) (0.33) (0.35) (0.43)

Alkalinity 40.10 39.55 39.62 39.62 39.77 39.36(0.95) (0.86) ( 1.32) ( 1.32) (1.42) (0.97)

pH 7.20 7.21 7.11 6.96 6.89 6.69(0.21) (0.19) (1.80) (0.41) (0.34) (0.31)

* mean values with standard errors in parentheses.

Table 2: Values* of haem atocr it and haemoglobin of the mudfish, Clarias gariepinus to sublethal concentrationsof malachite green for 10 weeks.

Concentrations Haematocrit (%)(ug.l I) Exposure period (weeks)

Start 2 4 6 8 100.00 34.30 32.15 31.65 28.75 .28.25 29.30

(0.80) (2.35) (0.85) (0.75) (1.25) (1.20)

5.00 34.65 28.95 28.20 26.30 27.45 27.30(0.55) (1.05) (0.30) (0.80) (2.55) (2.20)

10.00 33.30 27.85 25.95 25.25 26.30 26.65(0.80) ( 1.05) (0.1 5) (0.30) (0.80) (4.55)

15.00 30.70 26.95 22.80 24.25 24.75 25.70(0.20) (1.15) (0.30) (l.25) (1.55) (1.20)

20.00 30.30 26.30 22.45 22.75 22.55 23.75(0.80) (3.20) (1.55) (0.45) (1.45) (0.25)

25.00 29.20 27.55 19.95 21.55 22.55 23.75( 1.30) (0.65) (0.15) (0.45) (1.45) (0.25)

Haemoglobin (mg.l')0.00 10.93 9.00 9.13 9.58 9.76 9.83

(0.78) (0.60) (1.13) (0.51) (0.25) (1.18)

5.00 8.65 8.18 8.45 9.13 7.13 7.75(0.35) (0.18) (0.30) (0.88) (0.85) (0.75)

10.00 7.83 7.95 7.83 8.03 7.15 7.88(0.68) (0.20) (0.18) (0.63) (0.01) (0.83)

15.00 7.28 7.75 7.63 6.98 7.08 7.63(2.03) (0.23) (0.15) (0.64) (0.43) (0.18)

20.00 6.83 6.75 7.25 6.93 7.08 6.93(0.6,8) (0.20) (0.10) (0.08) (0.58) (0.43)

25.00 6.75 6.33 6.98 6.98 6.93 6.83(0.25) (0.18) (0.03) (1.08) (0.23) (0.85)

* mean values with standard errors in parentheses.

39

40

Table 3: Values" of erythrocyte and leucocyte counts or (he mud/ish, Clarias gariepinus to sublethal

4concentrations of malachite green for 10 weeks.

Concentrations Erythrocyte COWlt(106 mrl)(ug.l") Exposure period (weeks)

Start 2 4 6 8 100.00 2.39 .2.35 2.48 2.37 2.12 2.10

(0.03) (0.04) (0.07) (0.06) (0.11) (0.21)

5.00 2.33 2.00 1.91 1.87 1.97 1.96(0.04) (0.12) (0.02) (0.13) (0.03) (0.09)

10.00 2.32 1.86 1.76 1.73 1.87 1.85(0,07) (0,06) (0.04) (0.04) (0.03) (0031)

15.00 2.27 1.89 1.72 1.68 1.82 1.81(0.06) (0.11) (0.08) (0.07) (0.19) (0.24)

20.00 2.24 1.88 1.69 1.70 1.80 1.82(0.15) (0.04) (0.02) (0.06) ('UO) (0.37)

25.00 1.95 1.85 1.61 1.61 1.62 1.70(0.14) (0.14) (0.03) (0.03) (0.04) (0.09)

Leucocyte count (103 ml")0.00 56.00 54.25 51.74 51.26 49.51 52.24

(5.00) (4.75) (0.78) (3.59) (4.05) (3.72)

5.00 66.00 55.01 53.26 58.73 56.00 54.52(3.01) ( 1.05) (1.24) (2.27) (4.50) (4.06)

10.00 66.00 55.50 57.52 58.29 56.08 56.25(1.50) (1.02) (2.06) (6.18) (1.73) (7.25)

15.00 66.75 60.00 59.78 59.75 59.75 57.01(1.79) (2.50) (0.72) (0.70) (2.76) (4.00)

20.00 78.71 66.07 67.52 63.75 63.00 60030(1.57) (7.05) (3.51) (3.25) (6.05) (LSO)

25.00 79.75 75.50 79.56 67.72 64.52 57.00(1.57) (2.09) (2.50) (2.25) (2.25) (6.50)

* mean values with standard errors in parentheses.

41

Table' 4: Values" oflymphocytes, neutrophils andthrombocytes of the mudfish, Clarias gariepinus to sublethalconcentrations of malachite green for 10 weeks.

Concentrations Lymphocytes(ug.l") Exposure period (weeks)

Start 2 4 6 8 100.00 91.50 93.00 94.00 92.50 84.50 88.50

(2.51) (2.50) (2.06) (5.50) (5.52) (6.50)

5.00 88.50 86.00 78.50 85.00 84.00 88.00(2.51) (2.06) (6.59) (4.27) (2.00) (0.51)

10.00 88.50 79.50 81.00 81.00 88.50 93.50(2.50) (0.50) (2.00) (3.05) (1.06) (3.04)

15.00 86.50 79.00 71.00 81.00 80.00 89.50(4.53) (0.02) (3.06) (0.08) (1.00) (2.07)

20.00 79.50 79.50 79.50 79.50 81.50 84.50(0.52) (3.09) (4.05) (1.59) (1.51) (2.50)

25.00 78.00 79.00 76.50 76.50 77.50 82.00(0.04) (1.04) (1.50) (1.06) (1.05) (3.00)

Neutrophils (%)0.00 2.00 3.00 5.00 4.50 4.50 2.50

(0.12) (0.03) (0.01) (0.05) (0.25) (0.15)

5.00 6.50 8.50 5.00 5.50 3.00 4.00(0.05) (0.50) (0.02) (0.15) (0.00) (0.01)

10.00 9.50 12.50 11.50 6.00 4.00 3.00(0.45) (1.52) (0.35) (0.01) (0.00) (0.00)

15.00 10.50 12.50 16.50 7.00 7.50 3.00(0.45) (0.50) (0.05) (0.02) (0.05) (0.02)

20.00 17.50 8.00 8.00 5.00 4.00 4.00(0.25) (0.02) (0.10) (0.02) (0.00) (0.00)

25.00 17.00 12.50 7.50 9.50 3.50 5.00(0.20) (0.50) (0.06) (0.25) (0.15) (0.01)

• mean values with standard errors in parentheses.

Table 5: Values" of thrombocytes of the mudfish, Clarias gariepinus to sublethal concentrations of malachitegreen for 10 weeks.

Concentrations Thrombocytes (%)(ug.l") Exposure period (weeks)

Start 2 4 6 8 100.00 6.50 4.00 4.00 3.00 6.00 5.50

(0.15) (0.01) (0.01 ) (0.02) (0.03) (0.02)

5.00 5.00 5.50 6.50 9.50 13.00 8.00(0.02) (0.25) (0.15) (0.26) (0.29) (0.10)

10.00 4.50 8.00 6.50 8.00 7.50 7.00(0.51) (0.09) (0.07) (0.21 ) (0.15) (0.03)

15.00 3.00 8.50 11.50 12.00 13.00 8.00(0.00) (0.15) (0.01) (0.04) (0.10) (0.04)

'20.00 3.00 12.50 15.50 13.50 14.00 10.50(0.02) (0.52) (0.45) (0.06) (0.21 ) (0.55)

25.00 5.00 8.50 16.00 13.00 14.50 14.00(0.20) (0.26) (0.13) (0.10) (0.19) (0.15)

42

REFERENCESAPHA, AWWA and WPCF (American Public Health

Association, American Water Works Associationand Water Pollution Control Federation) (1989).Standard Method for the Examination of Waterand Waste Water. 17th Edition. APHA,Washington, D.C. USA. 1391 pp.

Blaxhall, P. C. and Daisley, K W. (1973). Routinehaematoiogical methods for use with fish blood.Journal ofFish Biology 5: 771-781.

Foster, F. 1. and Woodbury, L. (1936). The use ofmalachite green as a fimgicide and antiseptic.Progressive Fish Culturist 18: 7 - 9.

Gardner, G. R. and Yevich, P. P. (1970). Histologicaland haematological responses of an estuarineteleost to cadmium, Journal of FisheriesResearch Board of Canada 27: 2185 - 2196.

Glagoleva, T. P. and Malikova, E. M. (1968). Theeffects of malachite green on the bloodcomposition of young Baltic salmon. RybnoeKhozyaistra 45: 15 - 18.

Grizzle, J. M. (1977). Haematological changes infingerlings channel catfish exposed to malachitegreen. Progressive Fish Culturist 39: 90 - 93.

Johansson, M. L. and Larson, A. (1978). Effects ofinorganic lead on aminolevulinic acid dehydraseactivity and haematological variables in therainbow trout, Salmo gairdneri. Achieves ofEnvironmental Contamination and Toxicity 8:419-431.

Mulcahy, M. F. (1975j. Fish blood changesassociated with disease. A haematological studyof pike lymphoma and salmon ulcerative dermalnecrosis. In: The Pathology of Fishes (Eds.: W.E. Ribelin and C. Migaki). University ofWisconsin, Madison, USA. 925 - 944.

Nelson, N. C. (1974). A review of the literature onthe use of malachite green in fishes. U.S. Fishand Wildlife Services. Literature Review No. 74,11 pp.

Omoregie, E. (1995). Effects of petroleum on theNile tilapia and its helminth infection. Ph.D.Thesis, University of Jos, Jos, Nigeria. 152 pp.

Omoregie, E., Ufodike, E. B. C. and Keke, 1. R.(1990). Tissue chemistry of Oreochrom isniloticus exposed to sublethal concentrations ofGammalin 20 and Actellic 25 EC. Journal ofAquatic Sciences 5: 33 - 36.

Omoregie, E., Ofojekwu, P. C. and Eseyin, T. G.(1992). Effects. of sublethal ·concentrations ofmalachite green on the carbohydrate reserves ofthe Nile tilapia, Oreochromis niloticus. Journalof Aquatic Sciences 7: 53 - 58.

Omoregie, E., Eseyin, T. G. and Ofojekwu, P. C.(1994). Chronic effects of formalin onerythrocyte counts and plasma glucose of Niletilapia, Oreochromis niloticus. Asian FisheriesScience 7: 1 - 6.

Rqi, R. and Qayyum, M. A. (1984). Haematologicalresponses in freshwater fish to experimental leadpoisoning. Journal of Environmental Biology 5:53 - 56.

Roberts, R. J. (1978). Fish Pathology. BailliereTindallLondon, UK. 318 pp.

Sampath, K, Velamnial, S. Kennedy, 1. J. and James,R. (1993). Haematological changes and theirrecovery in Oreochromis mossambicus as afunction of exposure period and sublethal levelsofEkalus. Acta Hydrobiologica 35: 73 - 83.

Sharma, R. C. and Gupta, N. (1984). Carbon-tetrachloride induced haematological alterationsin Clarias batrachus (L.). Journal ofEnvironmental Biology 3: 127 - 131.

Storozhuk, N. G. and Guleva (1983). Quantitativecomposition and morphology of blood cells incoho salmon, Oncorhynchus kisutch exposed tomercury. Vopr Ikhttol 23: 845 - 853.

Wintrobe, M. M. (1978). Clinical Haematology. H.Kimpton, London, UK. 488 pp.

Wright, L. D. (1976). Effects of malachite green andformalin on the survival of largemouth bass eggsand fry. Progressive Fish Culturist 38: 155 -157.

Received: 26th March, 1998; Accepted 30September, 1998.