1. Measurements of pigment composition in the Mediterranean Sea, a contribution to the Sentinel-3 Cal/Val activities
F. Artuso1, D. Cataldi1, S. Marullo1, A. Lai1, F. Colao1, F. Angelini1, A. Palucci1, A. Di Cicco2, M. Sammartino2, S. Colella2, G. Volpe2, R. Santoleri2, C. Cristini3
1ENEA, Via Enrico Fermi 45, Frascati (Rome), Italy
2ISAC, CNR, Via Fosso del Cavaliere 100, Rome, Italy
3University of Tor Vergata, Rome-Italy
CLEO Workshop, ESA, Frascati

2. Outline and objectives
To understand the role of phytoplankton in biogeochemical cycles of the Mediterranean Sea
Characterization of phytoplankton through marine pigment analysis :
phytoplankton biomass abundance and its distribution (at surface and along the water column);- TChla
phytoplankton community structure: size classes (PSCs) and functional types (PFTs)-diagnostic pigments (DP)
phytoplankton degradation, fisiology-pheopigments and other accessory pigments (AP).
High Performance Liquid Chromatography (HPLC) analysis is the established reference technique for the analysis of chlorophyll a (Chl a) and accessory phytoplankton pigments
Data derived from HPLC pigment analysis can be used for:
1) satellite data product validation;
2) bio-optical algorithm development;
3) calibration of on board instrumentation (CTD fluorometer and Lidar fluorosensor).

3. Method Sampling on board:
-water samples collected in-situ at the surface and at different depths through a rosette equipped with Niskin bottles
-filtration through GF/F membranes (0.7µm pore size)
-fractionated filtration through polycarbonate membranes with 2µm and 20 µm pore size.
Extraction:
-extraction in 100% acetone ;
-sonication;
-centrifugation;
-syringe filtration (0.2µm filters).
RP-HPLC analysis (Protocol of Wright et al. 1991):
Refrigerated autosampler at 5°C;
C18 column , T=22°C;
DAD (diode array detector)
λ =440, 410 nm, spectral range: nm
Agilent 1260

4. 19’-butanoyl-oxy-fucoxanthin 19’ hexanoyl-oxy-fucoxanthin
Analysed pigments
Pigment
Abbreviation
Chlorophyllide a
Chlde a
Chlorophyll c3
Chl c3
Clorophyll c2
Chl c2
peridinin
peri
19’-butanoyl-oxy-fucoxanthin
19’-but
fucoxanthin
fuco
19’ hexanoyl-oxy-fucoxanthin
19’-hex
neoxanthin
neo
prasinoxanthin
prasino
violaxanthin
viola
diadinoxanthin
diadino
alloxanthin
allo
diatoxanthin
diato
lutein
lut
zeaxanthin
zea
Clorophyll b
Chl b
Clorophyll a
Chl a
a carotene
a-car
b carotene
b-car
Pheophorbide a
pheode
Pheophitin a
pheotin
Wright Protocol (1991):
-good separation of most pigments and critical pairs (i.e. lut/zea);
-no separation of Chl a and Chl b from their divinyl forms;
-Good performance metrics (Hooker et al. 2005, NASA report on HPLC analysis intercomparison experiment, SeHARRE-2)
Chromatogram
TChl a= Chl a+dv Chl a +all.+epim.+ Chlde a

5. HPLC data set and campaigns
Mediterranean Ocean Color
CAL/VAL DATA SETS
HPLC data set and campaigns
-1200 samples analysed by HPLC
Annual Mediterranean cruises:
organized by CNR-ISAC since 1997 in the framework of National and International Projects
Bio-optical measurements:- in-water profiles of:
downwelling irradiance (Ed)
upwelling radiance (Lu)
downwelling radiance (Eu)
IOPs
Chl profiles concurrently with in-water optical cast
WMED BIOOPT 2012
In situ chlorophyll-a data:
- CTD chlorophyll profiles
- Surface Chlorophyll (LIDAR) data along the ship track-ENEA
- HPLC measurements (ENEA)

6. WMED BIO-OPT 2014 Campaign Rel.ab.%= (DP/SDP)*100
Period: April 11-24,2014;
-59 sample stations;
-420 samples
Mean TChla= 0.29 mg/L
Tchla range= mg/L
Gulf of Lion
Alboràn Sea
Quality control (Aiken et al.2009)
Rel.ab.%= (DP/SDP)*100

7. cyanobacteria and prochlorophytes <2
Taxonomy: surface distribution of DPs and related PFTs
Pigments
Taxonomy
Zea
cyanobacteria and prochlophytes
Tchl b
green flagellates and prochlophytes
19’-Hex
chromophytes ,nanoflagellates
19’-But
chromophytes, nanoflagellates
Allo
cryptophytes
Fuco
diatoms
Peri
dinoflagellates
Vidussi et al. 2001
Pigments
Taxonomy
Dimension (mm)
Zeaxanthin
Zea
cyanobacteria and prochlorophytes
<2
Chlorophyll b+divinyl-Chl b
TChl b
green flagellates and prochlorophytes
19’hexanoyloxyfucoxanthin
19’-hex
Chromophytes nanoflagellates
2-20
19’butanoyloxyfucoxanthin
19’-but
Alloxantin
Allo
cryptophytes
Fucoxanthin
Fuco
diatoms
>20
Peridinin
Peri
dinoflagellates

8. DPs and related Phytoplankton dimensional classes (PSCs)
Relation between DPs and TChl a for PSCs evaluation
Uitz et al adapted for the Mediterranean Sea case by Sammartino et al. 2015

9. Surf. TChl a ranges from 4.0 to 0.15 mg/L
Cosimo campaign 2015
H24 experiment
Period: , 2015;
-55 sample stations;
-298 samples-3600 data
Surf. TChl a ranges from 4.0 to 0.15 mg/L
-51 samples at the following depths: sup (2m), 10m, 40m, 64m
-982 data on pigment composition
-TChl a mean conc.= mg/m3

10. Diagnostic pigments relative abundances
H24 experiment

11. DPs surface distribution: phyotplankton functional types (PFTs)
Peri rel.ab.%
Chl b rel.ab.%
Fuco rel.ab.%
Pigments
Taxonomy
Zea
cyanobacteria and prochlophytes
Tchl b
green flagellates and prochlophytes
19’-Hex
chromophytes, nanoflagellates
19’-But
chromophytes,nanoflagellates
Allo
cryptophytes
Fuco
diatoms
Peri
dinoflagellates
Vidussi et al. 2001
19’-But rel.ab.%
19’-Hex rel.ab.%
Allo rel.ab.%
Zea rel.ab.%

12. PSCs (Phytoplankton Size Classes)
exp= PSCs derived from TCHla in fract. samples
mod=PSCs derived from DPs (Sammartino et al. 2015)
micro %
nano %
pico %
nano %
pico %
micro %

13. PSCs (Phytoplankton Size Classes)
5 stations
21 stations

14. Conclusions and future perspectives
Measurements of pigment composition were used to derive information on phytoplankton community structure and distribution in the Mediterranean.
TChl a distribution recorded confirmed the oligotrophic character of the Med Sea, but higlighted also the presence of some eutrophic zones (Gulf of Lion, Alboràn Sea, the North Adriatic Sea influenced by the ouflow of the Po river).
DPs were used to evaluate the surface distribution of the main taxa (PFTs) and PSCs.
Accurate PFT identification should be performed through the development of empirical algorithms specific for the Mediterranean Sea case (A. Di Cicco, CLEO oral presentation).
PSC analysis results showed the dominance of nanoplankton.
Comparison between PSC determined through in-situ measurements on fract.samples (exp) and through DP (mod) remarked the need to develop new regional PSC empirical algorithms for coastal waters. A good match between the PSC data obtained with the 2 approaches was found for deep waters (picoplankton prevalence).
Future…
The HPLC pigment in-situ data will be part of the Italian Sentinel-3 CAL/VAL contribution.   The data can be used to evaluate the performances of OLCI algorithms and development of Mediterranean regional algorithms. Data of future campaigns can be employed for validation of Sentinel-3 products.

15. Thank you!