Physiological and histopathological effects of Bisphenol A .Bisphenol A is less soluble in water. For that reason, dimethyl sulfoxide (DMSO) was used as a medium to obtain proper distribution in the test solution (Chen, J., et al, 2015). Working solution of commercial grade Bisphenol A (97% pure) was prepared by dilution of stock solution double distilled water immediately prior to experimental use. Serial dilutions of the stock solution were prepared using previously aerated, copper free and stored tap water. The water was continuously aerated. This was prepared by dissolving BPA (50mg) in 100ml of DMSO and the desired concentrations of BPA in tap water were prepared by adding appropriate volumes of this stock solution into test aquarium. A static non-renewable bioassay was conducted in triplicate for each concentration with four animals in each tub. No water exchange was done and the fishes were not fed during the period of the experiment. Percentage mortality was recorded at 12, 24, 48, 72 and 96 h interval. Control group was subjected to acetone at the maximum acetone volume used in the dilution of the dose concentrations. The range of LC50 for H.fossilis (mean wt. 36.78 g) under given conditions was determined to lie between 5 and 10 mg/L for BPA. Hence, for the definitive test, concentrations such as 2, 4, 6, 8, 10, 12, and 14 mg/L of BPA concentration were selected. The test was conducted in triplicate for each concentration with 10 fishes in each tank. At the end of 96 h, the fishes that had survived were anesthetized with clove oil at 100 mg/L, sampled for blood, and processed for hematological analysis. The data obtained from the experiment was processed by probit analysis using a Microsoft Excel computer program.

1. Physiological and histopathological effects of
Bisphenol A on male and female reproductive
function of H.fossilis (Bloch, 1974).
Dr.Reddy. P.B
Govt.PG College. Ratlam

5. Endocrine disrupting effects of bisphenol A

6. Aims and Objectives
 To determine lethal concentration (LC5o) of BPA in Heteropneustes fossilis.
 To study the effects of the sub-lethal concentration of BPA on histopathology of
reproductive organs of both male and female, blood profile, enzymes and and hormone
analysis.
 To convey a framework for identifying research needs related to the health and ecological
effects of BPA.
 To understand the behavior of BPA in surface and subsurface environments, enabling
prediction of their fate and transport in support of long-term environmental management
and development of new science-based remediation strategies.
 Common indicators used for the prediction of the adverse effects of BPA on human health
and the environment as well as their reliability and accuracy in the risk assessment of those
adverse effects
Expected outcome
 It generates indispensable facts and information for policymakers.
 It supports the scientific knowledge on xenobiotics and endocrine disrupting chemicals
(EDCs).
 This investigation will facilitate to incorporate information from various scientific authorities
and can apply these models to solve complex environmental problems.
 The study will help to identify and solve the complex relationships between scientific
approaches to environmental issues and political, social, economic, and ethical perspectives
on the environment.
 This study will be useful to formulate a framework for identifying research needs related to
the health and ecological effects of plastics.

7. H.fossilis is a remarkably tough and long-lived species, and can unquestionably live for 20
years or more. It can inhale atmospheric air to a certain extent by swim bladder,
permitting it to live in some antagonistic circumstances. It also secretes a mucous that
supports to delay the dehydration of the skin, and can cover its gills completely.
Lives in freshwater, rarely found in brackish waters. This is chiefly a fish of ponds,
trenches, wetlands and swamplands, but it is occasionally found in muddy rivers. It is
capable to tolerate marginally brackish water. Its air-breathing apparatus permits the fish
to survive in almost any type of water.
Morphological identification of the fish was done based on descriptions of Talwar and
Jhingaran (1991), Froese, and Pauly (2012). We selected both mature females and males
fishes of total length bigger than 12cm and males with total length larger than 5.5 cm.
The sexes were identified by peripheral inspection of the urogenital papilla in females
(Hoar, W.S.,et al, 1984) and by macroscopic observation of the gonads.

8. Bisphenol A (BPA) is an organic synthetic compound with the chemical formula
(CH3)2C(C6H4OH)2 belonging to the group of diphenylmethane derivatives and bisphenols,
with two hydroxyphenyl groups. It is a colorless solid that is soluble in organic solvents, but
poorly soluble in water (0.344 wt % at 83 °C).
Chemical formula: C15H16O2
Structural chemical formula and molecule model.

9. Determination of Lethal Concentration (LC) 50 value:
Bisphenol A is less soluble in water. For that reason dimethyl sulfoxide (DMSO) was
used as a medium to obtain proper distribution in the test solution. A group of 10
healthy fishes were exposed to diverse concentrations of the Bisphenol A to
calculate the medium lethal concentration LC50 value using probit analysis method
(Finney, D. J, 1952). A semi-static system was used to expose the fish (average
weight of 36.78 g) to the test chemical. 20-L glass aquaria were filled with tap water
(copper free).
At the start, a range finding test was performed to decide the range to be followed
in the decisive test. In this test, the fish were exposed to a range of concentration in
logarithmic scale such as 1, 2, 3, 4, 5, 6, 7 and 8 g/L.
The range of LC50 for H.fossilis (mean wt. 36.78 g) under given conditions was
determined to lie between 5 and 10 mg/L for BPA. Hence, for the conclusive test,
concentrations such as 2, 4, 6, 8, 10, 12, and 14 mg/L of BPA concentration were
carefully chosen. Experiment was conducted in triplicate for BPA and for each
concentration with 10 fishes in each tank.
Microsoft excel computer program. LC50 values of BPA for H.fossilis were finally
determined by using an online tool (www.aatbio.com/tools/lc50). The LC50 values of
BPA for H.fossilis were found to be 7.1443 mg/L.

10. Experimental design:
 Fishes were exposed to different sublethal concentrations i.e. to 0.714 mg/L
(1/10),
1.148 mg/L (1/20) and 2.142mg/L (1/30%) (Group II, III and IV) for 45 days
using ten fishes in each aquarium.
 At the end of experiment (45days), blood from the control and BPA treated
fishes was accomplished by puncture of caudal peduncle using a sterile
disposable plastic syringe . We studied following parameters in both control
and experimental fishes
 Effects of BPA on swimming behavior
 Histopathology of testes and ovary
 Hematological Analysis: RBC,WBC,Ht, MCV, MCH, MCHC,
 Total plasma protein
 Plasma AST (SGOT) and ALT (SGPT)
 Testosterone (17β-Hydroxy-4-androstene-3-one) and Estradiol (E2 or 17β-
estradiol) assay.

13. Parameter Values Parameter
Temperature 0C 20.01± 0.2 Temperature 0C
Electrical conductivity (umho/cm) 1392.1±.15.1 Electrical conductivity (umho/cm)
pH 7.1± 0.11 pH
Alkalinity (mg/L) 96.02 ± 5.1 Alkalinity (mg/L)
Dissolved Oxygen (mg/L) 7.1± 0.2 Dissolved Oxygen (mg/L)
Total dissolved substances (mg/L) 119.1± 3.3 Total dissolved substances (mg/L)
Total Hardness (mg/L) 54± 0.9 Total Hardness (mg/L)
Nitrate (mg/L) 35.2± 2.1 Nitrate (mg/L)
Chloride (mg/L) 128.1± 3.1 Chloride (mg/L)
Table 6.Physicochemical parameters of the water used for experimental purposes (Drinking water specification:
(Drinking Water Standards of BIS (IS 10500: 1991).
The results of test water parameters revealed that the water conditions were most
suitable for fish and were functional for laboratory measures. The experimental
mediums did not exhibit any significant differences among various parameters.

14. Dose Mg/l Log of
concentration
Total
individuals
Mortality Mortality % Probit kill
0 0 10 0 0 0
2 0.3010 10 2 20 4.16
4 0.6020 10 3 30 4.48
6 0.7791 10 4 40 4.75
8 0.9031 10 7 70 5.52
10 1 10 8 80 5.84
12 1.0792 10 10 100 7.37
14 1.1461 10 10 100 7.37
16 1.2041 10 10 100 7.37
18 1.2552 10 10 100 7.37
20 1.3010 10 10 100 7.37
Table 7: Relation between the concentration of BPA and the percentage mortality of the fish
y = 5.2424x + 3.8182
R² = 0.9557
0
20
40
60
80
100
120
0 5 10 15 20
%
mortality
BPA concentration in mg/L
Fig. 4. Determination of Lethal Concentration (LC) 50 of BPA
for H.fossilis
%
Mortality
Linear
(%
Mortality
)
%
m
ort
ali
ty
BPA mg/L
mg/L
Fig. Determination of LC50 value of Bisphenol A for
Heteropneustes fossilis (http://www.ic50.tk/).

15. Group II Group III Group IV
Irritation, Gulping air at the
surface, Surface movement, erratic
swimming
Slow movement, hyper active in
the beginning but later become
inactive, stop feeding.
Fast opercular movements,
effortless predation, loss of balance
by upright hanging, Move towards
surface,
Mucus secretion, slow movement,
hyper active in the beginning but
later become inactive,
Loss of equilibrium, swimming at
the water surface, spiralling,
convulsions, Heavy mucous
secretion, increased rate of
opercular movement. forwarded
pectoral fin
Table 8. Behavioral changes in H.fossilis in response to exposure to sublethal concentrations of Bisphenol A.

18. Parameter Control (Group I) Group II Group III
RBC (million/cu mm) 1.72 ± 0.058 1.9 ± 0.104 1.44 ± 0.024

19. Parameter Control (Group I) Group II Group III
WBC (1000/cu mm) 25.17 ± 0.748 43.02 ± 0.299 42.06 ± 0.624

20. Parameter Control (Group I) Group II Group III
Haemoglobin (g/dl) 8.45± 0.32 7.81 ± 0.39 6.44 ± 0.11

21. Parameter Control (Group I) Group II Group III
Hematocrit (%) 17.46 ± 0.722 13.14 ± 0.201 10.84 ± 0.515

22. Parameter Control (Group I) Group II Group III
MCV(fL) femtoliters 106.81±3.1 95.14±1.4 83.61±1.7

23. Parameter Control (Group I) Group II Group III
MCH (pg/cell) 32.55±0.75 27.15±0.48 26.36±0.61

24. Parameter Control (Group I) Group II Group III
MCHC (g/dL) 38.86±0.65 39.48±1.14 31.12±0.82

25. Parameter Control (Group I) Group II Group III Group III
Plasma protein(µg/ml) 9.77±0.95 8.34±0.24 6.56±0.63 4.58±0.61

26. Parameter Control (Group I) Group II Group III Group III
INR (sec) 11.2 ± 0.33 21.3 ± 0.51 38.7 ± 1.09 75.8 ± 2.1

27. Parameter Control (Group I) Group II Group III Group III
AST (SGOT) (U/L) 60.61± 0.78 71.23± 1.13 88.94± 2.3 124.22± 3.4
ALT (SGPT) (U/L) 29.8± 0.31 34.8± 0.41 41.2 ± 0.51 49.6± 0.75

28. Experimental Group 17-estradiol (E2) (ng/ml
Male Female
Group I (Control) 0.52 2.4
Group I (1/10% BPA) 0.49 2.7
Group I (1/20% BPA) 1.8 4.9
Group I (1/30% BPA) 2.3 6.1
Table.6 Plasma levels of 17-estradiol (E2) in H.fossilis after 45 days of exposure to BPA.
Data are expressed as the mean ± SEM (n = 3 samples).*

29. Experimental Group testosterone (T) (ng/ml
Male Female
Group I (Control) 1.42 0.81
Group I (1/10% BPA) 1.94 0.74
Group I (1/20% BPA) 1.97 0.98
Group I (1/30% BPA) 0.54 1.56
Table.6 Plasma levels of testosterone (T) in H.fossilis after 45 days of exposure to BPA.
Data are expressed as the mean ± SEM (n = 3 samples).

30. DISCUSSIONS
 Bisphenol A (BPA) has been the topic of extensive scientific study and evaluation due its
commercial use in wide range of products since 70years.
 Bisphenol A is an endocrine disruptor (EDC) that mimics the structure and function of
estradiol hormone by binding and stimulating the oestrogen receptor as the natural
hormone.
The overall outcome of this research work is as follows:
 The 96-hr LC50 of BPA for the fish, H.fossilis was found to be 7.14 mg/l.
 The fish exposed to different graded concentrations of Bisphenol A (BPA) revealed
symptoms of many abnormalities in their behavioural reactions.
 The sub lethal treatment of BPA caused morphological abnormalities like discoloration,
cracks and necrosis of fins, eye deformities, lower lip extension and excess mucus
secretion all over the body of H.fossilis. The percentage of these abnormalities was
higher at higher concentration.
 Results clearly revealed that the gonads of catfish H.fossilis treated with sublethal
concentrations of BPA shown irregular variations in dose dependent manner.
 The major effects of sub lethal concentration of BPA on histopathology of testis of
Heteropneustes fossilis include necrosis, nuclear hypertrophy, reduced lumen,
degeneration, presence of melano-macrophage centres, atrophy, germ cell syncytia,
hypertrophy and pyknosis and vacuolated germ cells.
 The major effects of sublethal concentration of BPA on histopathology of ovary of
Heteropneustes fossilis includes atretic oocytes, ruptured zona radiata and karyoplasmic
clumping, necrosis and nuclear hyper trophy, cellular degeneration, egg debris, broken
zona radiata and fibrosis.

31.  The effects of sublethal concentration of BPA on haematology recorded an initial
increase in the values of all blood parameters like RBC, WBC and Hct contents in
Group II. However, a significant decrease in all haematological parameters was
observed in Group III and IV exposed with the higher dose.
 BPA caused a significant decrease in plasma total protein (TP) and significant and
gradual increase in all experimental groups. AST (SGOT) and ALT (SGPT) in dose
dependent manner.
 BPA in fish, H.fossilis caused significant increase plasma 17β-estradiol
concentrations in all experimental fishes of both sexes .
 The values of testosterone (T) (ng/ml) in both male and female were significantly
increased in all experimental fishes.
 In conclusion, the exposure of sexually immature H.fossilis to bisphenol-A (BPA)
triggered reproductive dysfunction by altering sex steroid homeostasis and gonad
development.
 The present research confirms that sub lethal concentrations of BPA induced
various behavioural, histopathological, haematological, metabolic and
reproductive anomalies in H.fossilis.

32. Conclusions:
Overall, it is evident that BPA affected major disruption to reproduction by affecting
haematology, enzymes, biochemical parameters and plasma steroids in H.fossilis
exposed to sub lethal concentrations of BPA, likely via estrogenic mechanisms.
Further recommendations:
The current scientific investigation has proven that BPA have adverse effects on H
fossilis. However, additional research is required to validate the molecular
mechanism of BPA action. An improved understanding is wanted to identify the
occurrence and mobilization of BPA during different stages of life cycle of animals
and more broad databases are essential on the working and activity of this
chemical. The present data is preliminary and additional work is wanted,
principally to expand extra knowledge on the methylation pattern of the genes
involved in reproduction.
The path displayed in this thesis may deliver the basic need and serve as a
framework for forthcoming studies in this direction. Better perceptions and its
effects to this chemical is possible only by improved methodology, developing
profound bioassays and identifying reliable biomarkers.

33. THANKS