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Toxic Effects of Deltamethrin and Permethrin on Gill Surface Ultrastructure of Anabas

testudineus : A Scanning Electron Microscopic Study

Article in Journal of Advanced Microscopy Research · June 2014

DOI: 10.1166/jamr.2014.1201

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 Copyright © 2014 American Scientific Publishers Journal of
All rights reserved Advanced Microscopy Research
Printed in the United States of America Vol. 9, 1–5, 2014

Toxic Effects of Deltamethrin and Permethrin on Gill

Surface Ultrastructure of Anabas testudineus:
A Scanning Electron Microscopic Study
Maisnam Sapana Devi and Abhik Gupta∗
Department of Ecology and Environmental Science, Assam University, Silchar 788011, Assam, India

Scanning electron microscopy of gill surface of Anabas testudineus exposed to sublethal concentra-
tions of two synthetic pyrethroids deltamethrin and permethrin were studied. Fish exposed to 0.035,
0.007 and 0.0007 mg l−1 deltamethrin, and 0.93, 0.093 and 0.0093 mg l−1 permethrin revealed
concentration-dependent manifestations of toxic effects of both the pesticides on the fish gill sur-
face ultrastructure. Deltamethrin affected gill surface morphology by causing swelling of filament
tips and distortion. The deep erosive lesions associated with deltamethrin toxicity were probably
caused by its cyanide moiety. Permethrin characteristically produced edematous lesions on primary
and secondary lamellae through possible interference with calcium metabolism. Both the pesticides
affected microridge structure with profuse mucus secretion at lower and obliteration of mucus gland
openings at higher concentrations. The study also reveals that deltamethrin toxicity is of concern
because of its ability to inflict damages at extremely low, environmentally relevant concentrations.
Keywords: Anabas testudineus, Scanning Electron Microscopy, Synthetic Pyrethroids, Erosive
Lesions, Edematous Lesions.

1. INTRODUCTION
The use of pesticides is considered as an effective mea-
sure for controlling pests, although they are highly toxic
to non-target organisms in the environment including
aquatic species. Among various groups of pesticides, syn-
thetic pyrethroids are often preferred because of their high
effectiveness and easy biodegradability, low toxicity to
birds and mammals, and less persistence in the environ-
ment. However, they are highly toxic to non-target aquatic
organisms including fish.1–3 Gill surface of fish is known
to experience a host of adverse morphological impacts
when subjected to different stressors including microbial
infections,4 industrial pollutants,5 acidic pH,6 pesticides,7
8
metals and other chemicals.9
10 Earlier, light microscopy
technique has been used to investigate the histopatho-
logical effects of toxicants including deltamethrin on
gill morphology of freshwater fish.9
11 Scanning elec-
tron microscopy (SEM) which provides a quasi-three-
dimensional image of surface ultrastructure and can clearly
detect any change under stress,12 has been utilized to inves-
tigate the effects of toxicants including pesticides on the
surface ultrastructure of fish tissues, including gills. There-
fore, the present study used SEM to examine changes
∗
Author to whom correspondence should be addressed.
RESEARCH ARTICLE
that occurred on the gill surface of Anabas testudineus on
exposure to deltamethrin and permethrin and also to find
out whether the effects on the surface ultrastructure of the
gill were concentration-dependent or all or none in nature.
If it was the former, then surface ultrastructural alterations
could be used increasingly as toxicological endpoints. Fur-
ther, permethrin, one of the compounds tested, is a type I
synthetic pyrethroid without a cyanide moiety in contrast
to the type II deltamethrin, which is cyano-substituted.
Possible differences between them in terms of the nature
of toxicity were, therefore, thought to be worth investi-
gating. The selected fish species Anabas testudineus is an
important food fish in India, and a local delicacy in its
eastern and northeastern parts. It is categorized as “Data
Deficient” by the IUCN.13
2. MATERIALS AND METHODS
2.1. Procurement of Fish and Pesticides
Anabas testudineus weighing 12 ± 2 g and 8–10 cm
long were collected from ponds and wetlands in Cachar
district, Assam, India, brought to the laboratory and trans-
ferred to glass aquaria after dipping in 0.1% potassium
permanganate solution to prevent infections and cure
existing ones, if any. They were fed commercial fish

J. Adv. Microsc. Res. 2014, Vol. 9, No. 2 2156-7573/2014/9/001/005 doi:10.1166/jamr.2014.1201 1

 Toxic Effects of Deltamethrin and Permethrin on Gill Surface Ultrastructure of Anabas testudineus Devi and Gupta

pellets and acclimatized for 15 days in unchlorinated

tap water that was conditioned by storing for 24 h
and aerated prior to the introduction of test fish. Water
was regularly aerated and renewed every 48 h dur-
ing acclimatization. Commercial grade deltamethrin
((S)--cyano-3-phenoxybenzyl(1R,3R-3-(2,2-dibromovinyl)-
2,2-dimethylcyclopropanecarboxylate) 2.8% EC (trade
name Decis-Agrevo India Ltd.) and permethrin(3-
phenoxybenzyl (1RS) cis, trans-3-(2,2-dichlorovinyl)-
2,2-dimethylcyclopropanecarboxylate) 25% EC (Trade
name Agniban-Devidayal Sales limited-an ISO 9001:2000
Company, Mumbai, India) were purchased from an agro-
chemical dealer in Silchar, India.

2.2. Sublethal Toxicity

For evaluating the sublethal effects of deltamethrin
and permethrin on gill morphology, fish were exposed
to 0.035 mg l−1 (50% 96 h LC50 ) deltamethrin and
0.93 mg l−1 (96 h LC50  permethrin for 96 h; 0.007 and
0.0007 mg l−1 deltamethrin, and 0.093 and 0.0093 mg l−1
permethrin (10% and 1% 96 h LC50 , respectively) for
3 weeks (21 days) in sublethal exposure. These concen-
trations were derived on the basis of the 96 h LC50 of
deltamethrin and permethrin for A. testudineus.14
Fig. 1. (a)–(c) Gill architecture of control Anabas testudineus: (a) (i)
primary lamellae (ii) secondary lamellae; (b) (i) and (ii) primary and
secondary lamellae at higher magnification; (iii) well-defined interlamel-
lar space between secondary lamellae; (c) (i) microridges (ii) mucus
gland opening. (d), (e): Gill of A. testudineus exposed to 0.035 mg l−1
deltamethrin (DM): (d) (i) fusion of primary lamellae (ii) clumping of
primary lamellae; (e) (i) deep erosive lesion on lamellar surface (ii)
sloughing of lamellar surface (iii) erythrocyte extrusions.

2.3. Processing of Tissue Samples for lamellae had eroded tips with reduced interlamellar space
Scanning Electron Microscopy between secondary lamellae (Fig. 2(b)). Epithelial cell
Both control and pesticide-exposed fish at the end of 96 h boundaries, mucus gland openings and parts of micror-
RESEARCH ARTICLE

exposure in acute and 21 day exposure in chronic test idge structure were severely damaged. Several erythro-
were anesthetized using 2-phenoxy ethanol and their gills cyte extrusions were found and the ridges had lost their
collected. Gills were preserved in 3% glutaraldehyde for
4 h and then in 0.1 M cacodylate buffer at 4  C. These
were dehydrated in a graded series of acetone (30–100%)
for 30 minutes each, placed in tetramethylsilane (TMS)
solution, kept under refrigeration for 10 minutes, air-dried
for 5 minutes,15 mounted on stubs with double-sided tape
and coated with a thin layer of gold in a sputter coating
unit. Coated specimens were then observed in a JEOL-
JSM 6360 scanning electron microscope and photographed
with computer-integrated software.

3. RESULTS AND DISCUSSION

In control A. testudineus, gills had branchial arches with
neatly arranged rows of primary lamellae which in turn
had rows of evenly spaced secondary lamellae (Figs. 1(a),
(b)). They also had prominent microridges on lamellar
surface and openings of mucus glands (Fig. 1(c)). Gills
exposed to 0.035 mg l−1 (50% 96 h LC50 ) deltamethrin
for 96 h showed clumping of primary lamellae and severe
bulging, twisting, and occurrence of deep erosive lesions Fig. 2. (a)–(c) Gill of Anabas testudineus exposed to 0.007 mg l−1 DM:
on their tips, with lamellar fusion of adjacent lamellae and (a) (i) clumping of primary lamellae; (b) (i) erosive lesion at lamellar tip
(ii) reduced interlamellar space between secondary lamellae; (c) (i) loss
erythrocyte extrusions (Figs. 1(d), (e)). In fish exposed to
of microridge structure (ii) near-obliteration of mucus gland opening (iii)
0.007 mg l−1 (10% 96 h LC50 ) deltamethrin, clumping of erythrocyte extrusion. (d), (e) Gill exposed to 0.007 mg l−1 DM after 1
primary lamellae and bulging of tip were less pronounced week in control condition: (d) (i) damaged primary lamellae (ii) clumping
than that in acute exposure (Fig. 2(a)). The primary of lamellar tips; (e) (i) and (ii) damaged microridge structure.

2 J. Adv. Microsc. Res. 9, 1–5, 2014

Devi and Gupta Toxic Effects of Deltamethrin and Permethrin on Gill Surface Ultrastructure of Anabas testudineus

Fig. 3. (a), (b) Gill exposed to 0.0007 mg l−1 DM: (a) (i) erosive lesions
on secondary lamella (ii) clumping of primary lamellae; (b) (i) and (ii)
lesions on microridges and mucus gland openings. (c), (d) Gill exposed to
0.0007 mg l−1 DM after 1 week in control condition: (c) (i) clumping of
lamellar tips; (d) (i) thick mucus film over microridges (ii) deterioration
of microridge structure.
normal architecture (Fig. 2(c)). Gills of this exposed fish
that were kept for one week in water without added pes-
ticide showed further progress of clumping, twisting and
bulging of lamellar tips with no sign of recovery. Sec-
Fig. 4. Gill exposed to 0.93 mg l−1 Permethrin (PM): (a) (i) dam-
ages on primary lamella (ii) reduction of interlamellar space between
secondary lamellae (iii) edema of primary lamella; (b) (i) edema of
secondary lamella (ii) erythrocyte extrusion (iii) fine linear rupture on
lamellar surface.
(Figs. 5(a), (b)). The microridged surfaces were swollen
and edematous (Fig. 5(c)). In fishes of this group kept
for one week in control conditions, edema and lamellar
damage continued unabated (Fig. 5(d)). The microridged
surfaces showed signs of returning to normalcy, although
the mucus gland openings remained ill-defined (Fig. 5(e)).
Fishes exposed to 0.0093 mg l−1 (1% 96 h LC50 ) per-
methrin showed less pronounced edema on primary and
secondary lamellae (Fig. 6(a)). Their microridged surfaces

RESEARCH ARTICLE
ondary lamellae also revealed marked erosions in their also retained better structural integrity, albeit with some
structure (Fig. 2(d)). However, there was partial restora-
tion of microridge architecture, although several damaged
patches persisted (Fig. 2(e)). In fishes exposed to the low-
est deltamethrin concentration of 0.0007 mg l−1 (1% 96 h
LC50 ), distortive effects on lamellar tips and smother-
ing of microridge architecture were still less pronounced,
although damages to secondary lamellae could be observed
(Figs. 3(a), (b)). After a one week recovery period, the
fishes showed relatively less deterioration of primary and
secondary lamellar structure, but with some apical clump-
ing (Fig. 3(c)). Microridges in many places were covered
with a thick layer of mucus (Fig. 3(d)). Fish exposed to
96 h LC50 permethrin (0.93 mg l−1 ) did not show conspic-
uous clumping and apical distortions of primary lamellae.
One of its major effects comprised generation of edema
in both primary and secondary lamellae. The edematous
tissue became soft and vulnerable to rupture, either in
life or during SEM processing and viewing (Fig. 4(a)).
Besides, the secondary lamellae also turned edematous
with concomitant narrowing of interlamellar space. The Fig. 5. (a)–(c) Gill exposed to 0.093 mg l−1 PM: (a) (i) clumping of
surface of primary lamella had fine linear ruptures with primary lamellae (ii) and (iii) erosive lesions on primary and secondary
erythrocyte extrusions at several places (Fig. 4(b)). In fish lamellae; (b) (i) and (ii) erosive lesion on primary and secondary lamel-
exposed to 0.093 mg l−1 (10% 96 h LC50 ), edema of pri- lae; (c) (i) edema of microridged surface and near obliteration of mucus
gland opening. (d), (e) Gill exposed to 0.093 mg l−1 PM after 1 week in
mary and secondary lamellae was observed, although the control condition: (d) (i) clumping of primary lamellae (ii) and (iii) ero-
magnitude was less than that in acute exposure. Damages sive lesions on primary and secondary lamellae; (e) (i) partially restored
to both primary and secondary lamellae could be seen microridge structure (ii) ill-defined mucus gland opening.

J. Adv. Microsc. Res. 9, 1–5, 2014 3

 Toxic Effects of Deltamethrin and Permethrin on Gill Surface Ultrastructure of Anabas testudineus
Fig. 6. (a), (b) Gill exposed to 0.0093 mg l−1 PM: (a) (i) and (ii) clump-
ing and edema of primary lamellae; (b) (i) mucus film over microridges
(ii) distortion of microridges. (c)–(d) Gill exposed to 0.0093 mg l−1 PM
after 1 week in control condition: (c) (i) edema of secondary lamellae
(ii) reduction of interlamellar space between secondary lamellae (iii) ery-
throcyte extrusions; (d) (i) mucus on microridged surface (ii) damaged
microridges.
distortions (Fig. 6(b)). However, the secondary lamellae in
the fishes of this group kept for a week in control con-
ditions recorded unabated progressive damage with ery-
RESEARCH ARTICLE
throcyte extrusions (Fig. 6(c)). The microridged surface
remained covered in several places with mucus (Fig. 6(d)).
Review of the findings of several studies conducted
on the effects of a wide range of toxicants and stres-
sors including metals,9
10
16–19 pesticides,7
8
20
21 other
chemicals,9
10
22 industrial effluents,5 low pH,6 and even
city sewage22 on gill morphology of freshwater fish
indicate that they produce effects that have often been
classified as lesions and reactions. The former comprise
necrosis and rupture of respiratory epithelium, while the
latter include hyperplasia, epithelial lifting, mucus secre-
tion, fusion and others. The latter are believed to be
defense responses of the fish to the stressors.20 It had long
been pointed out that the myriad structural anomalies were
generalized stress responses rather than toxicant-specific
expressions, and hence were common among a range of
toxicants and stressors.23 Nevertheless, some characteristic
effects of a given toxicant or a group of toxicants were
also identified.10 In the present study, some differences
could be discerned between the predominant morpholog-
ical effects of the two pyrethroids used, of which per-
methrin was a type I pyrethroid lacking a cyano group,
while the type II deltamethrin possessed a cyano moiety.
Cyanide is known to cause severe necrotic damage and
desquamation to the filamental epithelium resulting in an
irregular outer surface.10 Deep erosive lesions on primary
lamella caused by deltamethrin were, therefore, likely to
4 J. Adv. Microsc. Res. 9, 1–5, 2014
Devi and Gupta
be the characteristic effect of its cyanide moiety. The major
effect of permethrin, on the contrary, was the production
of edema in both primary and secondary lamellae. The lift-
ing of the lamellar epithelium increased the diffusion dis-
tance between water and blood, while the swelling of the
secondary lamella reduced interlamellar distance, thereby
impeding water flow. Among other toxicants, cadmium
was also found to produce similar edematous conditions
by increasing gill permeability because of displacement of
calcium from intercellular tight junctions.10 It is known
that permethrin, and not deltamethrin, increased the aver-
age frequencies of glutamate-mediated miniature excita-
tory postsynaptic currents (mEPSCs), which in turn was
depressed by removal of Ca from the extracellular solu-
tion. The increased mEPSC frequency was through the
alteration of intracellular Ca dynamics that was a char-
acteristic of permethrin toxicity.24 Further, the impact of
deltamethrin on voltage-gated calcium channels was char-
acterized by Ca2+ influx in a concentration-dependent
manner, while permethrin did not induce Ca2+ influx.2
When the effects of several pyrethroids on sodium, cal-
cium and chloride ion channels were analyzed, permethrin
and deltamethrin were found to belong to two distinct
mechanism groups.25 It is, therefore, probable that impair-
ment of calcium metabolism by permethrin resulted in the
characteristic edematous effects in primary and secondary
lamellae. Another important effect was the loss of micror-
idge pattern in both deltamethrin and permethrin exposure,
which was likely to adversely affect retention of protec-
tive mucus layer.10 It was suggested that when the gill
surface came in contact with a toxicant, mucus secretion
acted as a protecting device checking further penetration
of the toxicants.26 Obliteration of mucus gland openings
meant impairment of protective mucus secretion and fur-
ther increase in the vulnerability of gill epithelium to tox-
icants. In an alternative view, it has also been argued that
loss of microridges could also comprise a defense mech-
anism by reducing the membrane surface area.20 How-
ever, we observed that when microridge integrity was more
or less intact such as in 0.7 g l−1 deltamethrin and
9.3 g l−1 permethrin sublethal exposures, fish secreted
a large amount of mucus that may be interpreted as an
attempt to restore its normal gill function. Viewed in this
perspective, loss of microridge would appear to be a lesion,
and not a defense reaction.
4. CONCLUSIONS
Possession of a cyano moiety inflicted deep erosive lesions
on the gill lamellae exposed to deltamethrin. Permethrin’s
characteristic effect comprised edema in both primary and
secondary lamellae that might have been caused by the
pesticide’s interference with Ca dynamics. Gill surface
morphology responded to deltamethrin and permethrin in
a concentration-dependent manner, thereby establishing a
cause-effect relationship among pesticide exposure and
 Devi and Gupta Toxic Effects of Deltamethrin and Permethrin on Gill Surface Ultrastructure of Anabas testudineus
morphological anomaly. The study is also suggestive of the
toxicity of deltamethrin at extremely low concentrations
that may be considered environmentally relevant.
Conflict of Interest
The authors declare that they have no conflict of interest.
Acknowledgments: The authors are grateful to the
Head, Sophisticated Analytical Instrumentation Facility
(SAIF), North Eastern Hill University, Shillong, India, for
providing access to SEM facility. Maisnam Sapana Devi
is grateful to the University Grants Commission for the
award of a doctoral fellowship.
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RESEARCH ARTICLE
Received: 13 April 2014. Accepted: 5 June 2014.
5