Chapter- 5

PIGMENT ANALYSIS

5.1 Introduction

5.2 Materials and Methods

5.3 Results and Discussion

5.3.1 Chlorophyll a 5.3.2 Chlorophyll b 5.3.3 Chlorophyll c 5.3.4 Total Chlorophyll 5.3.5 Carotenoids 5.3.6 Pheophytin 5.3.7 Pheophytin : chlorophyll ratio

5.4 Summary

Co

nt

en

ts

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5.1 Introduction

A primary algal characteristic is the presence of photosynthetic

pigments- the chlorophylls, carotenoids and biliproteins. Chlorophyll a is

the primary photosynthetic pigment of all oxygen-evolving photosynthetic

organisms and is present in all algae and Cyanobacteria. Chlorophyll a has

two in vitro absorption bands; in the red-light region at 660-665 nm and at

lower wave lengths near 430nm. Chlorophyll b, although common in

higher plants, is found only in green algae, Euglenineae and certain other

minor groups. Chlorophyll b is a light-gathering pigment that transfers

absorbed light energy to chlorophyll a for primary photochemical reactions.

Chlorophyll c, consisting of three spectrally distinct components, is

probably an accessory pigment to photo system II (Wetzel 2001).

Algal pigment composition of pokkali and prawn fields of

N. Paravoor and Vypeen Island has not been studied so far. The adjacent

Cochin estuary has been studied extensively with regard to the various

groups of planktonic algae (George 1958; Qasim et al. 1969, 1972;

Gopinathan 1972; Gopinathan et al. 1984; Nair et al. 1975; Joseph and

Sreekumar 1993). Sivadasan and Joseph (1995) have studied the

photosynthetic pigments of benthic microflora in Cochin estuary. Renjith

et al. (2004) have studied the primary production and fishery potential of the

Panangad region of Cochin estuary and recorded the chlorophyll a content.

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Sanilkumar et al. (2009) had estimated the pigment composition of

microphytobenthos in Cochin estuary. Periphyton colonization on

different substrata, floral composition of the periphyton community and

its spatial and temporal variations in the Cochin estuary has been studied

(Sreekumar and Joseph, 1995a: 1995b). Periphytic algae have a significant

role in the primary production and contribute substantially to primary

production (Wetzel 1964) and these are responsible for about 90% of total

organic production (Wetzel 1990). Seasonal and spatial variations in

pigment composition of periphytic algae in pokkali and prawn fields of N.

Paravoor and Vypeen Island are presented in view of their importance in

primary production.

5.2 Materials and Methods

Quantitative determination of the pigments was done using

periphyton grown on glass slides of 25x75 mm size kept submerged at

different stations (APHA 1998). Pigments were extracted with aqueous

acetone and OD was measured with spectrophotometer (Hitachi U-2001).

Individual glass slides with periphyton colonies retrieved from different

stations were placed directly into 100 ml of a mixture of 90% aqueous

acetone and 10% saturated MgCO3 solution. The samples were frozen

protected from the light by wrapping in aluminum foil in the field and

kept steeped in acetone for 24 hours in the dark near 4o C. 3 ml. of

clarified extract was transferred to a 1 cm cuvette and absorbance read at

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750, 645, 630, 510 and 480 nm. The concentration of chlorophyll a, b, c

and carotenoids were calculated using the equations in Strickland and

Parsons (1972).

Cb = Chlorophyll b = 20.7 E6450-4.34 E6650- 4.2E6300

Cc = Chlorophyll c = 55 E6300-4.64 E6650-16.3E6450

Cp = Carotenoids = 7.6 (E4800-1.49E5100)

where, E is the absorbance at respective wave lengths. Each extinction

was corrected for a small turbidity blank by subtracting the 750 nm

reading.

Chlorophylls and carotenoids were determined in mg/m² using the

formula APHA (1998).

mg chlorophyll/m² = 2m substrate, of AreaL extract, of x volumeC

where Cb, Cc, and Cp are respective values of Chlorophyll

b, chl. c, and carotenoids. For the estimation of active chlorophyll a and

pheophytin a, the procedure was similar to that of chlorophylls as given

above. The extinction of the extract was measured at 665 and 750 nm.

Each 750 nm reading was subtracted from the corresponding 665 nm

and using the corrected values chlorophyll a and pheophytin a

concentration per unit surface area of sample was calculated (APHA

2005) as follows.

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Chlorophyll a, mg/m² = m² substrate, of area v )665-(665 26.7 ab ×

pheophytin a, mg/m² = m² substrate, of area

}665 - ){1.7(665 26.7 ba

where 665 b = extinction at 665 nm before acidification.

665 a = extinction at 665 nm after acidification

v = volume of extract, L. The value 26.7 is

the absorbance correction and 1.7 is the

absorption peak ratio.

5.3 Results and Discussion 5.3.1 Chlorophyll a

Chlorophyll a (Chl. a) is probably the most frequently measured

compound of biological origin in aquatic sciences. The ease of its

extraction and the relatively wide-spread occurrence of instruments for its

measurement make it an attractive surrogate for estimation of algal

biomass (Yacobi and Zohary 2010).

The seasonal and spatial variations of chlorophyll a at different

stations are shown in the Table 5.1. The annual mean concentration of

chlorophyll a in the study area was 18.80 mg m-2. The highest

concentration of chlorophyll a, 61.273 mg m-2 in the study area was

recorded at Pookad in June and the lowest, 0.393 mg m-2 at Pattanam

station during November. Chlorophyll values are generally taken as an

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index for standing crop of an ecosystem. During pre-monsoon the mean

chlorophyll a value was 17.425 ± 9.259 mg m-2 where as the monsoon and

post-monsoon values were 17.623 ±1 3.275 and 21.333 ± 13.302 mg m-2

respectively (Fig. 5.1). The Chl. a distribution followed the same trend of

standing crop showing the highest number during post-monsoon. The

concentration of Chl. a at different stations highlighted the magnitude of

periphyton bio-mass and its significance. The seasonal variations of

chlorophyll a values in the study area indicated that the post-monsoon

period was more suitable for periphyton growth.

Singh and Gaur (1989) reported Chl. a concentrations of 53.8, 34.58

and 59.34 mg m-2 for periphyton in a stream polluted with oil refinery

effluent at Digboi (Assam, India) in April , September, 1986 and January

1987 respectively. However Hansson (1992) had reported 30-270 mg m-2

Chl. a in Swedish Lakes and 20-400 mg m-2 in Antarctic Lakes. Joseph and

Pillai (1975) reported spatial seasonal variation in Chl. a values for the

planktonic algae in Cochin estuary. For the plankton, Chl. a was the lowest

during monsoon and the highest during post-monsoon. Sivadasan and Joseph

(1995) had reported the concentration benthic floral Chl. a from Cochin

estuary. The Chl. a concentration ranged from 57.26 mg/m² (post-monsoon)

to 78.36 mg/m² (pre-monsoon). However, in this study the periphytic Chl. a

showed maximum concentration during post-monsoon (Fig 5.1).

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Chlorophyll a

0.0005.000

10.00015.00020.00025.00030.00035.00040.00045.000

1 2 3 4 5 6 7 8 9 10Stations

mg/

m2 Pre-mon

MonsoonPst-Mon

Fig. 5.1

Sanilkumar et al. (2009) had reported the Chl. a concentration in the

upper 1 cm sediment ranged from 2.6 to 86.3 µg cm³ of microphytobenthos

in Cochin estuary. Rajesh et al. (2001) had observed the highest Chl. a

concentration during May and the lowest during January. They had

reported the sediment Chl. a concentration fluctuated from 0.39 mg g-1 to

1.48 mg g-1 with no clear pattern of seasonal variation, although higher

values were found in May, December and September in brackish water

pond, Southwest coast of India. Renjith et al. (2004), from the studies of

Panangad region of Vembanad Lake, had reported that the Chl. a varied

between 1 and 34.61 mg/ m3. Putz (1997) had observed variation in

periphytic Chl. a concentration at different areas. Mean periphytic Chl. a

content of the floating meadow zone was considerably lower varying

between 0.55 mg/m² and 44.1 mg/m². Periphyton Chl. a from Solimoes main

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stream oscillated between 0.0 mg/m² and 7.1 mg/m². Some days Chl. a was

undetectable, which indicated a heterotrophic attached assemblage

( Putz 1997).

Nayar et al. (2005) had reported the periphyton Chl. a ranging from 0.03

to 2.71 µg/ cm-2 and a mean of 1.32 µg cm-2 in Ponggol estuary, Singapore.

Bothwell (1983) reported Chl. a concentration ranging from 0.006 to

5 .0 µg cm-2 in periphyton studies in Thompson River, British Columbia.

Ishida et al. (2006) had reported the Chl. a values of epilithic algae

ranging from 1.7 to 112.5 mg m-2.

Mc Minn et al. (2007) had reported the areal Chl. a concentrations

varied from a minimum of 0.025 mg m-2 to a maximum of 5.18 mg m-2

with a mean of 2.16 mg m-2 in their experiments in eastern Antarctica.

Seasonal and spatial variations in the concentration of Chl. a were

examined using two way ANOVA (Table5.9). A significant difference in

Chl. a values were observed between different stations. However, there

was no significant variation noticed between seasons. Correlation between

the monthly mean values of Chl. a and hydrological parameters such as

dissolved oxygen, nitrate, nitrite, silicate, phosphate, pH, temperature and

salinity were analysed (Table 5.8). No significant correlation between

Chl. a and any hydrological parameters except temperature were

observed. Chl. a showed positive correlation to temperature.

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The low chlorophyll a values could be due to the type of dominant

algae in this community. Welch et al. (1988) pointed out that periphyton

communities dominated by Chlorophyceae always show the highest

chlorophyll a values. 75.6 % of the periphytic algae of the study area

comprised with diatoms.

Posey et al. (2006) has recorded the highest benthic Chl. a values

(350-375 mg m-2) in Cape Fear estuary, southeastern North Carolina,

where nitrogen and phosphorus concentrations were very high.

5.3.2 Chlorophyll b

Monthly mean values of Chlorophyll b (Chl. b) at different stations are

shown in Table 5.2. The values ranged from beyond detectable level to

20.197 mg m-2. The highest Chl. b concentration was recorded Cherai kayal

during the month of August. The annual mean concentration of Chl. b

recorded was 2.534 ± 4.001 mg m-2. Monsoon season showed the maximum

mean Chl. b concentration, 5.669 ± 5.449 mg m-2. 0.999 ± 1.491 and

0.935 ± 1.348 mg m-2 were the mean Chl. b concentration recorded during

post-monsoon and pre-monsoon respectively (Fig. 5.2). The concentration of

chlorophyll b is directly proportional to the biomass of Chlorophyceae and

Euglenineae, as only these are the algal groups that possess Chl. b (Wetzel

2001). Comparatively high values of Chl. b during monsoon showed the

luxuriant growth of these algal forms during the period. Heavy precipitation

due to south-west monsoon lowered salinity in the study area favouring the

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growth of freshwater algae like Stigeoclonium, Mougeotia, Oedogonium,

Spirogyra and Cladophora. The low concentration of Chl. b may be due to

the low distribution of Chl. b bearing microflora. 75.6 % of the periphyton

recorded in the study belonged to Bacillariophyceae. Diatoms do not carry

Chl. b (Wetzel 2001).

Cherai kayal located near the Arabian Sea showed the maximum

Chl. b concentration during August. Compared to the values of Chl. a,

Chl. b values were very low. During the monsoon the study area

maintained more or less fresh water conditions but salinity increased

gradually through post-monsoon and reaching the maximum during the

pre-monsoon which favoured the diatoms.

Chlorophyll b

0.000

2.000

4.000

6.000

8.000

10.000

1 2 3 4 5 6 7 8 9 10Stations

mg/

m2 Pre-mon

MonsoonPst-Mon

Fig. 5.2

However the Chl. b values were the maximum during monsoon and

gradually decreased through post-monsoon reaching the lowest during pre-

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monsoon. The monsoon favoured the growth Chlorophycean members and

this attributed the high Chl. b concentration during monsoon.

Sreekumar (1998) had reported concentration of chlorophyll b in

periphytic algae in Cochin estuary which varied from 0 to 51.98 mg m-2 and

an annual mean of 6.36 mg m-2. Sivadasan and Joseph (1995) have estimated

the chlorophyll b of benthic microflora in Cochin estuary. The monthly

average values varied from 2.01 mg m-2, in October to 14.85 mg m-2 in

August. The concentration of Chl. b was almost negligible at many stations

in the present study. This was due to the occurrence of diatoms which carry

no chlorophyll b. Sanilkumar et al. (2009) also reported a low

concentration of Chl. b in microphytobenthos of Cochin estuary.

5.3.3 Chlorophyll c

Monthly mean values of the chlorophyll c (Chl.c) at different

stations are summarized in Table 5.3. The annual mean concentration of

Chl. c was 18.827 mg m-2. The lowest concentration of Chl. c recorded

was below beyond detection at Pattanam station during July and the

highest, 95.410 mg m-2 at Pukkad during June. The average

concentrations of Chl. c during pre-monsoon, monsoon and post-monsoon

were 19.422 ± 9.259, 18.760 ±16.611 and 18.675 ± 8.865 mg m-2

respectively (Fig. 5.3). Both monsoon and post-monsoon seasons recorded

almost similar quantity of Chl. c concentrations and the pre-monsoon

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recorded the highest. With the increase of the salinity in backwaters there

was a corresponding increase in the diatom standing crop as indicated by

the higher values of Chl. c during pre-monsoon.

Chlorophyll c

0.0005.000

10.000

15.00020.00025.00030.000

35.00040.000

1 2 3 4 5 6 7 8 9 10

Stations

mg/

m2 Pre-mon

MonsoonPst-Mon

Fig. 5.3

Sreekumar (1998) had recorded the same pattern of distribution of

periphytic Chl. c in Cochin estuary. Sivadasan and Joseph(1995) had

reported the Chl. c concentrations in benthic microflora in Cochin estuary

ranged from 11.3 mg m-2 to 44.31 mg m-2. Sanilkumar (2009) had noticed

Chl. c concentration in phytomicrobenthos of Cochin estuary ranged from

0 to 14.29 µg/cm³ in the top most layers.

5.3.4 Total Chlorophyll

The monthly mean values of total chlorophyll (a+b+c) at different

stations are given in Table 5.4. The distribution was similar to that of

chlorophyll a, both being maximum in June and minimum during

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September in most stations (Fig 5.7 -5.9). Annual mean value of total

chlorophyll was 40.130 mg m-2 with pre-monsoon, monsoon and post-

monsoon 37.781, 41.677 and 40.922 mg m-2 respectively (Fig. 5.4). The

concentration of the chlorophyll can be taken as an index of the floral

composition (Gopinathan et al. 1984). The study area showed an annual

average of 18.79 ±12.129 mg m-2 of chlorophyll a and 40.162 ± 23.220 mg

m-2 total chlorophyll during the study period which indicated the presence of

fairly good quantity periphyton standing crop through out the year. Chl. b

and Chl. c are characteristic of green algae and diatoms respectively (Wetzel

2001). O’Reilly (2006) has reported the periphytic total chlorophyll in a

tropical lake, Zambia as 26 mg m-2 at a depth of 16 m.

Total chlorophyll

0.00010.00020.00030.00040.00050.00060.00070.00080.000

1 2 3 4 5 6 7 8 9 10Stations

mg/

m2 Pre-mon

MonsoonPst-Mon

Fig. 5.4

The comparative high values of total chlorophyll showed that the

prominent periphytic flora of the backwater comprised mainly with

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diatoms. This is evident from the floral composition of the study area

(Table 3.1). Diatoms formed 75.50 % where as blue green algae and green

algae comprised 13.87 % and 9.79 % of total periphytic algae respectively.

Thus the pigment compositions agree well with the composition of

periphytic flora of the backwaters.

5.3.5 Carotenoids

The monthly mean concentrations of carotenoids at different stations

are shown in the Table 5.5. The monthly mean concentration varied from

below level of detection to 40.332 mg m-2. The annual mean concentration of

carotenoids in the backwaters was 8.738 mg m-2. The post monsoon recorded

the maximum concentration and the monsoon recorded the minimum.

9.018 ± 5.898, 7.413 ± 8.299 and 10.111 ± 8.269 mg m-2 were the

concentrations of carotenoids recorded during pre-monsoon, monsoon and

post-monsoon respectively (Fig 5.5). The annual mean values of carotenoids

at different stations; Malippuram, Pukkad, Nedungad, Edavanakkad,

Kuzhuppilly, Cherai kayal, Kedamangalam, Chathanad, Pooyappilly and

Pattanam were 10.336, 8.393, 10.667, 7.606, 8.473, 12.258, 7.966, 6.734,

9.021 and 5.932 mg m-2 respectively. Cherai kayal station recorded the

highest single value of carotenoids (40.332 mg m-2) during October.

Sreekumar (1998) has recorded 80.39 mg m-2 maximum carotenoids

during November from the studies of periphytic flora in Cochin estuary.

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Sivadasan and Joseph (1995) had reported the annual mean benthic

concentration of carotenoids as 33.07 mg m-2 and ranged from 19.31 to

48.85 mg m-2.

Fig. 5.5

The ratios of carotenoids: Chl. a at all the stations were < 1 (Table 5.7).

The ratios <1 indicated the physiologically active state of periphytic flora

in the backwaters. The lower ratios of carotenoids to Chl.a indicate the

active state of benthic microflora (Sivadasan and Joseph 1995).

5.3.6 Pheophytin

The monthly mean values of pheophytin concentration of the

periphytic algae in the backwaters is shown in Table 5.6. The annual

mean concentration was 2.497 ± 1.725 mg m-2. The highest seasonal

concentration (2.909 ± 1.676 mg m-2) was noticed during post-monsoon

followed by a gradual decrease during pre-monsoon (2.517 ± 1.604 mg m-2)

Mg/

m2

Carotenoids

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and the lowest (2.082 ± 1.829 mg m-2) during monsoon (fig.5.6) The highest

concentration of pheophytin (8.327 mg m-2) was recorded from station 1

during January and the lowest at station 8 (0.121 mg m-2) during March.

The ratio of pheophytin to Chl. a (Table 5.7) varied from 0.129 to

0.145 with 0.133, 0.145 and 0.129 for pre-monsoon, monsoon and post-

monsoon respectively.

Sreekumar (1998) had reported a higher concentration of periphytic

pheophytin in Cochin estuary with the highest concentration 16.04 mg/m²

during monsoon and the lowest 8.78 mg/m² during post-monsoon.

Pheophytin

0.000

1.000

2.000

3.000

4.000

5.000

6.000

1 2 3 4 5 6 7 8 9 10Stations

mg/

m2 Pre-mon

MonsoonPst-Mon

Fig. 5.6

Sanilkumar (2009) had reported the highest benthic pheophytin as

13.03µg L-1 during post-monsoon in some stations in Cochin estuary.

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5.3.7 Pheophytin : chlorophyll ratio

The ratio of pheophytin to Chl. a concentration gives a general

indication of the physiological or grazing state of microalgal community.

High ratio (Range; 0.5 to 1) indicates a stressed or declined community,

while low rates (Range; 0 to 0.5) represent actively growing community

relatively free of grazing pressure (Bidigare et al. 1986; Light and Beardall

1998; Mundree et al. 2003). The monthly ratio of pheophytin to Chl. a in this

study ranged from 0.088 to 0.192 (Table 5.7). Thus the ratio suggests that a

physiologically healthy periphytic community was present in the study area.

The correlation analysis of pigments (Table 5.8) revealed that all of

them have significant correlation with each other except Chl.b (p< 0.01).

The strongest positive correlation (0.723) was noticed between Chl. a and

Chl. c. This is an indication of the predominance of periphytic diatoms in

this area. ANOVA of the spatial and temporal distribution of pigments

were also analysed (Table 5.9). There was no significant spatial and

temporal variation in the distribution of various pigments.

5.4 Summary

The quantitative determination of the pigments was done using

periphyton growth on glass slides. The annual mean concentration of

chlorophyll a in pokkali and prawn fields of N. Paravoor and Vypeen

Island was 18.794 ± 12.129 mg m-2. The post monsoon recorded the

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175

highest seasonal average, 21.333 mg m-2. The concentrations of

chlorophyll a reported were comparatively lower than recorded in Cochin

estuary (Sreekumar 1998; Sivadasan and Joseph 1995). This is due to the

hetrotrophic attached assemblage.

The annual mean value of chlorophyll b was 2.534 mg m-2. The

monsoon season recorded the maximum chlorophyll b concentration,

5.669 mg m-2. The concentration of Chl. b is directly proportional to the

biomass of Chlorophyceae and Euglenineae as only these algae possess

Chl. b. Comparatively high concentration of Chl. b during monsoon was

due to the high growth of Chlorophyceae and Euglenineae.

The annual mean value of chlorophyll c concentration was

18.953 mg m-2. Pre-monsoon recorded higher concentration. The values of

pigment concentrations agree to the floral composition described in

chapter 3. The annual mean value of total chlorophyll was 40.126 mg m-2

and the monsoon recorded the maximum value.

8.847 ± 7.591 mg m-2 was the annual mean concentration of

carotenoids recorded. Post-monsoon recoded the maximum value i.e.

10.111 mg m-2. The ratio of various pigments to Chl. a was less than 1.

The annual mean concentration of pheophytin was 2.503 ± 1.725 mg m-2.

The highest concentration was noticed during post-monsoon followed by

a gradual decrease during pre-monsoon and the lowest during monsoon.

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Table 5.7 Ratio of spatial variation of pigments to Chl. a at different stations

Pigments 1 2 3 4 5 6 7 8 9 10

Chlorophyll b: Chl a 0.062 0.078 0.178 0.088 0.171 0.138 0.152 0.104 0.148 0.347

Chlorophyll c: Chl a 0.062 1.293 0.880 0.842 0.789 0.958 1.153 0.961 1.041 1.214

Carotenoid: Chl a 0.374 0.475 0.493 0.394 0.420 0.559 0.551 0.461 0.590 0.503

Phaeophytin: Chl a 0.138 0.140 0.136 0.139 0.058 0.088 0.131 0.118 0.140 0.192

Table 5.8 Correlation of different pigments

Chl. a Chl. b Chl. c Caroteno. Pheophy.Pearson Correlation 1

Sig. (2-tailed) .

Chl. a

N 120 Pearson Correlation .071 1

Sig. (2-tailed) .440 .

Chl. b

N 119 119 Pearson Correlation .723(**) .010 1

Sig. (2-tailed) .000 .915 .

Chl. c

N 120 119 120 Pearson Correlation .722(**) -.071 .626(*

*) 1

Sig. (2-tailed) .000 .443 .000 .

Caroten-oids

N 120 119 120 120 Pearson Correlation .752(**) -.101 .541(*

*) .492(**) 1

Sig. (2-tailed) .000 .275 .000 .000 .

Pheophy-tin

N 120 119 120 120 ** Correlation is significant at the 0.01 level (2-tailed * Correlation is significant at the 0.05 level (2-tailed).

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Table 5.9 ANOVA of temporal and spatial variations of Chl. a, Chl, b, Chl. c, Total Chl., Carotenoides and Pheophytin

Chl. a Chl. b Chl. c Caroteno. Pheophy.

Between Groups 387.842 2 193.921 1.325 .270

Within Groups 17119.184 117 146.318

Chlorophyll_a

Total 17507.026 119

Between Groups 589.850 2 294.925 26.230 .000

Within Groups 1315.539 117 11.244

Chlorophyll b

Total 1905.389 119

Between Groups 13.383 2 6.692 .045 .956

Within Groups 17392.603 117 148.655

Chlorophyll c

Total 17405.986 119

Between Groups 341.427 2 170.713 .313 .732

Within Groups 63821.378 117 545.482

Total Chlorophyll

Total 64162.805 119

Between Groups 147.255 2 73.628 1.284 .281

Within Groups 6709.483 117 57.346

Carotenoids

Total 6856.739 119

Between Groups 13.724 2 6.862 2.358 .099

Within Groups 340.523 117 2.910

Pheophytin

Total 354.246 119

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185

Fig.

5.8

Sea

sona

l dis

trib

utio

n of

Chl

. a a

nd to

tal C

hl. a

t Stn

. 5,6

,7&

8

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…..

…..

Fig.

5.9

Sea

sona

l dis

trib

utio

n of

Chl

.a a

nd to

tal C

hl. a

t stn

. 9&

10