Scrutinizing impact of atrazine on stress biomarker : plasma glucose concentration of fresh water fish , grass carp , ( Ctenopharyngodon idella )

Herbicide praxis in agricultural fields to curb herbs is utterly toxic to non-target areas like aquatic bodies and impinge aquatic organism distinctively fish health through impairment of metabolism, sometimes leading to mortality. Therefore to contemplate the acute (short term) and chronic (long term) toxicological effects of atrazine, study was designed to scrutinize the potential detrimental effects of commercial utilization of chemical named; atrazine, on aquatic organisms through evaluating stress biomarker; plasma glucose (energy currency) of freshwater fish; grass carp, (Ctenopharyngodon idella)for 01(24), 02(48), 03(72), 04(96), 10(240), 15(360) and 15(600) days(hrs) under the dose 15μl/L, 13μl/L, 10μl/L and 08μl/L, 06μl/L, 04μl/L, 02μl/L respectively. For analysis of plasma glucose concentration the protocol haematological analyzer (Mindray BC2300 Hematology Analyzer) was followed in the laboratory. Above 15μl/L LC50 was recorded indicating grass carp sensitivity to atrazine. Control plasma glucose concentration was 42.3mmol/L. Paramount downturn during acute toxicity and chronic toxicity was observed after 01 day (24hrs/15μl/L) and 10 days (240hrs /06μl/L) exposure respectively among treated groups while in both groups with the declining of dose quantity an inclined in glucose concentration was observed. Downturn in concentration indicating the toxicity of atrazine to liver and upturn in concentration may be due to enhancing glycogen breakdown in the liver and consequently elevates blood glucose level in response of stress to cope with new environmental conditions. Our study is also purposive to fathom that atrazine is causing agent of acute and chronic health problem to aquatic organisms and induced stress in fish body which revealed pessimistic domination on ecosystem of the aquatic organisms.


Introduction
The environmental impact of herbicides is often more preponderant than what is intended by farmers who utilized it for the purpose to have better products of crops.98% sprayed insecticides and 95% herbicides, reached to other destination including air, bottom sediments, different foods, aquatic bodies like lakes, rivers, ponds, etc, and thus contaminate these areas [1, 2].Due to herbicides runoff to aquatic bodies, each year 6 to 14 million fishes killed, reported by United States Fish and Wildlife service and United States Department of Agriculture [1].Similarly 4,000 to 20,000 cancer cases were reported by National Academy of Sciences estimation due to ingesting herbicides contaminated foods and water [1].Herbicides can be carried to aquatic bodies via surface water and rain water runoff while in grazing areas, human settlements, undeveloped areas; it can be carried by wind, potentially affecting other species [3].Similarly herbicides can contaminate different regions near to the place of application by major routes that when it is sprayed so it may be drift outside from the application area, in soil it's found after leaching, due to runoff or due to spilled it may contaminate the aquatic bodies [2, 4, 5].Animals may be badly affected by eating herbicides contaminated foods, ex.when wild animals eat food after short time of spraying [6].According to the Fish and Wildlife service of US, they estimated that 72 million birds are killed by these herbicides per year by eating materials having herbicides drops [7].In the past amphibians population was declined and different reasons were presented for such decline but beside different reason the pesticide effect is also considered as responsible reason for such decline [8].Similarly serious threats to aquatic organisms are due to characteristic of herbicides that is of bioaccumulation and non-biodegradable nature of herbicides

Methods and materials Experimental fish and its maintenance
For the present study, Grass carp (Ctenopharyngodon idella) were selected as a model and were procured from Sherabad Hatchery, Peshawar, Khyber Pakhtunkhwa, Pakistan.During experimentation, two type of tanks were required that were, acclimatization tank and experimentation tanks.After selecting the healthy specimen, before it should transfer to acclimatization tank, it was treated with 0.2% KMno4 solution, to get rid of any dermal infection.Acclimatization process was continuing for about two weeks and then after two weeks the fish specimens were transferred to experimentation tanks for experimental purpose.There were present different experimental tanks, which was labelled according to days of treatment and concentration of herbicides.In both tanks, the fish were fed properly on every alternate day, and all other water quality parameters were in normal range, which was also checked in both tanks on every alternated day.

Experimental design
The main purpose of this experiment was to compare the control group concentration which was kept without treatment, with other groups which was treated with different doses on different days.So for this purpose, one group consisted of 05 fish, was considered as control group and was kept without treatment, while other 4 groups were made for acute toxicity analysis in which group 1 & 2 consisted of 05 fishes each and was treated for 01 day (24hrs) and 02 days (48 hrs) under the dose of 15µl/L and 13µl/L respectively.Similarly two other groups named as group 03 & 04 consisted of 08 fishes each and was processed for 03 days (72hrs) and 04 days (96 hrs) under the dose of 10µl/L and 08µl/L respectively.Likewise for analysis of chronic toxicity, 03 groups were made named as groups 01, 02 and 03, in which group 01 consisted of 10 fish specimen while group 02 and 03 consisted of 15 fish specimen each and these groups were treated for 10, 15 and 25 days (240, 360 and 600 hrs) under the dose of 06, 04 and 02 µl/L respectively.

Blood collection and preservation
Freshly anesthetized fish (anesthesia given to fish by chemical, MS 222) were used for collection of the blood from the caudal vein and sometime from direct puncturing of heart of fish.Hypodermic syringes, which were heparinized by few drops of heparine, for the purpose to prevent blood clotting, were used for the collection of the blood sample.[20, 21].EDTA and Gel tube were used for storing of the blood samples.As we know that blood biochemical parameters are analyzed form serum, so in that case, Gel tubes were found more beneficial because they have a special layer of gel which is helpful in isolation of serum form the blood cells through the centrifugation (3000 RPM).After centrifugation the serum was analyzed for plasma glucose concentration.Estimation of plasma glucose concentration Plasma glucose concentration in control group fish and in all treated groups fish, were analyzed by following the protocol of biochemical analyzer model; Merck micro lab 300.After obtaining the results, SPSS software was used to find out significant (P value) difference between control and experimental means.

Results
By comparing control group concentration (42.3µl/l) as shown in table 1, with concentration obtained after acute toxicity as shown in table 2 and figure1, maximum highly significant (P≤0.001)decline in concentration of plasma glucose was observed after treatment of fish for 01 (24hrs), 02 (48hrs) and 03(72hrs) days against the dose of 15µl/l, 13µl/l and 10µl/l respectively.Likewise highly significant (P≤0.01)decline in concentration of plasma glucose was observed after treatment of the fish for 04 (96hrs) days against the dose of 10µl/l as shown in table 2. Result clearly indicating that paramount downturn during acute toxicity observed after 01 day (24hrs) against the dose of 15µl/L while with the declining of dose quantity on the remaining days of exposure an inclined in glucose concentration was observed indicating the glycogenolysis process to cope with the stress environment.indicating the glycogenolysis process to cope with the stress environment.In the present study comparison of control group concentration and concentration obtained after exposure to atrazine revealed significance difference which was due to effect of atrazine.By comparing control group concentration (42.3µl/l) with concentration obtained after acute toxicity maximum highly significant (P≤0.001)decline in concentration of plasma glucose was observed after treatment of fish for 01 (24hrs), 02 (48hrs) and 03 (72hrs) days against the dose of 15µl/l, 13µl/l and 10µl/l respectively.Likewise highly significant (P≤0.01)decline in concentration of plasma glucose was observed after treatment of the fish for 04 (96hrs) days against the dose of 10µl/l.Similarly by comparing control group concentration (42.3 µl/l) as with concentration obtained after chronic toxicity, highly significant (P≤0.01)decline in concentration of plasma glucose was observed after treatment of the fish for 10 (240hrs), 15 (360hrs) and 25 (600hrs) days against the dose of 06µl/l 04µl/l and 02µl/l respectively.
Present study findings are justified by the findings of [30] whose found consequential decrease in serum glucose of fresh water fish Oreochromis niloticus and Chrysichthyes auratus againstatrzine, at Assiut, Egypt and indicated the responsible reason for such decrease was the toxicity of atrazine on liver [31].Similarly the present study also betoken the paramount downturn during acute toxicity observed after 01 day (24hrs) against the dose of 15µl/L while with the declining of dose quantity on the remaining days of exposure an inclined in glucose concentration was observed, likewise paramount downturn during chronic toxicity observed after 10 (240hrs) days treatment against the dose of 06µl/l while with the declining of dose quantity on the remaining days of exposure an inclined in glucose concentration was observed, indicating the glycogenolysis process to cope with the stress environment.So such findings are in agreement with the observation of [32] who studies the hematological parameters under the effect of clomazone herbicide and some biochemical parameters like protein and carbohydrate metabolism of silver catfish, Rhamdiaquelen under the effects atrazine and in their study he observed significant increase in glucose concentration in fish exposed to atrazine for long term.Same results were observed by different researcher [33-35] and all of them indicating the glycogenolysis process for inclining the glucose concentration to cope with the stress environment.Therefore this condition was induced by environmental pollution which produces stress and in response glycogen breakdown enhanced in the liver and consequently elevates blood glucose level called hyperglycemic condition [36].Likewise this secretion of catecholamines from the adrenal medulla, in turn enhances the breakdown of glycogen in the liver, which in turn elevate the blood glucose concentration led to hyperglycemic state, a response to stress environment.[37, 38].In contrast [39, 40]recorded a marked decrease in glucose level in Nile tilapia, Oreochromis niloticus and catfish, Chrysichthyes auratus exposed to atrazine and silver catfish, exposed to glyphosate herbicide.However [41] found that the glucose level of common carp, Cyprinus carpio exposed to simazine remained similar to the control group.This could be attributed to the difference in fish species, varied concentration of atrazine or the type of herbicide used.

Conclusions
It was concluded that paramount downturn during acute toxicity and chronic toxicity was observed after 01 day (24hrs/15µl/L) and 10 days (240hrs/06µl/L) exposure respectively among treated groups while in both groups with the declining of dose quantity an inclined in glucose concentration was observed.Downturn in concentration indicating the toxicity of atrazine to liver and upturn in concentration may be due to enhances glycogen breakdown in the liver and consequently elevates blood glucose level in response of stress to cope with new environmental conditions.Our study is also purposive to fathom that atrazine is causing agent of acute and chronic health problem to aquatic organisms and induced stress in fish body which revealed pessimistic domination on ecosystem of the aquatic organisms.