A pollutant of water resource

1. Ph.D., VIVA - VOCE EXAMINATION
MARCH 15 – 2021

2. P.G. & RESEARCH DEPARTMENT OF ZOOLOGY
KANDASWAMI KANDAR’S COLLEGE
VELUR - 638 182

3. INTRODUCTION
 Water is an essential abiotic factor for all the living organisms in this
world. Due to enormous industrialization and consumer utilization
creates major threat to the aquatic organisms and Humans.
 Plastic industries use various endocrine disrupting chemicals (EDCs)
for the synthesis of polycarbonate materials.
 The aquatic ecosystems are the ultimate sink for all kinds of
endocrine disrupting chemicals (EDCs).
 EDCs have the capacity to disturb the hypothalamic neural secretions
and functions of the exposed organisms that could be expressed
physiologically and metabolically altered way.
 Diethylene glycol dibenzoate (DGB) is an alternative phthalate
chemical widely used in adhesives, sealants, PVC pipes, elastomers
and vinyl flooring synthesis.
 DGB ((HOCH2CH2)2O) is an organic compound with physiochemical
properties such as odorless, colorless, and poisonous and a
hygroscopic liquid with sweet taste.
 DGB shows solubility in water, alcohol, ether and acetone. Diethylene
glycol dibenzoate has moderate acute toxicity against animals.

4. TAXONOMICAL CLASSIFICATION
COMMON NAME : ZEBRAFISH

5. OBJECTIVE OF THE STUDY
Objective of the study was concluded as follows.
Effect of Diethylene glycol dibenzoate in zebrafish was studied through
enzymes studies in the organs such as gills, liver, muscles and gonads
(ovary & testis) in this study.
Changes were observed and analysed specifically in biochemical,
histological and physical conditions, due to chemical (DGB) stress.
The Environmental Protection Agency (EPA) reported that EDCs
primarily trigger the androgen and estrogen receptors (AR&ER) either by
inhibition or activation.
Considering the report by the EPA, Effect of DGB on Reproductive
hormonal dynamics in Zebra fish were recorded using In silico analysis of
receptors by docking studies.
Docking study helps to understand the interaction of DGB with
Androgen and Estrogen Receptors through Ligand Binding Domains.

6. TOXICOLOGICAL AND BEHAVIORAL STUDIES
 Accumulated toxicant in the organisms altered the morphology and
the behavior of the fish as a response to the external toxic agent.
 EDC has the capacity to disturb the hypothalamic neural secretions
and functions of the exposed organisms, which cause physiological
and metabolic alterations in the organisms.
 The analysis of the toxic nature to the fish by exposure clearly
provides information about the chemical interaction with the
biosystems.
 It was found that environmental toxicants entered the biosystem by
membrane disruption and also altered the potential gradient of the
cytosol, which resulted in ROS formation. Increased oxygen radicals
in the cytoplasm developed stress.
 Behavioral studies were effective indices for the assessment of the
ecosystem for lethal and sub-lethal doses of chemicals.

7. TOXICOLOGICAL STUDIES
 The toxicity analysis in aquatic species based on acute studies provided
the data needed for the documentation of the hazardous nature of
chemical compounds
 Toxicologists paid a lot of attention to the LC50 analysis to study the
potentiality of the chemical compounds
 For the toxicity studies, different concentrations of DGB, such as
75ppm, 65ppm, 55ppm, 45ppm, 35ppm, and 25ppm were prepared and
infused in the individual testing vessel
 Seven consecutive days later, the deaths of the fish were recorded and
the mortality was analyzed by regression values with the help of SPSS
software. From these results, the sub-lethal toxicity doses of DGB for
Zebrafish treatment were finalized
 Sub-lethal dose exposure caused altered enzymatic processes, which
posed a serious threat to the survival of the fish by decreasing their
fecundity.
 The toxicity data was analyzed by a statistical method as described to
determine LC50 and 95% confidence intervals. The LC50 values were
expressed in ppm and for fish, the standard 96h LC50 was used.

8. Probit analysis of DGB in adult Zebrafish

9. BEHAVIORAL STUDIES
 Dissolved organic and inorganic pollutants were easily absorbed
by the fish tissues either directly through respiration or through
food.
 Based on the toxicity dose levels, three different sub-lethal
concentrations (1ppm, 5ppm and 10ppm) of DGB solutions were
prepared and added to the respective testing vessels. One control
was maintained separately without DGB.
 Before and after the feed supplements, the behavioral samplings
were observed.
 During the natural sunlight duration, i.e. from 9am till 3pm, the
control and the treated fish were observed.
 The alertness of the fish, especially during feeding, domination,
chasing others, submission, butting, lateral and frontal fin
movements, swimming, circling, chafing, mouth fighting and
fleeing between the treated and the control group fish were
compared
 The datas were interpreted and scoring was provided.

10. RESULT
 The fish behavioral studies were used as biomarkers, which analyze the
environmental toxic agents in the aquatic ecosystem and help to scrutinize
the organism’s physiological response.
 Behavioral changes reflected the health status of the individual organism.
 Chemical exposure caused stress to the fish with high level of alterations
in dominance, identification of feed, growth, recognition of predator and
avoidance.
 The exposed chemicals act as endocrine disruptors, which showed toxicity
and significantly reduced swimming, circling and frontal display behavior
of the male and the female fish.
 The treated fish showed high mucous secretion around the opercular
region and were twitching frequently.
 The fish with high concentration showed loss of equilibrium and the
absence of chafing between fish.

11. Gill Tissue - Histopathology of adult Male
Zebra fish after exposure to
diethyleneglycoldibenzoate

12. Histopathalogy Study of Gill Tissue in
Adult Male Zebra Fish
 Figure shows gill lamellar deformities of 1ppm DGB-exposed male
adult Zebra fish.
 It shows the congestion of the gill rakers and lamellar regions due to
the exposure to 5ppm DGB in the male gill tissues.
 Figure shows high degenerations and structural collapse of the gill
rakers and aggregations of mucous and chloride cells in the primary
lamellar region due to 10ppm DGB exposure.
 These results clearly proved the increased histoarchitectural
degradations associated with increased doses of DGB exposure to
the gill tissues of the adult male Zebrafish.

13. Gill Tissue - Histopathology of adult Female
Zebra fish after exposure to
diethyleneglycoldibenzoate

14. Histopathalogy Study of Gill Tissue in Adult Female Zebra Fish
 Figure shows gill lamellar deformities of 1ppm DGB-exposed male
adult Zebra fish.
 It shows the congestion of the gill rakers and lamellar regions due
to the exposure to 5ppm DGB in the male gill tissues.
 Figure shows high degenerations and structural collapse of the gill
rakers and aggregations of mucous and chloride cells in the
primary lamellar region due to 10ppm DGB exposure.
 These results clearly proved the increased histoarchitectural
degradations associated with increased doses of DGB exposure to
the gill tissues of the adult male Zebrafish.

15. Liver Tissue - Histopathology of adult
Female Zebra fish after exposure to
diethyleneglycoldibenzoate

16. The liver tissues of 1ppm DGB-treated group male Zebrafish showed
degradation of the hepatic cells with moderately congested sinus
venous
The liver tissues of 5ppm DGB-treated male Zebrafish showed
congested sinus venous with degenerating hepatocytes
At a high concentration (10ppm) of DGB, increased eosinophilic
infiltrated conditions were observed in liver tissues.
The hepatocytes were severely damaged with increased lipid droplets
and increased eosinophilic infiltrated conditions were also observed in
male Zebrafish liver tissues.

17. Liver Tissue - Histopathology of adult
Female Zebra fish after exposure to
diethyleneglycoldibenzoate

18. The liver tissues of 1ppm DGB-treated group Female Zebrafish
showed increased hepatic cell destruction and vacuolations.
The liver tissues of 5ppm DGB-treated Female Zebrafish shows the
apoptosis and the accumulation of ruptured cells at the portal vein.
At a high concentration (10ppm) of DGB, increased eosinophilic
infiltrated conditions in female fish of liver tissues.
The hepatocytes were severely damaged with increased lipid
droplets and increased eosinophilic infiltrated conditions were also
observed in male Zebrafish liver tissues.

19. Muscle Histopathology of adult Female
Zebra fish after exposure to
diethylene glycol dibenzoate

20. Histopathalogy Study of Muscle in Adult Female Fish
 The muscle tissues of the control female Zebrafish showed
regular arrangement of muscle bundles with regular intervals.
 Increased concentrations of DGB increased the shrinkage and
accumulation of lipid droplets.
 This represented the emergence of substrates to satisfy the
energy demand.
 Muscle exposed to the DGB caused abnormally arranged
muscular bundles (AB).
 Also the muscle fibers were shrunk, which resulted in increased
space intervals.

21. Muscle Histopathology of adult Male
Zebra fish after exposure to
diethylene glycol dibenzoate

22. Histopathalogy Study of Muscle in Adult Male Fish
 The muscle tissues of the control female Zebrafish showed regular
arrangement of muscle bundles with regular intervals.
 Increased concentration of DGB increased the shrinkage and
accumulation of lipid droplets.
 This represented the emergence of substrates to satisfy the
energy demand.
 Muscle exposed to the DGB caused abnormally arranged muscular
bundles (AB).
 Also the muscle fibers were shrunk, which resulted in increased
space intervals.

23. Testis Histopathology of adult Male
Zebra fish after exposure to
diethylene glycol dibenzoate

24. Histopathalogy Study of Testis in Adult Male Fish
 The testes of the control male Zebrafish showed
seminiferous tubules filled with spermatogonium (Sg), which
was surrounded by spermatozoa.
 The testes of 1ppm DGB-treated zebrafish showed
constriction of germinal epithelium
 Degenerated spermatogonium was also observed.
 5ppm and 10ppm DGB exposure developed an increased
vacuolation in the seminiferous tubules.
 It caused a separation of germinal epithelial layers from
spermatogonium.
 It resulted in reduced spermatocytes (Sc) and spermatozoa

25. Ovary Histopathology of adult
Female Zebra fish after exposure to
diethylene glycol dibenzoate

26. Histopathalogy Study of Ovary in Adult Female Fish
 Plate shows the ovarian sections of the control female
zebrafish, and they contained previtellogenic (PVO) and
mature oocytes (MO) filled with vitelline fluid surrounded by
the interlobular segments (ILS).
 Plates show the progressive disturbances of the ovary of
the female fish treated with 1ppm, 5ppm and 10ppm
respectively.
 Increasing atresia of the oocytes was observed in
increasing concentrations of DGB.
 Primary oogonium was also severely reduced, resulting in
disturbed vitellogenic activities in the ovaries of the treated

27. RESULT DISCUSSION
 The fish altered their gill morphology to withstand the
environmental changes
 Gills showed increased deformities due to the toxicant
exposure, and it also explained the potential interaction of the
toxicant with the fish vascular system
 The treated liver tissues showed progressive effects, such as
accumulation of lipids in hepatocytes and hepatomegaly in the
female zebrafish.
 In the treated males, the hepatic tissues showed increased
lipogenesis and inflammation.
 These results were in corroboration with the results of this
study as progressive incidences of liver and gill tissues exposed
to diethylene glycol dibenzoate (DGB) were observed.

28. REPRODUCTIVE HORMONAL DYNAMICS OF MALE AND
FEMALE ZEBRAFISH EXPOSED TO DIETHYLENE
GLYCOL DIBENZOATE
 In the fish brain, the hypothalamic neurons are responsible for
the synthesis and secretion of gonadotropin hormones
follicle-stimulating hormone (FSH) and luteinizing hormone
(LH) from pituitary gland.
 In zebrafish, the male hormones, androgen and testosterone,
are responsible for the development and maturation of
different parts of the male reproductive system
 Fish FSH promotes the growth and development of the
gonads, while LH regulates gametogenesis, ovulation and
spermiation till gamete maturation and release in female fish,
estrogen and progesterone promote follicle maturation and

29. Mean±SD values of LH hormones in male and female zebrafish
exposed to different doses of DGB

30. RESULTS
 Table shows the mean and standard deviation values of the male
and the female zebrafish exposed to different concentrations of
DGB compared with the LH results of the control fish
 In the male zebrafish, DGB exposure (1ppm, 5ppm and 10ppm)
showed significantly (P<0.05, F value =35.65) decreased levels of
LH hormones as 16.418±0.134, 13.405±0.200 and 11.246±0.108U/L
respectively
 In Female zebrafish, the LH hormones were significantly (P<0.05,
F value = 42.12) decreased when compared to the control as
21.646±0.069, 18.716±0.071 and 15.274±0.059U/L respectively
 When compared to the control male (18.446±0.166U/L) and
female (24.734±0.109U/L) fish, the LH level was reduced in the
DGB-treated fish.

31. Mean±SD values of FSH hormones in Male and Female
zebrafish exposed to different doses of DGB

32. RESULTS
 FSH level of the control male fish was found as 12.294±0.101U/L
(Table 5.2). On treating with DGB at 1ppm, the FSH level was found
as 9.656±0.083U/L, at 5ppm, the FSH level was 7.698±0.068U/L and at
10ppm, the FSH level was 5.394±0.049U/L.
 The FSH level of the control female fish was found as
11.506±0.074U/L, while after treatment with DGB at 1ppm, 5ppm and
10ppm, the FSH levels decreased to 8.714±0.071, 6.370±0.050 and
4.530±0.072U/L respectively
 One-way ANOVA results showed significantly decreased (P<0.05)
FSH levels between the mean values of the control and the treated
groups.
 The hormone levels decreased as the concentrations of DGB were
increased.

33. Mean±SD values of Androgen and Testosterone
Hormones in male zebrafish exposed to different
Doses of DGB

34. Mean±SD values of Estrogen and Progesterone
hormones in female zebrafish exposed to different
doses of DGB

35. DISCUSSION
 The disturbances in the sexual behavior of the zebrafish were
highly correlated to the fish hypothalamic hormones
 In zebrafish, EDCs directly affected the enzymes 17b-
hydroxysteroid dehydrogenase, 11b-hydroxysteroid
dehydrogenase and cytochrome P45017 responsible for estrogen
and androgen hormone production.
 DGB exposure in sub-lethal doses caused serious fluctuations in
the gonadotropin hormone titer levels in both the male and the
female fish supernatant, which resulted in significantly disrupted
sex hormone levels in the fish exposed to DGB compounds.
 The present study states that on exposure to DGB, the level of
sex steroid hormones in the male and the female Zebrafish got
reduced greatly.
 These results finally concluded that DGB acts as major EDC,

36. TO MEASURE THE IMPACT OF DGB MOLECULES ON THE ANDROGEN
AND ESTROGEN RECEPTORS OF ZEBRAFISH BY INSILICO ANALYSIS
Sequence selection and similarity search (BLAST search)
 Protein structure prediction
 Homology Modeling
 Protein Quality estimation
 Active site prediction
 Ligand selection and Pharmacophore Analysis
 Protein Docking
 Target Protein selection
 Prediction of physicochemical properties
 Secondary structure Prediction (SOPMA)
 Tertiary structure prediction and Homology Modeling
 Protein structure validation
 Chemical selection (DGB) and docking of hormone receptors (AR &
ER).

37. Protein Structure Prediction
 The selected target protein sequences were used to predict the
primary structure analysis using ProtParam tool to understand the
physico-chemical properties of the protein sequence to analyse the
physico-chemical properties.
 The secondary structure of the protein sequence was predicted
using SOPMA to understand the amino acids distributed based on
the α-helix, β-sheets, random coils and extended stand.
 The tertiary structure of the hormone receptors was predicted using
Swiss Model to design the three-dimensional structure of the target
protein receptors.
 The best template models were selected to predict the model
structures and note the quality scores of Q-mean.

38. Homology Modeling
 The target model three-dimensional protein structures were
designed using SwissPDBViewer software.
 Using homology modeling parameters such as the
Ramachandran plot observation to reveal the amino acid
breakages within complex structures.
 They were modeled using build and scan loop method to
create anchors to the amino acids, which develop peptide
bonds that form protein structure complex.
 Furthermore, to reduce the energy of the model protein
structure based on the bond length, bond angle, torsion
angle, electrostatic and Van der Waals interactions,
negatively charged groups were noted and complex

39. Ligand Selection and Pharmacophore Analysis
 The chemical structure of diethylene glycol dibenzoate was derived
from PubChem, Compound database (PubChem CID: 8437).
 The Pharmacophore analysis was done using Mol inspiration tool,
based on the Lipinski Rule of 5 to calculate the molecular property,
bioactive scores and enzyme inhibitor properties.
 This result helped understand the functional inhibition of the
phytochemical against the aldose reductase inhibitor.
 ADMET analysis was performed on the selected compounds using
admetSAR tool to screen the compound’s effect on physiological
parameters.
 Toxicity based parameters were predicted by Lazar toxicity
predictions server.

40. Molecular docking of the Hormone Receptors with DGB
using AutoDock 4.2

41. Chemical docking of Diethylene glycol dibenzoate with the
Hormone Receptors of zebrafish
Androgen
Receptor
Testosterone
Receptor
Estrogen Receptor A1

42. Estrogen Receptor B1 Progesterone Receptor
Based on the observation, the
chemical structure had good
bioactive properties, which were
highly agreeing with its drug-like
properties.
The chemical structure was a very
good bioactive to GPCR ligand and
nuclear receptor ligand properties,
which showed that it was very well
active at these receptor sites.

43. BIOCHEMICAL PARAMETERS ANALYSIS IN ZEBRAFISH EXPOSED
TO DIETHYLENE GLYCOL DIBENZOATE
 Oxidative stress has been the basic event in cellular and tissue
damage. Free radicals formed by the oxidative stress induce cell
death by necrosis or apoptosis
 The enzymes involved in the antioxidant defense system serve as
bioindicators of the contaminant-induced chemical stress
 Acid phosphatase (ACP),Alkaline phosphatase (ALP), Glutamate
oxaloacetate transaminase (GOT) and Glutamate pyruvate
transaminase (GPT), Superoxide dismutase (SOD), Catalase (CAT),
Sorbitol dehydrogenase were used to measure the tissue damage
caused by the toxicants and act as biomarkers of cellular impairment
and cell damage
 On exposure to stressors, fish modulate their metabolism and alter
the GOT, GPT, ACP and ALP activities

44. RESULTS - Acid Phosphatase
 The acid phosphatase (ACP) activity in the gill, liver, muscle and
gonad (ovary and testis) tissues of the male and the female adult
zebrafish exposed to different concentrations of DGB.
 In the control group, the male tissues showed increased ACP than
the female zebrafish.
 The treated groups of Male and Female fish was observed with the
ACP levels significantly, decreased with increased concentrations
of DGB when compared to the control samples
 This condition was observed due to the infiltration of the ACP
enzymes into the body fluids (serum) of the fish, which resulted in
decreased levels in the tested organs than the control

45. RESULTS - Alkaline Phosphatase
 Variations are observed in the mean level of alkaline phosphatase
(ALP) activity in the tissues of liver, gill, muscle, testis and ovary of
the male and the female Zebrafish exposed to DGB
 The fish in the control group had higher ALP level, while on
exposure to DGB at 1ppm, 5ppm and 10ppm, the ALP activities were
reduced when compared to the control group.
 Especially when compared to the gill, muscle, ovary and testis
tissues, the ACP and ALP levels were reduced drastically in the liver
tissues
 It indicates that DGB exposure resulted in tissue damage and cell
impairment, which in turn affected all the metabolic activities in the
fish.

46. RESULTS - Sorbitol Dehydrogenase
 Variations in the mean level of sorbitol dehydrogenase activity
in the gill, liver, muscle, testis and ovary tissues of the male and
the female zebrafish (Danio rerio) exposed to diethyleneglycol
dibenzoate were observed
 After exposure to 10ppm and 5ppm of DGB, the SDH activities in
the tissues were reduced when it is compared to control tissues
of the liver in Male and Female zebrafish
 The variance in the mean level was statistically significant at
P<0.01
 Overall results concluded that SDH, as a biomarker, indicated

47. RESULTS - Glutamic Oxaloacetic Transaminase
 Variations in the mean Glutamic Oxaloacetic Transaminase (GOT)
activity in the tissues of gill, liver, muscle, testis and ovary of the male
and the female zebrafish (Danio rerio) exposed to diethylene glycol
dibenzoate were observed
 10ppm, 5ppm and 1ppm concentrations of DGB exposure showed
gradually decreased GOT levels in all the tissues of Male and Female
zebrafish
 The statistical analysis of all the values was significant at P<0.01
 at 5ppm and 1ppm concentrations, a gradual decrease in GOT activity
was observed when compared with control samples

48. RESULTS - Glutamic Pyruvate Transaminase
 Statistical analysis of Glutamic Pyruvate Transaminase (GPT)
activity in the tissues of gill, liver, muscle, testis and ovary of the
male and the female zebrafish (Danio rerio) exposed to
diethylene glycol dibenzoate was recorded
 The data displayed in reveals that DGB has induced significant
variations in GPT concentration of various tissues of male
zebrafish at 1ppm, 5ppm and 10ppm
 In gills, liver, testis and ovary concentration of GPT was
significantly decreased but in muscles, the GPT concentration
gets increased significantly.
 The mean level of GPT in the gill, liver, testis, ovary tissues and
muscle showed a gradual decrease and increase in the GPT
activity respectively when compared to the control fish

49. RESULTS - Superoxide Dismutase
 The mean level of superoxide dismutase activity in various
tissues of the male and the female zebrafish (Danio rerio)
exposed to diethylene glycol dibenzoate at 10ppm, 5ppm and
1 ppm along with the control group
 The mean values of SOD activity in the male and female fish
gill, liver, muscle and testis tissues were observed as
decreased
 These results indicated that SOD activity reduced in all the
treated groups
 The overall result concluded that the SOD activity reduced
significantly (P<0.01) on exposure to DGB and the variation
was confirmed by Duncan post hoc testing

50. RESULTS - Catalase
 The variations in the mean values of the catalase activity in the
gill, liver, muscle, testis and ovary tissues of the male and the
female Zebrafish (Danio rerio) exposed to DGB were observed
 On exposure to 10ppm, 5ppm and 1ppm of DGB, the CAT activity
gradually decreased in the DGB-treated fish when compared to
the control fish
 DGB exposure reduced the CAT activity in all the tested tissues.
On observing the female fish, the CAT activity reduction was
lesser than in the male fish
 The overall results indicated that CAT activity reduced on
exposure to DGB and the variation was statistically significant
(P<0.01) in all the tissues

51. CONCLUSION
 This study was aimed at investigating the effects of sub-lethal doses of
phthalates on the hormonal titer, enzyme titer, histological changes and
subsequent changes in the biochemical constituents of the whole body tissue.
 in- silico analysis of the interaction or binding affinity of diethylene glycol
dibenzoate with the reproductive hormone receptors.
 The observations registered in this study conclude that diethylene glycol
dibenzoate alters the hormone and enzyme titer, as well as causes structural
changes at the cellular level.
 In- silico analysis reveals that diethylene glycol dibenzoate at low
concentrations could bind with androgen and estrogen β receptors.
 These results are the baseline data to signal the endocrine disrupting activity of
diethylene glycol dibenzoate in fish.
 These results are the records of initiation to eliminate the Endocrine disrupting
compounds from the usage and highlights the immediate need of alternate
source instead of Diethyleneglycoldibenzoate.

53. THANK
YOU
ALL