II. MATERIALS AND METHODS

- - MATERIALS AND METHODS

The test organisms selected for the present investigation include five

fresh water microalgae viz. (;hlorella ellipsoidea Gerneck, C:hlorococum humicola

(Naeg.) Rabenhorst, Scenedesmus bijuga (Turp.) Lagerheim, Ankistrodesmus

falcatus (Corda.) Ralfs and Haematococcus laccustris (Girod.) Rostafinski- all

belonging to the class Chlorophyceae.

2.1 Isolation of pure cultures of test organisms

The pure cultures of the test organisms were isolated from water samples

collected from different freshwater ponds in and around Kotlayam. Small quantities

of water samples were kept in sterilized petridishes enriched with a pinch of KNO,.

The samples were exposed to fluorescent light for incubation without any

disturbance. The isolation methods were based on bacteriological techniques (Stein,

1973; Gopinathan, 1982; APHA, 1980). The algal colonies were examined under

light microscope and identified with the help of morphological descriptions given by

Fritsch (1935) and Prescott (1982). The pure cultures of algae obtained were used

to establish stock cultures.

Systematic positions of the isolated algae (Fritsch, 1935) are as follows:

Chlorella ellipsoidea Gerneck

Division - Chlorophyta

Class - Chlorophyceae

Order - Chiorococcales

Family - Chlorellaceae

Chlorococcum humicola (Naeg.) Rabenhorst

Division - Chlorophyta

Class - Chlorophyceae

Order - Chlorococcales

Family - Chlorococcaceae

Scenedesmus bijuga (Turp.) Lagerheirn

Divls~on - Chlorophyta

Class - Chlorophyceae

Order - C.hlorococcales

Family - Coelastraceae

Ankistrodesmus falcatus (Corda.) Ralfs

Dlvlsion - C;hlorophyta

Class - C:hlorophyceae

: 19 ::

Order - ChYorococcales

Family - Selenastraceae.

Haematococcus laccustris (Girod.) Rostafinski

Division - Chlorophyta

Class - Chlorophyceae

Order - Volvocales

Family - Selenastraceae

2.2 Culture conditions

Different culture media like Miquel's medium (Miquel, 1980), Walne's

medium (Walne, 1974) and Ward and Parish medium (Ward and Parish, 1982)

were tried for culturing the test algae. Of these, Ward and Parish medium was

found to be most suitable for the algal growth and hence both mother cultures and

experimental cultures were ra~sed in this medium.

The composition of Ward and Parish medium is as follows:

Macronutrients

1. NaNO 3 - 25.5 glL

2. MgCI,. 6H,O - 12.2 g/L

3. Mg S0,.7H20 - 14.7 glL

4. Ca CI2.2H,O - 4.41 g/L

5. NaHCO, - 15.OglL

6. K,H PO, - 1.044 g/L

Micronutrients

1. COCI, - 0.78glL

2. CuCI, - 0.9 gI100 ml. Dilute 1 ml of this solution to one

litre for making stock solution.

3. a. H,BO, - 0.1855g

b. MnCI, - 0.26439

c. ZnCI, - 0.327g

d. Na, MOO,. H 0 2

- 0.00739

e. FeCI, - 0.0960g

f . Na,EDTA - 0.300 g

g. Micronulrient solution 1 and 2 - 1 ml each

h. Distilled water - 1 litre

The maintenance and test medium were prepared by adding one millilitre

of each macronutrient solution and one millilitre of micronutrient stock solution to

one litre double distilled deionised water. Both the stock cultures and experimental

cultures were marntained in "Borosil" conical flasks plugged with sterilized non-

absorbant cotton (plate2la). The stock cultures were recultured fortnightly. All the

cultures were illuminated with daylight fluorescent tubes with an intensity of 2000

lux. The ambient temperature ranged from 28 OC to 32 OC. The duration of light and

dark period was 10:14 hours. The cultures were shaken manually three to four

times a day to provide a uniform suspension.

2.3 Effluents

The effluents used for the exper~ments were collected from the following

factories.

21

1. Hindustan Newsprint Limited, Velloor, Kottayam.

2. Hindustan Organic Chemicals Limited, Irumpanam, Ernakulam and

3. Mc Dowells Distilleries, Varanad, Cherthala.

The selected algae were treated with different concentrations of the

effluents (0.05%, 0.1%, 0.2!i0/0, 0.5%, 0.75%, I .0% and 1.5%).

2.4 Algal bioassay procedures

Different species of algae were exposed to different concentrations of

effluents to study their effect on each alga. The bioassays were always of the static

type (Reish and Oshida, 1986).

Initially a range-finding test was conducted to determine the

concentrations of the test materials to be used in the definitive tests (Ward and

Parish, 1982 and Reish and Oshida, 1986). The concentrations selected for the

range - finding tests were 0.1%, I%, 10% and 100% of effluents and a control with

out the effluents. Each flask was inoculated with equal quantity of algal cells and

incubated for 96 hours. The growth in each test flask was compared with that

occurred in the control flask. Definitive tests were conducted using concentrations

selected from the results of the range - finding tests (Ward and Parish, 1982; APHA, 1980).

Clean, sterilized '1000 ml "Borosil" conical flasks were filled with 400ml

culture medium. Required volume of effluent was added to the medium with the

help of micropipettes. A known number of microalgal cells was inoculated from a

stock culture kept in exponential growth phase. The flasks were shaken thoroughly

and incubated. All the experimental concentrations and control were maintained in

triplicate. All the cultures were hand shaken to keep the cells in suspension. Duration

of the experiment was 21 days. Cell counts were made on alternate days and other

parameters were measured at 4 -day intervals. Experimental set up is as shown in

Plate 2.1 b.

2.5 Growth measurements

2.5.1 Cell number

To determine the growth in terms of cell number, 1 ml of sample was

taken from each conical flask. The cells were fixed in Lugol's iodine solution and

counted with a calibrated haemocytometer. Four counts were made from each

sample and the mean value was taken

2.5.2 Productivity

Primary production or productivity was estimated using light anddark

bottle method (Gaarder and Gran, 1927). Three samples - initial, light and dark

were taken from each flask and were analysed for the dissolved oxygen content

using Winkler method. The oxygen values were converted into their carbon

equivalents, using the following formula :

\JLB - VDB x 0.536 GPP (mg C IL ih) =

NPP (mg CIL Ih) = - PQ x N

VLB. VDB & VIB I= quantity of Na2S20, titrate values obtained from

titration of light, dark and initial bottles respectively,

N = incubation period

PQ = Photosynthetic quotient (1.25)

0.536 = Factor to convert mg of oxygen to mg of carbon

2.5.3 Pigment analysis

Determination of quantitative variation of algal pigments was done by

spectrophotometric analysis (Parsons eta/. , 1984).

10ml samples from the control and othertest concentrations were taken.

To the algal pellet 90% acetone was added and kept in dark in a refrigerator for 20

hours for the completion of p~gment extraction. Prior to spectrophotometric analysis,

all extracts were centrifuged for 10-15 minutes at 4000 rpm.

The absorbance of the clear pigment extract was measured against blank

at different wavelengths such as 480, 510, 630, 647, 664 and 750 nm. The

concentrations of various pigments were then calculated using the equation given

by Parsons et a/. (1 984).

2.5.4 Estimation of biochemical compounds

2.5.4.a Protein

The protein was estimated by heated biuret folin method (Dorsey et al.,

1978). This method consistently measures 90% of the total nitrogen of filtered algal

samples as protein - nitrogen without the need of mechanical disruption, as long as

the heating period in biuret is 100 minutes at 100°C.

The algal cells were concentrated by centrifugation. 'The volumes of the

reagents added were as per VVinkfors eta/. , (1984). The protein was extracted with

24 : :

appropriate reagents at 100°C in a water bath for 100 minutes. The intensity of

colour was measured at 660 nm. Protein - nitrogen values were reckoned by

interpolations from a standard graph, prepared with different concentrations of

Bovine Serum Albumin. The protein was then calculated by using a conversion

factor 6.58 generally accepted for most algal species (Dorsey eta/. 1978).

2.5.4.b. Carbohydrate

Carbohydrate determination was made using phenol-sulphuric acid

method reported by Kochert (1978) based on the procedure developed by Dubois

et a/. (1956).

10 ml of the sample was centrifuged and the algal pellet was homogenized

in one ml of 80% sulphuric acid (Myklestad and Haug, 1972) and the total amount

of carbohydrate in the solution was measured by phenol sulphuric acid method.

The absorbance was read at 485 nm. Standard curve was prepared with glucose

solution of known concentrations, by which unknown concentration of carbohydrate

was calculated.

2.5.5 Photomicrography

In order to visualize the morphological changes occurred in the algal

cells, which were exposed to effluents, photomicrographs of all the test species

were taken, using an inverted microscope (Leica, Germany) with Kodak 200 COlOUrfilm~.

2.5.6 Statistical analysis

Statlstlcal analys~s of the data was carried out by 3-way ANOVA to

compare the s~gniflcance in different parameters between species, between different

25

concentrations of the effluent and between different days and the first order

interaction in pairs of the three factors viz. species, concentration levels and days

(Snedecor and Cochran, 1967; Jayalakshmi, 1998). Student's t - test was applied

to compare the significance of difference between species in pairs, between days

in pairs and between concentrations in pairs for the different parameters (Fisher

and Yates, 1963; Sokel and Roholf, 1981). Trelli's diagram was used for the graphic

representation of the significance of observed differences in terms of t- statistic

values (Sanders, 1968).

Arithmetic method, Logarithmic method and Probit method were used to

calculate 96 hour EC,,values. Probit method was used to compute confidence limit

(Reish and Oshida, 1986).

2.6 Physico-chemical characteristics of water in the studied sites

With a view to understand the effect of effluents on phytoplankton in the

natural water sources, water samples were collected from three different study

areas. The study areas were designed on the basis of discharge of effluents from

factories Into the natural water sources.

Hindustan Newsprint Limited, Velloor, situated on the banks of

Muvattupuzha river, is discharging its effluent into the same (Plate 2.2). Hindustan

Organic Chemicals Limited Irumpanam, which is manufacturing phenol and acetone,

is giving out the effluent into Chitrapuzha river (Plate 2.3). The effluent from

Mc Dowells Distilleries, Varanad is sending out the effluent into Vembanad estuary

(Plate 2.4). In all the above study areas, four stations were fixed for the collection

of water samples (Fig 2.1 to 2.4).

The present study ,was conducted for a period of 'twelve months from

January 2001 to December 2001. In order to find out the seasonal variations, the

study period was categorized into three seasons viz. pre-monsoon (February to

May), monsoon (June to September) and post monsoon (October to January). The

samples were collected once in every fortnight and average values reckoned in

presenting the results.

Water samples were collected from the surface layers from the different

sampling stations. Samples were collected in polyethylene bottles, after washing

the bottles with the same water. The sampling was done uniformly between 07.00

hours to 09.00 hours.

2.6.1 Water temperature

The temperature of the water samples was measured using a centigrade

thermometer graduated from 0 to 50°C. Immediately after collecting the water sample

in a narrow mouthed polyethylene bottle, the thermometerwas introduced into the

water column up to 5cm.

Hydrogen ion concentration of the water sample was recorded

immediately after collecting the sample with a pH meter.

2.6.3 Dissolved Oxygen

The dissolved oxygen was estimated by Winkler method (Strickland and

Parsons, 1968). The dissolved oxygen was fixed with manganous sulphate solution

and alkali iodide azide reagent immediately after the collection of water samples.

2.6.4 Free CO,

Free CO, was determined with NaOH reagent and phenolphthalein

indicator (Golterman et al., 1978). 3-4 drops of phenolphthalein indicator was added

to the sample and titrated with NaOH till the appearance of a pink colour.

2.6.5 Alkalinity

The amount of acid required to titrate the bases of the given watersample

is a measure of its alkalinity. Bicarbonates, carbonates, and sodium hydroxides are

considered to be the chief bases in natural water. Alkalinity was determined based

on the procedure of Golterman eta/., (1978). The titration was conducted in two

steps with phenolphthalein and methyl orange indicators. Total alkalinity was

determined from the above titre values.

2.6.6 Hardness

The concentration of calcium and magnesium expressed as equivalent

to CaCO, is considered as a measure of total hardness and it was calculated

following APHA (1979). The calcium and magnesium ions of the samples were

titrated with ethylene diamine tetra-acetic acid disodium salt to form the stable Ca

EDTA, Mg EDTA. A small quantity of Eriochrome black-T added to the water sample

would lead to a soluble wine red complex with some of calcium and magnesium

ions. During titration EDTA will complex all of the Ca2+ and the solution will turn blue

at the end point.

2.6.7 Chloride

The chloride content of the water samples combines with the silver of

the silver nitrate reagent leading to a white precipitate of silver chloride. When all

: 28 ::

the chlorides are used up, the excess silver combines with chromate indicator to

produce a pinkish orange coloured silver chromate. The analysis was based on

Golterman eta/.. (1978).

2.6.8 Nitrite

Nitrite was estimated by Azo dye method (Bendschneider and Robinson,

1952). The determination is based on the classical Griess's reaction in which the

nitrification at pH 1.5 to 2.0 i s diazotised with sulphanilamide resulting in diazo

compound which is allowed to react with N-I-naphthyl ethylene diamine (NNED) to

form high coloured azo-dye as end product. The extinction of the end product is

measured at 543 nm in the spectrophotometer.

2.6.9 Nitrate

Nitrate was estimated following the method of Mullin and Riley (1955).

The nitrate present in the water is reduced quantitatively into nitrite by hydrazine in

the presence of copper ion as catalyst. The nitrite thus produced is determined by

diazotizing with sulphanilamide and coupling with NNED to form a highly coloured

azodye as an end product. The extinction of the end product is measured at 543 nm

in the spectrophotorneter~

2.6.10 Phosphate

The method of ph~~sphate estimation described by Murphy and Riley

(1962) and followed by Strickland and Parsons (1968) was used for the analysis.

The phosphate in the sample was allowed to react with a minimum molybdate forming

a complex hetropoly acid compound. The compound, reduced by ascorbic acid in the

29 :

presence of antimony1 tartarate as catalyst, produces a blue colour at the end point.

The absorbance of the final product was measured using a spectrophotometer

at 885 nm.

2.6.11 Silicate

Silicate was determined following the method described by Strickland

and Parsons (1968). The water sample was allowed to react with acid molybdate

solution, which resulted in the formation of silicomolybdate, phosphomolybdate and

arsenomolybdate complex. A reducing solution containing rnetol and oxalic acid

was added which subsequently reduced the silicomolybdate complex to produce

blue colour compound as end product.

2.6.12 Primary production

Primary production was estimated following the technique of light and

dark bottle method (Gaarder and Gran, 1927).

2.6.13. Total dissolved and suspended solids

The quantity of total dissolved solids is an estimate of dissolved organic

and inorganic matter in the sample. To determine the total dissolved solids, the

sample was filtered through a glass fibre filter to remove the particulate matter, and

the filtrate was evaporated. The weight of the residue was then calculated. In order

to estimate the total suspended solids, the glass fibre filter used in the previous

experiment was weighed after drying in an oven at 105'~.

, .. -..

i - I ' -. . .

2.6.14 Biochemical Oxygen Demand (BOD) . . , . , \. ~'~

, ,.. , " . * , '.<

-._ .,;.

Five day biochemical oxygen demand test was done follow;^ t?i&'i&ih&

of Young etal. (1 981). The dissolved oxygen of the sample was determined before

and after 5 days of incubation (Strickland and Parsons, 1968).

2.6.15 Chemical Oxygen Demand (COD)

The chemical oxygen demand is a measure of the total amount of oxygen,

which is required to completely oxidise all the oxygen matter in a sample to CO,

and H,O. It is based on the principle that almost all organic compounds in water

can be oxidised to CO, and water by the action of strong oxidising agents under

acid conditions. The excess chromate can be measured by back titration with ferrous

ammonium sulphate using ferroin indicator to detect the end point (Ruttanagosrigit

and Boyd, 1989).

2.6.16 Phytoplankton count

The samples were kept undisturbed for one day in darkness for

sedimentation. The supernatant was decanted. The sedimented portion was shaken

well and transfered carefully to a lOOml beaker. This was left undisturbed overnight.

The beaker was covered with an aluminium foil. The supernatant was poured into

another beaker (100ml) and was observed under light microscope.

PLATE - 2.1

a. Stock cultures of test organisms

A. Chlorella ellil~soidea Gerneck

B. Ankistrodesmus falcatus (Corda.) Ralfs

C . Scenedesrnus bijuga (Turp.) Lageheim

D. Haematococcus laccustris (Giropd.) Rostafinski

E . Chlorococcum humicola (Naeg.) Rabenhorslt

b. Experimental set up

PLATE - 2.1

PLATE - 2.2

a. Muvattupuzha river

b. Pulp-paper mill fasctory (Hindustan Newsprint Limited)

PLATE - 2.2

PLATE - 2.3

b. Chitrapuzha river

a Petrochemical factory (Hindustan Organic Chemicals Limited)

PLATE - 2.3

PLATE - 2.4

a. Cochin estuary

b. Distillery (Mc Dowells Distillery)

PLATE - 2.4

Fig. 2.1 Cochin estuary

Fig. 2.2 Muvattupuzha river showing sampling stations