Synergism between environmental

contaminants and parasitic infection in

Anuran amphibians

Under the Supervision of: Presented By:

Dr. Anirudha Giri Utsab Singha
Associate Professor, Environment and Human
Dept. of Life Science & Toxicology Laboratory
Bioinformatics, Dept. of Life Science &
Assam University, Silchar Bioinformatics, AUS
 Human beings are intricately
linked with the components of the
ecosystem

Therefore,

Human welfare is intimately

associated with

“Welfare of the ecosystem”

 It has been said that
“Healthy individuals make
healthy ecosystem”

Therefore,
An assessment of the biodiversity
will let us know about the health
of our ecosystem.
 Since it is not possible
to assess the health of each and
every individuals in an
ecosystem

Bioindicators are used as

surrogates
 It is generally agreed that
use of endogenous biota
as bio-indicator is a more realistic
option

and

if such species is ecologically

threatened the monitoring process
can address both hazard
identification and provide vital
data for their conservation.
 Keeping in this requirements -

Amphibians present an excellent

prospect
as bio-indicators in environmental
monitoring.
 Total species = 272
Endemic to India = 75
Red List Category Number of Species
Extinct 34
Extinct in the wild 1
Critically endangered 455
Endangered 768
Vulnerable 670
Near threatened 369
Least concern 2236
Data deficient 1382
Total number of species 5915
HABITAT LOSS CLIMATE CHANGE

TRADE
DISEASE
INVASIVE SPECIES
 However,

Environmental contaminants may

play a major role in
amphibian population decline
 In aquatic systems,
pesticides are a common type of
contaminants
and found to be present in
30–60% of shallow groundwater
and
60–95% of surface water.
 Since aquatic habitat is an
inseparable part in
the life history of amphibians,
exposure to pesticides is an
unavoidable reality
 Besides, as amphibians are
co-exposed to other stressors in
the environment like predation
risk, Parasitic infection etc. it
would be interesting to know their
effects on pesticide toxicity
 Materials and Methods
Larval collection Tadpoles of F. limnocharis
and rearing and P. Maculatus were used
for this Experiment

Acclimatized in laboratory
prior to exposure

Fejervarya limnocharis Gosner 26-28 stages tadpoles

were selected for the
experiment (Gosner 1960)

Standard laboratory
condition and food supply
Polypedates maculatus was made during experiment
 Materials and Methods
Parasitic Infection Snails of genus Helisoma were
Assay collected from eutrified pond &
then acclimatized in laboratory
condition

Metacercariae @
100X
 Experimental setup

The experiments were

1 2
performed in
polypropylene tubs Water and treatments
containing 2–8 liters of renewed alternate day
aged well water

Different experiments done as per

standard protocols
Images of micronucleated
blood cells
Predator Cue Exposure

Predator cue preparation and

exposure was done as described

by
Fraker, 2008
 Statistical Analysis

Survival analysis by Kaplan-Mayer product limit

analysis
Change in metamorphosis time and body weight;
Micronucleus frequency by Analysis of Variance
Determination of LC50 by Probit regression
Dose response relationship by Linear regression
F-Test was done to check for normality prior to
analysis
Analyses were made using SPSS 18.0 at 95%
Confidence interval
Fejervarya limnocharis
 Control
100%

Sodium Nitrate: 75%

Survival
50%
% Survival 25% No parasite
Parasite
(F.limnocharis) 0%
1 2 3 4 5 6 7 8 9 10 11 12 13
Time in Days

30 mg/L 40 mg/L
100% 100%

75% 75%

Survival
Survival

50% 50%

25% No parasite 25% No parasite

Parasite Parasite
0% 0%
1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13

Time in Days Time in Days

50 mg/L 60mg/L
100% 100%

75% 75%
Survival

Survival

50% 50%
25% No parasite 25% No parasite
Parasite Parasite
0% 0%
1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13
Time in Days
Time in Days
 Sodium Nitrate:
Body Weight
(F.limnocharis)

Without Parasite With Parasite

80
Body Weight at Metamorphosis (mg)

70

60

50

40

30

20

10

0
Control 30mg/L 40mg/L 50mg/L 60mg/L

Treatment in mg/L
 Sodium Nitrate: Time
to Metamorphosis
(F.limnocharis)

Without Parasite With Parasite

40
Time to Metamorphosis (Days)

35

30

25

20

15

10

0
Control 30mg/L 40mg/L 50mg/L 60mg/L

Treatment in mg/L
 Sodium Nitrate: Snout
to Vent Length
(F.limnocharis)

Without Parasite With Parasite

12

10
Snout to Vent Length (mm)

0
Control 30mg/L 40mg/L 50mg/L 60mg/L

Treatment in mg/L
Polypedates maculatus
 Control
100%

Sodium Nitrate: 75%

Survival
50%
% Survival (P. 25% No parasite
Parasite
maculatus) 0%
1 2 3 4 5 6 7 8 9 101112131415

Time in Days

30 mg/L 40 mg/L
100% 100%
75% 75%
Survival

Survival
50% 50%

25% No parasite 25% No parasite

Parasite Parasite
0% 0%
1 2 3 4 5 6 7 8 9 101112131415 1 2 3 4 5 6 7 8 9 101112131415
Time in Days
Time in Days

50 mg/L 60 mg/L
100% 100%

75% 75%
Survival

Survival

50% 50%

25% No parasite 25% No parasite

Parasite Parasite
0% 0%
1 2 3 4 5 6 7 8 9 101112131415 1 2 3 4 5 6 7 8 9 101112131415
Time in Days Time in Days
 Sodium Nitrate:
Body Weight (P.
maculatus)

Without Parasite With Parasite

80
Body Weight at Metamorphosis (mg)

70

60

50

40

30

20

10

0
Control 30mg/L 40mg/L 50mg/L 60mg/L

Treatment in mg/L
 Sodium Nitrate: Time
to Metamorphosis
(P. maculatus)

Without Parasite With Parasite

40
Time to Metamorphosis (Days)

35

30

25

20

15

10

0
Control 30mg/L 40mg/L 50mg/L 60mg/L

Treatment in mg/L
 Sodium Nitrate: Snout
to Vent Length
(P. maculatus)

Without Parasite With Parasite

12
Snout to Vent length (mm)

10

0
Control 30mg/L 40mg/L 50mg/L 60mg/L

Treatment in mg/L
Fejervarya limnocharis
 Control
100%

Sodium Arsenite: 75%

Survival
50%
% Survival (F. 25% No parasite

limnocharis) 0%
Parasite

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time in Days

50 µg/L 100 µg/L

100% 100%

75% 75%

Survival
Survival

50% 50%

25% No parasite 25% No parasite

Parasite Parasite
0% 0%
1 2 3 4 5 6 7 8 9 101112131415 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time in Days Time in Days

200 µg/L 400 µg/L

100% 100%

75% 75%
Survival

Survival

50% 50%

25% No
25% No
parasite
parasite
0%
0%
1 2 3 4 5 6 7 8 9 101112131415
1 2 3 4 5 6 7 8 9 101112131415
Time in Days
Time in Days
 Sodium Arsenite:
Body Weight (F.
limnocharis)

80 Without Parasite With Parasite

Body Weight at Metamorphosis (mg)

70

60

50

40

30

20

10
All Dead
0
Control 50µg/L 100µg/L 200µg/L 400µg/L

Treatment in µg/L
 Sodium Arsenite: Time
to Metamorphosis
(F. limnocharis)

Without Parasite With Parasite

25
Time to Metamorphosis (Days)

20

15

10

All Dead
0
Control 50µg/L 100µg/L 200µg/L 400µg/L

Treatment in µg/L
 Sodium Arsenite:
Snout to Vent Length
(F. limnocharis)

10 Without Parasite With Parasite

9
8
Snout to Vent Length (mm)

7
6
5
4
3
2
1
All Dead
0
Control 50µg/L 100µg/L 200µg/L 400µg/L

Treatment in µg/L
Polypedates maculatus
 Control
100%

Sodium Arsenite: 75%

Survival
50%
% Survival (P. 25% No parasite

maculatus) 0%
Parasite

1 2 3 4 5 6 7 8 9 10 11 12 13
Time in Days

50 µg/L 100 µg/L

100% 100%

75% 75%

Survival
Survival

50% 50%

25% No parasite 25% No parasite

Parasite Parasite
0% 0%
1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13
Time in Days Time in Days

200 µg/L 400 µg/L

100% 100%
75% 75%
Survival
Survival

50% 50%
25% No parasite
25% No parasite
Parasite
Parasite 0%
0%
1 2 3 4 5 6 7 8 9 10 11 12 13
1 2 3 4 5 6 7 8 9 10 11 12 13
Time in Days
Time in Days
 Sodium Arsenite:
Body Weight (P.
maculatus)

80 Without Parasite With Parasite

Body Weight at Metamorphosis (mg)

70

60

50

40

30

20

10
All Dead
0
Control 50µg/L 100µg/L 200µg/L 400µg/L

Treatment in µg/L
 Sodium Arsenite: Time
to Metamorphosis
(P. maculatus)

Without Parasite With Parasite

35
Time to Metamorphosis (Days)

30

25

20

15

10

5
All Dead
0
Control 50µg/L 100µg/L 200µg/L 400µg/L

Treatment in µg/L
 Sodium Arsenite:
Snout to Vent Length
(P. maculatus)

12 Without Parasite With Parasite

Snout to Vent Length (mm)

10

All Dead
0
Control 50µg/L 100µg/L 200µg/L 400µg/L

Treatment in µg/L
 Acute Toxicity

All the four compounds tested produced

significant mortality in a concentration dependent
manner over the 15-day study period

From the estimated LC50 values, endosulfan was

found to be the most toxic compound followed by
cypermethrin, malathion and glyphosate.

Predator cue exposure along with the test

chemicals increased the acute toxicity in all the
chemicals except for malathion
 Life-history Traits
 Malathion,endosulfan and cypermethrin
reduced the age at metamorphosis while
glyphosate had no effect.

 The decrease in metamorphosis time, by and

large, was associated with a lower body
weight with the exception of malathion.

 Glyphosate did not change either of these

parameters suggesting that different
physiological pathways are involved in acute
and sub-lethal toxicities of these chemicals.
 Life-history Traits
 Predator
cue exposure affected the time to
metamorphosis and body weight following
exposure to endosulfan or cypermethrin only
 Genotoxicity Study
 Allthe chemicals tested induced significant
genotoxicity as assessed by the micronucleus
test in the peripheral bold erythrocytes.

 The genotoxicity was dose and context

dependent.

 The highest frequency of micronucleus was

found to be at 48-h of the exposure of the
chemicals, which however did not
significantly change at later periods.
 Genotoxicity Study
 Thefindings have methodological
implications. For genotoxicity studies in
amphibians in aquatic system, 48-h exposure
period is the ideal condition for micronucleus
assay in contrast to the 24-h exposure period
used in mammalian studies.

 Predatorcue exposure increased the

frequency of micronucleus following
exposure to either malathion or
cypermethrin, whereas no such effect could
be seen for glyphosate or endosulfan.
 Conclusion
 Thepresent studies clearly indicate that the
pesticides tested can be considered as
significant risk factor for E. cyanophlyctis
inhabiting the agroecosystems as well as
others species using these environment for
breeding.

 The magnitude and extent of the effects may

differ among species, populations, life
stages, and may also be context dependent
since multiple cross stressors could interact
among themselves in additive or synergistic
manner.
 ACKNOWLEDGEMENT

I express my deep sense of gratitude and sincere thanks to my

supervisor Dr. Anirudha Giri of the Department of Life Science and
Bioinformatics, Assam University, Silchar for their constant guidance and
inspiration throughout the course of this investigation enabled me to
complete this study.

I thankfully acknowledge to Prof. S. Giri, Professor, Department

of Life Science and Bioinformatics for not only providing laboratory
facilities, but also for her valuable guidance and encouragement all
throughout my study.

The sincere help and support of my laboratory colleagues Dr.

Freeman Boro, Ms. Krishna Bhuyan. Mr. Arabinda Pathor and Mr. Indranil
Das are thankfully acknowledged.
Publications
Publications
 I have to acknowledge many people for
this day. However, I take this opportunity
to sincerely thank my supervisor and co-
supervisor for their constant guidance.

Besides, I am thankful to our HOD Prof. S.

Giri and Dean sir Prof. D. Kar for their
help and support.

I will always remain thankful to the

teachers of this department and my lab
colleagues for their help and support.