Toxic Effects of Morpholineum-4-Morpholine Dithiocarbamate and Heavy metals ( Cu and Zn ) On the Indian Major Carp Catla catla : A Biochemical Analysis

The present investigation intended to evaluate the independent and combined toxicological effects of synthetic fungicide Morpholine derivative of dithiocarbamate (MMDTC) and two metallic compounds viz. copper sulphate and zinc sulphate on the biochemical constituents like total proteins, total sugars and total lipids in the muscle, liver, brain and kidney of Catla catla. Biochemical studies were carried out in both the control (untreated) healthy fish and the fish exposed to sub-lethal concentrations of MMDTC, CuSO4, ZnSO4, MMDTC +CuSO4,, MMDTC + ZnSO4 and CuSO4+ ZnSO4 on 4th ,7th ,14th, 21st, and 28th day of exposure.


INTRODUCTION
General health of the fish is influenced by the physiological activities occurring in the body.The metabolic activities in the liver are crucial in determining the physiological state of the organism.Any toxic substance reaches soon the liver and causes deleterious effect resulting in disturbed physiological activities.The total carbohydrate, protein and lipid contents of the tissues are crucial for the normal health of the organism because there appears a balanced distribution of these three biochemical constituents; a reduction in one should be compensated by others.If not, the entire metabolic processes become abnormal showing its effects on various physiological activities and may even lead to death.Due to this reason, studies on tissue biochemistry have formed basic necessity to evaluate the health profiles of the organisms under stress.
Dithiocarbamate fungicides are used generally as seed dressing and protection of fruit, vegetable and ornamental crops from a variety of fungal diseases 1 .In an aquatic system pesticide concentration increases as it moves up the food chain because of biomagnifications and the animals accumulate non-biodegradable pesticides from the corresponding environment 2 .Pesticides acting as stress factor affecting the fish has been well documented 3 .
Copper and Zinc are essential trace elements required for the body, but at higher level toxic in nature.Exposure to copper can induce stress responses such as changes in the fish's ion regulation, olfaction and swimming performance 4,5 .A significant reduction in three biochemical constituents viz.protein,lipid and sugars of Tilapia under the influence of zinc has also been reported 6 .Several investigators have reported the toxicological effect of zinc in decreasing the protein content of common carp 7,8 .Elevated levels of heavy metals can cause death and mutation in animal populations.
In an aquatic environment the pollutants reach easily as runoff from pesticide residues and heavy metals like copper, mercury, nickel, lead and zinc pollute the aquatic system through industrial and municipal wastes.In such a scenario, the metal compounds react with the chemical pesticides to form complexes that may have entirely different properties than the original one.
The toxic kinetics of the pollutant may inflict some pathogenic effect on the organism depending on the rate of metabolic transformation and elimination.There is paucity of information on how the fish reacts to cumulative toxic synergism.
Hence an attempt has been made in the present study to know the effects of a synthetic fungicide, Morpholineum-4-Morpholine Dithiocarbamate (MMDTC), and two heavy metal compounds, Copper sulphate (CuSO 4 .5H 2 O) and Zinc sulphate (ZnSO 4 ) acting independently and combined on the important biochemical constituents of one of the commercially important Indian Major Carp, Catla catla.

Maintenance of fish Stock and feeding
Catla fingerlings of the same size (10-12 cm in length and 6-8 g in weight ) were procured from culture ponds of Tamil Nadu Fish Seed Farm, Poondi, Thiruvallur district and brought to laboratory in oxygen packs.The fish were acclimatized and maintained in ferro-cement tanks (3'L× 2'W×2'H) filled with bore water.The stock fish were fed with pelleted feed prepared with rice bran, groundnut oil cake, tapioca powder and mineral mixture 9 .The fish were fed daily with pelleted feed at 5% body weight in two split doses, in the morning and evening.Feeding was started one day after the fish were stocked and stopped 24 hr prior to experiment.

Selection of experimental Fish
Healthy fish without any observable pathological symptoms were chosen for the experiments and were maintained in disinfected glass aquarium tanks (2'L x 1'W x 1'H) filled with water at rate of 2 litres per fish.During the period of experimentation, the room temperature fluctuated from 30-32 0 C. The water used for the experiment had dissolved oxygen content of 4.4 -4.8 ml/l and salinity of 0.82 -0.84 ppm.The pH of water was in the range of 7.2 -7.4.

Procurement of toxicants
M o r p h o l i n e u m -4 -M o r p h o l i n e Dithiocarbamate (MMDTC) has insecticidal and fungicidal properties and used as a pesticide.It was prepared by mixing Morpholine and methyl alcohol in 1:2 proportions.Keeping the mixture in ice bath (<10 0 C), carbon disulphide is added drop by drop with continuous agitation.Later the mixture is evaporated in room temperature to get amorphous powder of MMDTC.
Copper sulphate (CuSO 4. 5H 2 O) 'AR' grade supplied by Sarabhai Merck and Zinc sulphate 'AR' Grade supplied by Qualigen were used for experiment.

Experimental Design
Fish were divided into groups of ten each and exposed to the different toxicants viz.MMDTC, CuSO 4 and ZnSO 4 independently and synergistically.In the synergistic toxicity experiment, the fish were exposed to two toxicants together (combined synergism).

Experimental Groups
Fingerlings of Catla catla were treated with pesticide and heavy metals individually and synergistically to determine 96 hr LC 50 values for the test toxicants.Groups of fish were maintained in separate tanks and considered as experimental groups which were categorized as follows: Group I: Independent Toxicity 1.

Determination of LC 50
To determine LC 50 for the different groups based on the cumulative percentage mortality at the end of 96 h of experimentation the standard graphic method was followed.One fourth of LC 50 values obtained from the above experiments was taken as the sub-lethal concentration (SLC).Apparently normal and healthy catla fingerlings were exposed to respective SLC of MMDTC (DTC), CuSO 4 , ZnSO 4 for independent toxic analysis, DTC + CuSO 4 , DTC + ZnSO 4. CuSO 4 + ZnSO 4 for combined toxic analyses.Suitable controls were maintained in normal bore well water for all the experimental groups, without dissolving the chemicals.

Biochemical Analyses
Biochemical parameters like total carbohydrates, total proteins and total lipids were analyzed on zero day control and on 4 th , 7 th , 14 th , 21 st and 28 th day after exposing the fish with the toxicants in the experimental groups of independent toxic exposure and combined synergistic toxic exposure.
For the third group involving successive synergistic toxic exposure, these analyses were done on zero day and 7 th day post exposure.
Four different tissues Muscle, Liver, Brain and Kidney were dissected out carefully and weighed using K-Roy Single pan electrical balance for biochemical investigation.The dissected tissues were kept in an ice box till taken out for homogenization.
Colorimetric method was followed for the biochemical analyses using Spectronic-21 (Bausche and Lomb) spectrophotometer.Total sugars was estimated by anthrone method 10 and the total protein content in the tissues was done by folin phenol method 11 while for the estimation of lipids the method of Bligh and Dyer 12 was followed.

Statistical Analysis
The data obtained were subjected to statistical analysis to arrive at Arithmetic Mean, Standard Deviation and standard error of mean using statistical package software SPSS version 16.To test the significance of differences observed between control and experimental groups, the data was subjected to students 't" test.
The total sugar (Table 1), protein (Table 2) and lipid (Table 3) content in the muscle, liver, brain and kidney tissues of C. catla healthy untreated and treated with MMDTC, CuSO 4 and ZnSO 4 for independent toxic effects and with MMDTC and CuSO 4 , MMDTC and ZnSO 4 and CuSO 4 with ZnSO 4 for syngergistic toxic effects were recorded at 4 th , 7 th , 14 th , 21 st and 28 th days.
The mean value of the carbohydrate content in muscle of the fishes treated with MMDTC was significantly higher on 7 th day, when compared to control group.In liver the elevated level of the sugars was noticed on 21 st day (p<0.001).A significant increase in levels of sugar content were observed in brain and kidney tissues on 14 th , 21 st and 28 th day.The heavy metal CuSO 4 exposed fish recorded a significant increase of the sugar content in muscle on 4 th and 7th day.In liver a higher significant value of the average was recorded on 7 th and 14 th day.A significant increase over the values of control fish on 4 th , 7 th and 28 th day was observed in the sugar content of brain and kidney.When the fishes were exposed to ZnSO 4 the sugar content of muscle and liver was greatly reduced on all the exposure groups except on 7 th day, where the significant increase was observed.However, a significant increase were recorded on 4 th and 21 st day in the brain tissues and 4 th and 7 th day in the kidney tissues.MMDTC when combined with CuSO 4 , and ZnSO 4 revealed the reduction of the sugar content in the muscle tissues on 4 th , 7 th and 14 th day.However, in liver tissues a significant increase in the average of the sugar content was observed on 14 th , 21 st and 28 th day in fishes exposed to MMDTC with CuSO 4 , while MMDTC with ZnSO 4 brought about a significant decline on 4 th , 7 th and 21 st day.In brain and kidney tissues the synergistic effect of MMDTC and CuSO 4 revealed significantly elevated level of sugars on 4 th , 21 st and 28 th day,while MMDTC with ZnSO 4 significantly decreased the sugar content in kidney tissues on 21 st day.Exposure of CuSO 4 in addition to ZnSO 4 resulted in a significant decrease in the carbohydrate content of the muscle and liver tissue on 4 th , 7 th and 21 st day.The additive effect of these two compounds effected elevated level of carbohydrates from 4 th to 14 th day in the brain and kidney.
The Protein content in the muscle of catla fingerlings exposed to MMDTC was significantly decreased on 4 th day and 28 th day while in the liver, brain and kidney tissues of treated fish showed a significant increase in the protein content on all the exposure periods.The heavy metal CuSO 4 treated fish revealed significant depletion of protein on 4 th and 21 st day in the muscle, liver, brain tissues and on 4 th day in the kidney tissues.A reduction in the protein content was recorded in the muscle and liver tissues of ZnSO 4 exposed fish on 14 th and 28 th days, while in brain and kidney tissues an increase was observed on 7 th .21 st and 28 th day.
The protein content in the muscle and liver tissues of combined toxic synergistic group MMDTC and metals viz.CuSO 4 and ZnSO 4 reduced on 4 th day and continued to rise on subsequent days up to 21 st day and again dropped on the 28 th day.In brain and kidney tissues the protein content of MMDTC+CuSO 4 exposed group showed a significant decrease on the 4 th day and MMDTC with ZnSO 4 group showed a significant increase from 7 th to 21 st days.When the two metallic compounds were combined together the fish recorded progressively increased protein content in all the tissues viz.muscle, liver , brain and kidney compared to the control.
Exposure to the toxicants MMDTC, CuSO 4 and ZnSO 4 independently and in combination with one another had effected a very significant reduction in the lipid content in all the tissues on the early period of exposure (4 th day).However, the lipid content of the muscle on the 7 th day decreased in the fish exposed to MMDTC independently and in combination with CUSO 4 and ZnSO 4 while an increased trends was observed in groups of fish treated with the heavy metals both independently and combined together.Contrastingly,the lipid content of liver showed a decreasing trend in the fish exposed to CuSO 4 and ZnSO 4 together on 21 st and 28 th day.The lipid content of brain and kidney tissues showed significant elevation in the lipid content in all the treated groups on all the exposure periods.

DISCUSSION
Pesticides are mainly used against pests of crop and disease causing vectors, but their improper use in agricultural practice has posed a serious threat to human life and his environment.Any variation in the environment acts as a stress on the organisms.When a pesticide or any pollutant reaches the aquatic ecosystem, the fish are exposed to severe stress and as a natural instinct, the fish tend to adapt themselves by reacting suitably to overcome the stress.When two toxicants act together, the stress shall be more and severe and cause irreparable damage.
In the present investigation, total sugar content in the muscle,liver, brain and kidney revealed a mixed trend in the different experimental groups and different days of the exposure.There was an immediate fall in the sugar content of the 4 th day exposed fish in the muscle and liver, showing a state of recovery on the 7 th day.The recovery trend was normal in DTC exposed fish, and slow in the CuSO 4 treated fish.In the synergistic experimental groups, recovery occurred from 21 st day onwards.In the brain and kidney of the 4 th day experimental groups the sugar content was more compared to control groups, which gradually reduced on 7 th day.The increased or decreased trend was more pronounced in the groups of fish exposed to CuSO 4 independently and synergistically with MMDTC and ZnSO 4 .This shows the severe toxic nature of CuSO 4 both independently and in combination with others.
Protein showed a trend of increasing and decreasing activity in the different experimental groups.As far as lipids were concerned there was a reduction in all the tissues of 4 th day experimental group showing trends of recovery on further days of exposure.Increase in the protein content was in accordance with the reduction of sugar content and lipid content suggesting gluconeogenic pathway utilizing lipid and proteins to compensate for the loss of sugars.
Depletion of tissue proteins in fishes exposed to various pesticide toxicants have been reported by many workers.Further, it has been reported that acute or chronic treatment of pesticides cause biochemical alteration in the organs involved in detoxification mechanisms 13,14,15 .Decreasing trend in total proteins was reported in the liver,brain and gill tissues of C. catla under sub lethal and lethal concentrations of fenvalerate 16 .A significant decrease was reported in the protein content in almost all tissues in Ctenophayngodon idellus when exposed to sub lethal and lethal concentrations of both the technical and 20% E.C. formulations offenvalerate 17 .Break down and synthesis of protein proceeds simultaneously in all the tissues.But during pesticide stress breakdown of protein occurs which acts as an alternative source of energy.The fish exposed to toxic stress stimulates protein metabolism 18 .During protein metabolism the removal of amino group from different amino acids was observed suggesting the elevated levels of amino acids in the fish exposed to pesticide 19 .
The loss of protein in the tissues of fish exposed to heavy metal stress may be due to excessive proteolysis to overcome the metabolic stress.Heavy metals may alter the protein concentration through impairing the synthesis and metabolism of protein, DNA and RNA as well as by altering the activity of lysosomal enzymes.It is possible that pollutant stress influences the conversion of tissue proteins into soluble fractions reaching the blood and hence, decrease in protein content might have observed in the tissues of liver and muscle.The metal binding protein usually binds the metal ions,preventing them from exerting toxic effects through binding to enzymes or other sensitive sites.However, if the rate of influx of metals into the cell exceeds the rate at which metallothionein or metallothionein-like proteins can be synthesized, there may occur a"spill over" of metals from the metallothionein or metallothionein like proteins due to the displacement of essential metals from metalloenzymes by non-essential metals 20 .
The changes observed in the protein content in different days of exposure may be due to the influence of exogenous factors like toxic environment.The loss of protein under stress to long period may be attributed to the utilization of amino acid in various catabolic reactions.Another probability is that there might have occurred blocking of the protein synthesis and proteolysis on exposure to chronic period of stress condition.Similar observations of decrease in the protein content were reported in the muscle of Channa punctatus treated with heavy metal 21 , in the muscle of Puntius stigma after exposure with pesticides , 22 , in the muscle, liver and intestine of Cyprinus carpio treated with textile mill effluent 23 , in the muscle of Oreochromis mossambicus due to the effect of phenol 24 , in Etroplus maculates exposed to Ekalux 25 and in Anabas testudineus under the influence of alloxan monohydrate 26 .
Heavy metals are metabolic inhibitors of animals.They exert toxic effects in the organism at tissue, cellular, sub-cellular and molecular levels.At molecular levels metals interact with protein leading to denaturation, precipitation, allosteric effects or enzyme inhibition.Depletion in total protein could be due to augmented proteolysis and possible utilization of their product for metabolic purpose, decline in protein content may be related to impaired food intake, increased energy cost of homeostasis,tissue repair and detoxification mechanism during stress 27. .

CONCLUSIONS
The study clearly indicates that the toxic effect of pollutants or toxicants will be more