26

3. MATERIALS AND METHODS

3.1. Experimental Species

Labeo rohita, commonly known as rohu is an Indian major carp. Rohu

naturally occurs in the river systems. It is an indigenous fish, native to India. The

colour of the body is bluish or greyish on the dorsal side. Rohu is a herbivourous

column feeder, eating mainly on phytoplankton and submerged vegetation. It is a

highly cultivable species, and contributes in the provision of animal protein in

India. It readily adapts to laboratory conditions, and it is easily available,

inexpensive and suitable for field studies. It can be cultured in polyculture,

monoculture and integrated fish culture.

3.2. Reason for selecting fish

Selection of a suitable organism will depend upon a number of factors

(APHA, 1976). Labeo rohita was selected for the present investigation based on

the following reasons.

1. It is an economically and commercially important fish.

2. It is readily available in good numbers throughout the year.

3. The fish spawn spontaneously throughout the year.

4. It is sensitive to environmental factors under consideration.

5. It is easily adaptable to lab condition.

6. It does not need much effort for maintenance.

7. It has resistance to disease and other causes which weaken the fish

(Transport and wintering).

8. It is an ideal animal for toxicity studies in aquatic biology.

27

3.3. Systematic Position

Kingdom : Animalia

Phylum : Chordata

Class : Pisces

Order : Cypriniformes

Family : Cyprinidae

Genus : Labeo

Species : rohita

Fig.1. The freshwater fish Labeo rohita

28

3.4. Selection of Herbicides

3.4.1. Atrazine

Atrazine is a chlorotriazine herbicide, an organic compound consisting of

an s-triazine-ring

IUPAC name

1-Chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine

Properties

Molecular formula : C8H14ClN5

Molar mass : 215.68 g mol−1

Appearance : colorless solid

Density : 1.187 gcm−3

Melting point : 175 °C, 448 K, 347 °F

Boiling point : 200 °C, 473 K, 392 °F

Solubility in water : 7 mg/100 mL

29

3.4.2. Glyphosate

Glyphosate is an organophosphorus herbicide, n-(phosphonomethyl)glycine.

IUPAC name

N-(phosphonomethyl)glycine)

Properties

Molecular formula : C3H8NO5P

Molar mass : 169.07 g mol−1

Appearance : white crystalline powder

Density : 1.704 (20 °C)

Melting point : 184.5 °C

Boiling point : decomp. at 187 °C

Solubility in water : 1.01 g/100 mL (20 °C)

log P : −2.8

Acidity (pKa) : <2, 2.6, 5.6, 10.6

30

3.5. Fish Acclimatization

The freshwater healthy fish, Labeo rohita of the weight (10 ± 1g) and

length (8 0.5 cm) were selected for the experiment and were collected from

Katherasan Aquafarm near Thanjavur. Fish were screened for any pathogenic

infections. Glass aquaria were washed with 1% KMnO4 to avoid fungal

contamination and then sun dried. Healthy fishes were then transferred to glass

aquaria (35 20 20 cm) containing dechlorinated tap water (Temperature –28

2°C; total hardness – 518 23mg/l; DO - 5.6 0.2 mg/l; salinity - 1.2 0.13 ppt

and pH - 7.8 0.04). Fish were acclimated to laboratory conditions for 10 to 15

days perior to experimentation. They were regularly fed with commercial food ad

libitum and the medium (tap water) was changed daily to remove faeces and food

remnants.

3.6. Experimental Design

3.6.1. Acute toxicity test

Toxicity tests were conducted in accordance with standard methods

(APHA, 1992). Stock solution of atrazine with a concentration of 1 ml per litre

(equivalent to 1 ppt) was prepared in distilled water and different dilutions were

prepared by adding required amount of distilled water. The stock solution of

glyphosate with a concentration of 1 ml per litre was also prepared in distilled

water and the desired degree of concentrations was prepared. Based on the

progressive bisection of intervals on a logarithmic scale, log concentrations were

fixed after conducting the range finding test. The fish were starved for 24 hours

prior to their use in the experiments as recommended by storage to avoid any

31

interference in the toxicity of herbicides by excretory products. After the addition

of the toxicant into the test tank with 10 litres of water having twenty fish,

mortality was recorded after 24, 48, 72 and 96 hours. Five replicates were

maintained simultaneously.

Percent mortality was calculated and the values were transferred into probit

scale. Probit analysis was carried out as suggested by Finney (1971). Regression

lines of probit against logarithmic transformations of concentrations were made.

Confidential limits (upper and lower) of the regression line with chi-square test

were calculated by a computerized programme for Finney‟s (1971) probit analysis.

3.6.2. Chronic toxicity test

Based on acute toxicity test (96h LC50 ) sublethal concentrations (10% and

30%) were derived for atrazine and glyphosate which were used as the

experimental concentration of the herbicides in the subsequent experiments. Ten

fish were exposed to each concentration for a period of 10, 20 and 30 days. A

control batch was maintained simultaneously and six trails were run.

Fingerlings Labeo rohita was used in this present investigation. The

fingerlings were maintained in the aquaria at room temperature 28 2°C with the

photoperiod LD 12:12. Medium sized, (10 g) healthy and active individuals were

selected from the stock aquarium and used for respiratory studies. Labeo rohita

were exposed to 10 and 30% sublethal concentrations for 96 hour LC50 of atrazine

and glyphosate in water medium. Control was maintained in water medium

without herbicide. At the end of 10, 20 and 30 days, oxygen consumption was

measured in both control and treated fish.

32

3.7. Estimation of Oxygen Consumption

A series of rectangular glass jars, each with one litre capacity were used as

aquarium. They were filled with water. Care was taken to avoid trapping of air

bubbles. Only one fish was introduced into each aquarium and a thick layer of

coconut oil was spread on the surface of the medium to prevent the contact of the

medium to the atmosphere and to prevent the fish from reaching the atmospheric

air.

Before starting the experiment, the initial oxygen content of water used for

the preparation of animal chambers was estimated by collecting a sample into a

narrow mouth, glass stoppered sample bottle of known volume following the

Winkler‟s method (Annon, 1984). A healthy fish was allowed to respire for one

hour in animal chambers. After one hour, samples from respiratory chamber were

taken into the sample bottle of known volume through siphon system and the

dissolved oxygen was estimated.

3.7.1. Determination of oxygen content of the sample

The initial oxygen content of water was determined by collecting

the sample in a narrow mouthed glass stoppered sample bottle of known volume.

To this 1 ml of manganous sulphate solution was added followed by addition of 1

ml of alkaline iodide solution. The bottle was stoppered and shaken vigorously and

kept in a dark place to prevent any photochemical reaction for about 15 minutes. A

few drops of conc. sulphuric acid were added into the sample bottle in order to

dissolve the precipitate. The precipitate was completely dissolved by shaking

33

vigorously. Twenty five ml of the sample was taken in a clean conical flask and the

liberated iodine was titrated against sodium thiosulphate using four to five drops of

starch as indicator. The disappearance of blue colour was taken as end point. The

burette values were tabulated. The final oxygen content of the respiratory chamber

was also determined in the same manner. Oxygen consumed by the fish was

calculated by finding out the difference between the initial and final oxygen

content in the animal chambers. Also the rate of oxygen consumption per gram

weight of the fish per hour was calculated and the values were expressed as ml

O2/gm/hour.

The dissolved oxygen content in the water was calculated using the

following formula:

O2 content ml/litre = K 200 volume of Na2S2O3 consumed 0.698

Volume of the sample titrated

Where K = Volume of sample bottle

Vol. of sample bottle – Vol. of reagents added

(0.698 is the conversion factor to convert parts per million to ml/litre)

(200 is the constant which is obtained by multiplying the equivalent weight

of oxygen and normality of Na2S2O3 and 100 ml).

3.8. Haematological studies

Fish was collected and gently wiped with a dry cloth to remove water.

Caudal peduncle was cut with a sharp blade and the blood was collected in a watch

glass containing EDTA, an anticoagulant (6% Ethylenediaminetetra acetic acid).

34

The blood was mixed well with the EDTA solution by using a needle and this

sample was used for determining the Red Blood Corpuscle Count (RBC), Total

Leucocyte Count (TLC) and Haemoglobin content (Hb), Packed Cell Volume

(PCV) or Haematocrit (Ht) and red cell indices.

3.8.1. RBC Count

For RBC count, a method devised by Yokayama (1974) and later modified

by Christensen et al. (1978) was followed. The standard RBC diluting pipette and a

1:200 dilution was used for the RBC count. Yokayama‟s fluid was used for

dilution. The diluting fluid consisted of two parts and it was prepared by mixing

800 mg of NaCl, 40 mg of KCl of 40% formalin, 250 mg of dextrose, 50 mg of

NaHCO3 and 40 ml of distilled water with an equal part of Wright‟ s stain.

Blood was drawn in to the pipette up to the 0.5 mark. The tip of the pipette

was wiped with a filter paper to adjust the volume exactly to 0.5 marks. The pipette

was immediately filled to the 101 mark with the diluting fluid. Partial rotation of

the pipette while it was being filled assured the complete mixing of the blood and

diluting fluid and prevented clotting. With it ends grasped between the thumb and

second finger the pipette was then shaken for 5 minutes, which ensured the

thorough mixing of the blood with diluting fluid.

The improved Neubauer counting chamber was used for counting the cells.

A glass coverslip was placed over the „H‟ groove on the chamber. Then the blood

was allowed to enter the chamber by simply touching to the edge between the

35

coverslip and the chamber where the blood spread under the coverslip by capillary

action. The counting areas divided into 25 squares were taken to count RBC

marked as E1-E8. Two minutes time was given for the corpuscles to settle down.

For counting the total number of RBC, the total number of cells in the five squares

was multiplied by 106

which give the total number of RBC per mm3

of blood.

3.8.2. Total Leucocyte Count (TLC)

For counting the total number of WBC, the pipette with white bead

was used. The number of cells present in the four large corner squares marked by

capital letter ‘L’ was counted and multiplied by 103

which give the total number of

WBC per cubic millimeter of blood.

3.8.3. Haemoglobin Content (Hb)

Haemoglobin determination is the quickest means for detecting anaemia.

However, many factors are known to influence the haemoglobin level. The Sahli

Hellige method was followed for haemoglobin determination. Sahlis pipette was

filled slightly above the 20 mm mark, the pipette was wiped with a filter paper or

cotton to remove excess blood and the volume was adjusted to exactly 20 mm3

by

blotting the tip. The blood was expelled into a calibrated (transmission) test tube

containing 2 ml of 0.1 N HCl. The pipette was rinsed several times in the acid

solution. The sample was allowed to stand for 15 minutes.

The principle behind the method is the conversion of haemoglobin to acid

haematin. The acid haematin was then diluted with distilled water till colour

36

matched with the colour of the standard in the haemoglobinometer. The height of

the column at which the colour match obtained gives the value of haemoglobin in

g%. Oxygen carrying of blood was calculated by multiplying the haemoglobin

content with 1.25, oxygen combining power of Hb /g (Johansen, 1970).

3.8.4. Preparation of Blood Smear

Blood samples were taken from fish by puncture of the caudal vessel. After

blood removal, blood smear were made immediately.Smears made from blood

samples were air dried for 1 hr and then fixed in 95% methonal at 4C (Michael and

Stoskope,1992). Slides were stained with with Leishman‟s stain and a cover slip

was placed on top using glycerol. The blood corpuscles were examined by phase

contrast microscopy and photographed.

3.9. Biochemical studies

3.9.1. Estimation of total Carbohydrates

The total carbohydrate content was estimated by the technique of Roe

(1955). A 10% homogenate of tissues or 0.1 ml of blood was prepared using 5%

TCA and this was centrifuged at 1000 x g for 10 minutes. 10 ml of anthrone

reagent was added to the supernatant and this was boiled for 10-15 minutes. The

contents were cooled in the dark ambient room temperature for 30 minutes. The

absorbance OD was read at 620 nm and total carbohydrate content is expressed as

mg/g of tissue.

37

3.9.2. Estimation for Protein

Protein was estimated by the method of Lowry et al. (1951).

1% tissue homogenates were prepared in 10% TCA and centrifuged at 1000 g

time for 15 minutes. The sediment was dissolved in 1 ml of 1N NaOH. To the

above, 5 ml of alkaline copper reagent was added. After 10 minutes, 0.5 ml of

Folin phenol reagent was added and rapidly mixed. The OD was measured after 30

minutes at 640 nm. Known concentrations of bovine serum albumen were

processed in the same manner to prepare a standard graph. Results were expressed

in mg/gm wet weight of tissue and for blood as gm/100 ml of blood.

3.9.3. Estimation of Total Lipids

Total lipids were extracted following a modified version of the method of

Folch et al. (1957). About 200 mg of tissue of 0.2 ml of blood was taken and to

this chloroform: methanol mixture (3:1 V/V) containing 0.01 percent of butylated

hydroxytoluene (BHT) as an antioxidant was added. The tissue suspension was

thoroughly homogenized and centrifuged at 5000 rpm. This process was repeated

twice and the supernatant was pooled, and this crude lipid residue was

concentrated at 40-45 C in a water bath.

3.9.3.1. Removal of Contaminants

For removing non-lipid contaminants, 20 ml of chloroform: methanol (2:1

V/V) mixture containing 4% water was added to the crude residue. The sample

was evaporated at 40 C in a water bath. This process was repeated thrice and the

residue was dissolved in 100 ml chloroform: methanol (2:1 V/V) mixture

containing 0.01 percent BHT and taken up in a separating funnel. 20 ml of normal

38

saline was then added. The funnel was swirled gently for a few minutes and

allowed to stand for 4-6 hours. The lower phase containing the lipids was collected

and again evaporated at 40 C. The residue thus obtained was dissolved in 2 ml

chloroform and stored at 4 C in a glass stoppered tube. The extract was used for

the estimation of total lipid, and frees fatty acids.

Gravimetric method was used to quantify total lipid. A volume of 0.5 ml of

the extract was added to pre-weighed planchet, evaporated at 60 C and further

dried in a desiccator. Planchet containing dried lipids was weighed daily until

constant weights were obtained. The total lipids were expressed in mg/gm wet

weight of tissue.

The experimental conditions were the same as described in the previous

chapter. Labeo rohita were exposed to each of the two sublethal concentrations (10

and 30%) of each pesticide, individually for a period of 30 days. For enzyme

studies, liver, intestine, gill, kidney and muscle tissues were sampled.

3.10. Enzyme studies

3.10.1. Estimation of Phosphatases

Acid and alkaline phosphatases were estimated following the procedure

outlined by Tennis Wood et al. (1976), a modified method of Bessey et al. (1946).

The tissues were homogenized in 0.25 M sucrose solution and centrifuged at

1000 g for 10 minutes. The supernatants were filtered and the filtrates were used

for enzyme analysis.

39

3.10.2. Acid Phosphatase

For estimation of phosphatases, clean glass tubes were taken and 0.5 ml of

4% p-nitro-phenol phosphate and 0.5 ml of 0.1 M citrate buffer were added into

each. The tubes were placed in a water bath at 37 C for 5 minutes. The reaction

was then initiated by adding suitable aliquot of tissue extract. Exactly after 30

minutes of incubation, the reaction was arrested by adding 3.8 ml of 0.1 N NaOH.

The reaction product, P-nitrophenol was measured spectrophotometrically at 415

nm against a reagent blank. The enzyme activity was expressed as mole of P-

nitrophenol formed/mg protein/hour.

3.10.3. Alkaline Phosphatase

In each test tube 0.5 ml of 4% substrate solution in equal volume of

alkaline buffer solution was taken. The tubes were incubated at 37 C in a water

bath for 5 minutes. The reaction was initiated by adding suitable aliquot of tissue

extract. The tubes were incubated for exactly 30 minutes after which the reaction

was stopped by adding 10 ml of 0.02 N NaOH. The colour developed was read at

410 nm against a reagent blank. Then 0.1 ml of concentrated HC1 was added to

each tube and mixed well. The OD was again measured against a reagent blank at

400 nm. The second reading was subtracted from the first reading where the P-

nitrophenol produced colour. The corrected OD was then read from the standard

curve. The enzyme activity was expressed as mole of P-nitrophenol/mg

protein/hour.

40

3.11. Histological methods

3.11.1. Light microscopic studies

Generally, most of the histological studies are made on the dead cells or

tissues. For microscopical studies, cells or tissues have to pass through the process

such as fixation, dehydration, embedding, sectioning and staining (Gurr, 1959).

Fixation

The term fixation means to immobilize. The fixing solution performs the

following functions. It prevents bacterial decay and autolysis of the cells, renders

the components of the cell stable, reduces the visibility of different cellular

components and prepares the cells for staining.

In the present study, on 10, 20 and 30th

day, fish were taken out, sacrificed

and the tissues of gill, liver, intestine and kidney were excised out. After cutting

them into small pieces they were transferred immediately to the fixative.

Chemical fixation

The choice of suitable fixative generally depends on the type of analysis

and the chemical nature of different cells. In the present study alcoholic bouin‟s

solution was used as fixative. The period of fixation was 1-3 days.

Picric acid – 1gm

5% Formaldehyde -60 ml

Glacial Acetic acid – 15 ml

80% Alcohol – 150 ml

41

The tissues were fixed in freshly prepared fixative and after the period of

fixation, they were transferred directly to 70% alcohol.

Dehydration

After the fixation, water molecules from the cells or tissues were removed.

These processes are known as dehydration.

In the present study progressive higher grades of alcohol prepared from

rectified sprit were used to dehydrate the tissues. The grades used for dehydration

were30%, 50%, 70%, 90% and 100% (absolute alcohol).

In the stepwise dehydration, the tissues were kept along with the

progressive alcohol grades for the following duration:30% and 50% grades for 5 to

10 hours: in 70% grade for 10 to 12 hours: 90% grade for 10 to 12 hours: in 90%

grade for about 15 hours and in 100% absolute alcohol grade for 6 to 8 hours.

Clearing and Fixation

Xylene was used as the clearing agent, because the tissues contain alcohol

so it has to be removed from the tissue to make it firm for the purpose of section

cutting. The tissues were kept in xylene for about 2 to 4 hours. Xylene was not

only bring about the infiltration of paraffin into tissues but also make them

transparent by removing their opacity. Hot and cold infiltration was done in under

to ensure that all the xylene/ air space is replaced by paraffin wax.

42

Embedding

In the process of embedding the material was embedded in certain

supporting media of sufficient hardness for cutting thin sections on the microtome.

Paraffin wax was used in the process of embedding. The embedding process

involves soaking of the tissue in molten wax at a standard temperature, coinciding

with the melting point of the embedding medium used.

Block making

After embedding the tissue with wax, it was cast into a block of paraffin.

This process is known as block making or casting of block. The mould of „L‟

pieces was adjusted in such a way to accommodate the object. The mould was

filled with molten paraffin wax. The impregnated tissue was placed in the mould

according to the plane of section needed. Immediately warm tip of the needle was

moved in the molten wax on all the slides of the tissue. This was done to remove

the air bubbles. The label carrying all the details of the tissue was fixed on one

side of the mould. Gently the air was blown on the mould; the wax forms a thin

layer on the surface. The mould was gently immersed in cold water so as to cool

the wax rapidly. When the block become solid it was removed from water.

Trimming the block

The prepared block was trimmed into correct shape for section cutting.

Mounting the block

The trimmed block was attached to a holder and inserted into the jaws of

microtome.

43

Section cutting

The attached trimmed block was cut into thin sections of desired thickness

of the microtome.

Mounting and spreading the ribbon

For mounting the material, glass slides were used. The slides were smeared

with adhesive Mayer‟s albumen because the section should remain fixed to the

slide while staining subsequently. The a small piece of ribbon was placed on a

slide and floods it with water by dropper. Then the slide was placed on hot plate to

heat the water; thereby the paraffin ribbons begin to stretch. Soon after the ribbon

was completely stretched the slides were removed from the hot plate and the water

was drained off and allowed to dry.

Labeling

By using a diamond pencil, the slides were labeled after the slide was

completely dried.

Staining Process

The slide was kept in xylene for 30 minutes to 1 hour to deparaffinize.

Then the slides were passed through the down grade series of

100%,90%,80%70%,50% and 30% ethyl alcohol and water. First, the slides were

stained in haematoxylin for 2 to 5 minutes and then they were washed in water.

After that the slides were dehydrated by passing through ascending order alcohol

series up to 70%alcohol. Secondly, the slides were stained with eosin for 2 quick

44

dips and then washed in a fresh grade 70% alcohol. Dehydration was done through

ascending grades of 80% , 90% ethyl alcohol and absolute alcohol I and II for 5 to

10 minutes. The slides were allowed in absolute alcohol for complete dehydration.

Then the slides were transferred into xylene I for clearing. One or two changes of

xylene were given and then finnaly mounted in DPX mountant.

3.11.2. Scanning Electron Microscopic Study

The gill arches were dissected out, washed repeatedly in 0.2M phosphate

buffer and then fixed in 3% gluteraldehyde. The dehydration was done in acetone

grades and was followed by critical point drying. Ultimately dried gills were

mounted on the stub and were sputter coated with gold in a gold coating unit

(thickness 100Ao) and were examined and photographed using JEOL JSM 6360

scanning electron microscope (SEM) Japan.

3.11.3. Transmission electron microscopic study

Processing of tissue for electron microscopy

Collection

Obtained good experimental results is the isolation of the tissue in as close

as possible to in vivo condition prior to placing it into the fixation medium.

Fixation

Fix in 3% glutaraldehyde and washed in buffer. Post fix by 1%

osmiumtetroxide and washed in buffer. This doubles fixation gives stability during

dehydration, embedding and during electron bombardment. Further, it also

45

provides staining contrast, decreased distortion, fix fine cellular ultrastructure,

suitable for animal material (for cell line after pre and post fixation blocked with

2% agrose then proceed as usual).

Dehydration

Dehydrated by ascending series graded alcohol (50% to 100%) and clearing

by propylene oxide.

Infiltration

Infiltrated by propylene oxide and epoxy resin.

Embedding

Embedded in siligonised rubber mould with exposed resin.

Polymerisation

Embedded mould kept in incubator at 60 c for 48 hours, cool down. Blocks

ready for sectioning.

Cutting

One micron thick sections cut through ultra microtome (Leica ultract UCT)

with glass knife and stained by toluidine blue. Exam is extremely useful when

sections are needed to give general idea of the orientation of the tissue and for

marking the areas of (able to cut ultrathin sections) interest in the blocks face prior

46

to further trimming of the blocks for ultra microtomy.Also, allow the quality of

fixation and embedding.

Ultrathin section (below 100 nm) cut through ultra microtome (Leica) with

diamond knife (Diatome). Ultrathin section are taken on copper grid and stained

(Double metallic) uranyl acetate and Reynold‟s solution (sodium citrate + Lead

mitrate) which gives contrast.

Transmission Electron Microscope

Sections transmitted in EM (Philips 201C by Nether land and

photographed.