Hinton & Lauren, 1990

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JMAU 90 1–9 ARTICLE IN PRESS

Journal of Microscopy and Ultrastructure xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Microscopy and Ultrastructure

journal homepage: www.elsevier.com/locate/jmau
2 Original Article
3 Toxic effects of glyphosate-based herbicide, Excel Mera 71

4 on gill, liver, and kidney of Heteropneustes fossilis under
5 laboratory and field conditions
6 Q2 Palas Samanta a , Aloke Kumar Mukherjee b , Sandipan Pal c , Debraj Kole a ,
7 Apurba Ratan Ghosh a,∗
a
8 Ecotoxicology Laboratory, Department of Environmental Science, The University of Burdwan, Golapbag, Burdwan 713104,
9 West Bengal, India
b
10 P.G. Department of Conservation Biology, Durgapur Government College, Durgapur 713214, West Bengal, India
c
11 Department of Environmental Science, Aghorekamini Prakashchandra Mahavidyalaya, Subhasnagar, Bengai, Hooghly 712611,
12 West Bengal, India
13
14 a r t i c l e i n f o a b s t r a c t
15
16 Article history: The effects of glyphosate-based herbicide Excel Mera 71 under field and laboratory condi-
17 Received 13 April 2015 tions were investigated to evaluate the pathological symptoms through light and electron
18 Accepted 2 January 2016 microscopic study in the gill, liver, and kidney of Heteropneustes fossilis (Bloch) for a period
19 Available online xxx
of 30 days. Histological alterations like hypertrophy and fusion in secondary lamellae, dam-
20 age in chloride cells were more prominent in laboratory conditions under light microscopy.
21 Keywords: Topological changes such as complete loss of microridges, swelling, and irregular arrange-
22 Field conditions
ment of microridges in the gills were prominent under scanning electron microscopic study
23 Glyphosate (Excel Mera 71)
under laboratory conditions. Transmission electron microscopy (TEM) study depicted vac-
24 Heteropneustes fossilis
25 Laboratory conditions uolation and degeneration in chloride cells, dilation in rough endoplasmic reticulum (RER),
26 Light microscopy and mitochondria in gill epithelium. The liver showed enlarged and pyknotic hepatocytes,
27 Transmission electron microscopy vacuolation, excess fat deposition, and necrosis under laboratory conditions, while enlarged
acentric nuclei, increased sinusoidal space, and less vacuolation in cytoplasm were observed
under field conditions. TEM displayed cytoplasmic vacuolation and a reduced number of
endoplasmic reticulum and glycogen droplets in the laboratory, but this was less pro-
nounced under field conditions. In the kidneys, loss of hematopoietic tissue, degenerative
changes in glomeruli, proximal and distal convoluted tubule, and epithelial cell lining of
the renal tubules were comparatively less prominent under field conditions. Under TEM,
epithelial cell necrosis, endoplasmic reticulum fragmentation, and mitochondrial degener-
ation were more prominent under laboratory conditions. The present study evaluated the
comparative toxicity under field and laboratory conditions under long-term exposure to
glyphosate herbicide and identified pathological responses as indicators in monitoring the
herbicidal contamination in aquatic ecosystems.
© 2016 Published by Elsevier Ltd. on behalf of Saudi Society of Microscopes.
1. Introduction 28
Histopathological study has been widely used for tox- 29
∗ Corresponding author. icity testing of the effects of xenobiotic compounds at the 30
E-mail address: [email protected] (A.R. Ghosh). suborganismal or organismal level, as well as evaluation 31
http://dx.doi.org/10.1016/j.jmau.2016.01.002
2213-879X/© 2016 Published by Elsevier Ltd. on behalf of Saudi Society of Microscopes.
Please cite this article in press as: Samanta P, et al. Toxic effects of glyphosate-based herbicide, Excel Mera 71 on
gill, liver, and kidney of Heteropneustes fossilis under laboratory and field conditions. J Microsc Ultrastruct (2016),
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2 P. Samanta et al. / Journal of Microscopy and Ultrastructure xxx (2016) xxx–xxx
32 of overall health of the entire population in the ecosys- CY212 (EM grade) of analytical grade were purchased from 90
33 tem. The advantage of using histopathological symptoms Merck Specialities Private Limited. Osmium tetroxide was Q4 91
34 in specific target organs like the gills, liver, and kidneys in purchased from Spectrochem (Mumbai, India). 92
35 environmental monitoring is that they are most effective

36 to study the vital functions, such as respiration, accumula- 2.2. Fish 93
37 tion and biotransformation, and excretion of xenobiotics in
38 fish [1,2]. Histological changes appear as prime responses Freshwater teleostean fish H. fossilis (Bloch) of both 94
39 to sublethal stressors, while topological characterization of the sexes with an average weight of 31.77 ± 3.440 g and 95
40 cell surface and subcellular organelles can be best analyzed total length of 16.58 ± 0.388 cm were procured from local 96
41 under scanning electron microscopy (SEM) and transmis- markets and were acclimatized under congenial labo- 97
42 sion electron microscopy (TEM), respectively. Furthermore, ratory conditions for 15 days separately in aquaria of 98
43 the alterations in cells and tissues in vertebrates, especially 250-L capacity. Fish were kept in continuously aerated 99
44 fish, are recurrently used as biomarkers in many studies, water with a static system and experiments were con- 100
45 but such changes also occur in all invertebrates inhab- ducted with a natural photoperiod (12-hour light/12-hour 101
46 iting aquatic basins, and are normally easier to identify dark) and at an ambient water temperature. During 102
47 than functional ones [3], which ultimately serve as warning acclimatization, the average values of water parame- 103
48 signs of damage to animal health [4,5]. Fish after expo- ters were analyzed: temperature 26.49 ± 0.127 ◦ C, pH 104
49 sure to these xenobiotic substances show several lesions 7.94 ± 0.04, electrical conductivity 392.22 ± 0.62 ␮S/cm, 105
50 in different tissue systems [6,7]. Gills [8,9], liver [4,10], and total dissolved solids 279.33 ± 0.69 mg/L, dissolved oxy- 106
51 kidneys [11] are the most suitable organs for histological gen 6.44 ± 0.05 mg/L, total alkalinity 204.0 ± 7.30 mg/L 107
52 analysis in order to determine the effects of contamina- as CaCO3 , total hardness 180.44 ± 3.74 mg/L as CaCO3 , 108
53 tion. During xenobiotic exposure, the toxicants break down sodium 24.45 ± 0.56 mg/L, potassium 5.33 ± 1.02 mg/L, 109
54 the adhesion between the epithelial branchial cells and orthophosphate 0.03 ± 0.001 mg/L, ammoniacal nitrogen 110
55 the underlying pillar cells, accompanied by collapse of the 1.66 ± 0.21 mg/L, and nitrate nitrogen 0.21 ± 0.030 mg/L. 111
56 structural integrity of the secondary lamellae and sub- After acclimatization, fish were divided into two groups: 112
57 sequent failure of the respiratory functions of the gills one group was maintained in field ponds situated at Crop 113
58 [12]. The liver is the central metabolic organ and plays a Research Farm premises of the University of Burdwan, 114
59 key role in biochemical transformations of the xenobiotic West Bengal, India and the other group in a laboratory 115
60 substances, which inevitably reflects on its integrity by cre- aquarium. The fish were fed once a day with commercial 116
61 ating lesions and other histopathological alterations in the fish pellets (32% crude protein, Tokyu) during both accli- Q5 117
62 liver parenchyma [13]. The kidneys perform an important mation and exposure periods. Therefore, the study was 118
63 function in maintenance of a stable internal environment carried out under two different experimental conditions: 119
64 and partially xenobiotic metabolism. field pond and laboratory, for a duration of 30 days. 120
65 Aquatic bodies are contaminated by several pesti-

66 cides, especially herbicides such as glyphosate, through 2.3. Experimental design 121
67 irrigation water and surface run-off. Among the non-
68 target aquatic organisms, fish represent the largest and 2.3.1. Field experiment 122
69 most diverse group of vertebrates that are chronically Fish were maintained in two groups in two separate 123
70 exposed to these substances continuously. Therefore, the adjacent fields: three control groups containing 10 fish 124
71 present study aimed to investigate the toxic effects of com- species in a cage in one field, and three glyphosate expo- 125
72 mercial formulations of the glyphosate herbicide, Excel sure groups containing 10 fish species in separate field 126
73 Mera 71 (Excel Crop Care Limited, Mumbai, Maharash- and cages for 30 days. The desired dose of 750 g/acre, 127
74 tra, India), at histopathological and ultrastructural levels corresponding to the concentration recommended for use 128
75 through changes in the gills, liver, and kidneys of the fish in rice culture, was dissolved in water and applied once. 129
76 Heteropneustes fossilis (Bloch) under laboratory and field It was sprayed on Day 1 of the experiment on the surface 130
77 conditions. of each glyphosate-treated cage. During experimentation, 131
glyphosate-treated and control fish were subjected to the 132
78 2. Materials and methods same environmental conditions. The cages were prepared 133
for the culture of the experimental fish species as per 134
79 2.1. Chemicals Chattopadhyay et al. [14], with some modifications. All the 135
cages were square in shape with an area of 2.5 m × 1.22 m 136
80 Commercial formulation of the glyphosate herbicide and height of 1.83 m (submerged height was 0.83 m). The 137
81 (Excel Mera 71, Excel Crop Care Limited) was used cages were framed by light strong bamboo. The four-sided 138
82 in both the experiments. Excel Mera 71 is the trade wall, floor of the cage, and top of the cage cover was fabri- 139
83 name of glyphosate herbicide in the Indian Market. cated with nylon net and was embraced by two polyvinyl 140
84Q3 Delafield’s hematoxylin stain, eosin yellow, xylene, DPX, chloride nets: the inner and outer bearing mesh sizes 141
85 amyl acetate, acetone, glutaraldehyde solution, sodium of 1.0 mm × 1.0 mm and 3.0 mm × 3.0 mm, respectively. 142
86 hydroxide, tricaine methonesulfonate, uranyl acetate (EM During the experimentation of 30 days, the field pond 143
87 grade), ethanol, disodium hydrogen phosphate, dihydro- water was analyzed: temperature 24.03 ± 0.203 ◦ C, pH 144
88 gen sodium phosphate, lead citrate (EM grade), epoxy resin 6.56 ± 0.087, electrical conductivity 347.00 ± 1.15 ␮S/cm, 145
89 (EM grade), paraformaldehyde (EM grade), and araldite total dissolved solids 247.67 ± 1.45 mg/L, dissolved oxygen 146
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P. Samanta et al. / Journal of Microscopy and Ultrastructure xxx (2016) xxx–xxx 3
147 7.0 ± 0.157 mg/L, total alkalinity 221.33 ± 3.53 mg/L 0.1 M phosphate buffer) for 12 hours at 4 ◦ C and then 202
148 as CaCO3 , total hardness 140.0 ± 2.31 mg/L as CaCO3 , post-fixed with 1% osmium tetroxide in phosphate buffer 203
149 sodium 63.40 ± 2.67 mg/L, potassium 15.96 ± 2.10 mg/L, (0.2 M, pH 7.4) for 2 hours at 4 ◦ C, dehydrated through 204
150 orthophosphate 0.24 ± 0.026 mg/L, ammoniacal nitrogen graded acetone, infiltrated and embedded in epoxy resin, 205
151 0.74 ± 0.111 mg/L, and nitrate nitrogen 1.66 ± 0.035 mg/L. araldite CY212. Ultrathin sections (0.5–1 ␮m) were then 206
cut by using a glass knife on an Ultracut E Reichart–Jung 207

152 2.3.2. Laboratory experiment ultramicrotome at a thickness of 70 nm, collected on naked Q9 208
153 Fish were segregated into two groups (control and copper-meshed grids, and contrasted with uranyl acetate 209
154 glyphosate-treated) and maintained in six aquaria (3 con- and lead citrate. The tissues were examined under a Tech- 210
155 trol and 3 treated), containing 10 fish in each aquarium in nai G2 high resolution transmission electron microscope at Q10211
156 the Ecotoxicology Laboratory, Department of Environmen- the Electron Microscope Facility, Department of Anatomy, 212
157 tal Science, University of Burdwan. The fish were exposed All India Institute of Medical Sciences, New Delhi, India. Q11 213
158 to sublethal doses of glyphosate, that is, 17.20 mg/L
159 in 40 L aquaria for a period of 30 days [15,16]. Doses 2.7. Ethical statement 214
160 were applied every alternate day, maintaining the same
161 water quality. During experimentation, water parame- The experiment was carried out in accordance with the 215
162 ters were measured: temperature 26.63 ± 0.120 ◦ C, pH guidelines of the University of Burdwan and approved by 216
163 7.93 ± 0.075, electrical conductivity 426.0 ± 5.93 ␮S/cm, the Ethical Committee of this University. 217
164 total dissolved solids 302.89 ± 4.69 mg/L, dissolved oxygen
165 5.06 ± 0.43 mg/L, total alkalinity 209.80 ± 10.50 mg/L 3. Results 218
166 as CaCO3 , total hardness 163.11 ± 3.04 mg/L as CaCO3 ,
167 sodium 37.76 ± 1.02 mg/L, potassium 7.26 ± 1.12 mg/L, 3.1. Gills 219
168 orthophosphate 0.04 ± 0.002 mg/L, ammoniacal nitrogen
169 7.09 ± 2.15 mg/L, and nitrate nitrogen 1.78 ± 0.263 mg/L Under light microscopy, the gills comprised lamel- 220
170 on average. The quality of the water was assessed as per lae (primary and secondary) composed of a cartilaginous 221
Q6 APHA [17].
171 skeletal structure, multilayered epithelium, and vascu- 222
lar system. Secondary lamellae were lined by squamous 223

172 2.4. Sampling epithelium. Between the secondary epithelia, the primary 224
lamella was lined by stratified epithelium. Secondary gill 225

173 After completion of the 30 days experiment, on Day
lamellae consisted of epithelial cells supported by pil- 226
174 31, the fish were collected from the control and treated
lar cells (Figure 1A). The most marked changes under 227
175 aquarium and pond and were anesthetized with tricaine
the laboratory conditions after glyphosate exposure were 228
176 methonesulfonate (MS 222) and the gills, liver and kidneys
hypertrophy and fusion of the secondary lamellae, and 229
177 were taken immediately after dissection and processed
severe damage to chloride cells (Figure 1B), while partial 230
178 separately for histological and ultrastructural analysis.
fusion of some lamellae in H. fossilis was prominent under 231
field conditions (Figure 1C). 232

179 2.5. Histopathological analysis
Topographical study by SEM revealed that each gill fil- 233
180 Fish gills, liver and kidneys from the control and treated ament in the control fish was composed of primary and 234
181 groups were collected and fixed in aqueous Bouin’s fluid secondary lamellae surrounded by stratified epithelial cells 235
182 solution, dehydrated through a graded series of ethanol, (Figure 1D). SEM showed complete loss of microridges 236
183 and finally embedded in paraffin. Paraffin sections were cut in some stratified epithelial cells in the gills, swelling 237
Q7 at 3–4 ␮m using a Leica RM2125 microtome. These sections

184
of microridges, and irregular arrangement of microridges 238
185 were then stained with hematoxylin and eosin. Histopatho- under laboratory conditions after glyphosate exposure 239
186 logical observations were made under a Leica DM2000 light (Figure 1E) but under field conditions, there were no signif- 240
187 microscope. icant alterations in stratified epithelial cells and microridge 241
structure (Figure 1F). 242
188 2.6. Ultrastructural analysis TEM observations of gill primary epithelium showed 243
general appearance of chloride cells supported by 244
189 For SEM, tissues were fixed in 2.5% glutaraldehyde tightly packed pavement cells under control conditions 245
190 in phosphate buffer (0.2 M, pH 7.4) for 24 hours at 4 ◦ C (Figure 1G). In gill epithelium of H. fossilis under laboratory 246
191 followed by post-fixation with 1% osmium tetroxide in conditions, TEM showed degenerative changes in chloride 247
192 phosphate buffer (0.2 M, pH 7.4) for 2 hours at 4 ◦ C, dehy- cells, fusion of microridges, dilation of RER and mitochon- 248
193 drated through graded acetone, subsequently by amyl dria, vacuolation of chloride cell cytoplasm (Figure 1H), 249
194 acetate, and subjected to critical point drying with liq- while damage to the tubular vascular network, dilation of 250
195 uid carbon dioxide. The tissues were then mounted on mitochondria, and vacuolation in the cytoplasm were the 251
196 metal stubs and sputter-coated with gold to a thickness of most significant changes under field conditions (Figure 1I). 252
197 ∼20 nm. The tissues were examined with a scanning elec-
Q8 tron microscope (Hitachi S-530) at the University Science
198 3.2. Liver 253
199 Instrumentation Centre, University of Burdwan.

200 For TEM, tissues were fixed in Karnofsky fixative (mix- Fish liver consists of hepatocytes that are polygonal 254
201 ture of 2% paraformaldehyde and 2.5% glutaraldehyde in and/or hexagonal cells with a centrally placed spherical 255
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Q16 Figure 1. Histopathological photomicrographs of gills of Heteropneustes fossilis under control conditions (Co), glyphosate-treated laboratory conditions
(GL), and glyphosate-treated field conditions (GF). (A) Normal structure of primary gill lamellae (PGL) and secondary (SGL) lamella under light microscopy
(Co, 400×). (B) Hypertrophy (arrow), fusion (white arrow) of SGL and distortion of chloride (oval) and pillar cells (broken arrow) under light microscopy
(GL, 400×). (C) Partial fusion of SGL (white arrow) and hypertrophy (arrow) under light microscopy (GF, 400×). (D) SEM showing normal arrangement of gill
Q17 rackers (GR) with primary gill lamellae (PGL) and stratified epithelial cells (SEC) on the PGL (Co, 200×). (E) Gill epithelium showing loss of MR in SEC (arrow)
under SEM (GL, 8000×). (F) Almost normal appearance of MR in SEC and excess mucin (M) droplets under SEM (GF, 5000×). (G) Gill epithelial cell under
TEM showing normal chloride cells (CC), pavement cells (PC) with prominent mitochondria (M) with apical pore (square) (Co, 3200×). (H) Degenerative
chloride cells (arrow), severe distortion in mitochondria (bold arrow), severe cytoplasmic vacuolation (broken arrow) with dilated endoplasmic reticulum
under TEM (GL, 2500×). (I) Vacuolation (broken arrow) and dilated mitochondria (bold arrow) under TEM (GF, 8000×).
SEM = scanning electron microscopy; TEM = transmission electron microscopy.
256 nucleus and a densely stained nucleolus and granular cyto- inflammation (Figure 2B), while vacuolation in the cyto- 262
257 plasm (Figure 2A). The most conspicuous histopathological plasm, enlarged acentric nuclei, and increased sinusoidal 263
258 alterations due to glyphosate toxicity identified by light space were observed under field conditions (Figure 2C). 264
259 microscopy in the liver of H. fossilis under laboratory condi- TEM of the liver showed normal appearance of hepa- 265
260 tions were enlarged and pyknotic hepatocytes, vacuolation tocytes with centrally placed prominent nucleoli and 266
261 in the cytoplasm, excess fat deposition, and hepatocytic cytoplasm that contained a large number of mitochondria, 267
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Figure 2. Histopathological photomicrographs of liver of Heteropneustes fossilis under control conditions (Co), glyphosate-treated laboratory conditions
(GL), and glyphosate-treated field conditions (GF). (A) Normal appearance of hepatocytes (HC) and compact arrangement around central vein (CV) with
distinct nucleus (N) under light microscopy (Co, 1000×). (B) Hypertrophied and pyknotic nuclei (white arrow), vacuolation in cytoplasm of hepatocytes
(broken arrow) under light microscopy (GL, 1000×). (C) Light microscopy showing vacuolation in cytoplasm (broken arrow) and appearance of sinusoidal
space (oval) (GF, 1000×). (D) Normal appearance of hepatocytes with large number of mitochondria (M), rough endoplasmic reticulum (RER) and glycogen
droplets (GY) under TEM (Co, 6300×). (E) Hepatocytes showing necrotic nucleus (arrow), vacuolation in cytoplasm (broken arrow) with reduced amount
of RER under TEM (GL, 2500×). (F) TEM showing hepatocytes with dilated mitochondria (bold arrow) and cytoplasmic vacuolation (broken arrow; GF,
5000×).
TEM = transmission electron microscopy.
268 RER, and glycogen (Figure 2D). Under laboratory con- glomeruli, PCTs and DCTs, and vacuolation in the epithe- 288
269 ditions, TEM showed reduced endoplasmic reticulum, lial cell lining of the renal tubules (Figure 3B), while mild 289
270 presence of necrosis, cytoplasmic vacuolation, and reduced changes in PCTs and DCTs and fat deposition in some places 290
271 glycogen (Figure 2E), while under field conditions, damage were noted in kidneys after glyphosate exposure under 291
272 was less than under laboratory conditions, which included field conditions (Figure 3C). 292
273 dilated mitochondria, large amount of endoplasmic reti- TEM of normal kidneys showed electron-dense 293
274 culum, fewer glycogen droplets, and less cytoplasmic mitochondria and nuclei and abundant vesicular struc- 294
275 vacuolation in some regions (Figure 2F). tures in the capillary epithelial cell cytoplasm (Figure 3D). 295
The kidneys showed epithelial cell necrosis, dilation, and 296
276 3.3. Kidneys fragmentation of the endoplasmic reticulum, degeneration 297
of mitochondria, and severe vacuolation under labora- 298
277 Histologically, fish kidneys comprised a large number tory conditions after glyphosate exposure (Figure 3E), 299
278 of nephrons and hematopoietic tissues. Nephrons con- but under field conditions, the kidneys showed normal 300
279 tained Bowman’s capsules and renal tubules. Renal tubules appearance of the nucleus with prominent nucleoli and a 301
280 consisted of proximal convoluted tubules (PCTs), distal large number of mitochondria (Figure 3F). 302
281 convoluted tubule (DCTs), and collecting ducts. The renal

282 tubules mainly consisted of columnar and cuboidal epithe- 4. Discussion 303
283 lial cells but a cross section were either oval or spherical
284 (Figure 3A). The most evident damage noticed in the The present study is believed to be the first attempt to 304
285 kidneys of H. fossilis under light microscopy under lab- report the toxicity of the glyphosate-based herbicide Excel 305
286 oratory conditions after glyphosate exposure were loss Mera 71 by histopathological and ultrastructural observa- 306
287 of hematopoietic tissue, degenerative changes in the tions through SEM and TEM in the freshwater teleostean 307
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Figure 3. Histopathological photomicrographs of kidney of Heteropneustes fossilis under control conditions (Co), glyphosate-treated laboratory conditions
(GL), and glyphosate-treated field conditions (GF). (A) Normal proximal convoluted tubule (PCT), distal convoluted tubule (DCT), Bowman’s capsule and
glomerulus (G) under light microscopy (C, 1000×). (B) Degeneration of PCT and DCT (arrow), vacuolation in hematopoietic tissues (broken arrow) and
loss of hematopoietic tissue (square) under light microscopy (GL, 1000×). (C) Light microscopy showing normal structure of PCT and DCT and vacuolation
(broken arrow; GF, 400×). (D) Normal appearance of kidney with electron-dense mitochondria (M) and nucleus (N) under TEM (Co, 4000×). (E) Dilation
and fragmentation of rough endoplasmic reticulum (square), degeneration in mitochondria (bold arrow) and severe vacuolation (broken arrow) under TEM
(GL, 5000×). (F) Normal appearance of kidney under TEM (GF, 6300×).
TEM = transmission electron microscopy.
308 fish, H. fossilis. However, Senapati et al. [18,19] reported that these alterations might interfere with normal respi- 331
309 histopathological alterations in the stomach and intestine ratory function and ultimately lead to impairment of the 332
310 of Anabas testudineus after almix herbicide exposure under general health condition of the fish. Under field condi- 333
311 laboratory conditions. tions, no significant alterations in stratified epithelial cells 334
312 The major changes were hypertrophy of the epithe- and microridge structures were observed after glyphosate 335
313 lial cells, fusion of some secondary lamellae, and severe exposure and this may have been due to natural habitat 336
314 damage to chloride cells. These alterations may be the and dilution effects. TEM showed degenerative changes in 337
315 early responses in the gills as a defense mechanism against chloride cells, fusion of microridges, dilation of RER and 338
316 toxic xenobiotic substances. Similar alterations in the gills mitochondria, and vacuolation in chloride cell cytoplasm 339
317 have also been reported in the fish exposed to metals of H. fossilis under laboratory conditions. Damage to chlo- 340
318 [20], and after acute exposure to insecticides [21]. Accord- ride cells can result in increased blood flow inside the 341
319 ing to Mallatt [8], such alterations are nonspecific and lamellae, causing dilatation of the marginal channel, blood 342
320 may be induced by different types of xenobiotic contam- congestion, or even an aneurysm [2,25]. Loss of micror- 343
321 inant. According to Arellano et al. [22] and Biagini et al. idge structures as well as fusion as observed in the present 344
322 [23], these histological alterations observed in fish gills can study was also reported by Mazon et al. [26] and Biagini 345
323 be considered as a rapid and valid method to determine et al. [23]. Mallatt [8] reported that the microridges were 346
324 damage caused by exposure to different xenobiotic sub- related to the retention of mucus on the epithelium to pro- 347
325 stances. The most conspicuous ultrastructural alterations tect gill epithelium against environmental alterations. The 348
326 in the gills of fish after glyphosate exposure under labo- appearance of vacuoles in glyphosate-exposed fish as seen 349
327 ratory conditions were the complete loss of microridges in the present study may impede gas exchange capacity 350
328 in some gill epithelial cells, swelling of microridges, and as well as indicate swelling of the mitochondria and RER 351
329 irregular arrangement of microridges. Similar observations [27,28]. Therefore, these histopathological and ultrastruc- 352
330 were reported by Schwaiger et al. [24] who suggested tural lesions in the gill morphology observed in the present 353
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354 study could lead to functional alterations and interference [37,47]. This may be due to either increased glycolytic activ- 415
355 in osmoregulation and the antioxidant defense system of ity to meet the energy demands imposed by enhanced 416
356 the gills [29]. metabolic activity [52,53] or reduced intestinal absorp- 417
357 The present study exhibited severe damage in the liver tion of carbohydrates [54]. Generally, the lesions displayed 418
358 tissue of H. fossilis including enlarged and pyknotic hepato- in the investigated cells, tissues, and/or organs represent 419
359 cytes, excess fat deposition, and hepatocytic inflammation an integration of cumulative effects of physiological and 420
360 along with vacuolation in the cytoplasm. Enhanced and biochemical contaminants, and therefore, can be linked 421
361 pyknotic hepatocytes observed in the present study were to the exposure and subsequent metabolism of chemical 422
362 also reported by Uguz et al. [30] who assumed that this contaminants [55]. Therefore, it can be interpreted that 423
363 may have been due to an increase in the DNA/RNA ratio, hepatocytes of laboratory-exposed fish showed stronger 424
364 which was also observed in carcinogenic cells induced structural alterations than hepatocytes of field-exposed 425
365 by 4-nonylphenol [31,32]. Jiraungkoorskul et al. [33] also fish, which ultimately indicates greater disturbance of the 426
366 noticed swelling of hepatocytes, nuclear pyknosis, severe cellular metabolism under laboratory conditions. 427
367 vacuolation in the cytoplasm, degenerative changes in the Due to central role of the kidneys in xenobiotic 428
368 cell membrane, and severe infiltration of leukocytes in the metabolism and excretion [56], and that like the liver, 429
Q12 liver of Orechromis niloticus after Roundup exposure. Rah-

369 they receive the largest proportion of the post-branchial 430
370 man et al. [34] reported severe necrosis, a large number blood, renal lesions might be expected to be good indi- 431
371 of vacuoles in the cytoplasm, and pyknotic nuclei in the cators of environmental pollution [21]. A large number 432
372 liver of Corydoras punctatus and A. testudineus exposed to of studies used histopathological characteristics of the 433
Q13 Diazinon 60 EC. Hued et al. [35] reported focal necrosis,

373 kidneys as indicators of aquatic pollution, especially by 434
374 infiltration of leukocytes, dilation of blood sinusoids, and herbicides [33,35], but studies on the glyphosate-based 435
375 vascular congestion in the liver of Jenynsia multidentata herbicide, Excel Mera 71, are rare. The present study 436
376 after Roundup intoxication under laboratory conditions. demonstrated the same histological changes in the kid- 437
377 Among the major cytopathological responses in the hepa- neys after exposure to glyphosate. Similar results have 438
378 tocytes of laboratory-exposed fish in the present study also been reported in different fish species after exposure 439
379 was the loss of cellular compartmentation, which might to different xenobiotic substances [21,33,57–59]. Nephro- 440
380 have been due to chemical attack on the cytoskeleton [36]. histopathological alterations in kidneys of H. fossilis may 441
381 In addition to this, it has also been assumed that poorly have been due to herbicidal stress as a compensatory 442
382 developed cellular compartmentation indicates severe response, and may also be correlated with disruption of 443
383 disturbance of cellular metabolism [37]. Hepatocytes of several biochemical and physiological pathways includ- 444
384 fish exposed to glyphosate under laboratory conditions ing endocrine disruption [60,61]. Several ultrastructural 445
385 generally showed greater disturbance of cellular com- alterations such as epithelial cell necrosis, dilation, and 446
386 partmentation than fish exposed under field conditions. fragmentation of endoplasmic reticulum, degenerative 447
387 Ultrastructural changes in the endoplasmic reticulum of changes in the mitochondria, and severe vacuolation under 448
388 fish exposed to other toxicants have also been described laboratory conditions were hard evidence of glyphosate 449
389 by Braunbeck et al. [38]. Alterations of the RER, includ- stress. Fischer-Scherl et al. [57] reported major ultrastruc- 450
390 ing proliferation, fragmentation, and vesiculation, are the tural changes such as degeneration and vacuolation in the 451
391 common response to xenobiotic stress [39–41]. Although epithelial cells, and fragmentation in the RER in the kid- 452
392 Braunbeck and Völkl [42] and Au et al. [43] correlated the neys of rainbow trout exposed to atrazine. Cytoplasmic 453
393 alterations of the RER with higher biotransformation capac- vacuolation has also been reported in the kidneys of gold 454
394 ity of hepatocytes, Ghadially [44] interpreted the dilation fish exposed to hexachlorobutadiene by Reimschüssel et al. 455
395 of endoplasmic reticulum cisternae as a result of enhanced [62] and Segnini de Bravo et al. [63] in two Venezuelan cul- 456
396 storage of proteins due to reduced secretory activity. Alter- tured fish, Caquetaia kraussii and Colossoma macropomum, 457
397 ation of RER also indicates the induction of mixed-function after triazine exposure. The presence of hyaline droplets 458
398 oxidase [45], which can also be interpreted as the morpho- in renal tubules indicates the occurrence of renal toxicity 459
399 logical counterpart of ethoxycoumarin-O-deethylase and after herbicide exposure [64]. 460
400 ethoxyresorufin-O-deethylase induction. Similar observa-

401 tions were also reported in rainbow trout after exposure 5. Conclusion 461
402 to endosulfan and disulfoton [46], and in the demersal

403 fish following intraperitoneal injection of benzo(a)pyrene In conclusion, the present study indicates that chronic 462
404 [43]. Enhanced lipid droplets in hepatocytes of exposed exposure to glyphosate induces histopathological and 463
405 fish species observed in the present study were also ultrastructural changes in gills, liver, and kidneys. The 464
406 reported by others [37,47–50]. This enhanced number of cytopathological lesions in all three tissues recorded 465
407 lipid droplets indicated a decline of protein synthesis in under two different exposure conditions demonstrate 466
408 the cytoplasm, which ultimately blocks the utilization of the cumulative physiological and biochemical effects 467
409 lipid–protein conjugation [51]. Glycogen content declined of glyphosate exposure. The effects were more pro- 468
410 in hepatocytes under both conditions but in the present nounced in laboratory-exposed than field-exposed fish, 469
411 study the maximum reduction occurred in the laboratory. which ultimately indicated greater disturbance of cellu- 470
412 Similar observations were reported in other fish species lar metabolism as well as serious structural alterations 471
413 such as Channa punctata, Oncorhynchus mykiss, Danio rerio, under laboratory conditions. Therefore, these histopatho- 472
414 and Liza ramada, following exposure to several toxicants logical including ultrastructural alterations under different 473
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474 environmental conditions for fish could be considered as [19] Senapati T, Samanta P, Mandal S, Ghosh AR. Study on histopatho- 546
475 sensitive biomarkers of xenobiotic exposure. logical, histochemical and enzymological alterations in stomach and 547
intestine of Anabas testudineus (Cuvier) exposed to Almix 20WP her- 548
bicide. Int J Food, Agri Vet Sci 2013;3:100–11. 549
476 Acknowledgments [20] Oliveira Ribeiro CA, Pelletier E, Pfeiffer WC, Rouleau C. Comparative 550
uptake, bioaccumulation, and gill damages of inorganic mercury in 551
477 The authors would like to thank the Department of tropical and Nordic freshwater fish. Environ Res 2000;83:286–92. 552
[21] Cengiz EI. Gill and kidney histopathology in the freshwater fish Cypri- 553
478 Science & Technology, Government of India for financial nus carpio after acute exposure to deltamethrin. Environ Toxicol 554
479 assistance. We would also like to thank the Head, Depart- Pharmacol 2006;22:200–4. 555
480 ment of Environmental Science, The University of Burdwan, [22] Arellano JM, Ortiz JB, de Canales KLG, Sarasuete C. Histopathological 556
alterations and induction of cytochrome P-450 1A in the liver and 557
481 for providing the laboratory and library facilities during the
gills of the gilthead seabream (Sparus aurata) exposed to 2,3,7,8,- 558
482 research. tetrachlorodibenzo-p-dioxin. Histochem J 2001;33:663–74. 559
[23] Biagini FR, David JAO, Fontanetti CS. The use of histological, histo- 560
chemical and ultramorphological techniques to detect gill alterations 561
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JMAU 90 1–9 ARTICLE IN PRESS

Contents lists available at ScienceDirect

Journal of Microscopy and Ultrastructure

3 Toxic effects of glyphosate-based herbicide, Excel Mera 71

Histopathological study has been widely used for tox- 29

∗ Corresponding author. icity testing of the effects of xenobiotic compounds at the 30

35 environmental monitoring is that they are most effective

65 Aquatic bodies are contaminated by several pesti-

77 conditions. of each glyphosate-treated cage. During experimentation, 131

glyphosate-treated and control ﬁsh were subjected to the 132

for the culture of the experimental ﬁsh species as per 134

cages were square in shape with an area of 2.5 m × 1.22 m 136

cut by using a glass knife on an Ultracut E Reichart–Jung 207

lar system. Secondary lamellae were lined by squamous 223

lamella was lined by stratiﬁed epithelium. Secondary gill 225

ﬁeld conditions (Figure 1C). 232

Q7 at 3–4 ␮m using a Leica RM2125 microtome. These sections

structure (Figure 1F). 242

general appearance of chloride cells supported by 244

199 Instrumentation Centre, University of Burdwan.

The kidneys showed epithelial cell necrosis, dilation, and 296

276 3.3. Kidneys fragmentation of the endoplasmic reticulum, degeneration 297

of mitochondria, and severe vacuolation under labora- 298

281 convoluted tubule (DCTs), and collecting ducts. The renal

Q12 liver of Orechromis niloticus after Roundup exposure. Rah-

Q13 Diazinon 60 EC. Hued et al. [35] reported focal necrosis,

400 ethoxyresoruﬁn-O-deethylase induction. Similar observa-

402 to endosulfan and disulfoton [46], and in the demersal

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