Ultrastructural Alterations in Oreochromis niloticus Exposed to Glyphosate-Based Herbicide, Excel Mera 71

Research Article

Austin J Environ Toxicol. 2016; 2(2): 1016.

Ultrastructural Alterations in Oreochromis niloticus Exposed to Glyphosate-Based Herbicide, Excel Mera 71

Samanta P1,2, Pal S3, Mukherjee AK4, Senapati T1 and Ghosh AR1*

1Department of Environmental Science, The University of Burdwan, India

2Department of Environmental Science and Ecological Engineering, Korea University, Republic of Korea

3Department of Environmental Science, Aghorekamini Prakashchandra Mahavidyalaya, India

4Department of Conservation Biology, Durgapur Govt. College, India

*Corresponding author: Ghosh AR, Department of Environmental Science, The University of Burdwan, India

Received: November 11, 2016; Accepted: December 08, 2016; Published: December 15, 2016


Oreochromis niloticus was exposed to Excel Mera 71 for 30 days both at field and laboratory conditions to investigate ultrastructural responses in stomach and intestine through light, Scanning and Transmission Electron Microscopy (SEM and TEM). Stomach showed damage in mucosal folds and gastric glands, fusion of gastric folds under laboratory condition but clumping of nucleus at the base of epithelial cell was observed in field. SEM study displayed cellular lysis, fragmentation of epithelial cells under laboratory condition, while excessive mucin droplets were observed in field. TEM displayed severe deformed mitochondria and Endoplasmic Reticulum (ER), vacuolation but under field condition vacuolations were prominent. Degenerative changes in Columnar Epithelial Cells (CEC) and lamina propria in intestine were observed under light microscopy but under field condition only damage in CEC were seen, while SEM study showed degeneration in CEC and microridge and huge mucus secretion under both conditions. Swelling of mitochondria, deformation and fragmentation in ER were seen under TEM in both conditions, but in field the degree of damages were comparatively less. Results clearly disclosed that responses were more profound in laboratory condition than field and these responses could be considered as potential biomarkers of exposure of agrochemicals like Excel Mera 71.

Keywords: Oreochromis niloticus; Columnar epithelial cells; Excel mera 71; Scanning electron microscopy; Transmission electron microscopy 


Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) is one of the most important commercially cultured fish species. They are very good species for aquaculture especially in developing countries such as Asia, Africa and America where there are high levels of animal protein deficiencies and integrated paddy-cum-fish-culture system has gained considerable attention in earlier times [1,2]. But recently in India such integrated paddy-cum-fish-culture system is almost non-existent because of increasing use of inorganic fertilizers and pesticides in rice fields causing deleterious effects on fish [3].

Weeds in crop fields are responsible for lowering production of rice and reducing the yield of approximately 60-70% [4]. Therefore, for protection of the crop health and increasing its productivity in terms of biomass use of herbicides becomes inevitable in one hand that considered as a beneficial tool or a pollutant, in other hand, due to its bioaccumulation and non-biodegradable properties. Simultaneously, its indiscriminate use in the agricultural fields might endanger the ecosystem as they are ultimately reach to the nearby aquatic ecosystem as well as fish farm which are in close proximity to the agricultural fields and also posing threats to the non-target aquatic organisms such as fin-fish and shell-fish having great economic importance to the human society. This may also contribute long term effects in the environment by changing the water quality [5].

Excel Mera 71, a glyphosate-based commercial herbicide formulation, is one of the most extensively used agrochemicals for controlling the broad leaved weeds and sedges both in the rice fields and aquatic ecosystem [6]. It is a non-selective and post-emergent modern, third generation herbicide of organophosphate group. They are highly efficacious, cost effective, practically non-toxic and degrade readily in the environment [7]. Glyphosate is a weak organic acid and chemical name is N-(phosphonomethyl) glycine and is usually formulated as the isopropylamine or trimethylsulfonium salt of glyphosate. The half-life of glyphosate in soil ranges between 2 and 197 days; a typical field half-life is of 47 days. Soil and climatic conditions also affect the glyphosate's persistence in soil. The median half-life of glyphosate in water varies from 4 to 91 days [8].

Use of cytopathological biomarkers in evaluation of the health status of fish exposed to xenobiotic substances has gained much attention throughout the world both in the laboratory [9,10] and field studies [11-13]. Histological analysis provides a rapid and efficient process to investigate the health of organisms exposed to a contaminated environment. Histopathological alterations are very sensitive to determine the cellular changes and allow examining the specific target organs, including stomach and intestine [14]. Furthermore, the changes observed in these organs are normally easier to identify than the functional ones [15] and serve as warning signs of damage to animal health [16]. Some study relating to use and effects of agrochemicals including the glyphosate in fish and aquatic invertebrates are available [17-24]; however, a little or no information is available on the effects of glyphosate on histopathological and ultrastructural responses in Oreochromis niloticus (Linnaeus) in the field condition. Nevertheless, field studies using histopathology and electron microscopy of fish as biomarker of aquatic contamination have not yet been reported. In the present study, an attempt has been made to investigate the histopathological and ultrastructural responses in stomach and intestine of the air-breathing, omnivorous freshwater teleost, O. niloticus to an exposure of Excel Mera 71 under field and laboratory conditions to establish some baseline information on the application of this herbicide.

Materials and Methods


Freshwater teleostean fish, Oreochromis niloticus (Linnaeus) of both the sexes with an average weight of 38.57±2.47 g and total length of 13.59±0.50 cm were acquired from local market and were acclimatized under congenial laboratory conditions for 15 days in aquaria of 250 L capacity. Fish were kept in continuously aerated water with a static system under natural photoperiod of 12-h light/12-h dark. During the acclimatization period, the average value of water parameters were as follows; temperature: 26.49±0.127oC, pH: 7.94±0.040, electrical conductivity: 392.22±0.62 µS/cm, total dissolved solids: 279.33±0.69 mg/l, dissolved oxygen: 6.44±0.05 mg/l, total alkalinity: 204.00±7.30 mg/l as CaCO3, total hardness: 180.44±3.74 mg/l as CaCO3, sodium: 24.45±0.56 mg/l, potassium: 5.33±1.02 mg/l, orthophosphate: 0.03±0.001 mg/l, ammoniacal-nitrogen: 1.66±0.21 mg/l and nitrate-nitrogen: 0.21±0.030 mg/l. Fish were fed once a day with commercial fish pellets (32% crude protein, Tokyu) during both acclimation and exposure periods. The experiment was carried out in accordance with the guidelines of the University of Burdwan and approved by the Ethical Committee of this University.

Field experimental design

After acclimatization, one set of fish were transferred to the field ponds situated at Crop Research Farm premises of the University of Burdwan. Fish were again divided into two groups as follows: control group containing 10 fish in three separate cages and exposure group also with 10 fish in separate cages for 30 days. The desired dose of 750 g/acre corresponds to concentration recommended for use in rice culture was dissolved in water and applied once [25,26]. It was sprayed on first day of the experiment on the surface of each glyphosate-treated cage. Glyphosate concentration in water was monitored according to Jan, et al. [27] during 30 days and it was 1.20±0.47 mg/l. For field experiment a special type of cage was prepared and installed separately at pond of Burdwan University Crop Research Farm, The University of Burdwan. The cages were prepared for the culture of the experimental fish as per Chattopadhyay, et al. [28] with some modifications. All the cages were square in shape having an area of 2.5x1.22 m2 and height of the cage was 1.83 m (submersed height was 0.83 m). The cages were framed by light strong bamboo. The four-sided wall, floor of the cage and top of the cage cover was fabricated with nylon net and was embraced by two PVC nets: the inner and outer bearing mesh sizes of 1.0x1.0 mm2 and 3.0x3.0 mm2 respectively. During the experimentation period (30 days) in the field, pond water had the following average values; temperature: 24.03±0.203oC, pH: 6.56±0.087, electrical conductivity: 347.00±1.15 µS/cm, total dissolved solids: 247.67±1.45 mg/l, dissolved oxygen: 7.00±0.157 mg/l, total alkalinity: 221.33±3.53 mg/l as CaCO3, total hardness: 140.00±2.31 mg/l as CaCO3, sodium: 63.40±2.67 mg/l, potassium: 15.96±2.10 mg/l, orthophosphate: 0.24±0.026 mg/l, ammoniacal-nitrogen: 0.74±0.111 mg/l and nitrate-nitrogen: 1.66±0.035 mg/l.

Laboratory design

After acclimatization, another set of fish were transferred to the laboratory aquarium. Fish were again divided into two groups (control and glyphosate-treated) and maintained in six aquaria, containing 10 fish in each aquarium in the Ecotoxicology Lab, Department of Environmental Science, The University of Burdwan: three for control and another three for treatment. Fish were exposed to sub-lethal dose of glyphosate, i.e., 17.20 mg/l in 40 L aquaria for a period of 30 days [29,30]. Doses were applied every alternate day. Glyphosate concentration in water was 16.88±1.69 mg/l. During experimentation, glyphosate-treated and control aquariums were subjected to same environmental conditions. During experimentation period, the average water parameters were as follows; temperature: 26.63±0.120oC, pH: 7.93±0.075, electrical conductivity: 426.00±5.93 µS/cm, total dissolved solids: 302.89±4.69 mg/l, dissolved oxygen: 5.06±0.43 mg/l, total alkalinity: 209.80±10.50 mg/l as CaCO3, total hardness: 163.11±3.04 mg/l as CaCO3, sodium: 37.76±1.02 mg/l, potassium: 7.26±1.12 mg/l, orthophosphate: 0.04±0.002 mg/l, ammoniacal-nitrogen: 7.09±2.15 mg/l and nitrate-nitrogen: 1.78±0.263 mg/l.


During experimentation period the quality of the water was assessed as per APHA [31]. After completion of the experiment i.e., 30 days the fish were collected both from the aquarium and pond as well as from control condition and were anesthetized with tricaine methanesulphonate (MS 222) and stomach and intestine were taken immediately after dissection and proceeded specific ways for histological, scanning and transmission electron microscopic study.

Histopathological analysis

Fish tissues namely stomach and intestine from control and treatment were collected and fixed in aqueous Bouin’s fluid solution, dehydrated through graded series of ethanol and finally embedded in paraffin. Paraffin sections were cut at 3-4 µm using Leica RM2125 microtome. These sections were then stained with Haematoxylin-Eosin (H&E). Histopathological observations were made under Leica DM2000 light microscope.

Ultrastructural analysis

For scanning electron microscopic study, tissues were fixed in 2.5% glutaraldehyde in phosphate buffer (0.2 M, pH 7.4) for 24 h at 4oC and then post-fixed with 1% osmium tetraoxide in phosphate buffer (0.2 M, pH 7.4) for 2 h at 4oC, dehydrated through graded acetone, subsequently followed by amyl acetate and subjected to critical point drying with liquid carbon dioxide. Tissues were then mounted on metal stubs and sputter-coated with gold with thickness of approximately 20 nm. Tissues were examined with a scanning electron microscope (Hitachi S-530) at University Science Instrumentation Centre of the University of Burdwan.

For transmission electron microscopic study, tissues were fixed in Karnovsky fixative (mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer) for 12 h at 4oC and then post-fixed with 1% osmium tetraoxide in phosphate buffer (0.2 M, pH 7.4) for 2 h at 4oC, dehydrated through graded acetone, infiltrated and embedded in epoxy resin, araldite CY212. Ultrathin sections (0.5-1 μm) were then cut by using a glass knife on an "Ultracut E Reichart-Jung" with the thickness of 70 nm, collected on naked copper-meshed grids and contrasted with uranyl acetate and lead citrate. The tissues were examined under TECHNAI G2 high resolution transmission electron microscope at Electron Microscope Facility, Department of Anatomy, AIIMS, New Delhi.

Citation: Samanta P, Pal S, Mukherjee AK, Senapati T and Ghosh AR. Ultrastructural Alterations in Oreochromis niloticus Exposed to Glyphosate-Based Herbicide, Excel Mera 71. Austin J Environ Toxicol. 2016; 2(2): 1016. ISSN:2472-372X