Biochemical changes induced by mercury in the liver of penaeid prawns Penaeus indicus and P. monodon (Crustacea : Penaeidae) from
Rushikulya estuary, east coast of India
Snehalata Das, Sunil K. Patro & B.K.Sahu*
Department of Marine Sciences, Berhampur University, Berhampur 760 007, Orissa, India Received 16 October 2000; revised 6 June 2001
The biochemical components viz. protein, lipid and carbohydrate of the liver of two important penaeid prawns were significantly reduced, following six days of exposure to 0.005 ppm and 0.01 ppm of mercuric chloride during various reproductive stages i.e. preparatory, prespawning, spawning, and postspawning. Liver protein recorded highest in contrast to lipid and carbohydrate irrespective of the species, sex and medium of exposure. Depletion percentage with respect to control for protein was less compared to lipid and carbohydrate and the maximum depletion was at 0.01 ppm Hg medium. The effect of mercury was more in Penaeus indicus than that of Penaeus monodon, the female species and prespawning stage.
Liver-lipid deleteriously affected the female P. indicus during spawning while carbohydrate affected it prominently during preparatory stage. Hg concentration of 0.01 ppm had much damaging effect on liver. The change caused due to test solutions in the biochemical constituents of the liver of the prawns indicate that female had more affected than male.
Non-degradable heavy metals are regarded as hazardous to the aquatic ecosystem for their environmental persistence and their ability for bioaccumulation. Mercury and its compounds are fairly known to be nonessential but highly toxic for living organisms, even at low concentration.
Distribution and transport of this metal in an aquatic ecosystem depend on different chemical forms.
Mercury, which enters the aquatic environment mostly in its inorganic form, may undergo transformation depending upon the microbial, chemical and physical characteristics of the ecosystem1. It is also known that Hg is biomagnified through the aqueous food chain2. Almost all the proteins and enzymes are potential target of the mercurials3. It has been reported that heavy metals affect various biochemical parameters of the liver of fish4,5. Significant input of mercury into the Rushikulya estuary from the geochemical processes as well as from industrial effluents from a nearby chlor-alkali plant, and its effect on the biota were studied earlier6,7. Although substantial amount of work has been carried out on mercurial effect on marine organisms in India7-9, very little information is available on this aspect in prawns of Rushikulya estuary. Because of increased mercurial
contamination in estuarine environment, the present investigation was aimed at understanding the biochemical changes due to Hg stress in estuarine penaeid prawns.
Materials and Methods
The effect of HgCl2 (manifested at 0.005 and 0.01ppm level) on biochemical constituents such as protein, lipid and carbohydrate in the liver of estuarine penaeid prawns like Penaeus monodon Fabricius and Penaeus indicus H. Milne Edwards (Crustacea: Penaeidae) was studied. The prawns were collected during 1997-98 from a relatively clean site of Rushikulya estuary (lat. 19°22’-19° 24′
N and long. 85° 02′-85° 05′ E) in the vicinity of a chlor-alkali plant at Ganjam, Orissa. Prawn specimens were collected during high tide period using Chinese dip/cast nets with the help of local fishermen and transported to the laboratory in live condition in polyethylene bags containing estuarine water. The adult specimens having length of 65.3 ± 2.1 mm and weight of 52.91 ± 3.15 g female. Liver of both the specimens were collected, washed and kept frozen till the analyses for the determination of initial protein, lipid and carbohydrate contents at environmental condition. In the present context, liver is considered as it provides better indication of the mercury toxicity than any other organ of teleostean
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∗For correspondence.
bijay_sahu@yahoo.com
fish Channa punctatus10. The habitat water quality parameters like temperature, pH, DO and salinity were determined by standard methods11. The prawns were acclimatized to laboratory condition at temperature 25 ± 2°C, pH 7.0 ± 0.1, salinity 20 ‰ and dissolved oxygen 5.1 ± 0.1 mg l-1. During acclimatization organisms were fed with mercury free artificial food to avoid cannibalism. The acclimatization period was seven days. Both the newly moulted species selected as test animals were exposed to the desired medium of HgCl2. The duration of exposure to HgCl2 was 6 days during different stages of reproduction i.e. I-preparatory (September-October), II–prespawning (December- January), III-spawning (February-March) and IV- postspawning (April-May). The different stages of reproduction were determined by macro and microscopic examination of animals12.
Stock solution was prepared by dissolving analytical reagent grade HgCl2 in distilled water. The test medium concentration was selected on the basis of Hg concentration present in the habitat of test organisms. Then the desired test solutions (0.005 and 0.01 ppm) were prepared by double filtered estuarine water (with below detection limit of Hg), following dilution technique13. The control was maintained in double filtered estuarine water without adding any mercury. All tests were carried out in glass aquarium of 20-liter capacity and fitted with aerators. The physico-chemical conditions of the test solution in the aquariums were maintained. The concentrations of metal were kept steady following standard methods14 for 50% static renewal bioassay. Both control and experimental animals were also fed as during the acclimatization period. Test solutions and waters were renewed before feeding. Each experiment was maintained in duplicate. After 3rd and 6th day of exposure, males and females from each aquarium were sacrificed for collection of their liver. These were washed in 0.6% saline solution and kept frozen till biochemical analyses. Subsequently the organs
were dried at 70°C in a hot air oven, grounded into powder using mortar and pestle, and weighed.
Amongst the biochemical constituents, lipid was estimated by methanol–chloroform method15, carbohydrate was estimated colorimetrically using phenol-sulphuric acid method16 and protein was estimated using Biuret method17. All biochemical estimations were conducted by UV-VIS spectrophotometer (ELICO, CL-54). The values were expressed in mg g-1 dry weight basis. Each value represents the mean value of three samples analysed.
Data were analysed using Students’ t-test for significance level.
Results and Discussion
The water quality parameters like temperature, pH, DO and salinity of the study area during Sept. 1997- May 1998 are presented in Table 1. Water temperature varied between 16.3 to 30.7°C, while salinity fluctuated between 5.9 to 29.7 ‰. Values of DO and pH ranged from 3.2 to 8.5 mg l-1 and 5.6 to 7.8 respectively. These data demonstrated the tropical characteristics of the estuary7.
The initial liver biochemical constituents (protein, lipid and carbohydrate) measured prior to acclimatization at environmental condition are presented in Table 2. Protein content (632-806 mg g-1) was highest among the biochemical components and a similar finding was reported earlier4,18. In Penaeus indicus the levels of lipid and carbohydrate were higher when compared to P. monodon. Female members were rich in protein and lipid compared to male members experimented.
Protein
Fluctuation of protein in the liver of prawns at different reproductive stages, after exposure of 3 days and 6 days to different concentrations of mercury chloride, is depicted in Fig.1. During the period of exposure, liver-protein content for P. monodon at control, 0.005 and 0.01 ppm Hg ranged from 646.3 to
Table 1⎯Physico–chemical characteristics ( X±SD, n=3) of Rushikulya estuary.
Experimental period Temp (°C) pH DO (mg l-1) Salinity (‰) I (Sept-Oct) 25.1 ± 1.2 6.2 ± 0.6 7.3 ± 1.2 6.7 ± 0.8 II (Dec-Jan) 18.6 ± 2.3 7.2 ± 0.3 5.3 ± 0.8 17.2 ± 1.2 III (Feb-Jan) 24.4 ± 2.1 7.7 ± 0.1 4.6 ± 0.5 23.4 ± 1.1 IV (Apr-May) 29.1 ± 1.6 6.9 ± 0.4 3.4 ± 0.2 28.3 ± 1.4 (16.3 – 30.7)* (5.6 – 7.8)* (3.2 – 8.5)* (5.9 – 29.7)*
*Range of physico-chemical characteristics
805.7, 602.9 to 778.4 and 574.1 to 724.3 mg g-1 respectively for male specimens, while for female species, it ranged from 665.9 to 8312, 611.9 to 789.7 and 603.7 to 771.3 mg g-1 respectively. Further at control, 0.005 and 0.01 ppm Hg these ranged from 641.5 to 801.3, 568.5 to 716.8 and 529.5 to 693.6 mg g-1 for P. monodon (male) while for female these ranged from 664.2 to 808.1, 572.9 to 729.7 and 548.9 to 711.7 mg g-1 respectively.
For both male and female organisms, liver-protein values were always higher at III or IV stages of reproduction but lower in stages II or I. Penaeus monodon exhibited higher content compared to P. indicus at both the concentrations of test solution.
Thus P. indicus was said to be more affected. Control values remained highest (831.2 mg g-1 in stage IV of female P. monodon) and 0.01 ppm Hg values were least (529.5 mg g-1 in stage I of male P. indicus) in contrast with the species and sex. This could be attributed to the reaction of mercury with -SH group on the cell membrane and impair its function at higher concentration19. Protein content indicated decline with the increase of time period incase of both the organisms irrespective of their sex, stages of
reproduction and exposure medium. This may be due to the recurring stress on the organisms with the increased concentration of the medium20. The control value increased from the initial environmental protein content of the prawns (Table 2). This could also be attributed for fair metabolic activities resulting from qualitative management of the control. But, the exposure medium liver-protein values decreased from initial protein contents. The change over of protein values from control in 3rd and 6th days’ of exposure are given in Table 3. Maximum depletion of protein level was observed during preparatory stage in male (14.7%) and female (18.7%) for P. monodon. Penaeus indicus depleted maximum during prespawning stage in male (19.4%) and in female (21.9%). At 0.005 ppm Hg, the protein content was occasionally found elevated during 3rd day in different stages (I, IV) for both the organisms. On the other hand, 6th day exposure values always showed depletion due to longer period of exposure. Increase of concentration of test solution led towards lethality to the organisms8, with decreased values during 6th day. This trend might be due to accelerated rate of accumulation with the increase of exposure medium5. These values for 6th day exposure
Fig. 1⎯Fluctuation of protein (mg g-1) in the liver at different stages of reproduction of penaeid prawns on 3rd and 6th day of exposure at desired concentrations of mercuric chloride.
were always higher compared to 3rd day values, stating much stress on the test animals. The change over in liver-protein values was compared using Students’ t- test. These results were not significant at 0.05 level, except for female P. indicus on 6th day (t = 0.39120, p<0.05). Penaeus indicus liver protein was much
affected as compared to P. monodon and was found to be more on females at 6th day observation in prespawning stage at higher exposure medium.
Lipid
Figure 2 depicts the lipid content in liver of the
Table 2⎯Initial values (X ± SD, n=3) bio-chemical constituents in the penaeid prawns from Rushikulya estuary Specimens Reproductive
stages
Protein (mg g-1)
Lipid (mg g-1)
Carbohydrate (mg g-1)
P.monodon I 632±5.2 716±6.4 80±1.0 96±1.5 38±1.3 32±1.0
II 671±6.3 684±5.6 81±1.3 86±1.2 35±2.1 33±0.5
III 763±8.1 765±7.2 103±2.1 106±2.4 44±1.5 42±2.4
IV 745±10.2 806±8.5 92±1.8 112±1.6 55±0.8 41±1.1
P.indicus I 615±5.6 675±6.8 84±1.2 99±1.8 40±1.4 33±0.7
II 640±6.1 650±4.6 88±0.8 90±1.6 36±1.8 34±1.6 III 725±7.8 756±5.9 109±2.2 114±2.7 49±1.2 43±0.3 IV 705±9.1 781±7.2 108±2.8 122±3.1 60±1.6 41±0.8 Table 3⎯The 3rd and 6th day biochemical changeover values (%) with respect to control after six days of exposure to
desired test solutions of mercuric chloride.
Penaeus monodon Penaeus indicus
0.005 ppm Hg. 0.01 ppm Hg. 0.005 ppm Hg. 0.01 ppm Hg.
Reproductive stages
3rd day 6th day 3rd day 6th day 3rd day 6th day 3rd day 6th day Protein
Male
I 4.3 10.4 9.3 14.7 9.8 13.4 16.6 19.4
II 3.1 7.4 7.9 13.8 10.2 15.2 12.8 18.7
III 3.4 7.2 7.9 12.1 8.4 11.4 9.8 13.4
IV -0.2 10.1 8.4 12.7 9.6 12.8 12.6 16.0
Female
I 4.4 6.8 6.7 18.7 -0.3 14.3 16.7 21.9
II 6.5 11.0 7.9 13.7 11.1 15.6 10.2 13.2
III 5.9 9.7 7.2 11.2 11.4 12.1 13.2 13.8
IV 4.5 7.6 6.7 9.5 7.9 11.1 10.2 13.2
Lipid Male
I 15.0 23.1 30.5 34.7 8.0 21.8 24.5 32.5
II 14.4 19.4 20.5 28.3 10.4 18.4 23.2 31.3 III 14.7 23.8 30.4 39.4 13.8 22.1 32.3 36.7
IV 0.3 8.4 4.2 12.9 1.0 11.6 3.8 14.7
Female
I 13.3 26.4 31.9 41.9 5.3 17.3 31.5 38.3
II 6.3 23.6 8.3 25.5 4.2 15.9 6.7 18.0
III 12.4 29.8 28.8 38.2 9.6 17.2 33.5 41.4
IV 11.3 14.7 17.8 20.8 4.7 9.7 11.1 18.1
Carbohydrate Male
I -1.0 18.7 9.4 24.9 5.7 24.7 11.0 29.3
II 3.1 19.3 9.6 21.4 8.4 23.4 9.7 24.1
III -0.6 9.0 5.7 18.7 2.4 13.8 6.1 20.9
IV 6.2 20.5 14.3 27.3 14.2 27.1 21.8 33.3 Female
I 3.6 18.3 8.5 25.0 6.3 23.7 28.2 49.4
II 2.3 17.6 8.9 23.4 -4.0 31.5 18.9 45.7
III -1.0 7.8 6.2 17.2 1.8 12.6 16.7 32.0
IV 3.0 14.7 6.1 22.4 11.9 15.7 26.2 36.9
“-” Indicates elevation (%) and others are depletion.
two studied animals. The lipid content in control varied from 90.8 to 122.6 (male) and 91.6 to 128.4 (female) and 91.5 to 126.2 (male) and 93.1 to 130.9 (female) mg g-1 for P. monodon and P. indicus respectively. At 0.005 ppm concentration, it ranged from 72.7 to 102.9 (male) and 78.3 to 112.5 (female) mg g-1 for P.
monodon and from 75.7 to 108.6 (male) and 82.5 to 121.8 (female) mg g-1 for P. indicus. Whereas, at 0.01 ppm Hg, it ranged from 61.7 to 98.9 and 65.3 to 105.5 mg g-1 for males and from 63.3 to 104.2 and 68.7 to 113.6 mg g-1 for females of P. monodon and P. indicus respectively. These data revealed that the increment of lipid content was inversely related to the increase of concentration of test solution.
Amongst the liver-lipid content of both the sexes at control, females were higher as compared to males at different reproduction stages for the test animals. A similar trend for liver-protein was noticed for 0.005 and 0.01 ppm in case of control. From the inter-species comparison it was also observed that P. monodon had fewer lipids as compared to P. indicus at every stage of reproduction indicating a just reverse trend to that of liver-protein. However, similar result was observed for period of exposure (3rd and 6th day) as much as protein.
The liver-lipid values at control increased, while declined in 0.005 and 0.01 ppm Hg medium for the test animals, as compared to that of the study environment (Table 2). During spawning at 0.01 ppm Hg, lipid content significantly reduced not only from control but also from the initial values at study environment4. The changeover in lipid content from control for the test animals are presented in Table 3. The depletion percentage was maximum in 6th day analysis and minimum in 3rd day analysis at both the test medium.
The maximum depletion (41.9%) for P. monodon (female) was worked out on 6th day analyses at 0.01 ppm. The changeover in lipid values in P. monodon was significant in male (t = 0.21220, p<0.05) on 3rd day and female (t = 0.23853, p<0.05) on 6th day. This clearly indicates that P. monodon could deleteriously be affected by test solution. Inter-stage depletion has not followed any specific order for both the sexes of the test animals. The depletion of lipid content may be due to decline in the lipid synthesizing capacity21. Carbohydrate
The fluctuation of carbohydrate content in the liver of both prawns is depicted in Fig. 3. In male
Fig. 2⎯Fluctuation of lipid (mg g-1) in the liver at different stages of reproduction of penaeid prawns on 3rd and 6th day of exposure at desired concentrations of mercuric chloride.
the maximum 68.7 mg g-1, minimum 31.3 mg g-1 and in female the maximum 54.7 mg g-1, minimum 30.1 mg g-1 carbohydrate were recorded on 6th day at control of P. indicus and on the same day at 0.01 ppm Hg of P. monodon respectively. During the test carbohydrate variation indicated that 3rd day observations were higher and 6th day were lower in case of each test animal. Higher medium marked low carbohydrate content than that of lower medium irrespective of sexes and specimens. The males were higher compared to female in both the concentrations of test solution during any time period of exposure. In P. indicus carbohydrate content was found to be more than P. monodon.
The control values were elevated with the increase of time. However, within the species and sexes, male and female attained highest at stage III and IV respectively for each of the animals under study.
This observation in control differed for protein and lipid in control. The observations in test mediums were different from the observations for protein and lipid of same medium. The levels of carbohydrate in control and during 3rd day in test medium (sometimes) were shown to be elevated when
compared with that of the initial liver-carbohydrate (Table 2). This could be the reason for availability of food in the environment21. The elevation of carbohydrate was noticed during 3rd day observation at 0.005 ppm (Table 3) because the concentration medium was almost safe level of Hg.
In this period depletion recorded was minimum (0.5%) in male P. monodon at stage I and maximum (49.4%) during 6th day in female P. indicus at stage I. The change over values for carbohydrate in P. indicus was noticed in male on 3rd (t = 0.33121, p<0.05) and 6th (t = 0.29025, p<0.05) day. As the concentration of the test solution raised, the percentage of depletion also raised with the increase of time period of exposure, but between the stages no such particular relation could be established.
In general livers of the studied prawns were proteinous (maximum 831.2 mg g-1)at control. The control values for every biochemical components were higher during 6th day of experiment as compared to 3rd day, but reverse was found at test medium.
Penaeus monodon liver has higher protein contents than P. indicus, but reverse was noticed in lipid and
Fig. 3⎯Fluctuation of carbohydrate (mg g-1) in the liver at different stages of reproduction of penaeid prawns on 3rd and 6th day of exposure at desired concentrations of mercuric chloride.
carbohydrate contents. Female biochemical content remained higher in case of protein and lipid while for male it remained higher in carbohydrate. Overall the liver-protein was less depleted as compared to liver- lipid and carbohydrate22.
In conclusion it could be stated that the liver protein had more damaging effect during prespawning stage in female than male of the both studied prawns, while liver lipid was deleteriously affected in female P. indicus during spawning.
However effect of test solution was more prominent in liver carbohydrate of female P. indicus during preparatory stage. Of the two sexes female was more affected than the male.
Acknowledgement
The authors are thankful to the HOD, Department of Marine Sciences, and Berhampur University authority for providing laboratory facilities.
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