Priya P and Sujatha L. B.

Department of Zoology, Pachaiyappa’s College, Chennai-600030. India.

Corresponding Author E-mail: priyaponmudi79@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/1341

Abstract

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, CuSO_4, ZnSO_4, MMDTC +CuSO_4,, MMDTC + ZnSO₄ and CuSO₄+ ZnSO₄ on 4^th, 7^th, 14^th, 21^st, and 28^th day of exposure.

Keywords

Catla Catla MMDTC; CuSO₄; ZnSO₄; Synergistic Toxicity;

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Copy the following to cite this article: Priya P, Sujatha L. B. Toxic Effects of Morpholineum-4-Morpholine Dithiocarbamate and Heavy Metals (Cu and Zn) on the Indian Major Carp Catla Catla: A Biochemical Analysis. Biomed Pharmacol J 2017;10(4).

Copy the following to cite this URL: Priya P, Sujatha L. B. Toxic Effects of Morpholineum-4-Morpholine Dithiocarbamate and Heavy Metals (Cu and Zn) on the Indian Major Carp Catla Catla: A Biochemical Analysis. Biomed Pharmacol J 2017;10(4). Available from: http://biomedpharmajournal.org/?p=18305

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 (CuSO₄.5H₂O) and Zinc sulphate (ZnSO₄) acting independently and combined on the important biochemical constituents of one of the commercially important Indian Major Carp, Catla catla.

Materials and Methods

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 30-32⁰C. 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

Morpholineum-4-Morpholine 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 (<10⁰C), 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. 5H₂O) ‘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, CuSO₄ and ZnSO₄ 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 LC₅₀ 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

MMDTC (DTC)

Copper sulphate (CuSO₄)

Zinc sulphate (ZnSO₄)

Group II: Combined Synergism

DTC + CuSO₄

DTC + ZnSO₄

CuSO₄ + ZnSO₄

Determination of LC₅₀

To determine LC₅₀ 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 LC₅₀ 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), CuSO₄, ZnSO₄ for independent  toxic analysis,  DTC + CuSO_{4  ,}DTC + ZnSO_4.  CuSO₄ + ZnSO₄ 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 method¹⁰ and the total protein content in the tissues was done by folin phenol method¹¹ 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.

Results

The  analysis of 96 h LC₅₀ by  arithmetic graphic method, showed   16 ppm (Fig. 1),  4.8 ppm (Fig.2) and  160 ppm (Fig.3) concentrations of MMDTC, CuSO₄ and   ZnSO₄  respectively. Further, the SLC (1/4^th of LC₅₀ value) for MMDTC, CuSO₄  and   ZnSO₄ were calculated as 4 ppm, 1.2 ppm and 40 ppm respectively for independent toxicity.

Figure 1: 96 hr LC50 for MMDTC   Click here to View figure

 

Figure 2: 96 hr LC50 for CuSO4   Click here to View figure

 

Figure 3: 96 hr LC50 for ZnSO4   Click here to View figure

 

The LC₅₀ value for synergistic toxicity  were calculated as 10ppm of MMDTC with 4.4ppm of CuSO₄  (Fig.4), 5ppm of MMDTC and 24ppm of ZnSO₄ (Fig.5) and 2.6 ppm of CuSO₄ with 10ppm of ZnSO₄ (Fig. 6). ¼ ^th  these values were calculated as SLC.

Figure 4: 96 hr LC50 forMMDTC + CuSO4   Click here to View figure

 

Figure 5: 96 hr LC50 for MMDTC + ZnSO4   Click here to View figure

 

Figure 6: 96 hr LC50 for CuSO4+ ZnSO4   Click here to View figure

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, CuSO₄  and   ZnSO₄  for independent toxic effects and with MMDTC and CuSO₄, MMDTC and ZnSO₄ and CuSO₄ with ZnSO₄ for syngergistic toxic effects were recorded at 4^th, 7^th, 14^th, 21^stand 28^th days.

Table 1: Independent and combined  toxic  effects of MMDTC,  CuSO_4, ZnSO₄ , MMDTC+ CuSO₄ , MMDTC + ZnSO₄ CuSO₄  + ZnSO₄on total sugar (Mg/G wet Wt) of Catla catla

Days of exposure	MMDTC (SLC-4ppm)
	Muscle	Liver	Brain	Kidney
Control	0.97 ± 0.11	26.25 ± 1.1	1.13 ± 0.15	2.86 ± 0.4
4th day	0.55 ± 0.13*	5.77 ± 2.13*	1.56 ± 0.1*	4.67 ±1.16*
7th day	2.18 ± 0.72*	26 ± 1.6a	2.38 ± 0.37*	1.87 ± 0.23*
14th day	0.45 ± 0.02*	26.35 ±  1.12a	3.05 ± 0.34*	6.65 ± 0.44*
21st day	0.57 ± 0.13*	31 ± 1.1*	2.26 ± 0.44*	4.22 ± 0.37*
28th day	1.07 ±  0.15a	21.55 ± 0.56*	3.91 ± 0.45*	3.21 ±0.3d
Days of exposure	CuSO4 (SLC-1.2ppm)
	Muscle	Liver	Brain	Kidney
Control	0.97± 0.11	26.25 ± 1.1	1.13 ± 0.15	2.86 ± 0.4
4th day	3.05 ± 1.25*	12.3 ± 3.01*	11.28 ± 2.55*	27.94 ± 2.7*
7th day	1.47 ± 0.28*	32.11 ± 2.45*	1.62 ± 0.23*	13.66 ± 0.88*
14th day	0.44 ± 0.02*	31.71 ± 1.25*	1.46 ± 0.3b	1.06 ± 0.09*
21st day	0.44 ± 0.03*	15.28 ± 0.48*	0.84 ± 0.03*	0.63 ± 0.03*
28th day	1.28 ± 0.27b	15.21 ± 0.52*	2.25 ± 0.2*	10.62 ± 0.8*
Days of exposure	ZnSO4  (SLC-40ppm)
	Muscle	Liver	Brain	Kidney
Control	0.97± 0.11	26.25 ± 1.1	1.13 ± 0.15	2.86 ± 0.4
4th day	0.88 ± 0.11a	9 ± 0.49*	2.46 ± 0.63*	3.16 ± 0.93a
7th day	1.09 ± 0.18a	32.98 ±1.9*	0.41 ± 0.02*	4.43 ± 0.56*
14th day	0.48 ± 0.08*	2.03 ± 0.42*	0.83 ± 0.05*	1.12 ± 0.1*
21st day	0.44 ± 0.02*	4.33 ± 0.52*	4.1 ±0.31*	2.05 ± 0.32*
28th day	0.51 ± 0.02*	3 ± 0.22*	0.44 ± 0.04*	1.02 ± 0.1*
Days of exposure	MMDTC+ CuSO4 (SLC-2.5ppm+1.1ppm)
	Muscle	Liver	Brain	Kidney
Control	0.97± 0.11	26.25 ± 1.1	1.13 ± 0.15	2.86 ± 0.4
4th day	0.65 ± 0.09*	3.55 ± 0.42*	3.09 ± 0.6*	4.53 ± 0.5*
7th day	0.46 ± 0.04*	5.96 ± 0.6*	0.47 ± 0.05*	0.76 ± 0.1*
14th day	0.45 ± 0.04*	34.32 ± 3.33*	1.15 ± 0.34a	1.15 ±0.36*
21st day	2.7 ± 0.6*	55.69 ± 2.3*	14.63 ± 2.3*	24.66 ± 1.97*
28th day	6.54 ± 1.75*	42.56 ± 5.81*	20.77 ± 4.03*	24.82 ± 4.78*
Days of exposure	MMDTC+ZnSO4  (SLC-1.25ppm+6ppm)
	Muscle	Liver	Brain	Kidney
Control	0.97± 0.11	26.25 ± 1.1	1.13 ± 0.15	2.86 ± 0.4
4th day	0.42 ± 0.05*	2.65 ± 0.6*	1.85 ± 0.59b	2.68 ± 0.49a
7th day	0.46 ± 0.05*	1.87 ± 0.47*	5.21 ± 0.4*	8.07 ± 0.64*
14th day	0.85 ± 0.08c	25.75 ± 2.5a	3.34 ± 0.51*	3.2 ± 0.51a
21st day	1.18 ± 0.52a	4.09 ± 0.54*	2.9 ± 0.5*	1.28 ± 0.28*
28th day	3.41 ± 0.66*	79.97 ± 1.95*	7.73 ± 1.4*	4.73 ± 1.04b
Days of exposure	CuSO4+ ZnSO4  (SLC-0.65ppm+2.5ppm)
	Muscle	Liver	Brain	Kidney
Control	0.97± 0.11	26.25 ± 1.1	1.13 ± 0.15	2.86 ± 0.4
4th day	0.44 ± 0.07*	2.86 ± 0.34*	3.8 ± 0.36*	15.65 ± 0.75*
7th day	0.44 ± 0.03*	23.4 ±1.6*	4.15 ± 1.21*	6.65 ±0.87*
14th day	5.83 ± 1.8*	36.27 ±3.64*	8.38 ± 1.9*	9.72 ± 1.85*
21st day	0.49 ± 0.08*	1.07 ±0.437*	0.5 ± 0.04*	0.76 ± 0.04*
28th day	3.07 ± 0.79*	9.9 ± 2.131*	2.13 ± 0.56*	1.01 ± 0.26*

Student t -Test  * P<0.001; ^a  P  N.S; ^b  P<0.01; ^c P<0.02; ^d p<0.05

Table 2: Independent and combined  toxic  effects of MMDTC, CuSO_4, ZnSO₄, MMDTC+ CuSO₄, MMDTC + ZnSO₄, CuSO₄  + ZnSO₄ on total protein (Mg/G wet Wt) of Catla catla

Days of exposure	MMDTC (SLC-4ppm)
	Muscle	Liver	Brain		Kidney
Control	85.38 ± 0.75	72.31 ± 6.8	53.71 ± 6.63		66.84 ± 4.28
4th day	80. 05 ± 3.79	106.21 ± 7.82	59.41 ± 6.22		131.46 ± 0.79
7th day	172.63 ± 2.74	154.23 ± 2.36	136.42 ± 1.84		194.73 ± 3.17
14th day	91.75 ±1.05	76.41  ±  1.33	60.25 ± 1.04		109.36 ±1.24
21st day	120.56 ± 1.59	117.84 ± 0.93	123.86 ± 1.09		203.17 ± 1.49
28th day	82.32 ±1.56	83.52 ± 1.92	94.85 ± 1.22		114.23 ±1.90
Days of exposure	CuSO4 (SLC-1.2ppm)
	Muscle	Liver	Brain		Kidney
Control	85.38 ± 0.75	72.31 ± 6.8	53.71 ± 6.63		66.84 ± 4.28
4th day	82.21 ± 2.018	38.21 ±1.41	27.52 ± 2.05		48.25 ± 1.18
7th day	92.08 ± 2.70	104.91 ± 2.61	87.04 ± 0.91		161.27 ± 1.29
14th day	96.88 ± 1.38	87.31 ±1.2	71.56 ± 0.91		121.39 ± 3.46
21st day	61.01 ± 1.27	55.95 ±1.5	50.17 ± 1.25		94.34 ± 2.89
28th day	118.79 ± 1.72	89.33 ±1.68	88.7 ± 2.17		166.31 ± 1.92
Days of exposure	ZnSO4  (SLC-40ppm)
	Muscle	Liver	Brain		Kidney
Control	85.38 ± 0.75	72.31 ± 6.8	53.71 ± 6.63		66.84 ± 4.28
4th day	99.78 ± 1.86	35.24 ±1.18	19.93 ± 1.54		8.61 ± 0.92
7th day	101.67 ± 0.58	112. 3 ± 1.37	65.93 ± 1.93		197.32 ± 1.2
14th day	30.44  ± 1.02	29.89 ± 0.84	64.51 ± 2.55		35.87 ± 0.6
21st day	65.69 ± 1.42	106.11 ± 2.25	102.28 ± 1.44		107.4 ± 0.99
28th day	72.01 ± 2.42	65.6 ± 2.01	75.12 ± 2.6		104.45 ± 2.8
Days of exposure	MMDTC+ CuSO4 (SLC-2.5ppm+1.1ppm)
	Muscle	Liver	Brain		Kidney
Control	85.38 ± 0.75	72.31 ± 6.8	53.71 ± 6.63		66.84 ± 4.28
4th day	30.79 ± 0.68	46.65 ± 1.2	32.01 ± 0.99		61.11 ± 0.82
7th day	77.64 ± 3.42	96.13 ± 2.5	93.52 ± 3.85		124.26 ± 4.19
14th day	107.32 ± 4.32	76.09 ± 5.13	83.81 ± 5.07		144.53 ± 5.25
21st day	152.95 ± 2.63	72.59 ± 4.3	60.83 ± 3.63		72.74 ± 2.83
28th day	144 ± 3.87	113.69 ±3.67	144.18 ± 7.62		144.08 ± 3.44
Days of exposure	MMDTC+ZnSO4  (SLC-1.25ppm+6ppm)
	Muscle	Liver	Brain	Kidney
Control	85.38 ± 0.75	72.31 ± 6.8	53.71 ± 6.63	66.84 ± 4.28
4th day	25.38 ± 0.66	71.67 ± 0.74	37.75 ± 1.25	117.15 ± 3.55
7th day	97.44 ± 3.21	128.22 ± 4.83	124.06 ± 4.58	125.65 ± 9.44
14th day	124.59 ± 2.84	87.21 ± 2.9	84.39 ± 3.16	114.36 ± 2.75
21st day	168.35 ± 3.2	125.23 ± 6.67	153.25 ± 4.71	112.84 ± 4.26
28th day	67.33 ± 1.343	90.15 ± 3.69	40.39 ± 2.88	100.06 ± 3.77
Days of exposure	CuSO4+ ZnSO4  (SLC-0.65ppm+2.5ppm)
	Muscle	Liver	Brain	Kidney
Control	85.38 ± 0.75	72.31 ± 6.8	53.71 ± 6.63	66.84 ± 4.28
4th day	102.84 ± 4.44	77.31 ± 1.05	48.59 ± 1.13	101.29 ± 0.74
7th day	108.62 ± 1.64	102.89 ± 1.56	82.39 ± 1.98	133.35 ± 3.68
14th day	120.08 ± 3.52	108.65 ± 4.26	108.48 ± 3.34	228.89 ± 5.46
21st day	174.88 ± 4.99	152.01 ± 6.69	161.79 ± 5.01	180.23 ± 5.12
28th day	112.35 ± 3.83	113.12 ± 3.58	101.5 ±2.28	96.3 ± 3.99

Table 3: Independent and combined  toxic  effects of MMDTC,  CuSO_4, ZnSO₄ , MMDTC+ CuSO₄ , MMDTC + ZnSO₄ CuSO₄  + ZnSO₄on total lipid (Mg/G wet Wt) of Catla catla

Days of exposure		MMDTC(SLC-4ppm)
		Muscle	Liver	Brain	Kidney
Control		31.62 ± 0.72	55.37 ± 1.81	42.11 ± 0.91	23.97 ± 1.21
4th day		8.63 ± 1.85 *	8.38 ± 1.08*	7.55 ± 1.39*	4.76 ± 0.55*
7th day		19.76 ± 1.17 *	102.56 ± 1.61*	85.24 ± 2.06*	143.19 ± 2.31*
14th day		61.61 ± 1.14 *	147.66  ± 1.39*	151.96 ± 1.23*	294. 81 ± 2.04*
21st day		118.4 ± 0.91 *	141.78 ± 1.59*	175.76 ± 1.79*	181.03 ± 1.26*
28th day		56.99 ±1.01 *	139.34  ± 1.38*	143.28 ± 1.81*	141.76 ± 1.36*
Days of exposure		CuSO4 (SLC-1.2ppm)
		Muscle	Liver	Brain	Kidney
Control		31.62 ± 0.72	55.37 ± 1.81	42.11 ± 0.91	23.97 ± 1.21
4th day		3.71 ± 0.24 *	33.75 ± 4.71*	10.3 ± 3.04*	2.7 ± 0.5*
7th day		114.67 ± 0.92 *	202.8 ± 1.77*	144.03 ± 2.17*	244.7 ± 4.71*
14th day		71.42 ± 0.85*	138.37 ± 0.85*	184.25 ± 5.72*	143.43 ± 1.59*
21st day		47.19 ± 0.84*	184.37 ± 1.67*	242.39 ± 1.62*	196.69 ± 2.41*
28th day		41.01 ±1.64 *	83.7 ± 2.67*	117.08 ± 1.53*	170.42 ± 1.34*
Days of exposure		ZnSO4  (SLC-40ppm)
		Muscle	Liver	Brain	Kidney
Control		31.62 ±0.72	55.37 ± 1.81	42.11 ± 0.91	23.97 ± 1.21
4th day		1.41 ± 0.25*	20.87 ± 0.86*	7.01 ± 0.13*	1.91 ± 0.29*
7th day		31.84 ± 0.72 a	110.92 ± 1.13*	181.44 ± 0.89*	85.44 ± 1.16*
14th day		47.48 ± 0.66 *	113.89 ± 1.78*	137.6 ± 1.25*	103.83 ± 1.34*
21st day		77.01 ± 1.21 *	131.23 ± 0.9*	242.16 ± 2.13*	191.13 ± 2.2*
28th day		76.13 ± 2.78*	99.93 ± 2.12 *	156.33 ± 3.16*	340.14  ± 1.31*
Days of exposure		MMDTC+ CuSO4 (SLC-2.5ppm+1.1ppm)
		Muscle	Liver	Brain	Kidney
Control		31.62 ±0.72	55.37 ± 1.81*	42.11 ± 0.91	23.97 ± 1.21
4th day		6.07 ± 0.12 *	27.84 ± 0.76*	5.13 ± 0.19*	3.11 ± 0.638*
7th day		27.18 ± 2.79 *	66.67 ± 2.06*	70.81 ± 1.95*	53.17 ± 2.63*
14th day		45.63 ±2.52 *	83.67 ± 1.82*	88.65 ± 3.58*	69.01 ± 2.67*
21st day		76.92 ± 3.29*	126.84 ± 5.47*	61.32 ± 4.71*	97.48 ± 3.41*
28th day		48.51 ± 4.49 *	96.24 ± 3.9*	172.52 ± 2.98*	100.73 ± 4.81*
Days of exposure		MMDTC+ZnSO4  (SLC-1.25ppm+6ppm)
		Muscle	Liver	Brain	Kidney
Control		31.62 ±0.72	55.37 ± 1.81	42.11 ± 0.91	23.97 ± 1.21
4th day		5.91 ± 0.45*	23.64 ± 0.72*	4.65 ± 0.44*	2.81 ± 0.41*
7th day		28.6 ± 3.47c	65.67 ± 4.59*	82.6 ±  5.44*	53.46 ± 3.75*
14th day		49.09 ± 4.07 *	96.44 ± 1.95*	134.31 ± 3.21*	93.48 ± 5.26*
21st day		81.88 ± 7.47 *	67.31 ± 3.75*	244.31 ± 4.02*	96.28 ± 3.80*
28th day		143.87 ± 3.09*	164.54 ± 2.35*	211.89 ± 2.14*	301.95 ± 5.78*
Days of exposure		CuSO4+ ZnSO4  (SLC-0.65ppm+2.5ppm)
		Muscle	Liver	Brain	Kidney
Control		31.62 ±0.72	55.37 ± 1.81	42.11 ± 0.91	23.97 ± 1.21
4th day		4.62 ± 0.46 *	22.85 ± 0.79*	3.84 ± 0.5*	2.06 ± 0.33*
7th day		53.27 ± 3.53 *	75.68 ± 2.78*	94.56 ± 1.96*	108.02 ± 3.56*
14th day		57.51 ± 3.13 *	158.26 ± 2.85*	167.08 ± 3.69*	6.41 ± 1.07*
21st day		48.46 ± 3.36 *	53.84 ± 4.06a	115.72 ± 5.11*	86.5 ± 3.85*
28th day		38.93 ±1.87 *	42.89 ± 1.89*	66.95 ± 4.03*	38.24 ± 6.65*

Student t -Test  * P<0.001; ^a  P  N.S; ^b  P<0.01; ^c P<0.02; ^d p<0.05

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 CuSO₄ 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  ZnSO₄ 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 CuSO₄, and ZnSO₄ 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 CuSO₄, while MMDTC with ZnSO₄ 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 CuSO₄ revealed significantly elevated level of sugars on 4^th, 21^st and 28^th day,while MMDTC with  ZnSO₄ significantly decreased the sugar content in kidney tissues on 21^st day. Exposure of CuSO₄ in addition to ZnSO₄ 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  CuSO₄ 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 ZnSO₄ 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. CuSO₄ and ZnSO₄ 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+CuSO₄ exposed group showed a significant decrease on the 4^th day and  MMDTC  with ZnSO₄  group  showed a significant increase  from 7^thto 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, CuSO₄ and ZnSO₄  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 CUSO₄ and ZnSO₄ 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 CuSO₄ and ZnSO₄ 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 CuSO₄ 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 CuSO₄ independently and synergistically with MMDTC and ZnSO₄.This shows the severe toxic nature of CuSO₄ 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 fenvalerate¹⁶. 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 Ekalux²⁵ 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 when they act together in a synergistic manner. The alterations of biochemical profiles of the test organism as observed in the present study may be used as non–specific biomarkers against anthropogenic stress.

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