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, 00044 Frascati (Rome), Italy 2ISAC, CNR, Via Fosso del Cavaliere 100, 00133 Rome, Italy 3University of Tor Vergata, Rome-Italy florinda.artuso@enea.it CLEO Workshop, ESA, Frascati 09.07.2016

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).

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: 250-700 nm Agilent 1260

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

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)

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=1.22-0. 05 mg/L Gulf of Lion Alboràn Sea Quality control (Aiken et al.2009) Rel.ab.%= (DP/SDP)*100

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

DPs and related Phytoplankton dimensional classes (PSCs) Relation between DPs and TChl a for PSCs evaluation Uitz et al. 2006 adapted for the Mediterranean Sea case by Sammartino et al. 2015

Surf. TChl a ranges from 4.0 to 0.15 mg/L Cosimo campaign 2015 H24 experiment Period: 03. 29-04. 14, 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.= 0.285 mg/m3

Diagnostic pigments relative abundances H24 experiment

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.%

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 %

PSCs (Phytoplankton Size Classes) 5 stations 21 stations

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.

Thank you! florinda.artuso@enea.it