1

Author version: J. Environ. Monit., vol.13(3); 2011; 614-620

Impact of salinity and pH on phytoplankton community in a tropical freshwater

system: An investigation with pigment analysis by HPLC

Parthasarathi Chakraborty*1, Tamoghna Acharyya1, P. V. Raghunadh Babu1 and

Debasmita Bandhyopadhyay1

1
National Institute of Oceanography (CSIR), India, 8-44-1/5, Plot No. 94, Chinawaltair,
Visakhapatnam, 530003, Andhra Pradesh, India, e-mail: [email protected]

Abstract:
An in vitro study was carried out to understand the effects of salinity shock and variation in pH on
phytoplankton communities in a tropical freshwater system of Godavari River (a major peninsular
river of India). The effects were assessed by pigment analysis using HPLC technique. Subtle changes
in the salinity of freshwater by one practical salinity unit (PSU) completely removed green algae
from the system and allowed cyanobacteria to come into dominance. The cyanobacteria were found
to tolerate higher osmotic stress until salinity reached a PSU of 16. The higher salinity tolerance
range of the cyanobacteria was attributed to enhanced synthesis of zeaxanthin as a protective
xanthophyll against the osmotic stress. However, the effect of pH was not as dramatic as salinity
where green algae and cyanobacteria from the same freshwater system showed a considerable
acclimation towards fluctuating pH. These findings are environmentally relevant to understand the
likely impact of salt water intrusion and pH variation on phytoplankton communities in a tropical
freshwater system.

Key words: Salinity shock; sea water intrusion, phytoplankton community; tropical
freshwater, pH shock
 2

Introduction:

Two potentially important factors that can regulate estuarine phytoplankton community and
biomass are salinity and pH. Salinity in an estuary is a dynamic entity that is chiefly regulated by the
river discharge, local rainfall and tidal amplitude. In an unperturbed estuary, different groups of
phytoplankton communities are adapted to withstand a certain range of salinity and therefore show
complex pattern of distribution along the salinity gradient of the estuary from the head to the mouth
1, 2
.
3, 4
It was reported that the distribution of phytoplankton along estuary gradients tends to favour
1
cyanobacteria and chlorophytes in brackish waters. However, Kies reported that mid-to-high
salinities in an estuary favour dinoflagellates and diatoms. Species diversity usually becomes very
2
low at high salinities. Rijstenbil reported that high salinities can be a lethal limit for many
phytoplanktons in estuaries.

Guillard 5 reported that the salinity change can result in osmotic stress on cells, uptake or loss of ions
and effects on the cellular ionic ratio in phytoplankton. To maintain osmotic balance due to frequent
alterations in salinity level result in an increased respiratory activity in phytoplankton. Inhibitory
effects on physiological processes of phytoplankton can follow changes in salinity.

Alterations in salinity level frequently result in increased respiratory activity to maintain osmotic
balance 6. A rise in NaCl levels in the medium has been shown to increase respiration rate and
7-9
decrease photosynthetic O2-evolution for two species of Scenedesmus . It has also been showed
that an increase in salinity immediately reduced rates of net carbon fixation by Nitzschia americana.

Much of our current knowledge of salinity effects is based on laboratory studies of cultured algae 10-
12
and on observations from field studies. Surprisingly only few studies have experimentally
determined the effects of changes in salinity on phytoplankton community structure for naturally
occurring phytoplankton assemblages.

Effect of salinity shock on species diversity of natural assemblages of largely freshwater

13
phytoplankton was studied by Floder and Burns . In their study, the transition to brackish water
conditions resulted in reduced phytoplankton diversity, especially during the first few days of
incubation. They attributed this effect to osmotic stress.

The salinity tolerance of phytoplankton differ and based on their tolerance extent they are grouped as
euryhaline (able to tolerate wide range of salinity) and stenohaline (having very narrow salinity
tolerance range) species. Any unnatural change in the salinity is strong enough to affect stenohaline
 3

phytoplankton sp. and could irreversibly change local phytoplankton community structure and
establish a new stable climax community.

Any drastic change in phytoplankton community (which is at or near the bottom of the food chain in
14-16
the aquatic system) can have serious ecological impact . Godavari estuary is one of the major
estuaries of the Indian east coast and recently a huge natural gas reserve has been identified in its
17
basin . Thus, it is expected that heavy dredging activity will be performed in the near future that
may facilitate tidal salt wedge to intrude further which may have serious ecological impact.

Rivers and estuaries are also prone to pH fluctuation due to poor buffering capacity of riverbed clay
mineral and fresh water and periodic tidal mixing with alkaline sea water. Variation in pH also can
affect phytoplankton growth in a number of ways 18-20. It can change the clay buffering system thus
influencing the availability of trace metals and essential nutrients 21, and inflict direct physiological
effects at extreme levels.

The main stimulus in this investigation was to find out how freshwater phytoplankton communities
respond when they encounter different levels of salinity shock and pH change. The distribution of,
and variations in, the phytoplankton community was assessed by quantitative determination of their
class specific marker pigments. Highly significant linear regressions were reported for chl a-total
biomass, fucoxanthin-diatoms, lutein-green algae and zeaxanthin-cyanophytes 22. High Performance
23, 24
Liquid Chromatography (HPLC) was used to identify and quantify these phytoplankton
communities by their pigment markers.

This study is environmentally relevant in global scale (a) to understand the likely impact of salt water
intrusion by dredging and predicted sea level rise on local phytoplankton community in estuary (b)
recognizing the effect of pH changes in freshwater.

Experimental Section

Study Area

Godavari is the third largest river in India. It has formed two major distributaries; Goutami and
Vasishta before debouching into the Bay of Bengal in the east coast of India. Freshwater sample was
collected at Gautami Godavari as it carries the majority of the discharge.

Sampling was done further upstream at Alamuru (40 km upstream from Yanam as shown in Figure
1) that represents typical freshwater which is not affected by tidal intrusion at any time throughout
the year. As high discharge in the river overshadows all other potentially important factors 25 that
may regulate phytoplankton production and succession, the sampling was carried out in September,
 4

when river discharge virtually stops and tidal effect plays a major role in the salinity values of the
estuary in Yanam but not enough to impart its effects as far upstream as Alamuru

Sampling and analysis

Surface water samples from Godavari River were collected at Alamuru station by using 5-L Niskin
bottles and finally transferring it to 20 L Nalgene bottles. Before starting the incubation experiment,
the exact salinity of the water samples were measured by Autosal (Guild line Autosal 8400B
salinometer) in ‘practical salinity units’ (PSU) which defines salinity in terms of the conductivity
ratio of a sample to that of a solution of 32.4356 g of KCl at 15◦C in a 1 kg solution according to the
definition of the International Association for the Physical Sciences of the Ocean (IAPSO). pH was
measured by a Metrohm auto titration unit.

99.5% pure sodium chloride (NaCl) from Merck India Ltd was used for salinity manipulation. The
salt treatment will be indicated by means of the term ‘NaCl’, which is the main component of the sea
salt.

Water samples were distributed into fourteen 1L bottles (NALGENE), five bottles in one set was
manipulated by adding NaCl in such a way that the salinity of freshwater of Godavari changed to 1,
2, 4, 8 and 16. To manipulate the salinity of the river water, NaCl was dissolved in a glass beaker
with the same river water and added all at once to the original water sample. Salinity change was
allowed to take place suddenly instead of gradual dissolution of crystal based on the hypothesis that
salinity change in the natural system might takes place by sudden intrusion of tidal front having high
salinity gradient.

Another set of five bottles were pH manipulated by adding ultrapure HCl ( Fluka) and NaOH which
resulted in pH values for the water solutions of 6.0, 6.5, 7.5, 8.0, 8.5. The remaining 2 pairs of
bottles were kept as controls.

All the experimental solutions (after adjusting the salinity and pH) were incubated for 5 days (120
hours). Incubation experiments were carried out (in triplicates) in transparent bottles under the
natural light source. Temperature was found to be ~25±2oC during the incubation experiment under
the natural condition.

Filtration and Extraction:

After five days of incubation, 0.5L of samples was filtered through Whatman GF/F filter paper
(0.7μm nominal pore size and 47mm diameter) under reduced vacuum. Filters were dabbed in tissue
paper to remove water. Each filter was cut into small slivers and was placed in heavy-walled 10 ml
 5

amber coloured culture tubes (Shimadaju: P/N 638-41462). 4mL of 90% Acetone was added to the
tubes using a dispensette. Each tube was covered and placed in an ultrasonic bath filled with ice
slurry to prevent heat accumulation. Ultrasonification (Cole Parmer, Model: 08893-26) of samples
enables disruption of cells and facilitates the extraction of the pigments. All the tubes were then
placed in a freezer (-20oC ) overnight. Sample slurry was then vortexed (GeNei India Pvt. Ltd.) and
clarified by pushing the contents through a nylon HPLC syringe catridge filter (PAL, P/N 4118) with
0.45um pore size.

HPLC Analysis

Analysis was performed by using Agilent 1200 HPLC system equipped with quarternary pump,
autoinjector, Peltier column thermostat, temperature controlled autosampler and chemstation
software. Pigments were detected with the diode array detector using 450 and 665 nm wavelength
(20 nm bandwidth was used in both cases). 665 nm was used to quantify chlorophyll-a, divinyl
chlorophyll-a, chlorophyllide-a, phaeophorbide-a and phaeophytin-a as they respond similarly and
strongly in this wavelength. All other carotenoids and xanthophylls were detected and quantified at
445nm.

An injector program was optimized to deliver sample extract and buffer composed of 28 mM
aqueous tetrabutyl ammonium acetate (TBAA) (AR Grade, Fluka) at pH 6.5 and methanol (GC
Assay 99.7% pure,Merck) in 90: 10 ratio. The sample extract and buffer was mixed automatically
within the sample loop which enabled effective retention of early eluting chlorophylls and lessened
26
peak distortion. The method proposed by Heukelem was adapted for pigment analysis during the
study. The method outlined below.

Column: ZORBAX eclipse XDB-C8, 4.6 × 150 mm (diameter by length); PN: 963967-906

Gradient: Binary gradient elution

A: 70/30 methanol/TBAA pH 6.5

B: methanol
Linear gradient from 5-95% solvent B in 22 min, isocratic hold of 95% solvent B
From 22-29 min, return to 5% solvent B at 31 minutes, equilibration for further
5 minutes (31-36 minutes)
Injection volume: 200 µl
Oven temperature: 60 degree celcius
Solvent flow rate: 1.1 ml/min
 6

In this study, zeaxanthin was used as a marker of cyanobacteria, fucoxanthin for Diatoms, lutein for
green algae and Chl a as overall total phytoplankton biomass. Presence of Merismopedia sp.
(colonial and coccoid cyanobacteria) in Godavari estuary (our unpublished data) gave us additional
confidence of using zeaxanthin as their marker pigment and to trace their biomass change.

Results and Discussion

The pH of the sample was measured immediately after collecting the sample by a Metrohm auto
titration unit and found to be 8.15. The pH of the sample was measured prior to the incubation
experiment and after the incubation of 60 hrs. No variation in the pH value was observed. The total
organic carbon (TOC) concentration was found to be 12.0 ± 0.6 mg. L-1. The major ion
concentrations such as NO2-1, PO4 3-, NH4+ ions were found to be 4.10 µM, 0.60 µM, and 3.40 µM
respectively. The total concentrations of Na, K, Ca and Mg obtained from the literature were (0.70 ±
0.17) mM, (0.18 ± 0.05 mM, (0.63 ± 0.13) mM, and (0.41 ± 0.12) mM respectively in Godavari
River. However, it is important to note that the concentrations of major cations presented here are the
27-29
average values obtained from the literature . Total trace metal concentrations in the natural
sample were analysed by ICP-MS (X-SERIES II, Thermo Scientific) and all the biologically
important metals were present in the range of nM concentrations. This indicates that the surface
water collected in Godavari River at Alamuru station (with no discharge from Dawleshawaram Dam)
was not contaminated with trace metals.

Effect of salinity on total biomass and phytoplankton communities in freshwater system.

Figure 2a shows that the maximum phytoplankton biomass in terms of Chl-a was in the control and
was equally contributed by green algae and cyanobacteria (as revealed from the zea/Chl-a and lut/
Chl-a ratio as shown in Table 1.

Increasing concentration of NaCl in the salt manipulated bottles caused consecutive decrease in the
total biomass and the effect became most pronounced at the salinity of 16.

Pigment derived phytoplankton community structure revealed some interesting features. No signal
was detected by HPLC for lutein which is a representative marker pigment for green algae in all the
salt treated samples at salinity of 1. This indicates that green algae which was present initially in
freshwater (concentration of lutein in control was 2.7 ± 0.1 mg.m-3) were very sensitive to subtle
changes of salinity. However, the concentration of zeaxanthin enormously increased with the
increasing salinity and maximum concentration of 25.6 ± 1.3 mg.m-3 was recorded at the salinity of 4
( Fig 2a, Table 1).Further treatment with higher concentration of NaCl (at the salinity of 8) reduced
the concentration of zeaxanthin compared to the salinity at 4. Phytoplankton biomass was severely
 7

reduced at a salinity of 16 PSU (Fig. 2a) and indicating their salinity tolerance maxima.
Importantly, in all the salt treated samples, zeaxanthin was higher than the control. A striking shift in
phytoplankton community took place where green algae sp. were completely replaced by more salt
tolerant cyanobacteria (Merismopedia sp.) whenever there was an increase of salinity in the
freshwater. In a similar laboratory based salinity enhancement experiment in Myall lakes system,
Australia 30, the reduction in the abundance of green algae at the salinity of 4-8 and Merismopedia as
the most abundant taxa at the salinity of 16 was reported. In our investigation, the tolerance maxima
of green algae and cyanobacteria was found to be 1 and 16 respectively which shows that the fresh
water phytoplankton community in Godavari river are quite sensitive towards salinity hikes.

Scatter plot of zeaxanthyn: Chl-a ratio against respective salinity manipulations gave a linear fit with
R2 value of 0.97 (Fig 2b). Table 1 reveals that increase in the zeaxanthin concentration was always at
the cost of Chl-a, which is clearly the most plausible explanation for the linear fit. Another plausible
reason for the reduction in over all Chl-a could be explained by the complete removal of green algae
which was initially contributing to Chl-a in the control but not at all in the salt manipulated samples.
It is known that intracellular reactive oxygen species (ROS) formation is triggered in hyper osmotic
condition 31. As a part of the antioxidative mechanism algae can increase the ratio of xanthophylls to
32, 33
light harvesting pigments . Zeaxanthin and ß carotene are such non-photosynthetic carotenoids
which protect the photosynthetic centre against the destructive singlet oxygen.

It is evident from Table 1 that with the increasing salinity level cyanobacteria combat the salinity
stress by synthesizing more zeaxanthin. Energy cost associated with the zeaxanthin synthesis is
probably balanced by the adaptability of cyanobacteria to tolerate wider range of salinity fluctuation
that helps them to occupy the niche left by the green algae (green algae disappeared from the system
at the salinity of 1). It is apparent that increase in zeaxanthin concentration by cyanobacteria to
combat the progressively higher salinity stress is not indefinite and after a certain salinity maxima
(16, in this study) the population can not tolerate the stress. The highest zeaxanthyn: Chl-a value
(Table 1) was observed at the salinity of 16. This observation suggests that thriving population of
cyanobacteria could only survive by synthesizing higher than average concentration of zeaxanthin
per cell.

ß-carotene is reported as a minor (1-10 %) marker pigment of cyanobacteria. In HPLC

chromatogram ß-carotene also found to follow similar trend like zeaxanthin however the former was
11
not quantified as it is nonspecific and widely distributed among the phytoplankton taxa .
Apparently ß-carotene also similarly responds with salinity stress and is synthesized by
cyanobacteria as a protective carotenoid.
 8

A second set of laboratory based incubation experiment was simultaneously conducted to understand
the fate of the fresh water phytoplankton in brackish (intermediate salinities of 4 and 6) water by
mixing virtually fresh (with salinity value of 0.02) upstream river water from Alamuru with the aged
coastal water from the Bay of Bengal (with salinity of 35) in appropriate proportion. Another aim of
this experiment was to compare the phytoplankton community and biomass in salt treated samples
with that of the manually mixed water parcels of different salinities. Table 1 shows that the mixed
water samples at the salinity of 4 accumulated phytoplankton biomass (Chl-a, 4.8 ±0.2 mg.m-3) was
comparable to salt added sample at salinity 4 (Chl-a, 5.0± 0.3 mg.m-3). Chl-a concentration
decreased to 3.5 ± 0.2 mg.m-3 at the salinity of 6. Pigment analysis revealed that the major
phytoplankton community is cyanobacteria followed by diatom as presented in Table 1 (identified
from the marker pigment zeaxanthin and fucoxanthin, respectively). No signal of lutein was
observed in the chromatogram indicating the disappearance of green algae once again at elevated
salinity. Presence of diatom in the salt manipulated mixed samples could only be explained by the
proliferation of diatom propagules in optimal salinity and nutrient as intact diatom cell neither could
come from 34-36 fresh water side (no signal of fucoxanthin was observed in fresh water) nor from the
aged coastal water. Higher phytoplankton biomass accumulation in the second experimental set
could be explained by the fact that diatom also contributed to Chl a along with cyanobacteria. In
contrast, in case of the salt treated samples (with salinity of 4, 8, 16) only lower numbers of
cyanobacteria could proliferate by synthesizing higher concentration of zeaxanthin at the expense of
Chl- a.

Daily time series observation study (2007-2008) at the head of this estuary revealed that
phytoplankton community is dominated by cyanobacteria followed by diatoms whenever the surface
salinity goes beyond > 1 (unpublished data).Such community scenario is in accordance with our
laboratory experiment and strengthens the hypothesis that intrusion of saline water can change the
phytoplankton community structure which may have serious ecological impact.

Effect of pH on total biomass and phytoplankton communities in freshwater system.

Figure 3 and table 2 shows the effect of pH on Chl-a concentration in the freshwater system.
Concentration of Chl-a gradually increased from acidic to neutral and reached at its maxima 9.35
mg.m-3 at the pH of 8.15. Carotenoid signature revealed that in all the pH manipulated samples lutein
concentration was much higher than zeaxanthin (average concentration of zeaxanthine was found to
be 2.16 ± 0.57 mg.m-3) indicating that green algae was dominating phytoplankton compared to
cyanobacteria. In the control having pH 8.15 lutein: zeaxanthin ratio was 1.08. At more alkaline pH
of 8.5, the ratio was not significantly different while in the pH range from 6 to 7.5, the average ratio
 9

of lutein: zeaxanthin was 7.57 ± 0.18 (Average ± 2 SD). This result is completely contrasting from
that of salinity manipulation where cyanobacteria always dominated the community. It appears that
green algae inhabiting in the fresh river water system have evolved through the ages to tolerate wide
fluctuation of pH ranges that is more common due to the poor buffering capacity of river bed clay
and fresh water. However the same green algae could not tolerate even subtle changes in salinity as
Alamuru station (from where the sampling was done) is still free of influence from tidal salt wedge.

There is some obvious enhancement in zeaxanthin concentration in the alkaline pH range which
prompts us to consider the possible adaptability of estuarine cyanobacteria with the periodic tidal
mixing with the alkaline seawater although their response was not as strong as observed in case of
salt treated samples.

It is quite evident from this study that phytoplankton community is affected by sudden salinity
changes. Complete exclusion of green algae in the salinity enhanced samples indicates that they are
stenohaline sp having very narrow salt tolerance range. This is probably because of the fact that the
green algal sp. present in the Godavari river estuary does not have potent osmoregulatory mechanism
thus vulnerable to local extinction for subtle salinity increment. Cyanobacteria seem to be well
adapted to withstand such salinity fluctuation as they are capable of enhancing the production of
zeaxanthin to combat ROS. Consequently, cyanobacteria can completely replace green algae in
freshwater because of intrusion of saline water in estuary. This shift in phytoplankton community
(which is at or near the bottom of the food chain in the aquatic system) can have serious ecological
impact. Cyanobacteria are well known for their nuisance bloom formation. There is no
documentation of toxic bloom formation by cyanobacteria in Godavari river estuary. However, it is
not possible to rule out possible consequence in the higher tropic level. Ultimate outcome could also
vary in different estuaries of the world depending on the salinity tolerance level of the local
phytoplankton and the extent of salt water intrusion.

On the other hand green algae seemed quite adaptable to pH fluctuation and are potentially more
tolerant to any natural or anthropogenic pH change. Further studies are being carried out to
understand the complex interaction of other biogeochemical factors with salinity and pH to shape
the phytoplankton community structure.

Acknowledgements

Authors are thankful to the Director, NIO for his encouragement and support. Author (P.
Chakraborty) dedicates this work to the memory of his beloved PhD supervisor Late Prof C. L.
Chakrabarti from Carleton University, Canada, who passed away on 1st January 2010.
 10

References
1. J. W. Rijstenbil, Netherlands Journal of Sea Research, 1988, 22, 291–300.

2. L. Kies, Limnologica, 1997, 27, 55–64.

3. M. Nakanishi, M. Monsi, Journal of Faculty Science University Tokyo, 1965, III 9, 19–42.

4. K. Muylaert, K. Sabbe, Journal of Marine Systems, 1999, 22, 133–149.

5. R. R. L. Guillard, 1962. Salt and osmotic balance. In Lewin, R.A. (ed.), Physiology and
Biochemistry of Algae: 929 Academic Press, New York and London.

6. S. Z. Qasim, P. M. A. Bhattathiri , V. P. Devassy, Marine Biology, 1972, 12, 200–206.

7. A. Mohamed, A. Shafea, Biologia Plantarum, 1992, 35, 423–430.

8. A. Flameling, J. Kromkamp, Journal of Plankton Research, 1994, 16, 1781–1791.

9. R. L. Miller, D. L. Kamykowski, Journal of Plankton Research, 1986, 8, 305–315.

10. L. M. Brown, Journal of Phycology, 1982, 18, 483–488.

11. S., R. Fujii, S. Yamamoto, Nakayama, S.Yasui , P. A. Broady, Phycological Research, 1999,
47, 65–69.

12. E. Thessen, , Q. Dortch, M. L. Parsons , W. Morrison, Journal of Phycology, 2005, 41, 21–
29.

13. S. Floder, C. W. Burns, Journal of Phycology, 2004, 40, 54–61.

14. S. R. Carpenter, J. F. Kitchell, J. R. Hodgson, P. A Cochran, J. J. Elser, M. M.Elser, D. M.

Lodge, D. Kretchmer, X. He, C. N. von Ende, Ecology, 1987, 68, 1863-1876.

15. R. T. Paine, American Naturalist, 1966. 100, 65-75.

16. G. A. Polisand, D. R. Strong, American Naturalist, 1996. 147, 813

17. http://www.ril.com/html/business/exploration_production.html

18. C. Y.Chenl, E. G. Durbin, Marine Ecology Progress Series, 1994, 109, 83-94.

19. Locke, W. G. Sprules, Hydrobiologia, 2000, 437, 1573-5117.

20. P. J. Hansen, Aquatic microbial ecology, 2002. 28, 279-288.

21. P. Chakraborty, Analytica Chimica Acta, 2010, 659, 137-143.

22. S.W. Jeffry, R.F.C Mantoura, S.W. Wright, Eds. 1997. 'Phytoplankton Pigments in
Oceanography: Guidellines to Modern Methods', UNESCO: Paris.
 11

23. R.F.C. Mantoura, C. A. Llewellyn, Analytica Chimica Acta, 1983. 151, 297-314

24. S.W. Wright, S.W. Jeffry, R.F.C. Mantoura , C.A. Llewellyn, T. Bjǿrnland, D. Repeta, N.
Welschmeyer, Marine Ecological Progress Series, 1991, 77, 186-196

25. V.V.S.S. Sarma, S.N.M. Gupta, , P.V.R. Babu, , T. Acharya , N. Harikrishnachari, K.

Vishnuvardhan, N.S. Rao, N.P.C. Reddy, V.V. Sarma, Y. Sadhuram, T.V.R.Murty, M. D.
Kumar, Estuarine, Coastal and Shelf Science, 2009, 85,515-524

26. V. L. Heukelem, C. S. Thomas, Journal of .Chromatography A, 2001, 910, 31-49.

27. P. Duggempudi, 1995. Behaviour of some dissolved constituents in estuarine and shelf
regions of Godavari (Bay of Bengal), M.Phil.Thesis, Andhra University, India.

28. P. Duggempudi, 1998, Studies on the distribution and behavior of some chemical constituents
in riverrine, estuarine and coastal waters of Godavari (Bay of Bengal), Ph.D. Thesis, Andhra
University, India.

29. G. Biksham, V. Subramanian, Journal of Hydrology, 1988. 103, 375-392.

30. M. Redden, N. Rukminasari, Hydrobiologia, 2008, 608, 87–97.

31. N. P. Rout, B. P. Shaw, Plant Science, 2001. 160, 415–423.

32. G. N. Harris, D. J. Scanlan, R. J. Geider, Journal of Phycology, 2005. 41, 851–62.

33. W. H. Van de Poll, , M. van Leeuwen, J. Roggeveld, A. G. J. Buma, Journal of Phycology,

2005, 41, 840–50.

34. C. Anil, , S. Mitbavkar, , M. S. D'Silva , S. Hegde, P.M. D'Costa, S. S. Meher, D.

Banerjee, Journal of Experimental Marine Biology and Ecology, 2007, 343, 37-43

35. M. R. McQuoid, K. Nordberg, Estuaries and Coasts, 2003, 26, 927-937

36. B. Worm, H.K. Lotze, U. Sommer, Oecologia, 2001, 128, 281–293

 12

Captions and Legends for Figures

Figure 1 Geographical location of the study area

Figure 2. (a)Variation in chl-a, zeaxanthin and lutein concentrations obtained from phytoplankton in
Godavari River at different salinity shock ; (b) Variation in zeaxanthin /chl-a at different salinity
shock

Figure 3. (a)Variation in chl-a, zeaxanthin and lutein concentrations obtained from phytoplankton in
Godavari River at different pH; (b) Variation in zeaxanthin /chl-a and lutein /chl-a ratios at different
pH
 13

Dowleswaram Dam
Kakinada
Bay
Alamuru

Yanam
Goutami Godavari

Kotipalli
Vasistha
Godavari
 14

Concentrations of marker pigments (mg.M-3)

30
20
chl-a
Zeaxanthin 2
R = 0.97
22.5 Lutein 15

Zeaxanthin : chl-a
15 10

7.5 5

0 0
Control 1 2 4 8 16 0 5 10 15 20
Salinity (PSU) Salinity (PSU)

(a) (b)

Figure 2
 15

Leutine : chl-a
Concentrations of marker pigments (mg.M-3)

2.00
20
Chl-a
Zeaxanthin

Marker pigment : chl–a

Leutine 1.50
15

10 1.00

5 0.50 Zeaxanthin : chl-a

0 0.00
Control 6 6.5 7.5 8 8.5 5.75 6.25 6.75 7.25 7.75 8.25 8.75
pH
pH
(a) (b)

Figure 3
 16

Table 1. Variation in concentrations of chl-a, zeaxanthin, lutein and fucoxanthin obtained from
phytoplankton in Godavari River with respect to the different salinity shock

Salinity (PSU)
Chl-a (mg. Zeaxanthin
Zeaxanthin Leutine Leutine/Chl-a
(manipulated by the
M-3) ( mg. M-3) /Chl-a ( mg. M-3)
adding NaCl)

Control 9.4 ± 0.3 2.5 ± 0.1 0.3 2.7±0.1 0.3

1 7.0 ± 0.2 12.3 ± 0.6 1.8 0.0 -

2 7.0 ± 0.2 20.6 ± 1.0 2.9 0.0 -

4 5.0 ± 0.3 25.6 ± 1.3 5.1 0.0 -

8 2.5 ± 0.1 15.9 ± 0.8 6.3 0.0 -

16 0.2 ± 0.0 3.6 ± 0.2 18.0 0.0 -

Salinity (PSU)
(manipulated by the Chl-a Zeaxanthin
Zeaxanthin Leutin Fucoxanthin
mixing freshwater ( mg. M ) -3
( mg. M-3) /Chl-a (mg. M-3) (mg. M-3)
and aged sea water )

4 4.8 ± 0.2 23.4 ± 1.2 4.9 0.0 0.5 ± 0.1

6 3.5 ± 0.2 10.2 ± 0.5 2.9 0.0 2.5 ± 0.1

Values presented as the mean ± 2 × standard deviation, n = 3

All the data are presented with 95.5% confidence interval.
 17

Table 2. Variation in concentrations of chl-a, zeaxanthin, lutein and fucoxanthin obtained from
phytoplankton in Godavari River with respect to the varying pH

Chl-a Zeaxanthin Zeaxanthin / Chl-a Leutine Leutine/Chl-a

pH
(mg/M3) (mg.M-3) (mg.M-3)

6.0 7.6 ± 0.4 1.8 ± 0.2 0.24 13.8 ± 0.7 1.82

6.5 7.5 ± 0.4 1.6 ± 0.1 0.21 11.7 ± 0.6 1.55

7.5 7.8 ± 0.4 1.9 ± 0.1 0.24 14.5 ± 0.7 1.86

8.0 8.6 ± 0.4 2.5 ± 0.2 0.29 13.1 ± 0.8 1.51

8.15 (Control) 9.4 ± 0.5 2.5 ± 0.2 0.27 2.7 ± 0.1 0.82

8.5 6.9 ± 0.3 2.5 ± 0.1 0.36 2.7 ± 0.2 0.38

Values presented as the mean ± 2 × standard deviation, n = 3

All the data are presented with 95.5% confidence interval.