HPLC-DETECTED PHYTOPLANKTON PIGMENT PROXIES - A CONTRIVANCE FOR PROMPT FORTITUDE OF PHYTOPLANKTON COMPOSITION AND ECOLOGICAL

RECONSTRUCTIONDr.Aneeshkumar Narikkatan* and Dr.Sujatha C.H

Department of Chemical Oceanography,

Cochin University of Science and Technology

Cochin 682016

Corresponding Author: Dr.Aneeshkumar Narikkatan

* Central Ground Water Board

Ministry of water recourses

NWHR, Jammu 180004

Email: akncusat@gmail.com

Contact No : 09419210601

Abstract: Fossil pigment distribution and their chemotaxonomy with respect to phytoplankton

biomass and hydrological background were analyzed in the sediments collected from in Cochin

back water systems by HPLC. Higher order association of the individual Pigment, PSC, PSP,

PPC and Bio mass proportion calculated. The picoplankton communities add significantly to the

estuarine biomass structure whereas micro plankton appears to be most abundant in

anthropogenic affected area. The systems were classified into allochthonous vs. autochthonous

using total chlorophyll derivative to total carotene (CD/TC) ratios. A vertical profile of the Ch a,

its degradation pigments, carotene and their ratios were find out vertically at the depth of 6 cm.

Key Word: HPLC, Phytoplankton, Marker pigments, Sediments, Ecology.

1.1 Introduction

The quality and intensity of the factors and processes influencing the aquatic ecosystem,

and their consequences are extremely complicated they causes high temporal and spatial

variability both in the chemical and biological characteristics of water masses. Thus, it is very

rarely possible to reconstruct the exact state of past ecosystems by means of short-term

monitoring data. Palaeorecords in estuarine sediments can be used for recording long-term

changes in land-use which includes biological, geochemical and lithological information stored

in accumulative deposits over certain time periods (Chambers, 1993; Leavitt, 1993; Hassan et

al. 1997; Lami et al.1997). An understanding of the phytoplankton biomass- A fundamental

importance for the biology and its contributing pattern with respect to the behavioral chemistry of

nutrients, organic matters (O M) like carbohydrate and proteins would provide a pre requisite

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knowledge for the biogeochemical process occurring in the Cochin estuarine system. The

phytoplankton biomass comprises of carotenoids, phytopigments (chlorophyll a, b and c) and its

degraded products. Often carotenoids and chlorophylls are the only fossil remains of

nonsiliceous algae and bacteria and are therefore of considerable value to palaeoecology and

palaeolimnology as an indicator for chemotaxonomic or photo physiological studies ( Barlow, 1993;

Claustre et al.1994) . Higher order association of the individual pigment (sums and ratios) permits the

formulation of variables that are useful to different perspectives. The pool of photosynthetic (PSC) and

photo protective carotenoids (PPC) are key steps for both photo physiological studies and total

amount of accessory (non-chlorophyll a) pigments (TAcc) are useful in remote sensing investigations

(Trees et al. 2000). Accessory pigments have either photosynthetic properties allowing the

phytoplankton cells to increase their light harvesting spectrum or a role of photo protection in

dissipating the excess of light energy received and reducing the oxidation that takes place due to

stress in conditions of strong irradiance. The ratios that can be derived from these pooled variables,

e.g. [PSC]/ [TChl a] are dimensionless and have the advantage for automatically scaling the

comparison of results from diverse environs.

Very little information is available on these aspects for their change in the back waters

characters and the neritic zones. Since in estuarine environments, phytoplankton is

quantitatively the second source of particulate organic matter after terrestrial inputs from soil

erosion which supply the highest quality food source and constitutes premier basis of the

estuarine food web. The present study aims to investigate the inference featured in the morpho

- dynamic system of the Cochin estuary to provide evidence for the influence of various

circulation features on phytoplankton community structure in terms of pigment biomarkers using

high-performance liquid chromatography (HPLC). Furthermore it aim in addressing the

relationship between phytoplankton community structure and the balance between regenerated

and new production in the various identified circulation features using a combination of pigment

data and photosynthetic activity data.

2 MATERIALS AND METHOD2.1 Study areaThis research article focuses on the Cochin backwaters situated on the south-west coast of

India from the northern extension (9040’ to 100 10’ N and 760 13’ to 760 50’ E) of the Vembanad

lake along the Kerala coast (figure 1). All the sites have been influenced by anthropogenic

nutrient inputs from varies sources and have frequently monitored and recorded the past five

decades. Each location represents a unique depositional environment with regards to

preservation conditions which make them ideal sites for studying the biomarker pigments and its

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application. The backwater system covers an area of approximately 300 km2 with one

permanent bar mouth maintained at 12m depth at Cochin and two seasonal openings during the

peak monsoon period. The estuary is wide (16 km) in the Vembanad lake area and several

narrow canals carry along with water flow municipal waste and other particulate organic matter

and empting into it. Several major rivers Periyar, the Muvattupuzha and Pampa discharge fresh

water into the estuarine system. This estuary is classified as a tropical positive estuary and

character is influenced by the rivers flowing into it including the strong tidal currents. Both these

phenomenon characterizes the hydrological conditions of the estuary. Vallarpadam is situated

next to Bolgatty Island on the west, and linked to the Ernakulum mainland via the Goshree

bridges. It is about 3.5 km in length in north-south direction and hosts a population of 10,000

people. Vallarpadam is 1 km away from Ernakulum mainland. Cochin harbor is a major natural

harbor.

Based on the salinity characteristics of the surveyed area, stations 1 to 7 are grouped

into three zones (Table 1). Zone a (station S1) is the riverine zone, where the salinity remains

less than 1ppt throughout the year i.e. remains as freshwater zone. Zone B (stations S2, S3 &

S4) is partially estuarine in character and saline in the pre- monsoon season. The stations in

these zones are canals and tributaries which are in the vicinity of industries and markets.

Stations S5, S6 & S7 grouped together as zone C, becomes saline in the pre-monsoon and

post-monsoon seasons and acts as a sink for the discharges from the Pampa and, Vembanad

rivers, along with effluents from the municipal and industrial wastes from the Cochin City.

Sample collection were made bimonthly from November 2005 to September 2007,

surface water sample were collected approximately 5 cm below the water surface. Bottom

samples were collected approximately 25 cm above the sediments. The depth at each sampling

site varied throughout the year depending on the season and tide, the intervals observed were

(S1) 5-10 m , (S2) 0 to 0.5 m ( only surface sample ) (S3) 1to 2m, (S4) only surface sample (S5)

2- 4 m , (S6) 3- 7m (S7) 1- 5 m. The highest water level was found in the monsoon season and

lowest in the pre-monsoon. Water samples grabbed from the sites using Niskin sampler.

Sediment samples were collected at four distinct locations of CBWS by using a Van Veen Grab

sampler and the samples were immediately packed into special plastic cooling boxes and

transported to the laboratory for preservation at -20oC till analyses. Sediment samples for

pigment analysis were collected during peak summer using a stainless steel corer. Samples

were analyzed with a minimum 8 cm through the core and stored in pre cleaned polyethylene

bags for processing and transferred to the laboratory and preserved at 4oC. The sediment

samples for pigment analysis were immediately transferred to 15 ml vials kept in ice bags in the

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dark and then stored in a -70oC freezer to render them more stable ( Yacobi et al. 1990).

Finally transferred for preservation under -80oC (SANYO Ultra low MDFU-3086 maintained at -

80oC) and then directly freeze-dried in Viotis BENCHTOP-2K SI 213489 Lyophilizer) at -40oC, 6-

8 hrs. All field and laboratory work was carried out in subdued light to minimize pigment

degradation. Color, odor, taste and pH were noted at site. Sample for physico chemical analysis

was treated and analyzed according to Standard method (Standard methods 1995; Grasshoff et

al. 1999).

2.2 Phyto - Pigment Extraction and HPLC Analysis

Total carotenoids and chlorophyll with their degradation products were determined by

spectophotmetric method (Parsons, 1984). The sediment sample after collection was stored at

-80oC until analysis. The freeze dried samples were homogenized prior to sub sampling. After

weighing (approx 0.1mg) each sub sample was extracted in 95% acetone with internal standard

(vitamin E) sonicated in an ice cold sonication bath for 10min, mixed on a vortex mixer allowed

to extract at 4oC for 20hr and vortexed again. Extracts were then filtered through 0.2μm teflon

syringe filters to remove cell and filter debris, transferred to HPLC vials and placed in the

cooling rack of the HPLC. 357 μl buffer and 143 μl extract were injected on the HPLC

(Shimadzu LC-10A HPLC System with LC solution software) using a pre treatment program.

The adapted HPL method was described earlier (Hooker et al. 2005) not separating -β

carotene. The detection wave length was 420 and 450 nm and the flow rate was 12.5μl min -1.

Identification was based on the retention time and peak shape i.e. through fingerprint matching

with known peak shape from the diode array spectral library created by running pure standard of

individual pigments ( DHI group Denmark). The concentrations of the pigments were computed

from the peak areas.

3. Result and discussion3.1 Hydrological back ground During the observation period, the bathymetry of the water body indicates that depth variation

occurs between 1.5 m and 6.0 m in most parts except the dredged channels which are

maintained at 10–13 m deep. The hydrographical conditions of the CBWS are greatly influenced

by seawater intrusion and influx of river water as indicated by the distribution of temperature and

salinity at surface and bottom (0.25 m above the estuary bed). There are three pronounced

seasonal conditions prevailing in this estuary, i.e. monsoon (June–September), post-monsoon

(October–January) and pre-monsoon (February–May). During the monsoon period the region

receives about 290-320 cm rainfall annually, of which, nearly 60% occurs during the southwest

monsoon season and the rest fall on north east monsoon. The estuary is connected to the

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Arabian Sea at two locations, Cochin (Latitude 9°58′ N) and Azhikode (Latitude 10°10′ N) during

December to April, a salinity barrier at Thanneermukkom virtually cuts off the tidal propagation

further south and modifies the circulation in the remaining part of the estuary. The onset date

and duration of the southwest monsoon vary from year to year and the quantity of rainfall

contributes to the variability of the estuarine water levels and flow. It is recognized that the water

quality plays an important role in selecting the phytoplankton community. Zone A is situated at

upstream has the recorded lowest salinity in the study area and a fresh water zone 0.01- 0.12

(avgas 0.07). Zone B designated in the back water zone where the salinity ranges from 0.02 to

13.97 (avg1.79). While in the seaward end of the study area the estuary, salinity ranges

between 0.1 and 35.64 (avgas 16.8). The highest salinity was recorded at pre and post

monsoon and the salinity decreases with the onset of monsoon and became poorly fresh water

in character. pH drops up to 5.42 ( river), 5.92 ± 0.3 (back waters) and 6.14 ± 0.5 ( estuary )

and increased to 8.16 ± 0.2 , river ; 7.20 ± 1.2 , back waters and 8.3 ± 0.2 in estuary. This

trend creates an alkaline behavior at pre monsoon and monsoon. Lowest pH 5.34 (October) and

highest pH 8.5(August) was found wing to the immediate monsoon onset at Zone B and C. High

acidic or alkaline effluents may release from the various neighboring industries located in the

proximity of Zone B in the rain fall time and there is a pH recovery was eminent in pre monsoon.

DO content in the study varies from 1.28 to 8.0 mg/L. Lowest oxygen was recorded in

pre monsoon thereafter a recovery of DO was observed in the Post monsoon season due to low

water temperature and considerable growth of algae, which may release appreciable amount of

oxygen as a result of photosynthetic activities. Dilution of sewage and deposition of organic

material in the lower reaches of canal and estuarine openings resulting in the lowering of

oxygen content in surface water during monsoon in Zone B.

Zone A has lowest nitrate whereas Zone B & C recorded the highest. A net addition of

nutrients in monsoon and post monsoon and a net removal in pre-monsoon was observed.

Earlier studies support this drift (De Sousa et al. 1981). Among these locations, sites S2 is

located near by a fish market and slaughtering units and always prone to higher nutrient

enrichment with high anthropogenic discharges (> 1.2 mg/L) irrespective of season. Spatially

nitrite ranges from not detectable level to 0.6mg/L. An increase of nitrate concentration from

river, back water then to estuary was noted. Highest value was reported at (S2) and S4 with low

O2 (<4.0 to 1.28 mg/L) with a foul smell during summer and winter season due to anthropogenic

pollution. High NO3 content was observed in this station indicating an upward mixing and

displacement of NO2- to NO3

- completely. Phosphate content showed highest in back waters

than in the estuary and least in the riverine samples. The lowest value is viewed at Muvattupuza

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(S1) and highest > 4.0 mg/L was at Champakkara - a back water site. Champakara (S2) as

mentioned earlier is a direct anthropogenic effected area which has recorded highest PO4

concentration (4.4 mg/L) throughout the study period. Phosphate showed maximum in the pre

monsoon than in the monsoon and lowest was accounted in the winter season. It was observed

that generally the nutrients were more enriched at the surface than in the bottom and often

these nutrient availability in the water column were not fully utilized by the phytoplankton and

high concentration detected in the surface column. Similar observation reported earlier that that

higher productivity in bottom waters in these tropical waters and low concentration of nutrients

at bottom layer in the same study area (Gopinathan et al. 2001; Nair et al.1988). The

concentration of chlorophyll a (pre monsoon to Post monsoon) ranges from 0.84 – 29.75 mg/m3.

Chl a value were high during the pre monsoon at all stations and ranged from 1.18 to 14.06

( avg 5.07) mg/m3 at river ( S1 ), 1.05 to 29.75 mg/m3 ( avg 5.87) at Zone B and 2.75 to17.97

mg/m3 ( 6.5) at Zone C. The highest Chl a value (29.75 mg /m3 ) was observed S2. Moderately

high and lower values were noticed in the back waters where a regular pattern was observed in

the estuarine and riverine sites. In general lower values observed in monsoon and highest

values recorded in the pre monsoon and post monsoon seasons. Chl b is an accessory pigment

of Chl a and same trend has followed as that of chl a. The highest values were recorded in

some pockets of Zone B and C at pre monsoons. The concentration ranged from 0.62 - 3.6;

0.59 – 9.98 and 1.32- 10.42 mg /m3 at Zone A, B and C respectively. Moderately high and low

values were observed at Zone B (Champakara) and Zone C (Bolgatty) in monsoon and pre

monsoon periods respectively. The concentration of Chl c fluctuated between 1.17-11.1 mg/m3

in Zone A, 0.16-14.13 mg/m3 Zone B and 0.98 - 10.67 mg/m3 Zone C.

3.2 Phytoplankton speciation Being eutrophic, primary production in the estuary is always high and is mainly

constituted by nanoplankton (<20 mm) community. Perusal of literature from last five

decades(1958-2009) stand in this area reveals that total of 700 species of flora and fauna

comprising 65- 194 species of phytoplankton , 135 species of zooplankton 199 species of

benthos, 150 species of fishes and 7 species of mangroves were recorded between 1958 and

2007 (Table 2). Of the various categories, the concentration of nanoplankton (<20 mm)

community largely composed of diatoms (Bacillariophyceae), is relatively high throughout the

year, around 70% of the total phytoplankton was contributed by Skeletonema costatum. In the

present study and earlier work of Aneeshkumar and Sujatha. (2012) reveals the presence of 43

diatoms (Bacillariophyceae) species, 2 chlorophyceae species and 40 dinoflagellates species

at station S1. 42 diatoms species, 2 chlorophyceae species and 4 dinoflagellates species at

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station S2 whereas 66 bacillariophyceae species, 1 chlorophyceae species , 4 dinofllagellates

species, 1 chrysophyceae species and 1 stroptophyceae species were identified at champakara

(S3) site. At station S4 Cheranellur, 52 diatoms species 2 chlorophyceae species and 6

dinoflagellates species were detected.

3.3 PigmentsChromatographic analysis revealed the presence of a wide range of pigments which

clearly exhibits spatial variability. Identification was based on the retention time and peak shape

through fingerprint matching with known peak shape from the diode array of spectral library.

These were created by running pure individual pigment standards. The concentrations of the

pigments were computed from the peak areas (Figure 2 and 3). Nearly 12 algal classes were

recorded by HPLC in this monograph belonging to pico phytoplankton (<2 μm), nano

phytoplankton and (2-20 μm) micro phytoplankton (20-200 μm) which are rarely detected by

classical method (Table 3). The phytoplankton pigment composition was significantly different in

each of the sites. The identified carotenoids include fucoxanthin (diatoms), diatoxanthin and

diadinoxanthin (diatoms, dinoflagellates), alloxanthin (chryptophytes), lutein (green algae and

higher plants), zeaxanthin (cyanobacteria) and peridinin synthesized by dinoflagellats

(Johansen, 1974). Chl b commonly is ascribed to green algae while the β-carotene and Chl a are

more general indicators of total algal abundance.

Marker pigments were categorized into photosynthetic pigments (PSP) and photo

protective carotene or pigments (PPC), Photo synthetic carotene (PSC), Total accessory

pigments (TAcc). These macro variables are composed of pigment sums and ratios for

reconciling inquiries applied to data bases from different regimes. Total pigments and Pigment

ratio were calculated and presented in Table 4.

Photo protective carotenoids (PPC) = [Allo] + [Diad] + [Diato] + [Zea] + [Caro]

Photosynthetic carotenoids (PSC) = [But] + [Fuco] + [Hex] + [Peri]

Photosynthetic pigments (PSP) = [PSC] + [TChl a] + [TChl b] + [TChl c]

Total accessory pigments (Tacc) = [PPC] + [PSC] + [TChl b] + [TChl c]

Total pigments (TPig) = [TAcc] + [TChl a]

The maximum PPC, PSC and PSP were observed at station Champakara (S3) than at

the estuarine site. This intensity of photo protecting chlorophylls and carotenoids at these

stations derived from large density of hydropytes, diatoms and cynobacteria (zeaxanthin),

cryptophyta (alloxanthin), chrysophyta and algae (-β carotene). Bacillariophyta,

prymnesiophytes, chrysophyta, raphidophytes and several dinoflagellates indicated by

fucoxanthin contribute largest to the PSC budget. The high concentration of PSC resulted from

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the inherent chromatic and light intensity adaptation property of phytoplankton cells to specific

light conditions. The photosynthetic pigments (PSP) are constituted by floating flora and all the

photosynthetic algae excluding prochlorophyts, green algae, euglenophyta and plants. The

lower PPC, PSC and PSP at station S2 (Cheranellur) were due to the low production of flora. A

moderate value observed at estuarine sites reveals indicated the mesotropic nature. The

pigment derived ratios (Table 4) are dimensionless and are used for scaling. The ratios are

almost the same in all studied sites which indicate the authenticity in elution and identification of

each pigment and can be used for universal applications.

3.4 Taxonomic pigments and size structure indicesTo derive size-equivalent pigment indices Diagnostic Pigment [DP] criteria were

introduced by Claustre et al. (1994) then developed by Vidussi et al. (2001) and recently

extended by Uitz et al. ( 2006) which roughly correspond to the biomass proportions relevant to

pico-phytoplankton (less than 2μm), nano-phytoplankton (between 2 and 20μm) and micro-

phytoplankton (greater than 20μm); [pPF] or pBP, [nPF] or nBP, and [mPF] or mBP,

respectively. These variables are equivalent to the Fp ratio defined as the biomass ratio of

phytoplankton involved in new production over total phytoplankton and as such are equivalent to

the f- ratio (new production/ total production) (Claustre et al.1994; Eppley and Peterson,1979).

This means that together with size significance some of the criteria defined here also have a

functional/ biogeochemical significance. In addition to pigment indices, macro variables are

composed of pigment sums and ratios are in turn key for reconciling inquiries applied to

databases from different oceanic regimes.

The taxonomic composition of phytoplankton influences many change in the

biogeochemical processes. Therefore phytoplankton biomass and its composition over the

continuum of phytoplankton size were also performed simultaneously. The diagnostic pigment

(DP), mPF, nPF and pPF were derived by adopting the method of Vidussi et al.(2001) and Uitz et

al. (2006). These taxa are then grouped into three size classes (micro-, nano-, and

picophytoplankton), according to the average size of the cells. Generally seven pigments are used

as biomarkers of several phytoplankton taxa: fucoxanthin, peridinin, alloxanthin, 19-

butanoyloxyfucoxanthin, 19-hexanoyloxyfucoxanthin, zeaxanthin and total chlorophyll-b. In order to

know the contribution of each community in the study area, data reduction was performed and size

structure indices were made as follows (Rajdeep et al.2006).

Pico plankton proportion factor [pPF] = ([Zea] + [TChl b])/ [DP])

Nano plankton proportion factor [nPF] = ([Hex] + [But] + [Allo])/ [DP])

Micro plankton proportion factor [mPF] = ([Fuco] + [Peri])/ [DP])

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Diagnostic pigments (DP) = ([PSC] + [Allo] + [Zea] + [TChl b])

The Composition within each class is determined at each site from the pigment-derived

criteria (Figure 4). In estuarine site S1, the pico phytoplankton was predominant (70.8 %) then

nano plankton (15.0 %) and finally micro plankton (14.2 %). 55.5 % pico, 20.3 % nano and 24.2

% micro phytoplankton were observed at station S2. Similarly 68.6% pico, 17.1 % nano and

14.3 % micro phytoplankton was noted at Cheranellur ferry (S4). In Contrast to these sites a

proportional difference was observed at Champakara (S3), composed of highest portion by

micro (64.3 %) followed by pico (22.4 %) and nano (13.3 %) phytoplankton. Earlier studies have

confirmed that small sized phytoplankton is an integral component in environmental monitoring

assessment of the plankton community though their relative contribution to the total community

varies with the abundance of large-sized phytoplankton (Legendre and Fevre, 1988; Cermeno

et al. 2006; Raimbault et al. 1988). The greater abundance of pico plankton at estuary than

Champakra, probably reflects the difference in micronutrient availability. Generally, nutrient

enrichment favors the growth of large phytoplankton while the production of small phytoplankton

(nano-) is mainly controlled by microzooplankton (cilites and flagellates) grazing ( Riegman et

al.1993; Jyothibabu et al. 2006). The results confirm the predominance of micro plankton

cells and diatoms in the station Champakara. Upwelling, tidal activities, southwest and north

east monsoon and river discharges will often reflect nutrient enrichment in estuary which

ultimately results in spurts of smaller planktons. Typically large-sized phytoplanktons have

greater potential to export organic matter through a short classical food chain whereas the

small-sized phytoplanktons are utilized by complex microbial food webs that favor the recycling

of organic matter ( Cermeno et al. 2006). More over exogenous nitrates from anthropogenic

inputs are principally used by large phytoplankton (micro phytoplankton) and mainly contributes

to new production while regenerated forms of nitrogen (ammonia and urea) are the likely source

for pico planktons and nano planktons (Goldman, 1993; Vidussi et al. 2001). The station S3

was one among the high discharge area of anthropogenic waste containing easily available

exogenous nitrate. The period of study was close to the end of the summer period and these

processes was therefore more pronounced at the former stations, supporting larger pico

biomass plankton. A significant contribution was also seen from the nanoplankton community.

Picoplankton communities are generally contributed by prochlorophyte and cyanobacteria are

often investigated in tropical oceans and most likely, represent systems associated with

regenerated production (Claustre et al. 1994).

A linear regression analysis performed between DP and Chl a (Figure 5 a) found a

significant correlation (r2 =0.84,), indicating that DP can also act as a proxy of phytoplankton

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biomass. An existence of similar correlation between DP and Chl a has previously been

reported at eastern Mediterranean Sea at southern Benguela and at south west coat of India

( Vidussi et al. 2001; Rajdeep et al. 2006; Barlow et al. 2005). However the DP/Chl a ratio may

change with variations in nutrient dynamics and prevailing light condition, the DP can still be

used as a surrogate of phytoplankton biomass and for identifying general trends (Rajdeep et al.

2006). A good correlation were observed between PPP and PSP &PSP and TChl a ( r2=0.9

and 0.99 respectively) (Figure 5 b&c). Significant linear correlations were found between Chl a

and the photosynthetic carotenoid; photosynthetic pigment and photo protective carotenoids.

3.4 Pigment as a tool for recent ecological reconstructions Total carotenoids provide some indication of tropic status and also some evidence for

the relative importance of allochthonous vs autochthonous detritus in the sedimentary organic

matter. In decaying leaves and soil organic layers, chlorophyll derivatives are ultimately better

preserved than carotenoids, even though the initial breakdown of chlorophyll is faster.

Information about the bio-production and degradation of organic matter in estuarine system can

also be obtained from the Total Chlorophyll derivative to Total Carotene (CD/TC) ratios.

Intensive production of phytopigments or faster degradation of carotenoids in eutrophic system

may be the most important factor in the formation of higher CD/TC ratio in sediments

(Swain, 1985; Sanger, 1988). Algal decay favors preservation of carotenoids and diminishes

the ratios. Owing to the bulk organic matter of autochthonous character present in eutrophic

zone while in oligotrophic zone (ratios are higher) allochthonous detritus from the drainage

basin is the major source. High ratios reflect a greater proportional input of allochthonous

detritus and possibly a greater degree of aerobic decomposition of the autochthonous

sedimentary organic matter. These phenomena are compatible with swallowing and invasion

of the aquatic system. The CD/TC values in the sediment cores differ sharply site by sites

(Table 5). Overall a high value of CD/TC was observed at back water sites and lower values in

the estuary. Similar trend in the mesotropic system was also observed by many researchers.

The input of allochothonus detritus from the drainage basin and decaying of organic matter and

lower sedimentation would cause better preservation of chlorophyll derivative. As pointed out

by many authors these conditions favor degradation of carotenoids than that of chlorophyll

derivatives results in back water region (Swain, 1985; Sanger, 1988). Lower ratios (0.69) at

S1 and (0.48) S2 are probably indicative of eutrophic trend with autochthonous plankton

production prevailing. The back water stations are characterized by moderate carbon, high total,

inorganic and organic phosphate compared to estuarine station indicating the relatively

moderate rate of degradation. Preservation pattern in these two systems (S3 and S4) are

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different which were coupled with long residence time due to low flow conditions that result in

an accumulation of organic matter in the surf zones compared estuarine zone. This observation

has been further confirmed by CHN analysis reveals that the percentage of nitrogen was very

low but moderate carbon and sulphur detected in back water stations. These confirm the

predominance of grazing activities in these stations both Nitrifying bacteria and Sulphur bacteria

would act an important role in this process.

Sedimentary Chlorophyll degradation products (SCDP) preserved and embedded in the

sediment have been examined earlier in many surface sediment core studies, but their

knowledge value in paleoecology and paleolimnology remains still unraveled. The degradation

and preservation pathways which are seem to be initiated by rather subtle differences in the

sedimentary environment. Factors influencing the sediment pigment record include photo- and

chemical oxidation as well as herbivore digestive processes in the water column during

deposition and post depositional degradation in the sediment. However various organisms

including bacteria, fungi, protozoans crustaceans, oligochaetes, etc were abundant in the

sediment phase as pheophytin a, pheophorbide a, chlorophyllide a, or in various enzyme

systems, operating over differing pH ranges, confine to promote chlorophyll degradation. In this

study a two step slow mechanism was proven, first the loss of Mg2+ ions resulting the formation

of Pheophytin a and then phytol in the side chain resulting in the formation of Pheophorbide a

(Aneeshkumar and Sujatha, 2012).

Vertical profile of the chlorophyll a, its degradation pigments and carotene were found

from surface to a (0 cm) depth to 6 cm deep (figure 6). The core is immediately sub sampled in

the interval of 2 cm (0, 2, 4 and 6 cm). Highest concentration was recorded at surface and the

concentration of pigment decreases from top to bottom. Highest concentration of surface

chlorophyll recorded at Champakara (S3), ranging 8.42-1.42 μg/g; 3.83-1.1 μg/g at S4; 4.09- 2.2

μg/g at S1 and lowest inventory pigment 1.64 -0.7 μg/g were recorded at station S2 from top to

bottom. Pheophytin as expected was nearly double in concentration compared to Chl a

concentration. Vertically (0 to 6 cm depth) it ranges from 1.62 to 0.62; 0.86 to 0.23; 16.27to2.36

and 3.99to 0.56 μg/g at stations S1; S2; S3 and S4 respectively. Total Carotenoids varied as;

8.61- 1.32 μg/g at S1; 2.30 – 0.5 μg/g at S2; 13.46- 6.22 at S3 and 7.68 - 1.32 μg/g at S4. The

concentration of pigments below 6 cm characterized by lowest inventory pigment and often

difficult for separation in ordinary method due to leached materials of substantial degradation

caused by the daily tidal base of the sediment. A rapid decrease in concentration from top to

bottom was observed at station S1 and S2 up to 2cm than the degradation found to be slow

11

down. The anthropogenic input and high oxic condition would help rapid degradation of the

pigment in surface and sub surface layers but anoxic conditions in the sub surface layers

retarding the degradation of pigment in bottom layers. Pheophytin to chlorophyll ratios at these

stations showed variations (Figure 6). Pheopigment-a/chlorophyll-a ratio at station S2 and S3

indicates a net increase in ratio at 2 cm and indicate a rapid degradation of chlorophylls. Rapid

deposition of fresh sediment and oxic conditions may be the prevailing reason. Beyond this

layer, degradation was found to be lower due to low availability of oxygen. Anoxic conditions in

bottom waters results primarily from reduced water exchange due to the bathymetry and highly

stratified waters. The CD (Chlorophyll derivative) to TC (Total Carotenoid) ratio support the

Pheophytin a to Chla ratio. The increase in CD to TC ratio indicates the slow degradation of

chlorophyll or degradation of algae. The low ratio indicates the preservation of carotenoids.

The pigment profiles at Champakara (S3) showed a distinct subsurface maximum

attributes preservation showing major production changes due to deterioration of redox

conditions. The site is characterized by low oxygen content < 2mg/L .Good oxygen conditions

observed previously could be explained the lower pigment rate in the bottom of the sediment

core. However, no significant trend was noted in the pheopigment- a/chlorophyll-a ratio at this

site indicating the preservation regime a fairly constant. Degradation products of chlorophyll

origin, especially pheophytin a was heavily dominated in the sediment pigment record. Low

salinity at this site results mostly large macrofauna, e.g. polychaetes, influencing much in the

sediment. Re suspension of sediment from dredging process and high sedimentation rate

observed for one meter per annum at monsoon and average sedimentation rate at

0.21cm/annum were often reflected for the limited preservation of pigments in the estuary. Re

suspended material in this steep-sided estuary experiences an extended period of exposure to

high oxygen concentrations and light intensity in the water column which in turn causes high

degradation of pigments.

4. Conclusion The article introduces chemical oceanographer and related researches and insight in

to the Chemotaxonomic account on sediment associated pigments. The Pigment profiles in the

sediment varied widely. The pigment profile distribution inferring the influence of environment

changes, past productivity and exchange mechanism of nutrients across the different stations.

The degradation product of Chl a in this study were found to be Pheophytin a greater than

pheophorbide a establishes a recent and advanced degradation state of the sediment.

The other carotenoid pigments reflect specific distribution along the different sites. 12

algal class were identified form of this taxonomic pigment. The Pigment indices provide an

12

useful insight into the functioning of ecosystem as the larger phytoplankton are known to be

generally dominant in nutrient- rich, productive waters whereas smaller phytoplankton are more

abundant under mesotropic conditions. The biomass proportions derived from the marker

pigments and DP (Diagnostic pigment) as defined above indicate that the picoplankton

community contributed significantly to the biomass structure in the estuarine waters whereas

micro plankton appear to be most abundant in the Champakara canal . The order of distribution

of phytoplankton observed in the study area was micro plankton (38.6%), pico plankton (32.7%)

and nao plankton (24.6%).

Acknowledgment We thank Director, School of Marine Sciences, CUSAT, Cochin and

Dr. N. Chandramohanakumar, Head, Department of Chemical Oceanography, CUSAT for

providing laboratory facilities and suggestions.

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Table1. Description of sampling site and Salinity in the study area

Zone Salinity Station ID Name of Sampling Site

A <0.1 mg/L S1 

Muvattupuzha (Surface)

Muvattupuzha (Bottom)

B 0.02-14 mg/L

S2 Champakkara canal (Surface)Champakkara canal (Bottom)

S3 Cheranellur west (Surface)

S4 Cheranellur ferry(Surface)Cheranellur ferry (Bottom)

C 0.01-35.0 mg/L

S5 Port-Taj (Surface)S6 Port-jetty (Surface)    Port-jetty (Bottom)  

S7 Bolgatty (Surface)  Bolgatty (Bottom)

17

Table 2 .Phytoplankton abundance in the study area (microscopic examination) for the last 5

decades.

Phyto plankton Group/Species No Species

Time of Sampling Reference

Bacillariophyceae,Dinofla

gellates,Cyanophyceae,C

hlorophyceae &

Filamemntous algae 194

1958-1975

Devessy and

Bhattathiri ,1974;

Gopinathan, 1972;

Kumaran, and Rao,1975

(Periphytic algae)  

…….Sreekumar and Joseph.

1995

Bacillariophyceae 66

Chlorophyceae 8

Cyanophyceae 2

Bacillariophyceae 89

1999 ICMAM PD,2002Dinophyceae 31

Chlorophyceae 2

Cyanophyceae 1

Bacillariophyceae 582001-02 Selveraj et al ,2003

Dinoflagellates 2

Bacillariophyceae,Dinofla

gellates & others

89 (Pre monsoon)

65 ( Monsoon)2003 Madhu et al. 2007

Bacillariophyceae 28

2006-07

Sanilkumar, 2009.

Dinoflagellates 9

Chlorophyceae 2

Bacillariophyceae 54

2007-08Dinoflagellates 19

Chlorophyceae 2

18

Table 3 Pigment and corresponding Algal Class identified during the study by marker signature

pigment by HPLC (Jeffrey et al, 1997)

Pigments Name of Algal Class Size Fraction Size μm

Chl a,b Green algae Picoplankton < 2

Zeaxanthin Cynobacteria Picoplankton < 2

Canthaxanthin Eustigmatophyta Nanoplankton 2--20

Fucoxanthin Prymnesiophytes Nanoplankton 2--20

Diatoxanthin Chrysophyta Nanoplankton 2--20

Peridinin Dinophyta Nanoplankton 2--20

Fucoxanthin & Diatoxanthin Bacillariophyta Microplankton >20

Peridinin Dinoflagellates Microplankton >20

Alloxanthin Cryptophyta Microplankton 2-200

Lutein Chlorophyta Microplankton 2-200

9'-cis neoxanthin Euglenophyta Microplankton 20-200

Fucoxanthin Raphidophytes Microplankton 20-200

9'-cis neoxanthin Prasinophyta Microplankton 20-200

19

Table 4 photosynthetic pigments (PSP) and photo protective carotene or pigments (PPC), Photo

synthetic Carotene (PSC), Total accessory pigments (TAcc) and Ratios in the study area

Accessory pigmentsStations

S1 S2 S3 S4

PPC 2.77 5.2 14.81 0.82

PSC 0.16 0.44 3.04 0.05

PSP 2.39 3.39 39.25 1.34

TAcc 4.86 8.3 54.06 1.99

TPig 5.81 9.99 65.07 2.64

Ratios        

Tacc/TCha 5.12 4.91 4.91 3.06

PPC/TPig 0.48 0.52 0.23 0.31

PSP/TPig 0.41 0.34 0.6 0.51

PSP/Tcha 2.52 2.01 3.56 2.06

Table 5.The CD/TC values in the sediment cores.

Stations S1 S2 S3 S4

CD/TC 0.69 0.48 1.75 1.34

Classification of

Tropic status Autochthonous Autochthonous Allochothonous Allochothonus

20

Figure 1. Map of Cochin estuary showing sampling sites

21

Figure 2 Distribution of Marker pigments in the study area

22

Figure 3 Distribution of Chlorophylls and its degradation pigments

23

Figure 4 Distribution of Phytoplankton Composition with in each class at each site from the

pigment-derived criteria

24

Figure 5 A linear regression analysis performed between DP vs TChl a (a); PPP vs PSP (b) and

PSP Vs TCha (c).

25

Figure 6 The Vertical profile of the Chlorophyll a, its degradation pigments, carotene and ratios

from surface to 6 cm deep

26

Figures

Figure 1. Map of Cochin estuary showing sampling Sites

Figure 2 Distribution of Marker pigments in the study area.

Figure 3 Distribution of Chlorophylls and its degradation pigments.

Figure 4 Distribution of Composition with in each class is determined at each site from the

pigment-derived criteria.

Figure 5 A linear regression analysis performed between DP and Chl a.

Figure 6 The Vertical profile of the Chlorophyll a, its degradation pigments, carotene and

ratios from surface to 6 cm deep.

27

Tables

Table 1.Description of sampling site and Salinity in the study area

Table 2 .Phytoplankton abundance in the study area (microscopic examination) for the last 5

decades.

Table 3.Pigment and corresponding Algal Class identified during the study by marker signature

pigment by HPLC (Jeffrey et al, 1997)

Table 4 .Photosynthetic pigments (PSP) and photo protective carotene or pigments (PPC),

Photo synthetic Carotene (PSC), Total accessory pigments (TAcc) and Ratios in the study area

Table 5.The CD/TC values in the sediment cores .

28