The spatial distribution of phytoplankton pigments in the surface sediments of the Kongsfjorden and Krossfjorden ecosystem of Svalbard, Arctic

Regional Studies in Marine Science 31 (2019) 100815

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The spatial distribution of phytoplankton pigments in the surface sediments of the Kongsfjorden and Krossfjorden ecosystem of Svalbard, Arctic ∗

Archana Singh , K.P. Krishnan National Centre for Polar and Ocean Research, Headland Sada, Vasco-Da-Gama, Goa 403804, India

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Article history: Received 22 January 2019 Received in revised form 12 June 2019 Accepted 22 August 2019 Available online 26 August 2019 Keywords: Biomarker Photoprotection Pigment degradation Pigment preservation Primary productivity Paleoecology

a b s t r a c t Kongsfjorden–Krossfjorden twin fjord ecosystem in Svalbard (Arctic), is facing enhanced warm Atlantic water intrusion from ocean as well as intensified melting from glaciers. It is an interesting ecosystem to study phytoplankton productivity under the two opposing processes. For phytoplankton productivity, pigments are used as potential photosynthetic biomass indicators and taxonomic biomarkers. However, due to labile nature of pigments, degradation — preservation distribution must be investigated to form essential framework for biomarker studies. With this motivation, we have undertaken the present work to study the spatial distribution of surface sedimentary pigments in the twin-fjord ecosystem. The pigments were analysed using high-performance liquid chromatography coupled with diode array detector. A total of fifteen pigments were identified, which belonged to different classes — chlorophylls, pheopigments and carotenoids. Chlorophyll-a and pheophorbide-a were major pigments with concentration up to 700–800 ng g−1 dry weight of sediment. Among carotenoids, fucoxanthin, diatoxanthin and prasinoxanthin were abundant with maximum concentration recorded up to 400– 600 ng g−1 dry weight. The distribution of the pigments varied significantly between the two fjords as well as in different regions of the fjord. Most of the pigments were significantly high in their concentration at Krossfjorden stations. Within Kongsfjorden, outer region showed higher concentration of pigments than inner. However, preservation was lesser for the outer fjord pigments than inner as confirmed by various pigment indices. Further, the overall distribution of phytoplankton communities derived from taxonomic biomarker pigments in Kongsfjorden–Krossfjorden sediments, was coherent with that of water during spring and summer. The distribution was dominated by diatoms, dinoflagellates, prasinophytes and prymnesiophytes. The surface sedimentary pigments were found capable of providing information about phytoplankton biomass and composition under environmental conditions existing in the region. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Fjord is a deep, narrow and elongated arm of ocean surrounded by steep land on three sides. Fjords are high latitude equivalent of estuaries, representing transition area from streaming glaciers to ocean. The opening towards ocean is called mouth and innermost region is called head of the fjord. At head, the fjord is fed by fresh water from glaciers and rivers, and subsequently merge into the ocean at mouth. Subsequent mixing of different water masses give rise to physicochemical gradients in fjord properties for example water temperature, salinity, nutrients,

Abbreviations: DP, Diagnostic Pigments; DW, Dry Weight; HPLC, High Performance Liquid Chromatography; PAR, Photosynthetically Active Radiation; PI, Photoprotection Index; PPC, Photoprotective carotenoids; PSC, Photosynthetic carotenoids; Tchl, Total chlorophyll-a ∗ Corresponding author. E-mail address: [email protected] (A. Singh). https://doi.org/10.1016/j.rsma.2019.100815 2352-4855/© 2019 Elsevier B.V. All rights reserved.

sedimentation and suspended matter (Schüller and Savage, 2011; Quiroga et al., 2016). The suspended matter consists of inputs from melting glaciers, rivers, chromophoric dissolved organic matter, phytoplankton and detrital matter (D’Angelo et al., 2018). The suspended matter influences optical properties of water column (Mascarenhas et al., 2017). It increases turbidity of surface water, especially at the inner region of the fjord (Hanelt et al., 2001). Subsequently, lightentering the water column through surface affects primary production at sub-surface depths (Madden, 1992; Chaudhuri et al., 2012). Thus, the primary production is vulnerable to the gradients in suspended matter and turbidity across the fjord (Sdrigotti and Welker, 2002; Blasutto et al., 2005). The primary production and associated phototrophic communities can be identified using phytoplanktonic pigments (Wiltshire, 2000). Bulk measurements of these pigments use spectrophotometry and fluorometry techniques (Lorenzen, 1967; Jeffrey and Humphrey, 1975). Individual communities can be identified using single-cell analysis techniques — microscopy and

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A. Singh and K.P. Krishnan / Regional Studies in Marine Science 31 (2019) 100815

Abbreviations DP DW HPLC PAR PI PPC PSC Tchl

Diagnostic Pigments Dry Weight High Performance Liquid Chromatography Photosynthetically Active Radiation Photoprotection Index Photoprotective carotenoids Photosynthetic carotenoids Total chlorophyll-a

flowcytometry (Li et al., 2002). However, the cell-based analyses are often time taking and require taxonomic expertise (Dunker et al., 2018). These limitations can be overcome by High Performance Liquid Chromatography (HPLC) coupled with UV–Vis detector, which is automated, fast and highly reproducible (Jeffrey, 1997). Moreover, HPLC can measure individual pigments accurately (Mantoura and Llewellyn, 1983). Among various phytoplanktonic pigments, chlorophyll-a is found in majority of algae and photosynthetic communities (Leavitt and Hodgson, 2001). Whereas, certain other pigments are taxonomically specific, for example, alloxanthin for cryptophytes and prasinoxanthin for prasinophytes (Stauber and Jeffrey, 1988; Sanz et al., 2015). The phytoplanktonic pigments undergo degradation in water, whose rate depends on light, oxygen, acidification and zooplankton grazing (Leavitt, 1993). The modified pigments subsequently deposit at surface sediments. The pigments may be less degraded, if they deposit quickly and get buried in the sediments (Florian et al., 2015). However, even after deposition, pigments may get further influenced by benthic algae, grazers and biological– chemical processes in the sediments (Hurley and Armstrong, 1991; Burford et al., 1994). Also, these pigments have variable relative degree of chemical stability based on their structures (Leavitt and Hodgson, 2001). Thus, the final amount of pigment in sediments will depend on modifications they undergo in water column, sediment–water interface and underneath sediments (Leavitt and Carpenter, 1990; Reuss et al., 2005). The present work chose to examine the surface sedimentary pigments in Kongsfjorden–Krossfjorden, a sub-Arctic glacial fjord ecosystem in Svalbard. Recent reports describe that it is facing intensified glacier melting and warm Atlantic water intrusions related to climate change events (Svendsen et al., 2002; Cottier et al., 2010). Earlier studies in the region discussed distribution and composition of phytoplanktonic pigments and related communities in the water column (Piquet et al., 2014; van De Poll et al., 2016; Bhaskar et al., 2016). In Kongsfjorden sediments, Bourgeois et al. (2016) analysed total chlorophyll-a and pheopigments using fluorometric technique. However, a detailed study addressing the major pigments from various classes — chlorophylls, pheopigments and carotenoids, has not been attempted for Kongsfjorden–Krossfjorden sediments. Aim of present study is to examine the pigments and composition of contributing phototrophic communities in surface sediments of Kongsfjorden– Krossfjorden. We tried to answer the following questions for the twin fjord ecosystem: (1) How do the pigment composition and concentration vary spatially (2) What is preservation condition of these pigments in surface sediments (3) Is it possible to use these pigments as indicators of primary productivity and community biomarkers.

Fig. 1. Map showing the study area and sampling stations in Kongsfjorden and Krossfjorden (Kn = Kongsfjorden stations, Kr = Krossfjorden stations).

2. Materials and methods 2.1. Study site and sampling The study site Kongsfjorden and Krossfjorden are located between 78◦ 40’–79◦ 30’ N and 11◦ 3’–13◦ 6’ E. Kongsfjorden is oriented from south-east to north-west, whereas Krossfjorden is from north to south (Svendsen et al., 2002). There is a wellmarked inner fjord with <100 m depth and a deeper outer fjord region with >100 m depth. For the present study, nine samples were collected from upper 10 cm sediment across the two fjords using van-veen grab, deployed from research boat M. S. Teisten during July, 2016. Six stations Kn 1 to Kn 6 were sampled from Kongsfjorden. Stations Kn 1 and Kn 2 with respective depths 46 m and 81 m, were located in the inner region of the fjord. Kn 3 (143 m) and Kn 4 (107 m) were in the middle region. Kn 5 (307 m) and Kn 6 (69 m) were in the outer region of the fjord (Fig. 1). Due to logistical constraints, we sampled only three stations from Krossfjorden — Kr 1 (212 m), Kr 2 (150 m) and Kr 3 (228 m), located in the inner-middle region. After collection, all the sediment samples were transferred to −20 ◦ C, stored in dark and transported to laboratory at NCPOR in dry ice for further analysis. The samples were treated and analysed for particle size and pigments. 2.2. Particle size analysis The sediment samples (3 g each) were dried at 100 ◦ C till constant weight was obtained. Hydrogen peroxide (15 mL) was added to the dried samples and kept undisturbed overnight for dissolution of organic matter. The mixture was heated at 50– 60 ◦ C to remove excess hydrogen peroxide and subsequently washed three times with milli-Q water. To the hydrogen peroxide treated samples, 10 mL 10% sodium hexametaphosphate was added as sequestrant and kept overnight. Resultant samples were wet sieved through 125 µm and the <125 µm fraction was dried completely. Samples were then subjected to particle size analysis using Beckman Coulter LS I3-320 Laser Diffraction Particle Size Analyser.

A. Singh and K.P. Krishnan / Regional Studies in Marine Science 31 (2019) 100815

2.3. Pigment extraction and analysis Samples in triplicate were taken for pigment extraction from each station. The samples were thawed at 4 ◦ C and extraction was performed rapidly under minimal light conditions to minimise photo-oxidation. One-gram sediment sample and 2 mL acetone (90%) (Merck-HPLC grade) were taken in a tube covered with aluminium foil, and vortexed. This homogenised mixture was sonicated at 38 kHz for 10 min in ice-cold bath. Resultant mixture was then kept at −20 ◦ C for 24 h to allow pigment extraction into the solvent. The mixture was then vortexed briefly and subsequently centrifuged for 10 min at 10000 × g and 4 ◦ C. The supernatant was separated and residue was again subjected to the extraction procedure to ensure complete extraction of pigments. After second extraction, both extracts were combined and filtered through 0.2 µm polyvinylidene fluoride syringe filter into HPLC vials. The vials were immediately placed in temperature controlled autosampler set at 4 ◦ C of HPLC Agilent 1200 series. The HPLC system was equipped with Agilent quaternary pump system, injector and diode array detector. The extracted pigments were analysed on HPLC using the protocol of Van Heukelem and Thomas (2001) with few modifications as outlined in Singh et al. (2017). In brief, analysis was carried out on a C-8 column (XDB-C8 Zorbax Eclipse, rapid resolution 4.6 mm × 100 mm, 3.5 µm). 100 µL of each sample was injected at a flow rate of 1.1 mL/min with column temperature set at 40 ◦ C. A reverse-phase gradient method was used with 70:30 ratio of methanol:28 mM ammonium acetate (pH = 7.2) as solvent A and 100% methanol as solvent B. A linear gradient of solvent A (95% to 5%) against solvent B from 5 to 95% was obtained in initial 20 min. It was followed by an isocratic flow of 5% solvent A and 95% solvent B for 10 min. In next 6 min, solvent proportion was returned to initial conditions (A = 95%, B = 5%). Resultant chromatograms were recorded and analysed for peaks. The peaks were identified as various pigments based on retention time and absorption spectra (190–900 nm), when compared to authentic pigment standards purchased from DHI Water and Environment, Denmark. 3. Results 3.1. Particle size of sediments Sediment particles were classified under three categories — clay (<4 µm), silt (4–63 µm), and sand (>63 µm) based on particle diameter. The sediments were predominantly composed of silt particles varying between 50.0%–70.9%. Proportions of clay (11.8%–32.7%) and sand (2.8%–37.5%) were lesser (Table 1). Mean and median size of the particles were between 12.6%–29.7 µm and 7.1–16.6 µm, respectively. Proportion of fine particles i.e. clay and silt was >80% at most of the stations except Kn 4 and Kn 6. Kn 4 and Kn 6 had 37.5% and 31.4% of coarser sandy particles, respectively. However, the composition of all the sediment samples was positively skewed (>+0.1) towards fine particles. 3.2. Pigments in sediments A total of fifteen different pigments belonging to three major classes — chlorophylls, pheopigments and carotenoids, were identified from the surface sediments. The chlorophylls included chlorophyll-a, chlorophyll-b, chlorophyll-c2 and chlorophyll-c3. The pheopigments–pheophytin-a and pheophorbide-a, were identified as degradation products of chlorophyll-a. Various detected carotenoid pigments are peridinin, 19-hexanoylfucoxanthin, fucoxanthin, prasinoxanthin, alloxanthin, diatoxanthin, lutein and zeaxanthin. Moreover, β -carotenea carotene, was also identified from the sediment samples.

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Chlorophyll-a was the most abundant pigment in the sediments, whose concentration varied between 103.9 ng g−1 and 796.5 ng g−1 dry weight (dw) of sediment (Fig. 2a). Highest average concentration of chlorophyll-a was observed at station Kr 1 (796.5 ± 34.2 ng g−1 dw) and Kr 3 (706.9 ± 193.2 ng g−1 dw). Within Kongsfjorden, chlorophyll-a showed maximum average concentration at middle fjord station Kn 4 (265.9 ± 5.4 ng g−1 dw) and minimum at outer station Kn 5 (103.9 ± 9.4 ng g−1 dw). For normally distributed pigment data, one-way ANOVA followed by Tukey’s post hoc t test was used. Chlorophyll-a concentrations differed significantly at Kr 1 and Kr 3 as compared to all other stations (p < 0.05). Among Kongsfjorden stations, significant differences (p < 0.05) were found in chlorophyll-a concentrations between Kn 4 and other stations- Kn 1, Kn 3, Kn 5 and Kn 6 at p < 0.05. Among pheopigments, pheophorbide-a was in the range 122.3–871.7 ng g−1 dw, with lowest concentration recorded at Kn 1 and highest at Kr 3. Within Kongsfjorden, highest pheophorbide-a concentration was at middle fjord (349.9–433.8 ng g−1 ) and lowest at inner fjord (122.3–135.6 ng g−1 ). The ANOVA analysis revealed that pheophorbide-a concentration at Kn 4 was significantly different from Kn 1 and Kn 2 at p < 0.05. Pheophorbide-a at Kr 3 was significantly different from all other stations, whereas at station Kr 1, it was significantly different from Kn 1, Kn 2, Kn 6 and Kr 2 (p < 0.05). Another pheopigments, pheophytin-a was observed in lower concentrations 57.2–211.3 ng g−1 dw as compared to pheophorbide-a (Fig. 2a). Pheophytin-a concentration at Kr 1 and Kr 3 differed significantly from all Kongsfjorden and Krossfjorden stations (p < 0.05). Ratio of chlorophyll-a and total pheopigments (pheophytin-a and pheophorbide-a) was calculated to assess the preservation conditions in surface sediments. The ratio was found to be >1 at Kr 1 and >0.5 at Kn 1, Kn 2, Kn 4, Kn 6 and Kr 3 (Fig. 2b). Overall, the ratio was higher for inner stations (0.70 to 0.86) as compared to middle (0.39 to 0.53) and outer (0.34 to 0.53). The ANOVA analysis showed that the chlorophyll-a:pheopigments ratio at Kr 1 was significantly different from all other stations except Kn 2 at p < 0.05. The ratio at Kn 1 was significantly different from Kn 4, and ratio at Kn 2 was significantly different from that at station Kn 5 and Kr 2 (p < 0.05). Other chlorophyll pigments for example divinylchlorophyll-a was observed in range 2.8–36.8 ng g−1 and 18.5–104.6 ng g−1 in Kongsfjorden and Krossfjorden, respectively. Chlorophyll-b was spotted at Kongsfjorden stations Kn 4, Kn 5 and Kn 6, and all three Krossfjorden stations in range 24.2 to 45.5 ng g−1 . It was not detected at inner basin stations Kn 1, Kn 2 and Kn 3 of Kongsfjorden. Chlorophyll-c2 and c3 were other chlorophyll pigments recorded at Kn 2, Kn 4, Kr 1 and Kr 3. Chlorophyll-c3 was also detected at Kn 3. Among carotenoid pigments, fucoxanthin was the most abundant pigment at station Kn 1, Kn 2, Kn 3, Kn 5 and Kr 1 (80–403 ng g−1 ) (Fig. 3a). Whereas at Kn 4 and Kr 3, prasinoxanthin (125– 492 ng g−1 ) and diatoxanthin (130–552 ng g−1 ) were found in abundance. To compare the carotenoid pigments at various stations, we normalised the concentrations with respect to chlorophyll-a. The normalised values of all carotenoids were low at inner stations Kn 1 and Kn 2 except fucoxanthin (Fig. 3b). Perdinin, 19-hexanoylfucoxanthin, alloxanthin, lutein, zeaxanthin and prasinoxanthin showed increasing trend from inner to outer region with highest normalised values recorded at Kn 5. In contrast, fucoxanthin was high at inner stations with concentration between 80–139.6 ng g−1 and normalised values between 0.6– 0.7. Fucoxanthin was low at middle and outer stations Kn 3, Kn 4 and Kn 6 (49.6 to 92.9 ng g−1 ; 0.3 to 0.5) and peaked at Kn 5 (102.2 ng g−1 ; 0.9). Similarly, for Krossfjorden stations, all carotenoids showed increasing trend from Kr 1 to Kr 2 and peaked

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Table 1 Composition of particles and related parameters of surface sediments from Kongsfjorden and Krossfjorden. Sl. No.

Stations

Colour

Clay% (<4 µm)

Silt% (4–63 µm)

Sand% (>63 µm)

Mean size (µm)

Median

Skewness

1 2 3 4 5 6 7 8 9

Kn 1 Kn 2 Kn 3 Kn 4 Kn 5 Kn 6 Kr 1 Kr 2 Kr 3

Red Red Red Brown Brown Reddish-brown Brown Brown Brown

17.1 26.0 27.7 12.5 24.6 11.8 32.7 27.5 26.0

64.1 67.5 64.1 50.0 70.9 56.8 64.5 67.8 66.5

18.7 6.5 8.2 37.5 4.4 31.4 2.8 4.7 7.4

23.0 14.2 13.6 29.7 15.6 28.0 12.6 13.7 15.3

12.8 8.4 7.8 15.9 9.2 16.6 7.1 8.3 9.1

1.7 2.4 2.3 2.0 2.4 1.7 2.3 2.2 2.0

Fig. 2. Distribution of (a) chlorophyll-a, pheophytin-a and pheophorbide-a; (b) chlorophyll-a : pheopigments ratio in surface sediments of Kongsfjorden and Krossfjorden.

at Kr 3 (maximum 19-hexanoylfucoxanthin at Kr 2) except fucoxanthin. Fucoxanthin was high at Kr 1 and Kr 3 (322.7–403.5 ng g−1 ; 0.4–0.5) and low at Kr 2 (30.2 ng g−1 ; 0.2). The ANOVA analysis was done for the normalised values of carotenoid pigments to check variability in distribution at different stations and regions. It revealed that fucoxanthin was significantly different between inner-middle and middle-outer station pairs Kn 2-Kn 4, Kn 3-Kn 5, Kn 4-Kn 5, and between outer stations Kn 5Kn 6 (p < 0.05). Prasinoxanthin, alloxanthin and zeaxanthin at station Kn 5 were significantly different from all other Kongsfjorden stations (p < 0.05). β -carotene was statistically different between outer and inner stations of Kongsfjorden at p < 0.05. 3.3. Correlation and linear regression We noted weak and non-significant linear regressions of chlorophyll-a with station depth (r2 = 0.156, p = 0.291) and median particle size (r2 = 0.141, p = 0.318). However, chlorophyll-a was significantly correlated to other pigments (r = 0.67–0.94) except β -carotene. Besides, β -carotene did not show significant correlations with pheophytin-a and divinylchlorophyll-a, however, well correlated with all other pigments. Further, the chlorophyll-a:pheopigment ratio was positively correlated with chlorophyll-a (r = 0.71, p < 0.05), divinylchlorophyll-a (r = 0.82, p < 0.01) and fucoxanthin (r = 0.77; p < 0.05) (Table 2). The pheopigment–pheophorbide-a was correlated to all pigments except divinylchlorophyll-a. Pheophytin-a, besides chlorophyll-a and pheophorbide-a, was also correlated with divinylchlorophylla, peridinin, 19-hexanoyl fucoxanthin, fucoxanthin (r = 0.79– 0.86; p < 0.01) and alloxanthin (r = 0.72; p < 0.05). Fucoxanthin showed significant correlation with peridinin, 19-hexanoylfucoxanthin and alloxanthin (r = 0.73–0.79; p < 0.05). Whereas, all other carotenoids including perdinin, 19hexanoyl fucoxanthin, prasinoxanthin, alloxanthin, diatoxanthin,

lutein and zeaxanthin were strongly correlated among themselves (r = 0.74–0.99, p < 0.001–0.01). 3.4. Pigment indices Photopigment indices were derived to assess contribution of photosynthetic and photoprotective pigments to the total pigment pool (Barlow et al., 2007). The carotenoid pigments were divided into photosynthetic carotenoids (PSCs) and photoprotective carotenoids (PPCs). The photopigment indices symbolised as Tchla, PSCs, PPCs, DPs (Diagnostic Pigments) and PI (Photoprotection Index) are described in Table 3. We observed high Tchla at middle Kongsfjordens stations (Fig. 4). PPCs and PI were lower at inner station than middle and outer stations in Kongsfjorden. PI was highest for outer Kongsfjorden stations. In Krossfjorden, the indices TChla, PPCs, PSCs and DPs were lower at Kr 2 than at Kr 1 and Kr 3. However, PI was in increasing trend from Kr 1 to Kr 3. 4. Discussion 4.1. Spatial distribution of particles and pigments The present study reports distribution and composition of particle size and pigments in the surface sediments of Kongsfjorden and Krossfjorden. The sediments were dominated by silt particles with no significant spatial variation. However, spatial distribution of pigments varied significantly in the fjords. Chlorophyll-a and pheopigments were in the range of 103–796 and 57–871 ng g−1 dw, respectively. Chlorophyll was measured fluorometrically by Bourgeois et al. (2016) in Kongsfjorden sediments, which reported values as low as 350 ng g−1 dw at inner region (August) and higher 3.3 × 103 ng g−1 dw at outer region (spring). In another fjord of Svalbard-Hornsund, sediments were reported with

Chlorophyll-a: pheopigments Chlorophyll-a: pheopigments Chlorophyll-a Pheophorbide-a Pheophytin-a Divinyl chlorophyll-a Peridinin 19-Hexanoylfucoxanthin Fucoxanthin Prasinoxanthin Alloxanthin Diatoxanthin Lutein Zeaxanthin B-carotene

Chlorophyll-a

Pheophorbide-a

Pheophytin-a

1.00 0.81** 0.94*** 0.91*** 0.85** 0.87** 0.96*** 0.67* 0.79* 0.67* 0.68* 0.70* 0.60

1.00 0.78* 0.56 0.94*** 0.85** 0.71* 0.93*** 0.94*** 0.94*** 0.92*** 0.92*** 0.91***

1.00 0.84** 0.79** 0.86** 0.82** 0.60 0.72* 0.64 0.63 0.62 0.59

Divinyl Chlorophyll-a

Peridinin

19-Hexanoyl fucoxanthin

Fucoxanthin

Prasinoxanthin

Alloxanthin

Diatoxanthin

Lutein

Zeaxanthin

B-carotene

1.00 0.6 0.73* 0.57 0.60 0.64 0.48

1.00 0.97*** 0.99*** 0.99*** 0.98*** 0.97***

1.00 0.95*** 0.97*** 0.99*** 0.94***

1.00 0.98*** 0.95*** 0.97***

1.00 0.97*** 0.98***

1.00 0.95***

1.00

1.00 0.71* 0.22 0.59 0.82** 0.38 0.46 0.77* 0.12 0.25 0.13 0.16 0.13 −0.01

*Indicates that the correlations were significant at p values <0.05. **Indicates that the correlations were significant at p values <0.01. ***Indicates that the correlations were significant at p values <0.001.

1.00 0.60 0.74* 0.91*** 0.37 0.53 0.36 0.39 0.44 0.27

1.00 0.82** 0.79* 0.95* 0.98** 0.95* 0.95* 0.95* 0.90*

1.00 0.75* 0.74* 0.81** 0.74* 0.78* 0.76* 0.75*

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Table 2 Correlations (r) between pigments obtained from the Pearson correlation analysis.

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A. Singh and K.P. Krishnan / Regional Studies in Marine Science 31 (2019) 100815

102 –104 ng chlorophyll-a by fluorometry (Drewnik et al., 2016) and 8.9 × 102 to 4.5 × 103 ng chloro-pigments by HPLC (Krajewska et al., 2017). However, accuracy of fluorometric method is often uncertain due to overlap of absorption and fluorescence bands of co-occurring pigments (Jacobsen and Rai, 1990; Kumari, 2005). Also, sediments for the present work were collected during summer, which marks end of the bloom with reduced productivity (Wiencke and Hop, 2016). Sediments reflect properties of overlying water. So, we tried to see the correlation of sedimentary pigments with particle size and station depth. However, the pigments were not correlated with station depth and particle size. Therefore, we anticipate role of other relevant factors in shaping the distribution of pigments. The spatial distribution of pigments was significantly different within the two fjords as well as between different regions of the fjord. The two fjords, despite being located in similar geographical setup have some characteristic differences. Both the fjords are strongly influenced by presence of various tidewater glaciers. Inner Kongsfjorden is affected by outflows from Kronebreen and Kongsvegen glaciers at head (Svendsen et al., 2002). On its northern coast, it is influenced by Conwaybreen and Blomstrandbreen glaciers. In Krossfjorden, Kr 1 and Kr 3 are located in the outflow region of Lilliehöökbreen glacier at head. Station Kr 2 is located in the region of other calving glaciers present along eastern coast (Svendsen et al., 2002). Different glaciers contribute variable amount of fresh water and organic matter, which may lead to differences in physicochemical properties of water column. For example, inner Krossfjorden surface water is often characterised with lesser turbidity as compared to inner Kongsfjorden (Piquet et al., 2010). Less turbid surface water allows deeper euphotic layer and may support higher phytoplankton growth. The same is evident from the observed higher pigment concentrations in Krossfjorden sediments than Kongsfjorden. Water column photosynthetically active radiation (PAR) and fluorescence also confirmed the same, where inner Krossfjorden water showed 4fold chlorophyll maximum values as compared to Kongsfjorden (Fig. 5). Within Kongsfjorden also, pigments were significantly different at the inner, middle and outer regions. Inner Kongsfjorden receives glacial inputs, which give rise to gradient in sedimentation and other properties like salinity, temperature and turbidity (Hop et al., 2002; Bourgeois et al., 2016). These gradients strongly influence primary productivity and hence relative distribution of phytoplanktonic pigments in different regions of the fjord. Kongsfjorden waters in outer region showed more primary production and pigments as compared to inner region (Hegseth and Tverberg, 2013; Piquet et al., 2014). However, we observed that the distribution of pigments in sediments was different from water. Outer sediments of Kongsfjorden were observed with lower average chlorophyll-a concentration than middle and inner stations.

Fig. 3. Carotenoid pigments (a) concentration profile and (b) ratio to chlorophyll a (w/w) at different stations of Kongsfjorden and Krossfjorden.

4.2. Degradation and preservation of pigments The conflict in the distribution of pigments in sediments and water may be understood from pigment degradation and preservation. Pigments are labile and can be degraded by light, oxygen and other factors (Kowalewska, 2004). So, pigments may degrade faster in less turbid, transparent and oxic environment. The turbid surface waters in the inner region, besides restricting photosynthesis, may also limit photo-degradation in sub-surface and deeper layers. Inner Kongsfjorden sediments were also observed with hypoxic and heterotrophic conditions (Sevilgen et al., 2014). Thus, we may expect relatively preserved conditions in inner region as compared to outer Kongsfjorden. On other hand, degradation may be faster in the outer fjord, which experiences deeper euphotic layer and better water mixing conditions.

Fig. 4. Concentration (µg/g) of Tchl, PSCs, PPCs, DPs and PI values at Kongsfjorden and Krossfjorden stations.

A. Singh and K.P. Krishnan / Regional Studies in Marine Science 31 (2019) 100815

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Table 3 Details of pigment indices calculated for the pigment data.

a b

Abbreviation

Indices

Pigments

Tchl PPC PSC DP PI

Total chlorophyll-a Photoprotective carotenoids Photosynthetic carotenoids Diagnostic Pigments Photoprotection Index

chlorophyll-a + divinyl chlorophyll-a alloxanthin + diadinoxanthina + diatoxanthin + zeaxanthin + caroteneb 19-butanoylfucoxanthina + fucoxanthin + 19-hexanoylfucoxanthin + peridinin PSC + alloxanthin + zeaxanthin + chlorophyll-b (diadinoxanthin + diatoxanthin + zeaxanthin)/chlorophyll-a

Not detected/analysed in the present study. Only β -carotene is considered among carotenes.

Fig. 5. Water depth profile of PAR and fluorescence at Kongsfjorden and Krossfjorden.

Another parameter that can be used to assess the preservation conditions in the sediments is ratio of chlorophyll-a and pheopigments (Reuss et al., 2005; Brock et al., 2006; Deshpande et al., 2014). Pheopigments are formed from chlorophylls upon grazing activities and senescence. Chlorophyll-a degrades to form pheophytin-a upon loss of magnesium atom. Subsequently, pheophorbide-a is formed after loss of phytol chain and various other side groups. We observed the ratio was >0.5 for six out of nine sampled stations. This indicated the pigments in surface sediments were relatively fresh and preserved despite all kinds of grazing and degradation processes. Kr 1 was the only station with the ratio >1, which may be considered as an indicator for freshly deposited pigments at the time of sample collection. Also, some of the less stable pigments like chlorophyll-c2 and c3 were absent in the outer region. Among different regions, higher ratios at inner fjord substantiates our earlier observation that it was more preserved than the middle and outer regions of Kongsfjorden. 4.3. Phytoplankton pigments as biomarkers Moreover, carotenoid pigments also showed significant spatial variability in different regions of the two fjords. We observed lower concentration of carotenoids at inner Kongsfjorden, which is in agreement with less productive inner fjord as compared to outer fjord. We found higher carotenoids at outer station Kn 5. This is expected as outer region of Kongsfjorden experiences highest phytoplankton biomass during peak bloom (Hop et al., 2002). One of those carotenoids-zeaxanthin was high in outer region and decreased successively towards inner side along the path of intrusion of Atlantic water. Zeaxanthin is usually found as major pigment in cyanophyta (cyanobacteria), prochlorophyta and rhodophyta (macroalgae) (Vidussi et al., 2001). The Atlantic water incursions transporting cyanobacterial species in the fjord

are reported by Piquet et al. (2014) as potential Atlantic water marker. Another outer station Kn 6 which does not fall in Atlantic water path, however, showed lesser zeaxanthin concentration. This confirms the relation of cyanobacterial entry with Atlantic water. In contrast to other carotenoids, fucoxanthin was high at inner Kongsfjorden stations. This may be due to shift of diatom community to the inner region during late spring (Piquet et al., 2014), and subsequent abundance in summer (van De Poll et al., 2016). Fucoxanthin, prasinoxanthin and diatoxanthin were the most abundant carotenoids that suggested diatoms, prasinophytes and cryptophytes as main source phytoplankton derived organic matter. However, caution must be taken while using taxonomically diagnostic carotenoids to estimate contribution of different algal groups. Because stability varies substantially among pigments and also, many of the carotenoids are found in more than one taxonomic group (Jiang et al., 2017). For example, fucoxanthin and peridinin from diatoms and dinoflagellates are generally more labile than alloxanthin (cryptophytes), zeaxanthin (cyanobacteria) and lutein (green algae) (Chen et al., 2016). Fucoxanthin is found as a major pigment in bacillarophyte (diatoms), dinophyte (dinoflagelllates), chrysophyta, haptophyte, rhodophyta and prymnesiophytes (Leavitt, 1993). Diatoxanthin is found as trace pigment in bacillarophyta (diatoms), dinophyte (dinoflagelllates), chrysophyta, euglenophyta and raphidophyte. Peridinin and 19-hexanoylfucoxanthin are specific markers for dinoflagellates and prymnesiophytes, respectively. Other than 19-hexanoylfucoxanthin, chlorophyll-c2, c3 and fucoxanthin as major and β -carotene and diatoxanthin as trace pigments are also present in prymnesiophytes. In the surface sediments, however, 19-hexanoylfucoxanthin was lower than fucoxanthin, indicating that diatoms were major source of fucoxanthin. Prevalence of diatoms, dinoflagellates and prymnesiophytes (P. pouchetii) is

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A. Singh and K.P. Krishnan / Regional Studies in Marine Science 31 (2019) 100815

a known occurrence during pre-bloom, peak-bloom and postbloom in Kongsfjorden and Krossfjorden waters (Leu et al., 2006; Hodal et al., 2012). Correlation studies revealed that most of the carotenoids were positively correlated with each other, except fucoxanthin. The correlation among carotenoid pigments may indicate co-occurrence of representative phytoplankton communities. Positive correlation of chlorophyll-a and pheopigments with carotenoid pigments re-affirms that the dominant phytoplankton communities are contributing majorly to the algal biomass. Benthic algae and macroalgae forms significant part of Kongsfjorden ecosystem. Their contribution to the sediment chlorophyll-a pool cannot be overlooked. Macroalgae in Kongsfjorden include chlorophytes, rhodophytes and phaeophytes (Hop et al., 2016), which contain pigments chlorophyll-a, b, c, fucoxanthin and zeaxanthin (Gordillo et al., 2006). However, benthic microalgae and macroalgae are substantial mainly in coastal, subtidal and intertidal regions (Woelfel et al., 2010; Sevilgen et al., 2014; Krajewska et al., 2017). 4.4. Phytoplankton pigment indices Pigment indices provide estimation of physiological condition of the phytoplankton community resulting from environmental and trophic conditions (Barlow et al., 2007). We observed PPC > PSC in the middle and outer stations of Kongsfjorden, indicating light stress conditions. The PPC index (ratio to chlorophylla) showed an increasing trend from inner to outer fjord. The PPC index was increasing from inner to outer region as water irradiance was increasing. This confirmed that photoprotective carotenoids are produced by phytoplankton in response to the light fields (Moreno et al., 2012). PI describes environmental stress conditions for example, intensive irradiance and low nutrient concentrations (Barlow et al., 2007). High PI values in the outer fjord followed by middle fjord as compared to inner fjord emphasises high stress conditions, especially light, in outer fjord. 5. Conclusion Based on pigment distribution in surface sediments, Krossfjorden was more productive than Kongsfjorden. Within Kongsfjorden, middle and outer regions had higher pigment concentration than inner, which is a familiar distribution in water during bloom and post-bloom period. Further, the phytoplanktonic community distribution derived from biomarker pigments was also coherent with that of water during spring and summer, as reported by various studies in the region. The biomarker pigments for diatoms, dinoflagellates, prasinophytes and prymnesiophytes communities confirmed their abundance in the fjords. At contrast to the overall pigment distribution, inner region was found to be more preserved than the outer. This was supported and conformed by occurrence of higher chlorophyll-a to pheopigments ratio at inner region, and higher protective carotenoids and photoprotection index at outer region indicating photo-stress conditions. Thus, the information gathered from the surface sedimentary pigments was consistent and useful despite the labile nature of pigments. In future, temporal studies using pigments from sediment cores may be undertaken for the Kongsfjorden–Krossfjorden region on the basis of spatial distribution described in the present work. Acknowledgements The authors wish to express their gratitude to Dr. M. Ravichandran, Director, National Centre for Polar and Ocean Research (NCPOR) for providing laboratory facilities and work platform. We would also like to thank Dr. John Kurien, NCPOR for facilitating Particle Size Analysis on Laser Diffraction Particle Size Analyser. We thank Ministry of Earth Sciences, India for funding this research. This is NCPOR’s contribution number J-28/2019-20.

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