ThesisPDF Available

Toxicological assessment of chromium using Hydra vulgaris as model organism

August 2022

DOI:10.13140/RG.2.2.13484.54403

Thesis for: Master of Science in Biotechnology
Advisor: Dr. Chinnamani PrasannaKumar

Authors:

P.R.S Yoganathan

National College, Tiruchirappali

Chinnamani PrasannaKumar

Kristu Jayanti College

In the recent times use of vertebrate animal models in toxicity testing of heavy metals entities has come under criticism and surveillance in view of lack of relevance as surrogates to humans and also ethical considerations. Though in vitro and in silico tools can obviate animal models in toxicity testing, use of some simple organisms, the sentience level of which is not a big issue, as animal model is also considered pertinent. Hydra, a simple freshwater cnidarian is already a model organism for studies in developmental biology, and is also thought to be suitable for environmental toxicity assessments. In the present study, we have tested chromium, an essential microelement, is known to be toxic to aquatic life at concentrations higher than that could be tolerated. We worked out the LC50 of chromium adopting the morphological assay. Effective sub-lethal doses were fixed and tested for toxicity adopting regeneration and feeding assays. Since the anatomy of Hydra is such that all the cells in its two body layers can potentially be continuously exposed to the toxicant present in the water, the present study justifies another of its application viz., environmental risk assessment. DNA barcoding, a molecular identification tool used for species identification in the present study has also confirmed the identity of the animal as H. vulgaris.

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Toxicological assessment of chromium using Hydra vulgaris as model organism

Dissertation submitted to

NATIONAL COLLEGE (AUTONOMOUS)

affiliated to

Bharathidasan University, Tiruchirapalli

in partial fulfillment of the requirements for the award of the degree of

MASTER OF SCIENCE IN BIOTECHNOLOGY

S.YOGANATHAN

Admission No. BTPS20004

Under the Guidance of

Dr. Chinnamani Prasannakumar

Associate Professor,

DEPARTMENT OF BIOTECHNOLOGY & MICROBIOLOGY

(Supported under DBT-PG TEACHING & DBT-STAR COLLEGE Schemes)

NATIONAL COLLEGE (AUTONOMOUS)

(Nationally Re-accredited with „A+‟ Grade by NAAC)

(College with Potential for Excellence)

TIRUCHIRAPALLI-620001

AUGUST 2022

NATIONAL COLLEGE

Dr. CHINNAMANI PRASANNAKUMAR, M. Phil., Ph. D. (DST-INSPIRE fellow)

Associate Professor

PG & Research Department of Biotechnology and Microbiology

National College (Autonomous) (Estd. 1919)

(Nationally Re-Accredited at "A+" Grade by NAAC)

(A College with Potential for Excellence)

Tiruchirapalli - 620001, Tamil Nadu, India.

https://www.nct.ac.in/dept-biotech.html

+91-9865929945

micropras@gmail.com, cprasanna@nct.ac.in

CERTIFICATE

This is to certify that the thesis entitled Toxicological assessment of chromium using

Hydra vulgaris as model organism submitted by Mr. Yoganathan S, for the award of the

Degree of Master of Science in Biotechnology is based on original studies carried out by her

under my supervision.

The thesis or any part thereof has not been submitted for any other degree or diploma in

any university or institution.

Place: National College

Date: (CHINNAMANI PRASANNAKUMAR)

Date: ………….

DECLARATION

I, S. YOGANATHAN hereby declare that the Dissertation, titled Toxicological

assessment of Chromium using Hydra vulgaris as model organism submitted to the

National College (Autonomous), Tiruchirapalli in partial fulfillment of the

requirements for the award of the Degree of Master of Science in Biotechnology, is my

original work executed between March, 2022 & August, 2022, under the supervision

and guidance of Dr. Chinnamani Prasannakumar, Associate Professor,

Department of Biotechnology & Microbiology, National College (Autonomous),

Tiruchirapalli, and it has not formed the basis for the award of any Degree / Diploma /

Associateship / Fellowship.

Signature

ACKNOWLEDGEMENT

It is indeed an immense privilege and honor to extend my deep sense of gratitude to Dr.

M. A. Akbarsha, Research Co-ordinator, National College (Autonomous), and Former

Director, Mahatma Gandhi-Doerenkamp Center (MGDC), Bharathidasan University,

Tiruchirappalli, for being a constant source of inspiration and providing all the facilities

for the successful completion of this project work.

I would like to thank Dr. M. S. Mohamed Jaabir, Head, Department of Biotechnology

& Microbiology, National College(Autonomous),, Tiruchirappalli, for his valuable

support.

I would like to express my thanks to Dr. Chinnamani Prasannakumar, Associate

Professor, Department of Biotechnology & Microbiology, National

College(Autonomous), Tiruchirappalli, for being a perfect mentor.

I offer my heartfelt thanks to Mr. Ankit Dilware, Research Scholar, Mahatma Gandhi

Dorenkamp Center (MGDC), Bharathidasan University, Tiruchirappalli for the timely

help during the project work.

Last but not least I express my deep sense og gratitude to my beloved parents and my

friends for their care, love,optimistic, support, encouragement, and blessing, without

which this work would not have became a reality.

S.YOGANATHAN

TABLE OF CONTENT

S. No.

Topic

Page No.

Abstract

Abbreviations

List of Figures

List of Tables

Chapter-1: Introduction

Chapter-2: Review of Literature

Chapter-3: Scope and work plan

Chapter-4: Material & Methods

Chapter-5: Results

Chapter-6: Discussion

Chapter-7: Summary

Conclusion and future thrust

References

Toxicological assessment of Chromium using Hydra vulgaris as model

organism

ABSTRACT

In the recent times use of vertebrate animal models in toxicity testing of heavy metals

entities has come under criticism and surveillance in view of lack of relevance as surrogates

to humans and also ethical considerations. Though in vitro and in silico tools can obviate

animal models in toxicity testing, use of some simple organisms, the sentience level of which

is not a big issue, as animal model is also considered pertinent. Hydra, a simple freshwater

cnidarian is already a model organism for studies in developmental biology, and is also

thought to be suitable for environmental toxicity assessments. In the present study, we have

tested chromium, an essential microelement, is known to be toxic to aquatic life at

concentrations higher than that could be tolerated. We worked out the LC50 of chromium

adopting the morphological assay. Effective sub-lethal doses were fixed and tested for

toxicity adopting regeneration and feeding assays. Since the anatomy of Hydra is such that all

the cells in its two body layers can potentially be continuously exposed to the toxicant present

in the water, the present study justifies another of its application viz., environmental risk

assessment. DNA barcoding, a molecular identification tool used for species identification in

the present study has also confirmed the identity of the animal as H. vulgaris.

Keywords : Hydra, Model organism, chromium, Morphological variations, Sub-lethal dose,

DNA barcoding

LIST OF ABBREVIATION

Abbreviation

Definition

Hydra Medium

Acridine Orange

Chromium

NOEC

No Observed Effective Concentration

LOAEL

Lowest Observed Adverse Effect Level

µg

Microgram

Effective concentration

Hours

Centimeter

µL

Microliter

Milliliter

LC50

Lethal Concentration 50

mg/L

Milligram per Liter

µg/L

Microgram per Liter

NaCl

Sodium chloride

KCl

Potassium chloride

MgSO₄

Magnesium chloride

CaCl2

Calcium chloride

NaOH

Sodium hydroxide

ROS

Reactive oxygen species

PBS

Phosphate buffered saline

BSA

Bovine serum albumin

LIST OF FIGURES

Fig

number

Title of the figure

Page no

Fig 1

Anatomy of Hydra.

(A) Whole animal Hydra.

(B) The bilayered cellular organization of a Hydra polyp

Fig 2

Phylogeny of metazoans with focus on Cnidarians

Fig 3

The multiple aspects of biology that can be addressed to the hydra

model system

Fig 4

Hydra regeneration capacities

Fig 5

A summary of the approaches developed or adapted in our lab to

investigate the toxicological effects of nanoparticles (NPs) &

Heavymetals.

Fig 6

Artemia salina hatching

(A). The Artemia cyst after absorbing water

(B).Newly hacted Artemia.

(D). Artemia double washing using petridish.

Fig 7

BOD Incubator

Fig 8

Stereozoom Dissection Microscope

Fig 9

Hydra culture bowl ( Pyrex culture dishes)

Fig 10

Toxic effect of chromium in Hydra as revealed in its morphology

Fig 11

Chromium-induced impairment of Hydra regeneration.

Fig 12

Effect of Chromium on Hydra reproduction.

Fig 13

Effect of chromium on feeding behavior in Hydra.

Fig 14

Fluorescent microscopic image of ROS and apoptosis in hydra

Fig 15

AO stained cells of chromium treated live animal observed in a

fluorescent microscope.

Fig 16

Phylogram analysis

List of Tables

Table

No.

Title of the table

Page

Table..1

Toxicity of Chromium to aquatic organisms

Table..2

Scoring system devised by Wilby (1988) for assessing the

morphological damage.

Table..3

Scoring system devised by Wilby (1988) for assessing the toxicant

induced inhibition of regeneration of gastric region.

Table..4

Lethal concentration value of chromium.

Table..5

Median score recorded at each chromium concentration in different

exposure time.

Chapter 1

1. General Introduction

1.1 Background

Since environmental awareness has become a pressing issue and a popular

movement, great efforts have been expended by individuals and organizations to evaluate the

anthropogenic impact on the ecosystems and the humans. Aquatic ecosystem is a vital part of

the environment, and human activity greatly influences the aquatic systems. This is especially

true with respect to introduction of potentially harmful substances such as heavy metals,

cosmetics, nanoparticles and polycyclic aromatic hydrocarbons (Collins et al., 2008;

Kahru and Dubourguier, 2010; Hahn, 2011; Brausch et al., 2012; Zeeshan et al., 2016). An

aquatic ecosystem is vulnerable to anthropogenic contamination as it is the ultimate sink to

all kinds of chemicals. This is a serious issue since large aquatic ecosystems are chemically,

and biologically, predisposed to excess of chemicals which inflict toxic damages to aquatic

flora and fauna. The aquatic ecosystem is fragile, and chemicals with relatively longer

retention time pose threat to hydrobionts. Further, the many hydrobionts are highly sensitive

in which one or more life stages may be particularly susceptible to the influence of toxic

contaminants (Hodkinson and Jackson, 2005; Burger, 2006).

The environment is marred with chemicals, and it is estimated that there are more

than 80,000 chemicals in commercial use today, with another 2000 being introduced each

year for which there is little to no toxicological data (Rand, 2010). Thus, living beings, be it

humans or other life forms, are being continuously exposed to chemicals which could be of

potential risk to them. Chemicals can be natural or anthropogenic, could be organic or

inorganic, and are indispensible part of our daily life. Virtually every manufactured product

used in our homes, gardens, fields and industries contains chemicals, either synthetic or

natural. Chemicals in medicines and cosmetics are another example of diverse use of

chemicals. Chemicals are present as food additives, coloring agents and as preservatives

(Dix et al., 2007). They are used for agricultural purposes such as insecticide or pesticide to

combat fungi, weeds and insects in our fields.

In our homes they are used as disinfecting agents (biocides) and are found in numerous

products such as solvents, paints, furniture, plastics and air fresheners. Ultimately all these

chemicals find their fate in getting dumped into aquatic bodies either though industrial

effluents, agricultural run-offs or domestic wastes (Hahn, 2011; Stankovic et al., 2014).

Heavy metals are considered as an important class of chemical contamination to the

environment (Stankovic et al., 2014). Unlike other chemical contaminations heavy metal

contamination is one of the most bothering issues of environmental health; once they are

released they remain persistent for a very long time. Heavy metals are ubiquitous in nature,

non-biodegradable with long retention time in the environment (Rainbow, 2012). Heavy

metals are metallic elements, which have high atomic weight and a density at least 5 times

greater than that of water. Their multiple industrial, domestic, agricultural, medical and

technological applications have led to their wide distribution in the environment, causing

concerns over their potential effects on human health and the environment. Cadmium,

mercury, lead and arsenic are considered as the most toxic environmental agents and appear

in the World Health Organization‟s list of 10 chemicals of major public concern. Other

examples include manganese, chromium, cobalt, nickel, copper, zinc, selenium, silver,

antimony and thallium (IARC, 2006).

Metals, in general, can be classified into two groups based on their function in

biological systems, i.e., essential metals and non-essential metals. Heavy metals such as

copper, chromium, mangenese, zinc, and cobalt are essential metals required by organisms in

trace amounts for many physiological processes (Reeves and Baker, 2000) and are toxic only

when present in excess (Blaylock and Huang, 2000; Monni et al., 2000). The heavy metals

are generally present as cofactors of metalloenzymes which are required for performing many

physiological processes. Approximately half of the enzymes in eukaryotes have metal in the

core center. Heavy metals induce cytotoxic, genotoxic and reprotoxic effects in hydrobionts,

and demand careful investigation and monitoring procedure from various national and

international agencies

Chromium, like other widely distributed heavy metals, has been the subject of

increasing research to determine and ultimately to control its concentration in freshwater,

estuarine and coastal habitats (Sobral and Widdows, 1997). Chromium, in trace quantities, is

an essential metal for many biological processes. However, it poses detrimental effect when it

is present in excess quantity (Davenport and Redpath, 1984). Chromium toxicity is reported

in a variety of aquatic species, marine as well as freshwater. Invertebrates in general are more

sensitive to chromium than fish genera with detrimental effects on survival, morphology,

development, feeding, growth, and population dynamics. Elevated concentrations of

chromium commonly produce toxic effects such as induction of reactive oxygen species

(ROS) in trout hepatocytes (Krumschnabel et al., 2005), interference with whole body ion

regulation (Lauren and McDonald, 1987; Sloman et al., 2003), and DNA damage in PC12

cells (Kawakami et al., 2008). Chromium exposure may cause systemic damage to the whole

organism and populations of aquatic animals. However, the mode of action of chromium in

aquatic organisms, especially invertebrates, is still poorly understood. Chromium exists in

several oxidation states, but the most stable and common forms are Cr(0), the trivalent

Cr(III), and the hexavalent Cr(VI) species. Hence, study of the effects of chromium at

organismal level with manifestations at molecular systems of invertebrates is highly relevant

and desirous.

Chromium may enter the natural waters by weathering of Cr-containing rocks, direct

discharge from industrial operations, leaching of soils, among others. In the aquatic

environment Cr may suffer reduction, oxidation, sorption, desorption, dissolution, and

precipitation. The aqueous solubility of Cr(III) is a function of the pH of the water. Under

neutral to basic pH, Cr(III) will precipitate and conversely under acidic pH it will tend to

solubilize. The forms of Cr(VI) chromate and dichromate are extremely soluble under all pH

conditions, but they can precipitate with divalent cations. Chromium has been demonstrated

to be toxic to vertebrate as well as invertebrate species. Much of our knowledge of chromium

toxicity is based on animal models and mammalian cell line studies (Lison et al., 2001;

Simonsen et al., 2012).

Advances in life sciences have revolutionized testing of environmental agents,

especially heavy metals, by incorporating techniques emerging from the field of molecular

biology, bioinformatics, biotechnology, etc. Some of the most promising techniques include

assays based on in vitro models, high throughput testing, omics approaches, systems biology,

computational modeling and non- mammalian/invertebrate models. The technological

advancements render testing of environmental agents more mechanistic, less expensive and

time-saving. From these perspectives understanding of toxic mechanisms relies on

identifying genes and gene products (e.g., genomic DNA, mRNA transcripts and/or proteins)

that are targeted for disruption by the xenobiotics that we are likely to encounter. Developing

informative, sensitive and rapid assays to screen for ecotoxicity has, therefore, become a

priority.

The use of model organisms belonging to a lower level of the phylogenetic tree is probably

the best alternative in the context of environmental testing. First and foremost, these model

organisms do not invoke ethical concerns because they are of no or less sentience and can be

used as biomarkers for anthropogenic agents. In general both vertebrates and invertebrates

are used for environmental risk assessment of chemicals and for a variety of ecotoxicological

studies in the laboratory and in the field. However, with the growing public pressure to

replace or minimize the use of vertebrates in ecotoxicity testing with effective alternatives so

as to reduce suffering of the animals without jeopardizing environmental safety is gaining

momentum. Although these organisms are evolutionarily and morphologically very distant,

they exhibit physiological and biochemical properties similar to humans which are useful for

toxicity testing. It is widely accepted that no organism is universal for evaluating toxicity for

different class of chemicals. Hence, a search for newer organism with same or advantageous

properties is always waiting for. Hydra, a cnidarian, has the potential to be developed into an

alternative model organism for environmental monitoring and risk assessment.

1.2 Hydra as a model organism for environmental toxicity assessment

Hydra is a freshwater organism belonging to the phylum Cnidaria and class Hydrozoa.

Hydra arose very early in evolution somewhere around 540 million years ago. It is

phylogentically placed in the metazoon group that form the sister group to all bilaterian

animals. The polyp measures roughly 2-3 mm diameter and 5-20 mm long. Hydra has

contributed to many facets of science including developmental biology, stem cell biology and

environmental toxicology. The body plan of the animal is simple, and radially symmetrical

with oral end and aboral ends (Fig. 1). The oral end has the mouth at the tip of the hypostome

which is dome-shaped and is surrounded by a whorl of tentacles. The aboral end comprises

the basal disk (Bossert and Galliot, 2012). Ectoderm and endoderm are separated by an

acellular matrix called the mesogloea (gray) (Fig. 1). All epithelial cells in Hydra are

myoepithelial, with myofibers on the basal side (red). In ectodermal epithelial cells (green),

the fibers are oriented longitudinally, and in endodermal epithelial cells (pink) they are

oriented circumferentially (ring muscle). Most interstitial cells and nematoblast clusters are

located between ectodermal epithelial cells. Neurons are found in both the endoderm and

ectoderm. Sensory neurons are located between epithelial cells and connect to ganglion

neurons (purple), which are at the base of the epithelium on top of the myofibers and

sometimes cross the mesogloea. Different types of gland cells, most of which are found in the

endoderm, are intermingled between the epithelial cells (Technau and Steele, 2011).

This spurred interest in Hydra research to use it as a tool for understanding many

biological phenomena. A recent orthologome analysis showed that Hydra shares at least 6071

genes with humans, in contrast to Drosophila and C. elegans, which share only 5696 and

4571 genes, respectively, with humans (Wenger and Galliot, 2013).

One fascinating property of Hydra is its ability to regenerate from a fragment of

tissue or from dissociated cells. Infact, Hydra is the highest form of invertebrate that has the

capability for whole body regeneration (Pardos et al., 1999). The high regenerative capacity

of Hydra is solely due to the epithelial cells that are fully capable of complete body

regeneration regulated by tightly controlled developmental programs (Bosch, 2007.

Fig. 1 Anatomy of Hydra. (A) Whole animal Hydra. (B) The bilayered cellular

organization of a Hydra polyp (Technau and Steele, 2011).

The animal, because of its simple body organization, ease of culture, low animal husbandry

cost, and requirement of less space, has staked claim to be a suitable candidate for

environmental toxicity testing. The development of omics strategy in Hydra; sequencing of

Hydra genome; generation of transgenic lines; and conserved molecular repertoires such as

autophagy, apoptosis, etc., have reinforced the strength of Hydra as a model for

environmental studies. Hydra has been extensively used for studying the teratogenicity and

embryotoxicity of numerous chemicals and its popularity to assess the impacts of

environmental pollutants has increased. Hydra has been used to assess the impacts of

numerous environmental pollutants including heavy metals, pesticides, persistent organic

pollutants (polychlorinated biphenyls, Azo dyes and endocrine-disrupting chemicals),

pharmaceuticals, nanomaterials, and industrial and large human settlement effluents such as

municipal wastes and drainages (Beach and Pascoe, 1998; Quinn et al., 2012; de Jong et al.,

2016). It has been found to be among the most sensitive animals tested for metals and

certain effluents, comparing favorably with more standardized toxicity tests. In more recent

times, molecular investigations of inorganic nanomaterials and heavy metals on DNA

damage, ROS production, cellular defense and apoptosis showed the unlimited potential of

Hydra in understanding toxicity (Quinn et al., 2012; Zeeshan et al., 2016). Hydra has been

extensively used and is regarded as a model organism in aquatic toxicology; and with the

recent developments the scope has been invigorated. With a view to strengthen these claims,

in the present study attempt has been made to standardize/revisit the modalities for testing the

chromium toxicity in the Hydra model.

Chapter 2

2. Review of Literature

2.1. Aquatic toxicology

The challenges in aquatic toxicology is immense considering that virtually all

manmade chemicals have the potential for entering aquatic system either directly or

indirectly. The chemicals entering aquatic environments alter the well-being of organisms,

and perturb its community structures. The task becomes complicated by the presence of

hundreds, and perhaps thousands, of xenobiotics that may influence biological mechanisms at

any level (O'Connor and Huggett, 1988; Collins et al., 2008). The effects of chemicals on

aquatic ecosystems may be deciphered from biological and biochemical responses to

pollutant exposure and also through the incidence of pollution related pathologies in

organisms. The use of bioindicators to monitor aquatic pollution is more advantageous than

using analytical assays based on water quality tests or chemical speciation (Zhou et al.,

2008). Bioindicators provide direct picture of the effect of the pollutant which can be

observed in vivo with scope of molecular investigations also. Thus, the use of bioindicators is

definitely a first step towards understanding and comparing the detrimental effects of the

chemicals and hence, employed by many international agencies to establish their

management guidelines and environmental laws (Hodkinson and Jackson, 2005; Burger,

2006).

The effects of pollutants on hydrobionts may be acute and/or chronic, and the

remediation process may be difficult, costly, and long term (Hodson, 1988; Zhou et al.,

2008). Assessment of chemical toxicity in aquatic organisms is done mainly by conventional

static or semi-static tests for short periods. Assays based on changes in the morphology,

behavior, feeding, development and regeneration, etc., manifest the physiological and

behavioral responses on pollutant exposure to hydrobionts. In addition to it, the ease of

molecular study empowers in vivo testing to understand the mechanisms of action of a

pollutant and, hence, makes environmental risk assessment accurate, precise and scientific-

driven (Hodkinson and Jackson, 2005; Stankovic et al., 2014).

With the advent of industrialization, the environment is exposed to thousands of chemicals

with potential harmful effects on the organisms including human beings. These chemicals are

being manufactured in large quantities and used for various purposes. Among the various

kinds of pollutants, heavy metal pollution seems to be the most persistent one (Rainbow,

2002; Stankovic et al., 2014). They are ubiquitous in nature and non-biodegradable with long

retention time in the environment. Heavy metals are easily soluble in water, bioavailable and

strongly bind to sulphydryl groups of proteins. Numerous studies conducted on the

detrimental effect of metallic pollutants have documented that when the metal concentration

exceeds a reasonable level for each species, the animal enters into a toxic situation that

manifests a wide range of effects and responses at all levels (Monni et al., 2000; Blaylock and

Huang, 2000).

2.2 Chromium

Chromium, one of the most common ubiquitous pollutants in the environment,

does not occur naturally in the pure metallic form. The element is present in divalent [Cr(II)],

trivalent [Cr(III)], and hexavalent [Cr(VI)] oxidation states, with Cr(VI) and Cr(III) being the

most stable forms. Chromium enters into various environmental matrices (air, water, soil)

from a wide variety of natural and anthropogenic sources in the Cr(III) or Cr(VI) form. The

health hazards associated with exposure to Cr are dependent on its oxidation state, ranging

from the low toxicity of the metal form to the high toxicity of the hexavalent form. Trivalent

Cr plays an important role in glucose metabolism by serving as a cofactor for insulin action.

Hexavalent chromium is a toxic industrial pollutant and classified carcinogen possessing

mutagenic and teratogenic properties. All Cr(VI)-containing compounds were once thought

to be man-made, with only Cr(III) naturally ubiquitous in air, water, soil, and biological

materials. Recently, however, naturally occurring Cr(VI) has been found in ground and

surface waters at values exceeding the World Health Organization limit for drinking water of

50 μg of Cr(VI) per liter.

Generally, the aquatic environment is the ultimate sink for metal pollutants. Several

industrial applications like leather tanning, electroplating, and corrosion protection

contaminate ground water , whereas surface waters are polluted by discharges from

manufacturing processes and cooling towers . On the other hand, the combustion of fossil

fuels and manufacturing processes of iron and steel industries release Cr into the atmosphere

in particulate form. Most of the Cr in air will eventually settle and end up in waters or soils.

When released to land, Cr compounds bind to soil and are not likely to migrate to ground

water. In water, however, these compounds are very persistent as sediments, with a high

potential for accumulation of Cr in aquatic life. In its dissolved form, Cr is present as either

the anionic trivalent Cr(OH)3 or hexavalent CrO42−.

Hexavalent chromium is a toxic industrial pollutant and classified carcinogen

possessing mutagenic and teratogenic properties. All Cr(VI)-containing compounds were

once thought to be man-made, with only Cr(III) naturally ubiquitous in air, water, soil, and

biological materials. Recently, however, naturally occurring Cr(VI) has been found in ground

and surface waters at values exceeding the World Health Organization limit for drinking

water of 50 μg of Cr(VI) per liter. In many Cr(VI)-exposed fish, certain species have

demonstrated extra sensitivity whereas others are tolerant. More in-depth studies in

toxicodynamics and toxicokinetics are needed, however, to establish an exact cause-effect

relation. The scientific data discussed in this review provide a basis for understanding the

potential impact and for advancing our knowledge of the eco-toxicological effects and risk

assessment of Cr.

2.3 Physicochemical properties of chromium and its principal ions.

Chromium (atomic number 24, relative atomic mass 51.996) occurs in each of the

oxidation states from - 2 to +6, but only the 0 (elemental metal form), +2, +3 and +6 states

are common. Divalent chromium (+2) is unstable in most compounds as it is easily oxidized

to the trivalent form by air. Accordingly, only the trivalent Cr[III] and hexavalent Cr[VI]

forms are important for human health. Valid generalizations of the biological effects of

chromium in its elemental form can not be made.

it is of great importance to realise that the +3 and +6 oxidation states have very different

chemical and hence biological properties. The relationship between the hexavalent and

trivalent states of chromium is described by the equation:

Cr2O7 2- + 14H+ 6 electrons = 2Cr½[III] + 7H2O + 1:33 eV

Thus, the difference in electric potential between Cr[VI] and Cr[III] reflects the strong

oxidizing potential of hexavalent chromium and the substantial energy (1.33 eV) required to

oxidize the trivalent Cr to the hexavalent form. Thus, oxidation of Cr[III] never occurs in

biological systems. In contrast, reduction of Cr[VI] occurs spontaneously in the organism

unless present in a insoluble form. For example, in blood, Cr[VI] is rapidly reduced to

Cr[III]. Thus, once Cr[VI] has penetrated the membrane of the red blood cell it is reduced and

Cr[III] becomes bound to cellular constituents making it unable to leave the erythrocyte.

Table 1. Toxicity of Chromium to aquatic organisms

S.NO

Test

Organisms

Time of

Exposure

Test Conditions

and Organism

Size (if available)

96 h LC50 Values

of Cr

Concentration

(mg/l)

References

Labeo rohita

96hrs

Renewal,

(27.5 ± 1)°C

39.40

[[53], [2]]

Pimephales

promelas

96hrs

Flow through;

25 °C

61.00

[54]

Channa

punctatus

96hrs

Static;

(29.8 ± 1)°C

50.00

[[90], [2]]

Salvelinus

fontinalis

96hrs

Flow through;

12 °C

59.00

[2]

Catla catla

96hrs

Static; (28 ± 1)°C

100.00

[2]

Carassius

auratus

96hrs

Renewal; 26–

28 °C;

85.70

[[49]

Salmo

gardnerii

24hrs

Renewal; early

stage fry

44.00

[58]

Cirrhinus

mrigala

96hrs

Static; Age

120 days

113.35

[50]

Cyprinus

carpio

96hrs

Static; Age-

120days

128.89

[61]

10.

Labeo bata

96hrs

Static; Early stage

fry

7.33

[56]

11.

Puntius sarana

96 h

Static; Early stage

fry

10.37

[56]

12.

Colisa

fasciatus

96 h

NMa

60.00

[62]

2.4 Chromium Toxicity

Beside the role of Cr (III) in metabolism of glucose, fats and proteins in animals and

humans it has distinct toxicological features. In humans and animals high level of chromium

(VI) in drinking water causes tumors in stomach. Cr (VI) can be reduced to Cr (III) and in

this form toxicity is not found as it cannot be transported inside the cells. Cr (VI) enters many

types of cells and under various physiological conditions produces reactive intermediates that

may interrupt cellular integrity and numerous functions. The aquatic toxicology of Cr

depends on both biotic and abiotic factors. The biotic factors include the type of species, age

and developmental stage. The temperature, concentration of Cr, oxidation state of Cr, pH,

alkalinity, salinity, and hardness of water constitute the abiotic factors. Moreover, lethal and

sub-lethal concentrations of the metal and its speciation also determine the sensitivity of the

individual organism.

2.5 Abiotic And Biotic Factors

Chromium toxicity in aquatic ecosystem depends on both biotic as well as abiotic

factors. Biotic factors consist of age, developmental phase of an individual and also type of

species. While the abiotic factors comprises concentration and oxidation state of Cr,

temperature, pH, alkalinity and hardness of water. Furthermore, Chromium toxicity is

directly related with the concentration and temperature, any increase in these parameters

boosted its toxicity i.e. raised with the increase of concentration as well as temperature but

declines with increasing salinity and sulfate concentration. Cr toxicity is also influenced by

the pH of the water. In contrast to seawater, interstitial waters and estuaries may experience

variation in pH and represents different levels of chromium toxicity. The concentration of

chromium in lakes and rivers ranges from 1 to 10 ug/L and EPA proposed level for protection

of aquatic life and human health are 50 to 100 ug /L. Numerous fish species have fatal effect

of chromium like lymphocytosis, anemia, eosinophilia, bronchial and renal lesions. Its high

concentration can harm the gills of fish that swim near the point of disposal of metal products

in surface waters. This section of review evaluates the acute and chronic hazardous effects of

Cr to numerous fish species.

2.6 Hydra as a model organism

Hydra is a freshwater animal which belongs to the phylum Cnidaria, class Hydrozoa,

order Hydroida, family Hydridae. The phylum Cnidaria consists of four distinct groups,

anthozoans (corals, sea anemones), scyphozoans and cubozoans (jellyfish), and hydrozoans

(Hydra, Obelia). Hydra arose very early in evolution something around 540 million years;

and are phylogenetically placed in the medusoan group that forms the sister group to all

bilaterians (Fig.2) (Galliot and Miller, 2000). The figure shows the evolutionary relationships

among sponges, bilaterians (protostomes and deuterostomes), and the five classes in the

phylum Cnidaria. The phylum Cnidaria is the largest and a diverse group consisting of 8000–

9000 species; the genus Hydra has a little more than 30 species (Augustin et al., 2010). In this

connection it is important to mention, that because of the lack of taxonomically distinctive

feature, phylogenetic classification of species in Hydra genus has remained debatable.

Although few phylogenetic studies have been conducted and scientists agree to the

classification made by Campbell (1983, 1987, 1989) who proposed that genus Hydra consists

of four species groups, viridissima group, braueri group, vulgaris group, and oligactis group,

based mainly on the morphological characters.

Fig. 2. Phylogeny of metazoans with focus on Cnidarians (Augustin et al., 2010).

Hydra is a true cosmopolitan and is found in all the continents except Antartica

(Jankowski et al., 2008). Hydra is a sessile animal and lives in calm and clean water rather

than fast moving or dirty water. The animals are generally found attached to the submerged

twigs or leaves. Under unfavorable conditions such as predator alert, temperature variations

or lack of food the animals get detached from the surfaces and undergo a somersault

movement which is governed by a neuromuscular system (Jankowski et al., 2008; Bossert

and Galliot, 2012). Hydra is a voracious eater and feeds on lower invertebrates such as

cyclops, rotifers, daphnia and Artemia (Bode, 2011; Bossert and Galliot, 2012).

Hydra has been a subject of research for more than 250 years. It has contributed to the field of

biology in unlocking complex mechanisms of pattern formation, cell-cell interaction,

morphogenesis, etc.(Fig 2).

Fig 3 : The multiple aspects of biology thet can be addressed to the hydra model system (Galliot.2012).

Hydra, like other cnidarians, is based on two epithelial layers i.e., the outer ectodermal cells

and the inner endodermal cells. Each layer is a single cell thick, which extends throughout the

animal. The two layers are separated by an acellular substance called mesoglea which very

much mimics the function of a vertebrate extracellular matrix (ECM) (Sarras, 2012). The

epithelial cells of these two layers have stem cell properties which divide continuously and

differentiate at the body extremities. A third distinct pool of cells, interstitial stem cells are

located in the interstices of the epithelial cells of both layers and are distributed along the

body column but absent at the body extremities. Interstitial stem cells differentiate to give

rise to the majority of cell types in Hydra such as neurons, nematocytes, gland cells and

gametes. Ectodermal stem cells terminally differentiate into “battery cells” in the tentacles and

into mucous cells in the basal disk. As a result, a dozen cell types exists in Hydra which

differentiate from these three distinct stem cell population (Bosch, 2009). Therefore, it

appears that the stemness of Hydra is located in the body column whereas the head and foot

regions contain cells that are mitotically inactive and terminally differentiated (Bosch, 2009)

(Fig.4).

Fig. 4. Hydra stemness. Hydra has three independent stem cell lineages: ectodermal epithelial cells,

endodermal epithelial cells, and interstitial stem cells. (A) Ectodermal epithelial cells. (B) Endodermal

epithelial cells. (C) Interstitial stem cells.(Bosch, 2009).

The cell types of Hydra have distinctive roles which are required for

physiological, behavioral and molecular functioning. A single animal is composed of

50,000 to 100,000 cells which distribute among a dozen cell types. Hydra, though a simple

organism has a well developed neuronal system. The animal does not have a central nervous

system like in vertebrates but a nerve net is spread throughout the body as diffuse plexus.

High density of sensory, ganglion and motor neurons were observed at the body extremities

(hypostome and basal disk) (Koizumi, 2007). As a result, the polyps possess coordinated

movement such as prey-catching and feeding responses, touch and light responses.

Nematocytes are cells characteristic of the phylum cnidara as they “sting” the prey upon

contact. Nematocytes which are located on ectoderm also play a role in locomotion and

defense of Hydra. The nematocytes have a capsular structure called nematocyst, which eject

toxin into the prey. The process of discharge is one of the fastest events known in the animal

kingdom and generates an acceleration of more than 5 million g (Holstein and Tardent, 1984;

Nuchter et al., 2006).

One of the most amazing features of Hydra is that it is practically immortal. Under

controlled physicochemical environment and unlimited food, the polyp can survive years

reproducing new buds without showing any signs of aging (Martínez and Bridge, 2012). This

amazing feature is essentially due to the pool of stem cells which continuously divide and

differentiate. The Hydra tissue is dynamic in nature where new cells continuously form but

interestingly the animal does not increase in size. The new cells displace the old cells which

get sloughed off from the body extremities (head and basal disk), and are also to the budding

region to form buds. Thus, the animal maintains the size by a strict balance between the cell

production and cell loss. This combination of uniform growth and local cell loss leads to

continuous movement of new tissue either up or down of the body column. The doubling

time of interstitial cells and epithelial cells are 1.5 days and 3-4 days, respectively. As a

consequence, an individual healthy Hydra has all its epithelial cells replaced within a week

and in 20 days the entire Hydra gets replaced with new cells. This unique ability has led

scientists to propose the lack of senescence in Hydra. It was reported that individual Hydra

monitored for a period didn‟t show any signs of aging or reduction in budding rates (Martinez

and Bridge, 2012).

Another fascinating property of Hydra is its ability to regenerate from a piece of

tissue or from dissociated cells . Any isolated fragment of the Hydra body can regenerate into

an animal; although the animals regenerated are small but can grow like a normal Hydra over

a period of time. Even if Hydra is dissociated into a suspension of cells that if subsequently

centrifuged, these cells form aggregates and regenerate into viable polyps (Gierer et al.,

1972). In fact, Hydra is the highest form of invertebrate that has the capability for whole

body regeneration. The high regenerative capacity of Hydra is solely due to the epithelial

cells that are fully capable of complete body regeneration regulated by tightly controlled

developmental programs (Bode, 2011). Regeneration in Hydra represents a beautiful

experimental system for studying de novo pattern formation, the process of cellular

reorganization and regulation of signaling molecules.

The genome of Hydra has been sequenced and many genes were conserved to human but

most surprising was the fact that genes which were lost in popular model organisms like

Drosophila melanogaster and Caenorhabditis elegans during evolution were present in the

Hydra (Chapman et al., 2010). This spurred interest in Hydra research to use it as a model

organism for understanding many biological phenomena. A recent orthologome analysis

showed that Hydra shares at least 6071 genes with humans, in contrast to Drosophila and

C. elegans, which share only 5696 and 4571 genes, respectively, with humans (Wenger &

Galliot, 2013). The Hydra genome is (A+T)-rich which like in vertebrates contains

approximately 20,000 protein-coding genes and major bilaterian signaling pathways,

including, transforming growth factor-β, Hedgehog, receptor tyrosine kinase and Notch are

conserved.

2.7 Hydra toxicity testing

Hydra is a highly suitable model system for aquatic ecotoxicological studies. Thanks

to the simple organization of the organism and application of modern tools which rendered

not only medium throughput screening of chemicals but also facilitate understanding their

mechanisms of action. Hydra has been used to assess the toxic potential of organic as well as

inorganic chemicals used for domestic and/or industrial purposes. Toxicity of wastewaters

from sewage treatments plants, industrial effluents or water samples collected from lakes or

creeks near to mining regions were also appraised in Hydra. Organic chemical tested were

herbicides, insecticides, bacteriocides, endocrine disrupting chemicals, dyes, polychlorinated

biphenyls, industrial solvents, oil dispersants, mycotoxins, flocculant blocks, cosmetic

substances and formulations or active ingredients of pharmaceuticals.

Hydra offers multiple endpoints that can be used to evaluate the toxicity of

environmental agents. These endpoints are morphology, regeneration, developmental

toxicity/teratogenicity, population growth rate/asexual reproduction, feeding, histology,

ultrastructural analysis, enzyme activity, microarray, transcriptional and translation studies.

Physical endpoints based on progressive morphological changes provides LC50, EC50/SEC50,

NOEC, LOEC, and toxicity threshold [TT=(NOECxLOEC)1/2]. Hydra shows abnormal

morphology in stepwise alterations starting with tentacle getting clubbed, to body shortening

with tentacles retraction representing a tulip flower and finally body disintegration

representing mortality. Tulip stage and onward to disintegration are the lethal reaction of

the body and is used to calculate LC50.The clubbed tentacles stage determines EC50 or sEC50.

Most of the researchers used abnormal morphology to measure the impact of the chemical on

Hydra. But since the year 2000 studies adopted Wilby scoring method to assess the impact of

chemicals on morphology (Wilby, 1988). The Wilby method follows the same morphological

change but here score was assigned for each change. As such score 8 represents the clubbed

tentacles stage, score 5 tulip, and score 0 disintegration or mortality.

Regeneration of isolated gastric region or reaggregated cells of Hydra resembles

embryonic development due to the ontogenic similarity observed with higher organisms. All

of the known developmental events or phenomena considered subject to abnormal

development have been observed in this organism (Johnson et al., 1982). In initial

toxicological studies, regeneration was measured by the use of dissociated cells to make an

artificial embryo (Johnson et al., 1982) and later Wilby (1988) recommended the use of

dissected gastric sections [located below the hypostome (mouth) and above the budding

region] consisting of mitotically active multipotent stem cells to study regeneration.

Morphological changes are observed using a binocular microscope and the degree of

regeneration assessed. Today this is most commonly done using Wilby‟s (1988)

classification, with a score ≤5 corresponding to lethality (Pachura-Bouchet et al., 2006).

Hydra have the ability to reproduce both sexually and asexually. Asexual

reproduction occurs by budding which results in rapid production of a large number of

genetically identical organisms (Pollino and Holdway, 1999). This has the advantage for

toxicity testing as the lack of genetic variation allows increased reproducibility with decreased

coefficient of variation (Beach and Pascoe, 1998). The Hydra population reproduction

toxicity test method determines the maximum concentration at which a chemical that has

no statistically significant effect over certain period (7 days or more) on the population

growth as measured by increased number of buds with the no observable effect concentration

(NOEC) and lowest observable effect concentration (LOEC) determined (Stebbing and

Pomroy, 1978; Holdway, 2005). The mean relative population growth rate (K) is calculated

and is defined as:

K = ln (ny) – ln (nx)/T

where nx is the number of Hydra at the beginning of the first day (tx), ny is the number of

Hydra after y - x days (ty) and T is the length of the test period in days (ty – tx).

Feeding behavior is ecologically significant because of its direct effects on

reproduction, vis-a-vis population growth and, therefore, a useful endpoint to study toxicity in

aquatic ecosystems (Juchelka and Snell, 1994). Toxicant modification of feeding behavior

could eventually lead to reduced survival and reproduction, resulting in adverse consequences

at the population level (Halbach et al., 1983; Kooijman and Metz, 1983). The feeding

reaction has been successfully used in several studies to examine the sub-lethal toxicity of

several compounds including heavy metals (Beach and Pascoe, 1998; Quinn et al., 2007) and

pharmaceuticals (Pascoe et al., 2003; Quinn et al., 2008b; Quinn et al., 2009) with the sub-

lethal response investigated using feeding bioassay proved to be considerably more sensitive

than that recorded in lethal studies (Beach and Pascoe, 1998).

The present situation of information about the ecotoxic consequences of

nanotechnology is quite limited and should be extensively documented. Hydra was used in

this case to analyse the harmful potential of nanoparticles utilizing several endpoints. Hydras

models allow the evaluation of any nanomaterial's impact in vivo, ex vivo, and in vitro

utilizing a variety of experimental techniques (Fig. 6). Nine metallic particles were classified

as very toxic (copper zinc iron oxide, indium tin oxide, holmium (III) oxide), toxic (nickel

zinc iron oxide, samarium (III) oxide, erbium (III) oxide, SWCNT), and harmful (yttrium

iron oxide: titanium dioxide, strontium ferrite; fullerene–C60) based on the 96-hour LC50.

The H. attenuata assay was shown to be worthy of inclusion in future NP testing batteries

(Blaise et al., 2008).

Fig 6: A summary of the approaches developed or adapted in our lab to investigate the

toxicological effects of nanoparticles (NPs) and Heavymetals.

Methods for toxicity study using Hydra

Chapter 3

3. Aims and Scope of the Study

3.1 Rationale

Toxicology is undergoing a burgeoning change with introduction of robust systems

for precise and reliable assessment of the potential toxicity of each of the thousands of

chemicals that are/could be present in the environment (Collins et al., 2008). Many scientists

who pursued the translation of NRC‟s “Toxicity testing in the 21st century: vision and a

strategy” and the REACH Legislation have emphasized the use of non-classical model

organisms for risk assessment of chemicals in the context of relevance or otherwise of the

data generated and the number of chemicals the testing all of which using the conventional

animal models may never be achieved (Zeeshan et al., 2014). Hydra, a cnidarian, also has the

potential to stake its claim an alternative model organism for environmental monitoring and

risk assessment.

3.2 Definition of the problem

Heavy metals are a specific group of substances in the context of chemical risk

assessment that are of concern from the point of view of the aquatic environments. Due to

their widespread dispersal in agricultural, industrial and domestic runoffs, they are of concern

in the global scale (Rainbow, 2002). Indeed some of the metals, that are otherwise present in

the environment, associate in the biochemical and physiological processes of living

organisms. Chromium is such metals which are of great physiological significance and

industrial application. However, at levels above permissible, these metals may cause

deleterious effects to fauna and flora of the water bodies. There are several studies

implicating water-borne chromium in inflicting toxicity.

3.5 Scope of research work

The need for non-mammalian / invertebrate experimental systems for toxicity testing

is increasing because of growing concern about animal welfare and environmental safety.

With this background, we adopted a systematic approach to prove Hydra to be a convenient

model organism for in vivo testing looking at end points that are morphological,

developmental and molecular and to better understand the mechanisms of action of chromium

with special intention of developing Hydra an appropriate in vivo model organism for looking

at toxicant-induced apoptosis.

3.4 Objective

The primary motivation for this piece of work was the rapidly emerging shift in

thinking about the use of new technologies to evaluate human health and environmental risks

in the 21st century. Science, technology, and innovative thinking are all essential tools for

addressing the variety of global concerns regarding environmental health risks. The new

technology in our case- Hydra as a model organism- presents an exciting opportunity to

extend beyond traditional risk assessment approaches and to better understand the effects of

chemicals on humans and the environment. Thus, following are the objectives of the research

in this thesis.

3.5 General objective:

The thesis aims at elucidating the mechanism of chromium toxicity in Hydra with

special emphasis on ROS-mediated molecular and cellular changes. In doing so, the outcome

of the study substantiates the claim that Hydra is a suitable in vivo model for risk assessment

of chemicals.

3.6 Specific objectives

The specific objectives with regard to toxicity testing of Chromium (Chapter IV) in Hydra

are:

 To standardize and culture Hydra feed (Artemia salina)

 To maintain and standardize Hydra cuture.

 To determine the morpho-physiological changes adopting microscopic examination of

abnormal morphology and to determine the acute toxicity (LC50).

 To examine the chronic effect on the development and reproduction

 To investigate the effect on population growth and to examine the structural organization

of feeding apparatus.

 To determine the apoptosis in Hydra adopting acridine orange Staining.

 DNA barcoding, a molecular identification tool used to species identification.

4. MATERIALS AND METHODS

Culture of Hydra

4.1 Artemia hatching & harvesting

Hatching

Although the Artemia neuplii hatch in a wide range of salinities as long as

sufficient oxygen present, there survival is depends upon the suitable ionic balance. The

Artemia hatching was carried out in a Griffen Beaker of 1 liter capacity. 500 ml of tap water

was added to the beaker and then 2g of NaCL was added and mixed properly. Then the

aearation stone was placed in the bottom of the beaker, and it was attachecd to the low

pressure air pump. A „ pinch‟ of dried artemia eggs are sprinkled over the surface, than the

eggs were mixed by stirring with glass rod, and were aerated by turning on the aquarium air

pump. The yield were obtained after 24 hrs @ room temperature.

Harvesting

After 24 hours the air pump is turned off to collect hatched Artemia, which in a few minutes

accumulate closest to the light at the bottom of the bottle, while unhatched eggs rise to the

surface. Hatched Artemia form an orange mass that is collected with a 25 ml long glass

pipette. Care should be taken to harvest only the hatched Artemia as Hydra cannot digest

unhatched eggs. Procedure to desalt hatched Artemia: transfer them in a petri dish prefilled

with Hydra medium (HM), let them accumulate at the bottom; repeat the transfer procedure

through two other 2 petri dishes also prefilled with Hydra medium.The complete procedure

takes about 20 minutes. Finally Artemia that are now 100% hatched and desalted can be

collected in a small beaker.

4.2 Test organisms and culture methods

Preparing Hydra Medium (HM)

Various mineral salts are added to high-quality water to produce a Standard

Hydra Medium. The “M” medium from (Muscatine and Lenhoff, 1965) modified by

(Sugiyama and Fujisawa, 1977) is widely used and easy to prepare. The composition of 100x

hydra medium contains 1 mM calcium chloride, 0.1 mM magnesium sulfate, 0.1 mM

potassium chloride, 1 mM sodium chloride and 1 mM Tris Base (pH 7.4). The chemicals

were dissolved in autoclave double distilled water. The pH of the medium was adjusted with

1M NaOH to 8.0. The care was taken to dissolve CaCl2 & MgSO4 seperately and then added

to the remaining solution to avoid precipitation. Then the volume was made up to 1 litre with

double distilled water. Depending on the need, the stock was diluted to 1x using double

distilled water as diluent.

Feeding

Hydra was maintained in glass crystallizing dishes containing hydra medium

(composition as given in the appendix) at 18°C with 12 hour light/dark cycle and fed daily

with freshly hatched Artemia salina nauplii, free of saline. Using a Pasteur pipette the

suspension of nauplii spread quickly and evenly throughout the glass dishes of Hydra. the

glass dishes were swished so that proper distribution of the nauplii take place. Sufficient

Artemia was supplied so that the poyps fed to repletions within 20 minutes. In the general, 3-

4 nauplii on average per Hydra were added in the culture tray. For maintaining healthy

culture collections, culture is fed regularly in mornings.

Cleaning and maintenance

One to four hours after feeding, a first wash is accomplished by pouring the

contents of the culture bowl into a 1 liter beaker filled with normal culture medium (2 liters if

the culture is large). Swirl the beaker and remove the Hydra at the center with a large glass

pipette and transfer them to a 600 ml beaker. Swirl the beaker and remove the Hydra at the

center. Repeat the process in a third 600 ml beaker then return the cleaned Hydra to the

original culture dish. Periodically all Hydra should be removed from the stock dish so that it

can be thoroughly cleaned as described previously. Approximately 8 hours after feeding,

Hydra will expel undigested remains of the Artemia. A second washing is therefore strongly

recommended. A perfect routine would be to feed Hydra at 8 am, first wash at noon and final

wash at the end of the day. The final wash can be accomplished in the same way as the first

but examination of the culture dishes and the Hydra health should make it clear when a

complete change of culture dish is necessary. A plastic scraper can be used to gently separate

all Hydra from a dish so that they can be transferred to another.

The Pasteur pipettes used to transfer Hydra should be stored in a test tube with ethanol

then rinsed with water or HM before use. If multiple strains are present, culture bowls and

pipettes should be color coded with tape so that strains are not co-mingled. Indeed Hydra

frequently adhere to the body of the pipette and then might inadvertently be transferred from

one culture to another if pipettes are used interchangeably. Between uses the dishware should

be washed with a small amount of detergent, rinsed extensively with water to remove any

trace of detergent, rinsed in ethanol and allowed to dry completely so that the ethanol

evaporates before reuse.

4.3 Acute exposure and morphological study

Acute toxicity testing in Hydra was conducted according to Murugadas et al.

(2016). Briefly, 10 polyps without bud were placed in small petri-dishes containing 8 mL of

Hydra medium and incubate in Bod incubator. The polyps were continuously exposed to

twelve different concentrations of chromium ranging from 1μg/L to 65μg/L for 96 h and the

progressive changes in morphology were recorded at every 24 h interval using a stereo-zoom

dissecting microscope (Carl Zeiss, Jena, Germany) enabled with a camera (ERc5 s). A score

of 10 was assigned to healthy polyps and 0 to the animals that had undergone disintegration;

animals with scores 9–1 indicated altered morphology to different levels, in that order (Table

2). Median lethal concentration 50 (LC50) was analyzed for each time point based on the

observed median score and the values were calculated adopting PROBIT analysis. LC50

determination enabled fixing the sub-lethal concentration to assess the low-dose level toxicity

of chromium in respect of regeneration and feeding behavior of Hydra. The experiments were

always conducted in triplicates.

Table 2: Scoring system devised by Wilby (1988) for assessing the morphological damage.

Score

Morphology of polyp

Extended tentacles and body reactive

Partially contracted, slow reactions

Clubbed tentacles, body slightly contracted

Shortened tentacles, body slightly contracted

Tentacles and body shortened

Totally contracted, tentacles visible

Totally contracted, no visible tentacles

Expanded, tentacles visible

Expanded, no visible tentacles

Dead but intact

Disintegrated

Sample preparation

Chromium stock solution (100 mg/L) was prepared in Hydra medium using analytical base

potassium dichromate (K 2Cr 2O7).The solution was vortex properly and stored in the

refrigerator. The freshly prepared working solution was obtained every time it was used from

the stock solution. The experiments were performed in cell culture 6 well plates with 8ml of

hydra medium along with the desired test chemical concentrations.

4.4 Hydra regeneration assay

To this study 24 hours starved non-budded healthy hydra were selected to assess the impact

of chromium exposure on the potential for regeneration, two sub-lethal concentrations (T1

and T2) respectively, which did not induce much an adverse effect on Hydra's morphology,

were chosen based on the 96 h LC50 value. A group of 15 animals was bisected using a sterile

medical scalpel and the lower portion of body column was allowed to regenerate the missing

upper parts in the presence of sub lethal concentrations of chromium. The amputated body

parts were kept in 15 mL of Hydra medium and observed for 96 h. The progress of

regeneration was monitored using the stereo-zoom dissecting microscope, and score was

assigned according to Ambrosone et al. (2012). The scores for regeneration assay are shown

in Table 3.

Table 3: Scoring system devised by Ambrosone et al. (2012) to measure the head

regeneration in hydra

Score

Polyp Morphology

Fully regenerated

Emergence of tentacles

No regeneration

Lethal / Disintegrated

4.5 Hydra population study

Growth and population study was performed according to Ambrosone et al. (2012). Polyps of

same developmental stage were picked from the stock culture for experiment. Five Hydra

(one bud each) were exposed to T1, T2, or control for 24 or 48 h followed by wash in Hydra

medium and placed individually in petri dishes. During the next 14 days the polyps were fed,

counted and recorded daily. The buds produced were counted at every 24 h interval, and the

growth rate constant (K) of exponentially growing animals was determined using the

equation ln(n/ n0) ¼ kt, where n is the number of animals at time t and n0 is the number of

animals at t0. Population doubling time (t2) was determined when the number of animals (n0)

at time T doubled to that at the beginning of the experiment (n), i.e., when n/n0 ¼ 2, t ¼ t2

(population doubling time).

4.6 Feeding assay

The ability of untreated and chromium (sub-lethal concentrations)-treated animals to catch

and ingest the prey was recorded in this study. Animals exposed to T1, T2, or control were

placed in 6-well plates, and each well contained 3 mL of the Hydra medium. After the

treatment period (24 h exposure), freshly hatched Artemia salina nauplii or 10 mM reduced

glutathione (GSH) was challenged against Hydra and Hydra's ability to catch the prey, and

writhing of tentacles in response to reduced glutathione were recorded using the stereo-zoom

dissecting microscope equipped with an Axiocam ERc5s camera. . The polyps were

examined for the ability to catch, kill, or ingest the prey. The duration of the observation was

10 min, wherein recording was performed during the first 3 min, and the animals were only

observed during the next 7 min.

4.7 ROS

Assay principle:

Reactive Oxygen Species can be induced by some stress conditions like exposure to oxidant

or drugs. This fact leads to oxidative stress. ROS induce damage in DNA, protein and lipids

with important consequences in cells. Cell permeant reagent 2‟-7‟dichlorofluorescin

diacetate (DCFH-DA) is a fluorogenic dye that measures hydroxyl, peroxyl and other ROS

activity in the cell. After cell uptake, DCFH-DA is deacetylated by cellular esterases to a

non-fluorescent compound, which is later oxidized by ROS into 2‟-7‟dichlorofluorescein

(DCF). DCF is a fluorescent compound which can be detected by fluorimeter, flow

cytometer or fluorescence microscope with a maximum excitation and emission spectra of

485 nm and 535 nm respectively.

Method:

Intracellular ROS generation in Hydra treated with chromium was assessed using 2′,7′-

dichlorofluorescein diacetate (H2-DCFDA, Molecular Probes). Treated and untreated animals

were washed in PBS and incubated for 1 h in dark with 10 mM H2-DCFDA followed by

wash in PBS and finally relaxed in 2% urethane. Animals were immediately observed in the

fluorescent microscope and photographed.

Intracellular ROS levels Polyps Hydra treated with chromium was assessed using 2′,7′-

dichlorofluorescein diacetate. The healthy non-budded hydras were picked for this study, and

then they starved for 24hrs. Hydra (one bud each) were exposed to T1, T2, or control for 24

or 48 h. At the end, the treated wells were replaced with HM media. At the same time hydras

were washed three times without remove any of them. the hydras were carefully transferred

to the 0.5ml tubes with minimal amount of HM media. The HM were gently removed from

the well without discharging hydras. Then hydras were washed and macerated in (100 μL )

pre chilled PBS (10 μL/Hydra). The cell lysate was prepared in PBS by homogenizing hydra

using a 1mL insulin syringe. The test samples were prepared in the 96well plate with 200µl

of Bradford and 5µl of cell lysate were added and it mixed well using different tips.The

protein concentration was determined using Bradford reagent with bovine serum albumin

(BSA) as the standard. Estimated lysate was added the volume and it was made up to 100µl

with PBS.30µl of H-DCFDA dye was added thoroughly mixed without the formation of

bubbles. Cell suspensions were incubated in 10 mM H2DCFDA and the fluorescence was

measured using a fluorometer (PerkinElmer, USA). ROS levels in treated animals were

expressed as fold-change over control. All experiments were performed in triplicate.

4.1 Whole mount acridine orange staining-Hydra

Principle

Apoptosis, the programmed cell death is characterized by numerous

morphological changes. Since it is implicated in many pathological and physiological

conditions it becomes obligatory to have reliable methods for detecting cell death. Acridine

orange is a cationic acidophilic dye, which specifically stains apoptotic cells within

phagocytic vacuoles with low pH value (Schwarz and Wittekind, 1982; Clerc and

Barenholz, 1998). It penetrates living cells and fluoresces green/yellow excitation at 450-

490 nm. Live cells appear uniformly green, Early apoptotic cell fluoresces green and

contains bright green dots in the nuclei as a consequence of chromatin condensation and

nuclear fragmentation. Late apoptotic cells stain orange and show condensed and often

fragmented nuclei. Necrotic cell stain orange but have a nuclear morphology resembling

that of viable cells, with no condensed chromatin.

Procedure

Hydra were exposed to T1, T2, or control and subjected to acridine orange (AO) staining of

whole animal and were performed. Groups of 10 animals each were treated with T1 and T2

concentrations of chromium for 48 h,& then treated animals were washed five times in

Hydra medium for 5 minutes each. The animals were then transferred to a glass slide. A

drop of acridine orange (3.3 µM) was added to polyp and was kept in dark for 15 minutes.

Stained animals were washed four times in Hydra medium for 5 minutes each. 2% of

urethane was added to slide for animals to relax. Animals were observed under

epifluorescent microscope and photographed.

4.9 Molecular identification of Hydra vulgaris

4.9.1 DNA isolation and PCR

Hydra samples were preserved in 95% ethanol and commercially outsourced (Barcode

Biotechnologies Pvt. Ltd., Goa, India) for Cytochrome C oxidase subunit I gene (COI)

sequencing. Briefly, the DNA was isolated from Qiagen blood & tissue DNA isolation kit

and purified using Qiagen filter column. The purified DNA was confirmed through 1.2%

agarose gel electrophoresis and used as a template for PCR. The forward primer LCO (5'-

GCTCAACAAATCATAAAGATATTGG-3') and reverse primer HCO (5'-

TAAACTTCAGGGTGACCAAAAAATCA-3') was used in the PCR reactions which

consists of 1x reaction buffer (Thermo Scientific), 2.5 mMMgCl2, dNTPs, 2 pmol of each

primer, 1U Taq polymerase (Thermo Scientific), and approximately 50 ng template DNA in a

total volume of 25μl. Also Ready-to-Go PCR beads were used for PCR amplifications,

adding the same amounts of primers and template DNA, and ddH2O in a total reaction

volume of 25μl. PCR amplification used the following protocol: 95°C for 2 min; 29 cycles of

94°C for 30s each, 49°C for 30 s; 72°C for 2 min; with the final extension at 72°C for 8 min.

The PCR products were checked using a 1.5% agarose gel. Prior to sequencing the PCR

products were enzymatically purified using ExoSAPit (Thermo Scientific) following the

manufacturer‟s recommendations.

4.9.2 DNA sequencing and sequence analysis

Samples for bi-directional DNA sequencing were prepared on a 96-well plate, with DNA

template, primer and ddH2O. Bi-directional sequencing of the mtDNA COI fragments was

done using the same PCR primers.Sequencing was performed using an ABI3730xl Genetic

Analyzer (Applied Biosystems).

Sequence inspection and editing of each raw sequences (abi1 files) files of the forward and

reverse primer were assembled individually and inspected for sequencing errors using the

software Sequencher vs 5.0. The resulting text sequences were exported in nexus and fasta

formats, and assembled in sequence data matrices, which were used for further analyses.

Sequences were subjected to BLAST analysis and aligned using CLUSTAL X software.

Along with reference sequences collected from NCBI, phylogenetic analyses were conducted

in MEGA X using the Kimura 2-parametric distance model.

5. Results and Discussion

Results

5.1 Hydra culture and maintenance

Artemia salina hatching

Brine shrimp (Artemia salina) culture was standardized in the lab. It was observed

that Artemia cysts hatched in a wide range of salinities as long as sufficient oxygen was

present. Both too high and low levels of salt concentration inhibited brine shrimp hatching.

Brine shrimp hatching was optimised in artificial seawater (3.8% sodium chloride solution)

under artificial light with full aeration (Browne et al., 1991). The dried brine shrimp absorbed

water and required a circular shape before hatching, a pre step to hatching.(Figure 7) and

within 24 hours under optimal condition developed into Instar 10 stage animal (Figure 7).

The hatching temperature is important. Satisfactory yields were obtained after 24 hours at 22

24°C. If desired, the yield may be increased somewhat by incubating at 22-24°C for 30 hours,

or at 30°C for a shorter period of time. Too many eggs on the surface resulted in a low.

Overall hatch and many due to excess oxygen demand (Browne et al., 1991). Thus, the

practice was to sprinkle a pinch of brine shrimp for good yield.

A B

Figure 6: (A). The Artemia cyst after absorbing water, (B) Newly hacted Artemia.

Artemia Harvesting

Artemia are strongly attracted to light. A lamp was used at the corner of the beaker which

resulted in accumulation of live Artemia towards the light source. Collection was performed

within 25 min otherwise mortality started which was due to depleted oxygen level as

collection was performed in non-aerated condition. This maximizes getting healthy nauplii

and minimizes taking un-hatched eggs (Browne et al., 1991). A typical collection set-up

consist of light source and a hatched culture of Artemia nauplii container (Fig; 7).

Hydra culture and maintenance

Hydra were cultured in a reconstituted water and virtually unlimited numbers of

healthy Hydra were reared under controlled conditions in the laboratory with relative ease.

Hydra were cultured in Hydra medium consisting of different salts ingredients and it was

maintenance in Pyrex culture dishes (about 500 Hydra per 25 cm x 25 cm dish)(Lenhoff,

1983)(Fig 10). On feeding Hydra regularly at alternative day with newly hatched Artemia,

Hydra doubling time was 1-4 days. Healthy Hydra were attached to the base and side wall of

the beaker. After 2 hours of feeding, Hydra were washed with Hydra medium (HM) and were

maintained at 16°C in a BOD incubator (Fig. 8).

Fig : 7 (C) . Artemia culture collection set-up (D). Artemia double washing using petridish.

Figure 10 : Hydra culture bowl ( Pyrex culture dishes)

Figure 8 : BOD Incubator Figure 9 : stereozoom dissection microscope

5.2 Acute toxicity and morphological changes

The acute toxicity of chromium was performed for 24, 48, 72 and 96 h in

Hydra magnipapillata. Toxicity test was carried out in six well plate formate wherein 10

animals per well were taken and exposed to 8ml of hydra media containing the respective

chemicals at defined concentration. The toxicity was measured according to the Wilby et al.,

(1988) scale the score 5 and below considered as a death (Fig 3A). Initial range finding was

undertaken to select the maximum exposure level. The exposure was carried out according to

the logarithm dose. After determining the exposure limit Hydra were exposed to a nominal

concentration of 1 to 65 µg/L. The maximum concentration at which all animals died after

24, 48, 72 and 96 h treatment period were 1 to 65 µg/L, respectively (Figure 3B). All the

expriments were performed in triplicate. Probit analysis was perform to determine the 96 h

lethal concentration (LC50). The values of LC50 are presented in Table 4. Good regression

coefficient for was obtained (Table 5). The effect of chromium on Hydra was dose-and time-

dependent. In addition to it, 96-h LC 10, 20, 30 and 40 were also estimated using probit

analysis. The value of the lethal concentrations are present in table 4.

Concentration

LC10

LC20

LC30

LC40

LC50

Chromium

5.55

6.45

7.42

8.71

10.45

Table 4 : Lethal concentration value of chromium for 48hrs.

Morphological change

Changes in Hydra morphology were observed after 24, 48, 72, and 96 h of exposure

using a stereomicroscope ( Fig 10). A wide range of concentrations were taken for assessing

the morphological change. All control animals survived the experiment period. At each

observation time the individual animals were assigned a score dependent upon the structural

appearance as described in the assessment key (Fig 11). The median scores recorded at each

test concentration for chromium is shown in Table 5 and the representative image is

presented in Figure 12. The median scores of animals decreased both with increasing

exposure concentration and exposure time. Initial sign of toxicity was observed with

shortening of tentacles, to a more severe damage manifested as shortening of body and

tentacles .Lethality was induced with total body contraction (score 5) to tissue disintegration

(Score 0) (Figure 11). It was also observed that 96 h exposed animal showed marked

morphological change at higher concentration as compared to other time points. Statistical

difference between control and treatment was calculated using GraphPad Prism 9 software by

applying non-parametre Kruskal-wallis and Dunn's test. Significant difference was observed

for the highest concentration (65 µg/L) for all exposure periods (Table 5).

Table.5 : Median score recorded at each chromium concentration in different exposure time.

Conc(ug/ml)

Total no hydras

24h

48h

72h

96 h

6**

4***

0****

3**

2***

0**

0****

0**

0***

0****

*statistical significant with control p<0.05), as revealed in Dunn‟s test

**statistical significant with control p<0.01), as revealed in Dunn‟s test

***statistical significant with control p<0.001), as revealed in Dunn‟s test

Fig. 11. Toxic effect of chromium in Hydra as revealed in its morphology. A)

Representative images of Hydra showing progressive morphological changes on exposure to

chromium. Score 10 indicates healthy polyps. The progressive structural alteration is

presented by numerals in decreasing order. Score 5-0 is indication of lethality. The scoring

(Table 5) was performed according to Wilby (1988). Scale bar, 0.5 mm. B) Animals were

exposed to different concentrations of chromium and the median score were determined for

different time points. Chromium exposure caused dose- and duration-dependent toxicity as

evident in the decrease of score for both increase of time and concentration. Multiple

comparison of significance was performed applying non-parametric Kruskal-Wallis test

and Dunn‟s test (*p<0.05;**p<0.001). C) Median lethal concentration (LC50) is

presented for different time points.

Score 10 Extended

tentacles & body

reactive

Score 9 Partially

contracted, slow reactions

reactive

Score 8 Clubbed tentacles, body

slightly contracted reactive

Score 7 Shortened

tentacles, body slightly

contracted

Score 6 Tentacles and body

shortened

Score 5 Totally contracted,

tentacles visible

Score 3 Expanded,

tentacles visible

Score 0 Disintegrated

5.3 Regeneration assay

The median regeneration scores for isolated digestive regions were observed in a

stereo microscope after 24, 48, 72, and 96-h of exposure. To have a comparative study of the

effect of acute concentration and sub lethal concentration (5ug and 10ug)and it denoted as T1

nad T2 respectively, Hydra were exposed to LC50 and 1/10 and 1/100th value of LC. All

animals regenerated the experimental period. At catch observation time the individual

animals were assigned a score dependent upon the structural appearance as described in the

assessment key . It was observed that regeneration was concentration-dependent. For

Chromium LC30 exposed animal did not fully regenerated. 1/10 and 1/100 of LC50

concentration animal regenerated and regeneration was much in rapid for 1/100of LC50. A

graphical representation of chromium.

Fig 12. Toxicological impact of chromium on hydra regeneration:

% of animal (n = 45) p.a 72hrs % of animal (n = 45) p.a 96 hrs

Score Score

Fig. 12. Chromium-induced impairment of Hydra regeneration. (A) Representative

images of regeneration of gastric region. Median scores of regenerating tissue ranged from At

72h and 96h post-amputation animals were examined for viability and regeneration stage:

stage 3 indicates complete inhibition of regeneration; stage 1 indicates the presence of

tentacle buds; stage 2 indicates new emerging tentacles.. Scale bar, 0.5 mm. Untreated

animals regenerated within 96 h showing developed body and elongated tentacles. T1

treatment delayed the developmental process (score 2 for 96 h) and T2 completely inhibited

regeneration (score 3 for 96 h).(B) Comparison of regeneration effect of chromium at two

different time exposure.Significance was determined against control and multiple comparison

was performed applying non-parametric Kruskal–Wallis test and Dunn's test (*p<0.05).

5.4 Reproductive toxicity

Untreated animals demonstrated a typical linear increase in counts over time but exposure

to T1 or T2 decreased the rate of budding .At 48 h, T1 treatment affected the budding upto

day 1, whereas with T2 treatment the effect lasted for 4 days when the animal counts

remained almost unchanged before rising linearly (Fig. 13). At 24 h, the population growth

rate (K) was impacted at the beginning of the experiment (day 4) which was restored to near

normal by the end of the experiment (day 13) for T1 treatment but not for T2 (see values of

K in ( Fig. 13). At 48 h, the K values were significantly low for both T1 (p<0.05) and T2

(p<0.001). The increase of K value at the end of the experiment indicated the ability of the

polyp to restore the control mechanisms but the values were still significantly lower than in

untreated animals suggesting a chronic inhibitory effect of the treatment, which was revealed

in the analysis of population doubling time as well, wherein at 48 h incubation T1- and T2-

treated animals took nearly 5 and 6 days, respectively, to double as compared to control

animals which required only 4 days (Fig.13B).

Fig. 13. Effect of Chromium on Hydra reproduction. A) Following 24 or 48 h of

chromium exposure different parameters of reproduction were examined. Growth rate curve

shows a linear increase in population of animals treated for 24 h which was significantly

affected for 48 h incubation period. Hydra doubles in an uncontaminated environment within

4 days. Animals exposed for 48 h exhibited significant difference in doubling time between

groups.

5.5 Feeding assay

Feeding assay was performed to determine whether the structural aberration of

tentacles led to impairment of feeding or not. Feeding behavior typically comprises catching

and killing of the prey, bending of tentacles towards the mouth and ingestion of the prey.

Untreated animals readily captured Artemia nauplii and immediately killed them. Moments

thereafter the tentacles bend towards the mouth followed by opening of the mouth and

ingestion of the prey (fig. 14), Untreated animals readily captured Artemia nauplii, which

were immediately killed and then tentacles bent toward the mouth followed by the opening of

the mouth and ingestion of the prey. The ingestion of all nauplii took only 1mints. T1-treated

animals also readily captured the prey, but after the ingestion of 2 or 3 nauplii, the animals

stopped the ingestion, and by 10 min, the remaining nauplii dropped-off from the tentacles.

(Movie M2*; Fig.14 ). Animals in the T2 group displayed a similar ingestion capacity but

with a reduced ability to kill the prey where many a times the nauplii touched the tentacles

but were not ensnared. The falling of Artemia nauplii in T2 was quicker than in T1 treatment.

(fig 14).

Fig.14. Effect of chromium on feeding behavior in Hydra. (A) Animals show a

reduced entangling ability as observed from the dropped-off Artemia.

5.6 Induction of ROS generation:

Using the non-fluorescent dye 2, 7-dichlorofluorescin diacetate, the ability of chromium to

cause oxidative stress was discovered (H₂-DCFDA). ROS buildup causes H₂-DCF to be

oxidised, which produces highly fluorescent DCF, which was seen as green fluorescent

punctae in the polyps. DCHFDA fluorescence was barely visible in untreated animals

(Fig.16). In the treated animals 5 and 10 µg/L, there was a bright fluorescence (Fig.16). The

ectodermal layer of treated animals showed punctated DCF fluorescence, along with an

increase in fluorescence in the gastrodermal layer (as opposed to controls).

The highest level of ROS generation was seen in 10 µg/L animals exposed for 24 hours,

and it was concentration and time-dependent. In comparison to the control, the increase was

1.5 times greater for 5 µg/L and 2.5 times greater for 10 µg/L. Quantitative analysis of the

DCF fluorescence in hydras cell lysate provided additional evidence of the rise in ROS

generation (Fig.16).

a) Control b) 5µg/L c) 10µg/L

Figure 15. Fluorescent microscopic images of ROS and apoptosis in hydra following

exposure to chromium. Representative images of ROS generation in hydra following

chromium exposure. Induction of ROS generation was revealed in the punctated green

fluorescence.

Figure 15. Quantification of intracellular ROS generation in hydra following chromium

exposure. ROS level is expressed as fold change in the fluorescence in comparison with

untreated polyp. Significance between the control and the treatment groups were performed

by adopting two way ANOVA with Dunnet‟s multiple comparison test (**p<0.01).

5.7 Whole mount acridine orange staining-Hydra

To understand the mode of cell death acridine orange staining was performed on

animal exposed to LC50 and 1/100 of LC50 value of metals. The effect was observed for

animals exposed to 48-h. The results obtained at different exposures indicate the initiation of

apoptosis in a dose dependent manner. Appearance of green and faint orange dots in the

animals (Fig. 16) increased with increase of exposure which is an indication of the formation

of apoptotic bodies.

Fig.16. AO stained cells of chromium treated live animal observed in a fluorescent

microscope. (A,B) Control animal shows minimal apoptosis. (C) Signs of massive cell death

(bright green and punctated fluorescence) appear for T1 treated animals. (D) Animal at a

higher magnification shows punctated images through-out the body column. (E) T2 treated

polyps show a dramatic increase in apoptosis as indicated by orange spots. (F) Animal at a

higher magnification reveals the punctated AO-positive cells indicating phagocytosis.

5.8 Molecular identification of Hydra vulgaris

The COI sequences shared 100% of similarity with Hydra vulgaris (GenBank Acc. no.:

KY452027) sequenced at the Developmental Biology, Agharkar Research Institute, G.G.

Agarkar Road, Pune, Maharashtra 411004, India. In the constructed COI phylogram, the

sequence of the present study was placed within the same clade of various COI sequences of

H. vulgaris collected from the Genbank (Fig. 17), confirming the identification of the hydra

as H. vulgaris.

Fig 17: The Neighbor-Joining method tree with the percentage of replicate trees in which the

associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the

branches. The tree is drawn to scale, with branch lengths in the same units as those of the

evolutionary distances used to infer the phylogenetic tree. This analysis involved 9 nucleotide

sequences. There were a total of 456 positions in the final dataset. The COI sequence

produced in the present study was indicated by the strain name NCT1001 at the tip of a

branch.

6. Discussion

Effects of Chromium on aspects such as morphological changes, reproduction, feeding

and budding of a few species of Hydra have been investigated earlier (Stebbing and Pomroy,

1978; Beach and Pascoe, 1998; Karntanut and Pascoe, 2002). All these studies demonstrated that

Chromium is toxic to Hydra but the mechanism underlying the toxicity was not deciphered. This

study adopted a systematic approach to underpin the organismal, population, molecular and

biochemical manifestations of chromium toxicity in Hydra vulgaris. The Chromium LC50 for H.

vulgaris, as determined in this study, is comparable to that for other Hydra species (Beach and

Pascoe, 1998; Karntanut and Pascoe, 2002). Hydra exposed to T2 dose of Chromium for 48 h

showed remarkable morphological changes which were not observed in those exposed to T1. The

pre-eminent morphological changes began with formation of clubbed tentacles (score 8),

followed by shortening of body and tentacles (score 6), finally leading to whole body

disintegration (score 0) are by and large similar to observations on other Hydra species (Hydra

viridissima and Hydra oligactis) except for the reported detachment of tentacles (Beach and

Pascoe, 1998; Karntanut and Pascoe, 2002). Hydra vulgari and Hydra oligactis are closely

related yet distinct species due to difference in morphology, development, physiology, and

ecology (Hemmrich et al., 2007). Expression of transcriptionally restricted genes confers

species-specific differences in tentacle formation. In addition, the shape and size of nematocytes

differ between different species of Hydra (Khalturin et al., 2008). It is likely that these

differences provide for chromium to act differently between the two species resulting in tentacle

detachment in H. oligactis and not in H. magnipapillata. However, a detailed investigation in

this regard is required to substantiate this inference.

The regeneration assay revealed chromium-induced developmental deformity in Hydra.

Interestingly, T1 exposure, which did not produce morphological alterations, affected the

regeneration profoundly by inhibiting the development of the body wherein the tentacles

remained stumpy as was reported in Aiptasia pulchella (Howe et al., 2014). However, the

deformity in grass shrimp was observed at a dose above 1 mg/L, i,e., 100 times that to which

Hydra were exposed in this study, which could be attributed to the protective embryonic coat

present in the shrimp embryo (Rayburn and Fisher, 1999), which restricts entry of chemicals into

the body, whereas in Hydra the gastric region, also called embryo, has no such protective coating

except for the outer cuticle. In fact, because of the simple diploblastic body plan, with water

from the medium bathing the ectoderm and also circulating in the gastro vascular cavity, all cells

of Hydra are potentially exposed to the toxicant which provides scope for exposure level lower

than in the shrimp embryo. Embryos of zebrafish showed morphological and developmental

deformities in response to chromium exposure (0.01–1mg/L). Decreased hatching success,

increased yolk sac area and concomitant decrease in length of individuals as compared to controls

suggested slower development (Johnson et al., 2007). The results are comparable since the

exposure levels at which chromium caused deformity in zebrafish embryo are similar to those in

this study (above 0.06 mg/L). Thus, the developmental deformity can be attributed to induction of

ROS, DNA damage and activation of apoptosis (Barjhoux et al., 2012; Kong et al., 2013).

Further the long term effect of sublethal concentrations of chromium on population

growth which is was examined. Hydra reproduces by budding from the gastric region at the age

of about 4 days which is dependent on proliferation of epithelial cells and interstitial cells

(Bosch and David, 1984). Population growth rate constant (k) is a direct representation of

budding/asexual reproduction. Delay in budding after 24 h exposure was observed at the

beginning of the experiment (4d) which was restored to that in untreated animal by the end of the

experiment (14d). It could be explained that as Hydra produced more buds the metal-encased

cells were sloughed off from the body extremities or passed-on to the progenies thus diluting the

effect of chromium (Ambrosone et al., 2014). With increase in incubation period (48 h) and dose

(T2) population growth rate was severely retarded. The results here are comparable to the

observation in the sea anemone Aiptasia pulchella, in which 28 day exposure led to reduction in

the total number of developed juveniles (Howe et al., 2014b). In cnidarians heavy metals are

known to interfere with osmoregulation, behavior and reproduction. The decrease in growth

following sub- lethal chromium exposure has been demonstrated in crustaceans Gammarus pulex

(Maund et al., 1992) and Asellus aquaticus (de Nicola et al., 1988). Since tissue turnover is

dependent on the duration of epithelial cell cycle (Bosch and David, 1984) it is assumed that

increasing chromium concentrations would have delayed the normal epithelial cell cycle of

Hydra which in turn would have affected the reproduction and therefore the population doubling

time.

Out of the many culpable reasons for impaired reproduction one could be the consequence

of impaired feeding (Weeks and Rainbow, 1991). Battery cell complexes (BCCs) are each

epitheliomuscular cell enclosing nematocytes, and neurons (Hufnagel et al., 1985). In T1-

exposed animals the nematocytes were disorganized whereas in T2- exposed animals the

nematocytes were severely depleted with loss of characteristic BCC. Loss of nematocytes with

increasing doses of chromium is in agreement with the findings of Ambrosone et al. (2014), who

reported depleted BCCs on exposure to silicon nanoparticles accounts for altered feeding

response. This suggests that the loss of nematocytes in chromium-exposed Hydra impairs

feeding and ultimately affects reproduction by nutrient deprivation.

We observed that chromium treatment affected reproduction in Hydra in a dose and

duration-dependent manner. Previous studies have demonstrated that environmental factors such

as toxic agents, scarcity of food, and temperature variations affect reproduction in Hydra

(Schaible et al., 2011; Zeeshan et al., 2016). Untreated Hydra doubled within 3-4 days due to the

prolific epithelial stem cells division leading to the formation of a new organism in a process

called budding (Bosch, 2007). The increase in number of Hydra with time was typically linear

when numbers are expressed as logarithms and is determined by ln(n/n0)=kt (Bosch and David,

1984). The present study revealed that the number of animals over the first few days (see k and

ln n/no values) remained unchanged. However, with passage of time Hydra appeared capable of

neutralizing the inhibitory effects of chromium (see the increase in k values) but the values were

still lower than in control, indicating the lasting effect of chromium. This neutralizing property

may be due to the reactivation of epithelial stem cell division and differentiation, the impairment

of which explains the decrease of population (Ambrosone et al., 2014). An overall reduction in

the total number of live offspring and developed juveniles, as caused by chromium treatment, has

been reported for A. pulchella also (Howe et al., 2014a).

The nematocysts in the tentacle of Hydra and the associated neurons are housed within

single epithelio-muscular cells, and this structure is called battery cell complex (BCC) (Hufnagel

et al., 1985). These nematocytes are directly concerned with feeding and defense. T1 treatment

altered the BCCs in the backdrop that no morphological indication of toxicity was observed.

Chromium at the higher concentration (T2) caused shortening of the tentacles and altered the

ring-like morphology of BCCs. The results here are in agreement with recent findings of

chromium treatments in Hydra (Zeeshan et al., 2016). Low doses of chromium caused „severe‟

tentacle retraction in A. Pulchella (Howe et al., 2014b). Heavy metal intoxication has been

reported to affect feeding rate, and such disruptions of physiology bring about imbalance in

growth and reproduction (Weeks and Rainbow, 1991; Roddie et al., 1994; Howe et al., 2014b).

Feeding assay in Hydra was performed to explore how the structural impairment affects feeding

behavior. Feeding occurs in the following sequence, catching and killing of prey; bending of

tentacles towards the mouth; and ingestion of the prey (Ruch and Cook, 1984). In this study,

untreated animals paralyzed and ingested all the prey instantly, which indicated controlled

discharge responses of nematocytes and a normal feeding behavior. T1 treatment (polyps

showing normal morphology but altered BCCs) demonstrated reduced killing and ingestion

capability accompanied by incidents of prey falling-off from the tentacles. On T2 exposure the

falling off was even quicker than T1. Similar report of polyps failing to entangle and kill artemia

was observed for pulsed magnesium exposures (Prouse et al., 2015). Among the different types

of nematocytes in Hydra, desmonemes and stenoteles are responsible for entangling and killing

the prey, respectively (Ruch and Cook, 1984). Morabito et al. (2014) reported that treatment with

heavy metals such as zinc, chromium, cadmium and lanthanum significantly compromised the

nematocytes‟ discharge and the effectiveness of the toxin (Morabito et al., 2014).

Acridine orange is a fluorescent dye that is widely used to specifically highlight apoptotic

cells in a variety of organisms. Hydra were exposed to acute and sublethal concentration of

metals. chromium LC50 value showed animal was throughout stained with AO with appearance

of both bright green and red spots. AO stains both live and dead cells and emits colour

depending on the stage of apoptosis. Green cells indicated cells which are engulfed by

phagocytic cells and whose membrane are intact (Cikala et al, 1999). Red spots indicated cell

death which is in coherence with as Mpoke and Wolfe (1997) work. The authors reported

similar results on tetrahymena. The theory goes like AO is used as vital pH indicator in the

living cell (pH transition from alkalescent to weak-acid is manifested in changing of AO

spectrum from green area to red). Since the cytoplasm acidulation is a relatively reliable

apoptotic hallmark, AO is used in this way to project apoptosis. Sublethal concentration of

chromium produced early apoptosis as seen in the presence bright green spots. It was observed

that Chromium LC50 produced marked level of cell death as compared to animals exposed to

the other concentrations. These results are in comparison to genotoxicity assessment wherein

cromium LC50 showed highest number of dead cell as compared to any other group.

Chapter 6

7. Summary

The increasing worldwide contamination of water bodies with thousands of synthetic and natural

chemical compounds is one of the key environmental problems. Besides numerous organic

compounds entering aquatic ecosystems, heavy metal input continues to be rising. Some heavy

metals may transform into the persistent metallic compounds with high toxicity, which can be

bio-accumulated in the organisms and magnified in the food chain, thus threatening human

health. Various studies on chromium toxicity have been performed in many aquatic species

detailing the effect on survival, morphology, development, feeding, growth and population

dynamics. on the other hand, has been relatively less studied despite being known as an

environmental toxicant. Most of the studies for the chromium metals entailed physiological

effect and few of molecular studies were taken up that too in fish species and not in

invertebrates. As a result, the molecular mechanism of chromium toxicity are poorly understood.

One of the most important steps in achieving it is to find an appropriate model organism

which can help elucidate both acute and chronic toxicities of water pollutants with reproducible

results, shed light on developmental toxicity and understand the mechanism of toxic action at

various levels of biological organizations (i.e., molecular, cellular, whole organisms and

population). In this way, this model organism will also be of great importance for chemical

regulation in marine environments

Thus, the research in this thesis was aimed at finding insights to demonstrate the potential of

Hydra in risk assessment of heavy metals. Chromium at sub-lethal exposure were implicated in

inducing multiple toxicities. The metal produced adverse effects on morphology, survival and

reproduction in a manner dose-dependent. Asexual reproduction was significantly inhibited

either due to direct of the metals or due to the loss of nematocytes responsible for feeding.

Adopting AO staining on live animals cellular macerates of Hydra apoptosis was demonstrated

at the whole animal and nuclear levels for both metal exposures..

The salient features of the findings are presented below:

 The present investigation entailed study on a different Hydra species, Hydra vulgaris at a

greater depth.Chromium was studied for the first time in Hydra vulgaris.

 The LC50 of chromium for different time points ranged from 0.5 ug/L and 0.10 ug/L,

respectively.The concentrations of the metal to which Hydra were exposed could

potentially be present in freshwater bodies.

 The morphological changes to chromium exposures showed a similar pattern which began

with the formation of clubbed tentacles, followed by shortening of body and tentacles, and

finally, whole body disintegration. However, the latter was quick for chromium exposure.

 Two sublethal doses were selected from the results of 48h exposure and were denoted as

T1 and T2. For chromium, 0.5 and 0.10 5 µg/L, corresponded to T1 and T2, respectively.

T1 induced no visible morphological changes whereas T2 induced moderate morphological

changes.

 Control animals completely regenerated exhibiting normal morphology like elongated

body, extended tentacles, hypostome and pedal disc. Exposure to T1 dose delayed the

developmental progress of Hydra as was observed from animals with contracted buds and

no basal disk. Exposure to a higher dose, T2, completely inhibited regeneration and

resulted in tissue disintegration.

 Also, the population doubling time of treated animals was significantly increased as

compared to control animals. Since tissue turnover is dependent on duration of the

epithelial cell cycle, it is assumed that metal treatment would have delayed the normal

epithelial cell cycle which in turn affected the reproduction.

 The nematocytes in the tentacle of Hydra and the associated neurons are housed within

single epithelio-muscular cells, and this structure is called battery cell complex (BCC). The

nematocytes are directly concerned with feeding and defense. In general, loss of

nematocytes was observed for chromium metal treatments. However, complete collapse of

BCCs was observed for chromium treatment.

 Feeding assay showed that untreated animals demonstrated normal behavior such as

catching, killing and ingesting the prey within 30 secs from the time of contact with the

prey. Chromium treated animals demonstrated reduced killing and ingestion capability

with incidence of prey falling-off from the tentacles. Thus, the study demarcated that

alteration of structures responsible for feeding (nematocytes) resulted in disorganized

behavioral response (feeding), the consequence of which was reflected in reproduction.

 Sequencing of COI gene of study animal showed that the animal belong to H.vulgaris

The results show that H.vulgaris responds to chromium exposure with changes in morphology,

histology, regeneration, reproduction and feeding, hence could be an ideal modal for chromium

toxicity testing.

8. Conclusion and future thrust

 Since the present study has proved that H. vulgaris could be used as an alternative animal

model for metal toxicity testing, this work could be expanded for testing other compounds

such as pesticides, etc.

 The present work has created a Hydra culture facility at PG and Research Department of

Biotechnology and Microbiology, National College and opens up an opportunity to explore

many hydra associated works such as microbiome, bio-prospecting, etc.

Artemia culture

H. vulgaris being cultured

 Hydra cultures produced in the presents study and being maintained at Department of

Biotechnology and Microbiology, National College are available to other users for academic

and research purposes. The Hydra culture can be requested here

https://www.ncccc.in/index.php/hydra-vulgaris/

 In near future, more Hydra species from natural environment should be obtained and

acclimatized to laboratory conditions to explore the feasibility of using other Hydrazoans as

model organisms.

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Hydra -A Powerful Lab Model for Life Science Teaching and Research

Article

Full-text available

Dec 2023

Chinnamani PrasannaKumar

Hydra, a unique freshwater diploblast, not only boasts a well-defined body plan and an intricate nervous system but also stands out for its possession of stem cells. Revered as one of the oldest model organisms in biological research, Hydra defies aging and retains embryonic traits even in adulthood. Its remarkable regenerative prowess adds to its mystique, making it an invaluable resource for unraveling the evolution of complex animal forms. This article sheds light on Hydra's extraordinary features, emphasizing its potential significance in both scientific exploration and life science education.

Hydra as a research model

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Jan 2024

Effects of heavy metals on antioxidants and stress-responsive gene expression in Javanese medaka (Oryzias javanicus)

Article

Full-text available

Apr 2009
Comp Biochem Physiol C Comp Pharmacol

The differential expression of eight genes encoding stressor biomarkers was investigated by real-time quantitative PCR in liver tissue extracted from Javanese medaka after exposure to six heavy metals for 24 h. OjaCAT transcription increased in a dose-dependent manner during exposure to Cd, Cu, and Zn, but significantly decreased after exposure to Ag, Cr, and Ni. OjaCYP1A transcription decreased drastically on exposure to all heavy metals tested. OjaG6PD transcription increased dramatically after exposure to low doses of Cu and Zn, but decreased at high concentrations of these elements. No prominent changes in OjaG6PD transcription were observed after exposure to Ag, Cd, Cr, or Ni. OjaGPx mRNA expression was induced in the liver following exposure to Ag, Cd, Cu, and Zn, but suppressed following exposure to Cr and Ni. Exposure to all heavy metals increased transcription of OjaGR and OjaGST in a dose-dependent manner. OjaSOD transcription increased during exposure to Ag, Cd, Zn, and Cr, but showed no change in response to Cu and Ni exposure. OjaUB expression was induced by all doses of exposure. The transcriptional responses of these genes to heavy metal exposure will provide the basis for a multi-biomarker system that can be used for the biomonitoring of aquatic environments.

Alternative model organisms for toxicity testing and risk assessment

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Full-text available

Jan 2014

Developmental Effects of the ToxCast™ Phase I and II Chemicals in Caenorhabditis elegans and Corresponding Responses in Zebrafish, Rats, and Rabbits

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Oct 2015
ENVIRON HEALTH PERSP

Background: Modern toxicology is shifting from an observational to a more mechanistic science. As part of this shift, high-throughput toxicity assays are being developed using alternative, non-mammalian species to prioritize chemicals and develop prediction models of human toxicity. Methods: The nematode Caenorhabditis elegans (C. elegans) was used to screen the EPA's ToxCast™ Phase I and II libraries, containing 292 and 676 chemicals respectively, for chemicals leading to decreased larval development and growth. Chemical toxicity was evaluated using three parameters: a biologically defined effect size threshold, half-maximal activity concentration (AC50), and lowest effective concentration (LEC). Results: Across both the Phase I and II libraries, 62% of the chemicals were classified as active up to 200 µM in the C. elegans assay. Chemical activities and potencies in C. elegans were compared to those from two zebrafish embryonic development toxicity studies and developmental toxicity data for rats and rabbits. Concordance of chemical activity was higher between C. elegans and one zebrafish assay across Phase I chemicals (79%) than with a second zebrafish assay (59%). Using C. elegans or zebrafish to predict rat or rabbit developmental toxicity resulted in balanced accuracies, the average value of the sensitivity and specificity for an assay, ranging from 45 - 53%, slightly lower than the concordance between rat and rabbit (58%). Conclusions: Here we present an assay that quantitatively and reliably describes the effects of chemical toxicants on C. elegans growth and development. We found significant overlap in the activity of chemicals in the ToxCast™ libraries between C. elegans and zebrafish developmental screens. Incorporating C. elegans toxicological assays as part of a battery of in vitro and in vivo assays provides additional information for the development of models predicting a chemical's potential toxicity to humans.

Evaluation of the developmental toxicity of citrinin using Hydra attenuata and postimplantation rat whole embryo culture

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Jan 1990

Fluorescent nanocrystals reveal regulated portals of entry into and between the cells of Hydra

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Jan 2009
PLOS ONE

Multi-scale biomarker evaluation of the toxicity of a commercial azo dye (Disperse Red 1) in an animal model, the freshwater cnidarian Hydra attenuata

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Mar 2016

Acute (24 h, 48 h, 72 h) and chronic (7 days) tests have been performed to evaluate the effects of the commercial azo dye Disperse Red 1 (DR1) using various biomarkers in the freshwater invertebrate Hydra attenuata. Morphological changes have been selected to calculate ecotoxicological thresholds for sublethal and lethal DR1 concentrations. A multinomial logistic model showed that the probability of each morphological stage occurrence was function of concentration, time and interaction between both. Results of oxidative balance parameter measurements (72 h and 7 days) suggest that polyps set up defense mechanisms to limit lipid peroxidation caused by DR1. DR1 exposure at hormetic concentrations induces increase of asexual reproductive rates. This result suggests (1) an impact on the fitness-related phenotypical traits and (2) trade-offs between reproduction and maintenance to allow the population to survive harsher conditions. Changes in serotonin immuno-labeling in polyps showing alterations in feeding behavior suggest that chronic DR1 exposure impaired neuronal processes related to ingesting behavior in H. attenuata. This ecotoxicity study sheds light on the possible serotonin function in Hydra model and reports for the first time that serotonin could play a significant role in feeding behavior. This study used a multi-scale biomarker approach investigating biochemical, morphological, reproductive and behavioral endpoints in Hydra attenuata. This organism is proposed for a pertinent animal model to assess ecotoxicological impact of pollutant mixtures in freshwater environment.

ROS dependent copper toxicity in Hydra-biochemical and molecular study

Article

Mar 2016
COMP BIOCHEM PHYS C

Copper, an essential microelement, is known to be toxic to aquatic life at concentrations higher than that could be tolerated. Copper-induced oxidative stress has been documented in vitro, yet the in vivo effects of metal-induced oxidative stress have not been extensively studied in the lower invertebrates. The objective of the present study has been to find the effect of ROS-mediated toxicity of environmentally relevant concentrations of copper at organismal and cellular levels in Hydra magnipapillata. Exposure to copper at sublethal concentrations (0.06 and 0.1mg/L) for 24 or 48h resulted in generation of significant levels of intracellular reactive oxygen species (ROS). We infer that the free radicals here originate predominantly at the lysosomes but partly at the mitochondria also as visualized by H2-DHCFDA staining. Quantitative real-time PCR of RNA extracted from copper-exposed polyps revealed dose-dependent up-regulation of all antioxidant response genes (CAT, SOD, GPx, GST, GR, G6PD). Concurrent increase of Hsp70 and FoxO genes suggests the ability of polyps to respond to stress, which at 48h was not the same as at 24h. Interestingly, the transcript levels of all genes were down-regulated at 48h as compared to 24h incubation period. Comet assay indicated copper as a powerful genotoxicant, and the DNA damage was dose- as well as duration-dependent. Western blotting of proteins (Bax, Bcl-2 and caspase-3) confirmed ROS-mediated mitochondrial cell death in copper-exposed animals. These changes correlated well with changes in morphology, regeneration and aspects of reproduction. Taken together, the results indicate increased production of intracellular ROS in Hydra on copper exposure.

Some effects of metal ions on the freshwater organisms, Hydra oligactis and Chlorohydra viridissima

Article

Jan 1986

Identifying Hydra Species

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Jan 1983

Richard D. Campbell

Hydra species differ in morphology, development, physiology, and ecology. Repeatability of experiments therefore will depend upon correct identification. I outline a procedure for distinguishing between the types of hydra used most frequently in research. Three major criteria are used: (1) presence or absence of symbiotic algae, (2) arrangement of tentacles on buds, and (3) shape of the holotrichous isorhiza nematocyst.

Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment (ed. I. Raskin)

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B.D. Ensley

Toxicological assessment of chromium using Hydra vulgaris as model organism

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