Introduction

The  environmental  pollution  due  to  extensive  usage  of  the  pesticides  without  proper  management  has  toxic  effects  on  survival  of  aquatic  animals  because  some  of  these  toxic  pesticides  like  organochlorine  and  organophosphorus  may  present  in  the  environment  for  long  periods,  often  unchanged.  They  accumulate  in  liver  and  fatty  tissues ¹.  Poisoning  risk  depend  on  dose,  toxicity,  duration  of  exposure  and  sensitivity.  Organochlorine  and  organophosphorus  are  most  widely  used  pesticides.  The  fishes  exposed  by  these  pesticides  are  reach  to  human  being  by  food  chain  which  has  great  nutritive  values.  Now  a  days  these  highly  toxic  pesticides  are  replaced  by  less  toxic  pesticides  like  carbamate  and  herbicides.

Carbaryl  (1-naphthyl  1-N-methyl  carbamate;  sevin)  is  a  carbamate  insecticide  introduced  in  1956.  It  is  a  good  substitute  of  some  organochlorine  pesticides ². Small  amount  of  carbaryl  have  caused  adverse  effect  on  birds,  fish,  tadpoles,  salamenders,  shrimp,  bees  and  other  animals.  The  main  effect  of  carbaryl  include  reduced  production  of  eggs,  reduced  ability  to  run,  deformed  legs,  reduced  swimming  speed  and  mortality.

A  herbicide  2,4-D  sodium  salt  (sodium  salt  of  2,4-dichlorophenoxyacetic  acid)  used  against  pest,  undesirable  herbs  and  agricultural  diseases  were  found  to  have  adverse  effects  in  the  non  target  environmental  animals  and  plants ³.  The  2,4-D  is  the  most  widely  used  herbicide  in  the  world  (Industry  Task  Force  Research  Data).  It  is  low  toxicity  for  microorganism,  fish,  insects,  domastic  and  wild  animals  and  humans  at  low  concentrations  but  acute  poisoning  causes  the  toxic  effect  in  fish,  domastic  and  wild  animals  causing  apathy,  anaemia  of  mucous  membranes  and  drop  of  body  temperature ⁴.  High  does  also  causes  changes  in  blood  parameters ⁵,  necrosis  of  liver,  hyperplasia  of  Bowman^,s  capsule  and  adenomas ⁴.  The  fish  showed  marked  depletion  in  liver  glycogen,  collection  of  glycoproteins  in  the  blood.  The  2,4-D  sodium  salt  also  effects  can  lead  to  gradual  decreases  in  fish  population  through  increased  predation,  reduced  reproduction,  diseases  and  starvation  and  morphological  and  histochemical  changes  in  tissues  and  organs  has  been  also  demonstrated  on  exposure  of  2,4-D  sodium  salt ⁶.  The  aim  of  present  work  to  study  the  effect  of  carbamate  (carbaryl)  and  herbicide  (2,4-D  sodium  salt)  on  haematological  parameters  on  a  catfish,  Heteropneustes  fossilis,  at  different  dose  and  time  intervals.

Materials and Methods

Live  specimen  of  fish,  H. fossilis  (Weight  16.32+1.30  gm,  length  11.30+1.30  cm)  were  procurred  from  the  local  market  and  brought  to  the  laboratory  in  15  litre  plastic  bucket.  They  were  acclimatized  to  the  laboratory  condition  for  10  days  in  dechlorinated  water.  The  water  used  during  experiments  was  analysed  as  per  the  standard  method ⁷.  The  methods ⁸  were  used  to  calculate  24,  48,  72  and  96h  LC₅₀  values  and  95%  confidence  limits.  The  presumably  harmless  (safe)  concentrations  of  both  the  toxicants  were  estimated  by  the  formula  of ⁹.  The  stock  solution  of  toxicants  were  prepared  in  distril  water  and  the  toxicants  used  were  of  technical  grade  (95  to  98%  pure)  and  purchased  from  Sigma  Chemical  Company,  Mumbai.  The  fish  were  exposed  to  acute  (1/5^th  of  96h  LC₅₀  value),  subacute  (1/10^th  of  96h  LC₅₀  value)  and  sublethal  (1/15^th  of  96h  LC₅₀  value)  concentrations  for  the  acute  (96h),  subacute  (10-20  days)  and  sublethal  (30-60  days)  term.  A  parallel  group  of  control  fish  was  maintain  in  the  toxicant  free  tapwater.  The  physico-chemical  characteristics  of  tapwater  were  (pH  7.8,  dissolve  oxygen  8.40  mg/L^-1,  total  hardness  270.50  mg/L^-1  as  CaCO₃  and  BOD  16.20  mg/L^-1).  On  completion  of  fixed  exposure  periods,  caudal  peduncle  of  fish  were  cut  off.  The  free  flowing  blood  from  the  caudal  artery  was  directly  collected  for  the estimation of haematological parameters.

The  total  RBC  and  WBC  counts  were  made  by  improved  Neubaurer  hemocytometer  and  haemoglobin  content  (g%)  determined  by  Sahlin-Hellige  method  using  0.1  N  HCl.  Packed  cell  volume  (PVC)  was  measured  by  the  method  using  75×1.00-1.25  mm  capillary  tubes.  Clotting  time  of  blood  was  determined  by  the   capillary  tube  method  as  used  in  clinical  haematology .  The  results  were  subjected  to  statistical  analysis  by  student’s  ‘t’  test ¹⁰.

Results and Discussion

The  LC_o,  LC₅₀  and  LC₁₀₀  values  with  95%  confidence  limits  for  24,  48,  72  and  96h  for  both  the  toxicants  are  presented  in  Table  Ia  and  Ib .  The  comparative  values  of  96h  LC_50,  safe  concentration  and  fraction  of  96h  LC₅₀  value  of  these  toxicants  are  also  given  in  table  Ic.  On  exposure  at  acute,  subacute  and  sublethal  concentrations  of  both  the  toxicants  for  acute  (96h)  and  both  short  (10-20  days)  and  long  (30-60  days)  terms,  noticeable  changes  were  observed  is  haematological  parameters  of  the  exposed  fish  (Tables  2,  3  and  4).  The  haematological  parameters  play  an  important  role  in  diagnosis  of  diseases  in  fish  following  different  stress  condition.  Thus  haematology  can  be  considered  as  an  essential  index  to  general  health  status ¹¹.  The  results  also  showes  that  carbaryl  is  more  toxic  than  2,4-D  sodium  salt.

Total RBC Counts

The  decrease  in  erythrocytes  in  the  catfish  during  carbaryl  and  2,4-D  sodium  salt  exposure  indicates  an  inhibited  production  of  red  blood  cells  caused  by  increased  erythrocytes  destruction ¹².  The  development  of  erythropenia  in  the  catfish  could  be  due  to  toxicants  interference  with  haemopoesis  and/or  alteration  of  cell  membrances  by  hydrolysis  of  acetylcholine  in  the  body  fluids  by  cholinesterase  of  erythrocytes ¹³.  The  carbaryl  and  2,4-D  sodium  salt  induced  erythropenia  were  also  reflected  by  reduced  hemoglobin  content  in  this  study.  The  reduction  in  total  RBC  counts  may  be  due  to  microcyctic  or  normocytic  anaemia  as  suggested  by Dutta¹⁴ , Tuschiya¹⁵.  These  observations  are  in  close  agreement  with  the  findings  of  other  workers  ^16,17,18  who  reported  similar  changes  in  H.  fossilis  after  exposure  to  different  water  pollutant  and  GOAW  effluent.

Total WBC counts

The  studies  of  total  and  different  leucocyte  counts  have  been  suggested  as  an  indicator  of  stress  in  fish  ¹¹.  Significant  increase  was  observed  in  total  WBC  counts  of  the  catfish  at  acute,   subacute  and  sublethal  concentrations  of  carbaryl  and  2,4-D  sodium  salt  at  acute  (96h)  and  short  (10-20  days)  term  but  no  remarkable  changes  were  observed  at  long  (30-60  days)  term  exposure  to  2,4-D  sodium salt.

Leucocytosis  was  also  observed  in  the  other  teleostean  fishes  at  post  exposure  to  toxicants ^{12,16,17,18,19, 20}.  The  WBCs  are  inextricably  involved  in  the  regulation  of  immunological  function¹⁴  and  a  prolonged  exposure  of  H.  fossilis  to  a  toxicant  may  inflict  immunological  deficiencies  where  the  toxicant  may  work  as  an  antigen.  The  rise  of  total  WBC  counts  at  different  cocentrations  of  carbaryl  and  2,4-D  sodium  salt  may  be  due  to  malfunctioning  of  haematopoeitic  system  caused  by  intoxication  of  carbaryl  and  2,4-D  sodium  salt.

Haemoglobin content

Significant  reduction  in  haemoglobin  content  of  the  catfish  resulted  post  exposure  to  acute,  subacute  and  sublethal  concentrations  of  carbaryl  and  2,4-D  sodium  salt  for  96h  and  short  (10-20  days)  term  but  long  term  exposure  to  these  toxicants  at  the  subacute  and  sublethal  levels  did  not  reduce  haemoglobin  content.

The  anaemia  in  C.fasciatus  exposed  to  lead  was  identified  as  haemolytic  on  the  basis  observed  lysis  of  the  erythrocytes  with  concomitant  decrease  in  haemoglobin  content  and  haematocrit ²¹.  Anaemia  can  be  caused  by  a  number  of  pathological  conditions  and  in  this  case  it  was  similar  to  those  noticed  in  Coho  Salmon,  Oncorhynchus  Kisutch  following  exposure  to  sublethal  levels  of  total  residual  chlorine  ²²  and  in  other  teleosts  exposed  to  pulp  mill  and  GOAW  effluents ^16,23.  Erythropenia  was  also  reflected  by  the  reduced  hemoglobin  content  of  the  blood  as  well  as  by  marked  increase  in  sedimentation  of  erythrocytes.  The  decrease  in  haemoglobin  content  of  fish  on  exposure  to  carbaryl  and  2,4-D  sodium  salt  implicates  haemodilution  and  resulting  increase  in  cell  size.  This  is  accomplished  by  either  cellular  swelling  or  mortality  of  small  immature  cells.  Therefore,  anaemic  state  of  H.  fossilis  after  metal  treatment  may  also  be  attributed  to  inhibitat  ion  of  erythropoesis ²⁴ coupled  with  enhanced  rate  of  erythrocyte  destruction  ²⁵,  distrubed  Hb  sysnthesis  and  haemodilution  ²⁶.

Haematocrit (P.C.V.)

The  catfish,  H.  fossilis  in  the  present  study  showed  significant  decreased  in  haematocrit  (PCV%)  following  acute  (96h)  and  short  (10-20  days)  term  exposure  to  acute,  subacute  and  sublethal  concentrations  of  carbaryl  and  2,4-D  sodium  salt  but  no  effect  at  long  term  to  subacute  and  sublethal  concentrations.  Changes  in  haematocrit  value  in  fish  exposed  to  different  environmental  stressors  or  chemicals  have  been  reported  by  several  workers  ^{16,17,18, 19, 20, 27}.  Aldrin  indudced  erythropenia  was  reflected  by  reduced  haemoglobin  content  and  haemotocrit  as  well  as  rapid  sedimentation  of  erythrocytes.  These  changes  have  also  been  reported  in  Channa  punctatus  and  Gopy  melanostomus  post  exposure  to  some  insecticides  of  OC  group. Other worker^17,16  also  reported  decreased  level  of  haematocrit  in  fish  exposed  to  different  concentratons  of  detergent  and  GOAW  effluent.  Grizzle²⁸  attribute  the  decrease  in  haematocrit  and  haemoglobin  content  in  fish  to  impairment  of  gas  exchange  by  the  gills.  The  changes  in  haematocrit  value  in  this  study  might  have  occurred  due  to  a  slight  hypoxia,  during  exposure  to  both  the  toxicants.

Clotting  Time

The  fish  exposed  to  acute,  subacute  and  sublethal  concentrations  of  carbaryl  and  2,4-D  sodium  salt  for  acute  (96h)  and  short  (10-20  days)  term  intervals  exhibited  significant  delay  in  blood  coagulation.  Similar  response  was  also  observed  by  several  workers  after  exposure  of  catfish  to  pesticides,  dyes,  detergent  and  metals  ^{16,17, 18,19,27}.  Several  workers  have  reported  an  inverse  relationship  between  thrombocyte  count  and  blood  clotting  time  in  several  fish  species  ^29,30.  In  the  present  study,  it  seems  that  the  catfish  developed  thrombocytopenia  which  led  to  a  concomitant  increase  in  the  clotting  time  of  the  blood.  Perhaps  toxicosis  of  these  toxicants  triggers  a rapid  mobilization  of  the  haemostatic  system  and  the  fish  normally  appears  to  deal  with  it  by  adjusting  blood  clotting  time  and  thrombocyte  concentations.  The  fish  not  showing  any  significant  changes  in  clotting  time  at  long  (30-60   days)  term  exposure  to  subacute  and  sublethal  concentration  of  carbaryl  and  2,4-D  sodium  salt.

 Reference

1. Duke T.W. and Wilson A.J. Jr., Chlorinated hydrocarbons in livers of fishes from the northeastern Pacific Ocean. Pestic Monit J, 5,228(1971).
2. Ribera D., Narbonne J.F., Arnaud C. and Denis M.S., Biochemical responces of the earthworm Eisenia fetida Andrei exposed to contaminated artificial soil, effects of carbaryl. Soil Biol. Biochem. , 33, 1123-1130 (2001).
3. Sarikaya R., Yilmaz M., Gul A., The acute toxic effect of 2,4-D Tench (Tinca tinca L., 1758). J. Inst. Sci. Technol. Gazi Univ. ,15,289-296 (2002).
4. Loktionov V.N., Toxicology of derivatives of dichlorophenoxyacetic acid (the herbicide 2,4- D). Veterinarija 11,61-63 (1986).
5. Prokofyeva T.V., Acute toxicity for fowls of dialen ( a combination of 2,4-D and dicamba) herbicide. Veterinarija 5,56-58 (1988).
6. Cope O.B. , Contamination of the freshwater ecosystem by pesticides. J. of Appl. Ecol., 3b, 33-44(1966).
7. APHA, American water works association and water pollution control federation. (24th Ed.) Washington. Publ . Hlth. Associ., 801-823 (1998).
8. Litchfield, J.T. and Wilcoxon F., A simplified method of evaluating dose effect experiment. Pharmacol. Exp. Ther., 96 , 99-113 (1949).
9. Hart W. B., Doudoroff and Greenbank J., The evaluation of the toxicity of industrial wastes, chemicals and others substances to freshwater Fish. Atlantic Refining Co. Phil., 317 (1945).
10. Sokal R. B. and Rohef F. J. ,Biometry, 2^nd. Ed. W.H. Freeman and company, San Francisco. Calif. (1981).
11. Blaxhall P.C. and Daisley K.W., Routine haematological methods for use with fish blood. Fish. Biol., 5, 771-781 (1972).
12. Van Vuren J.H., Merwe J., M.V. and Preez Du. H.H., The effect of copper on the blood chemistry of Clarius batrachus (C.) Environ., 22, 212-215 (1994).
13. Varadaraj G., Subramanian M.A. and Nagarajan B., The effect of sublethal concentration parameters of Oreochromis mossambicus (Peters). Environ. Biol., 14 , 321-325 (1993).
14. Dutta H.M., Dogra J.v.V., Singh N.K., Roy P.K., Nasar S.S.T., Ashikari S., Munshi J.S.D. and Richmonds C., Malathion induced changes in the serum protein and hamatological parameters of an  India catfish, fossilis (Bloch). Bull. Environ. Contam. Toxicol, 49 , 91-97 (1992).
15. Tuschiya K., Hand book on the toxicology of metals. Elsevier/North Holland Biomedical Press. Amsterdam (1979).
16. Chandra P., Srivastava D.K. and Srivastava A.K., Effect GOAW effluents on certain haematological parameters of a catfish, Heteropneustes fossilis (Bloch). Ecophysiol. Occup. Hlth., 4 , 197-200 (2005).
17. Pandey P., Detergent induced biochemical, haematological changes and reproductive physiology of an Indian catfish, fossilis (Bloch). Ph.D. Thesis, V.B.S. Purvanchal University, Jaunpur, U.P. (2000).
18. Srivastava S.K., Studies of lead toxicity on reproductive and liver functions & role of thiamine as detoxicant in a freshwater catfish, fossilis. Ph.D. Thesis, V.B.S. University, Jaunpur (U.P.) (2002).
19. Ramalingam V., Vimaladevi V., Narmadharaji and Prabhakaran P., Effect of lead on haematological and biochemical changes in freshater fish, Cirhina mrigala. Poll. Res., 19 , 81-84 (2000).
20. Verma G.P. and Panigrahi P., Effect of agrofen on blood parameters of Oreochromis mossambica (P). Nat. Acad. Sci. India, 68B (1) , 29-36 (1998).
21. Srivastava A.K. and Mishra S., Blood dyscrasia in a teleost, Colisa fasiatus after acute exposure to sublethal concentrations of lead. J. Fish. Biol., 14 , 199-203 (1979).
22. Buckley J.A., Whitmore C.M. and Matsuda R.I, Changes in blood chemistry and blood cell morphology in Coho salmon ,Oncorhynchus Kisutch following exposure to sublethal levels of total residual chlorine municipal waste water, J. Fish. Res. Board Can., 33,776-78 (1976).
23. Leay D.J., Effects of 12h and 25 days exposure to kraft pulp mill effluent on the blood and tissue of juvenile Coho salmon, Oncorhynchus hisutuch. J. Fish Res. Board Can., 30 , 305-400 (1973).
24. Gardner G.R. and Yevich P.P., Histological and haematological responses on an esturine teleost to cadmium Fish. Res. Biol. Canada, 7,2185-2196 (1970).
25. Crandall A. and Goodnight C.J., Effect of sublethal concentration of several toxicants in the common guppy, Lebistes Reticulars (P.). Trans. Amer. Fish. Soc., 82, 59-73 (1963).
26. Larsson A., Bengtsson B.E. and Savanberg O., Some haematoligical and biochemical effects on fish. In : Effect of pollutants on aquatic organism (Lockwood, A.P.M. Ed.). Cambridge Univ. Press New York ,35-45 (1975).
27. Srivastava R.K., Singh H.S. and Srivastava S.J., Physiological changes in a freshwater catfish, fossilis following exposure to cadmium (Cd). Ecophy. and Occup. Health., 1 , 235-244 (2001).
28. Grizzle Jam., Haematological changes in fingerlings channel catfish exposed to malachite green. Prog. Fish Cult., 39,90-92 (1977).
29. Casillas E. and Smith L., Effect of stress on blood coagulation and haematology in rainbow trout, salmo gairdneri,J. Fish. Bio., 481-491 (1977).
30. Srivastava A. K. and Singh N. N., Haematological changes in fossilis following exposure to aldrin. J. Natocon. 6,85-88 (1994).