SESAME

BOUM cruise

Protocols for sampling and analysis Responsible scientist: Thierry Moutin

FIRST PRIORITY PARAMETERS: 1 SALINITY 1. Details of Institute: INSU/CNRS Division Technique Person reporting results: Claudie Marec Email: claudie.marec@ipev.fr 2. Parameter measured salinity 3. Sampling and conservation procedures: Conductivity sensors (SBE4) are installed on a SEABIRD CTD SBE911+; data is acquired in real time at 24scan/sec. 4. Outline of the method used: 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory Two conducitivity sensors are installed, so, any drift can be outlined immediately when comparing the real time data during the casts. Pre-cruise and post cruise calibration of the conductivity sensors are performed with an accuracy of 0.001 to 0.002 psu. During deep casts salinity samples will be collected and analysed with a Portasal Guildline salinometer onboard the ship. 6. Precision and detection limits Range: 0.0 to 7 Siemens/meter Resolution: 0.00004 S/m @24 samples per second Initial accuracy: +/- 0.0003 S/m Response time: 0.060 second Stability: 0.0003S/m/month 7. References Woce Operations Manual, part3.1.3: WHP Operations and Methods. (Woce report N°68/91, July 91) Protocols of the Joint Global Flux Study (JGOFS) Core Measurements. (IOC Manuals and Guides N°29, UNESCO 1994) 2 TEMPERATURE 1. Details of Institute: INSU/CNRS Division Technique Person reporting results: Claudie Marec Email: claudie.marec@ipev.fr 2. Parameter measured temperature

3. Sampling and conservation procedures: Temperature sensors (SBE3plus) are installed on a SEABIRD CTD SBE911+; data is acquired in real time at 24scan/sec. 4. Outline of the method used: 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory Two temperature sensors are installed, so any drift can be outlined immediately when comparing the real time data during the casts. Pre-cruise and post cruise calibration of the Temperature sensors are performed with an accuracy of 0.0005°c. 6. Precision and detection limits Range: -5°c to 35°c Resolution: 0.0003°c @24 samples per second Initial accuracy: +/- 0.001°c Response time: 0.065+/-0.010 second 7. References Woce Operations Manual, part3.1.3: WHP Operations and Methods. (Woce report N°68/91, July 91) Protocols of the Joint Global Flux Study (JGOFS) Core Measurements. (IOC Manuals and Guides N°29, UNESCO 1994) 3 BEAM ATTENUATION 1. Details of Institute: INSU/CNRS Division Technique Person reporting results: Claudie Marec Email: claudie.marec@ipev.fr 2. Parameter measured Beam attenuation (or light transmission) 3. Sampling and conservation procedures: A Wetlabs CSTAR transmissiometer is connected to a SEABIRD CTD SBE911+ as an auxiliary sensor. The C star measures light transmittance at a single wavelength over a known path (25cm); The light source is a LED and its wavelength is 660-670nm. Losses of light propagation through water can be attributed to both scattering and absorption. Suspended particles, phytoplankton, bacteria and dissolved organic matter contribute to the losses sensed by the instruments. Data (volt output) is acquired in real time at 24scan/second. 4. Outline of the method used: 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory

Recent factory calibration coefficients are used for real time acquisition. with the following formula . % transmission= (Vsig-Vd)/(Vref-Vd) Tr= e-cx

c=-1/x (ln((Vsig-Vd)/(Vref-Vd)) 6. Precision and detection limits Path length 25cm Sensitivity:1.25mV Beam divergence: 0.7deg in water Wavelength: 470nm Response time: 0.167 second 7. References 4 OXYGENE 1. Details of Institute: Laboratoire de Microbiologie Geochimie, Ecologie Marine, LMGEM , UMR CNRS 6117. Person reporting results: Dominique Lefèvre Email: dominique.lefevre@univmed.fr 2. Parameter measured Dissolved oxygen 3. Sampling and conservation procedures: Sampling and conservation according to Aminot and Chaussepied (1983) and Grasshoff (1984), Culberson 1991, Dickson (1996). Following procedure is based on the WHP Operations and Methods (Dickson, 1996). Seawater samples were collected with Niskin bottle attached to the CTD-system. Seawater for oxygen measurement was transferred from Niskin sampler bottle to volume calibrated flask (ca. 100 cm3) using silicon tubing. Three times volume of the flask of seawater was overflowed. Temperature was measured by digital thermometer during the overflowing. Then two reagent solutions (Reagent I, II) of 1 cm3 each were added immediately into the sample flask and the stopper was inserted carefully into the flask. The sample flask was then shaken vigorously to mix the contents and to disperse the precipitate finely throughout. After the precipitate has settled at least halfway down the flask, the flask was shaken again vigorously to disperse the precipitate. The sample flasks containing pickled samples were stored in a laboratory until they were titrated. Samples are stored in water and dark container at constant temperature waiting for titration. 4. Outline of the method used: Reagents: Pickling Reagent I: Manganous chloride solution (3M) Pickling Reagent II: Sodium hydroxide (8M) / sodium iodide solution (4M) Sulfuric acid solution (5M) Sodium thiosulfate (0.02M)

Potassium iodate (0.001667M) Instrumental details:

Analysis is carried out using a photometric endpoint detector coupled with a Methrom® burette (Williams and Jenkinson, 1982). Titration is run automatically using a Microsoft visual basic ® home made software.

Reagents are added using precision micropipette Acura 865 Socorex® Borosilicate bottles were calibrated using gravimetric calibration procedure

(Dickson and Goyet, 1994, SOP 13). O2 calculation:

• [O2] (moles.dm-3) = Cthio(Vthio - b)/(4*(Vf(t)-(v1 + v2))) - (103*7.6 10-8)/(Vf(t)-(v1 + v2))

with :

• -Vthio : thiosulfate volume used for the titration (cm3) • -Vf(t) : Seawater volume sampled (cm3) at sampling temperature. • -Vf(t) = (Vf(20°C) * (1 + [[alpha]]v*(t - 20)))*f with [[alpha]]v = 1,0 10-5 (f:

thermal expansion coefficient for borosilicate) and f=1.00105 (buoyancy correction).

• -Cthio : thiosulfate concentration (mol.dm-3) • -b : blank reagents volume (cm3) • -v1 + v2 : reagents (1 and 2) volume (2 cm3). • -7,6 10-8 : O2 added with the reagents. • • According to Murray et al. (1968), oxygen solubility, at PA=1 (pO2=0,2080)

is equal to :

• 1,67 cm3 O2 @ STP.dm-3 for MnCl2,4H2O 600 g.dm-3 • 0,040 cm3 O2 à STP.dm-3 for NaI 600 g.dm-3 and NaOH 320 g.dm-3.

Adding v1 cm3 of reagent 1 and v2 cm3 of reagent 2 lead to an O2 increase of:

(v1*1.67 + v2*0.04)/1000 cm3 O2 @ STP, i.e. : (v1*1.67 + v2*0.04)/(1000*22391) moles O2. v1 = v2 = 1 cm3, the added O2 amount from the reagents is 7.6 10-8 moles.

• Conversion to µmol kg-1

• [O2] (umol.kg-1) = [O2] (mol.dm-3).106/[[rho]](S,[[t]](S,t,p,0),0)

-[[rho]](S,[[t]](S,t,p,0),0) is the density of seawater at atmospheric pressure, depending on salinity and temperature when pickled [[t]].

5 . Quality control / Quality assurance (QC/QA) routinely employed within the laboratory

Quality control is carried out by titrating 20 oxygen bottles from the same bulk of water. This allows assessing the quality of the bottles used as well as the quality of the whole titration procedure.

Standardisation of the process is based upon a potassium iodate (KIO3) solution prepared to a known normality (0.02 N). Solutions of the KIO3 standard are then used to determine the exact normality of the sodium thiosulfate (Na2S203.5H2O) titrant (~0.2 N). Cross checked standardisation are made using KIO3 standard (CSK Standard, Wako Chemicals Gmbh). 6. Precision and detection limits

Precison on O2 concentration determination should be around 0.03 µmol dm-3 Detection limit: 5 µM

7. References Aminot A. ; Chaussepied M. 1983. Manuel des analyses chimiques en milieu marin. Handbook of chemical analysis in marine environnent. Editor Centre National pour l'Exploitation des Océans. Pp. 395. Carpenter, J.H., 1965. The Chesapeake Bay Institute technique for the Winkler dissolved oxygen method. Limnol. Oceanogr. 10. 141-143. Culberson, C. H., 1991. Dissolved oxygen. WOCE Operations Manual, Part 3.1.1 : WHP Operations and Methods, WHP Office Report WHPO 91-1, WOCE Report No. 68/91. Dickson, 1996. Determination of dissolved oxygen in sea water by Winkler titration. WHP Dickson, A. and Goyet, C. (1994).DOE Handbook of Methods for the Analysis of the Garcia and Gordon, 1992. H.E. Garcia and L.I. Gordon , Oxygen solubility in seawater: better fitting equations. Limnol. Oceanogr. 37 (1992), pp. 1307–1312 Grasshoff, K., 1983. Determination of oxygen. In: Methods of Seawater Analysis. 61-72. (Eds) Grasshoff, K., Ehrhardt, M. and Kremling, K. Verlag Chemie, Weinheim. Millero F. J. and A. Poisson, 1981. International one-atmosphere equation of state of seawater. Deep Sea Res., 28, 625-629. Murray, C. N., J. P. Riley, and T. R. S. Wilson, 1968. The solubility of oxygen in Winkler reagents used for the determination of dissolved oxygen. Deep Sea Res., 15, 237-238. Strickland, J.D.H. and Parsons, T.R., 1968. A practical Handbook of Seawater Analysis. 23-28. Fish. Res. Bd. Can. Bull. 167. Various Parameters of the Carbon Dioxide System in Sea Water, Version 2. Williams P.J. leB. and Jenkinson N.W. (1982) A transportable microprocessor-controlled precise Winkler titration suitable for field station and shipboard use. Limnol. Oceanogr. 27: 576-585. Winkler, L. W. 1888. The determination of dissolved oxygen in water. Chem Ber. Deutsch Chem. Gos., 21, 2843. 5 CHLOROPHYLL A 1. Details of Institute: INSU/CNRS Division Technique Person reporting results: Claudie Marec Email: claudie.marec@ipev.fr

2. Parameter measured Chlorophyll a estimate from in vivo fluorescence 3. Sampling and conservation procedures: A CHELSEA Aquatracka 3 fluorometer is connected to a SEABIRD CTD SBE911+ as an auxiliary sensor. The light source is a Xenon lamp. Excitation wavelength is 430nm and Emission wavelength is 685nm. Data (volt output) is acquired in real time at 24scan/second. 4. Outline of the method used: 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory Recent factory calibration coefficients are used for real time acquisition with the following formula . Conc= (Ax 10output)-B Where: Conc= fluorophor concentration in µg/L Output= Aquatracka output in volts HPLC measurements of chlorophyll will be used to correct these data. 6. Precision and detection limits Range: 0.01µg/L to 100µg/L Pulse rate: 5.5Hz 7. References 6 NUTRIENTS (except Silicate) 1. Details of Institute: Laboratoire Arago – UMR 7621 – BP44 66651 Banyuls sur Mer cedex Person reporting results: Mireille Pujo-Pay Email: pujopay@obs-banyuls.fr 2. Parameter measured Nitrate, Nitrite, Phosphate, Ammonia 3. Sampling and conservation procedures: Sampling in polyethylene vials for nitrate, nitrite and phosphate and in glass bottles for ammonia. Vials are rinsed 3 times before collection of samples. No conservation procedure as samples are immediately analyzed on board after sampling 4. Outline of the method used: Pre-treatment and conservation techniques: Instrumental details and analysis:

After sampling, Nitrate (NO3), nitrite (NO2) and phosphate (PO4) are immediately analysed on a Axflow/Bran Luebbe III autoanalyseur according to classical methods (Wood et al., 1967, Tréguer and Le Corre, 1975) After sampling, reagents are immediately added in samples for ammonia determinations. Ammonia is then analysed by fluorimetry according to Holmes et al (1999) on a spectrofluorometer JASCO FP-2020. 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory Participation to different international Intercalibration exercises (Somlit, EIL, last RMNS 2006 International Intercomparison Exercise for Reference Material of Nutrients in Seawater in a Seawater Matrix 2006 ) Use of standards OSIL 6. Precision and detection limits Nitrate ± 0.01µM Nitrite ± 0.002µM Phosphate ± 0.002µM Ammonium ± 0.01µM 7. References

Holmes R. M., Aminot A., Kérouel R., Hooker B.A., Petersen B.J. (1999) A simple and precise method for measuring ammonium in marine and freshwater ecosystems, Can. J. fish. Aquat. Sci., 56, 1801-1808 Tréguer, P., and P. Le Corre, (1975). Manuel d'analyses des sels nutritifs dans l'eau de mer. Laboratoire d'Océanographie Chimique, pp. 110, Université de Bretagne Occidentale, Brest, 1975. Wood, E.P.K., F.A.J. Armstrong, and F.A. Richards (1967). Determination of nitrate in seawater by cadmium cooper reduction to nitrite, Journal of Marine Biological Association of United Kingdom, 47, 23-31.

7 SILICATE

1. Details of Institute: LOB UMR6535 Person reporting results: Karine Leblanc Email: karine.leblanc@univmed.fr 2. Parameter measured Si(OH)4 –Orthosilicic acid 3. Sampling and conservation procedures: 20 ml samples will be collected from the Niskin bottles, filtered onto 0.2 µm polycarbonate filters and stored at 4 °C until analysis in PE vials. 4. Outline of the method used: -Pre-treatment and conservation techniques: 20 ml samples will be collectef from the Niskin bottles, filtered onto 0.2 µm polycarbonate filters and stored at 4 °C until analysis in PE vials. This ensure that no

dissolution of particulate biogenic silica occurs in the vials and eliminates the need for added preservative products such as HgCl2 which often brings contaminants into the samples. -Instrumental details and analysis: Si(OH)4 concentrations will be determined using the colorimetrical method of Strickland and Parsons (1972) on a spectrophotometer using a 5 or 10 cm cuvette. 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory This method is used in routine at our laboratory. An international intercalibration exercise perfomed last year showed very good agreement between our results and the consensus values. 6. Precision and detection limits The precision of analyses is ± 35 nM and detection limit is 50 nM. 7. References Strickland, J.D.H. and Parsons, T.R. (1972). A practical handbook of seawater analysis. Fisheries Res. Board of Canada Bull., 167, 310.

SECOND PRIORITY PARAMETERS: 1 CARBONATE SYSTEM

1. Details of Institute: Laboratoire IMAGES (institut de Modélisation et d’Analyses en GéoEnvironnement et Santé), Bat B Université de Perpignan Via Domitia 66860 Perpignan Person reporting results: Franck Touratier Email: touratie@univ-perp.fr 2. Parameter measured Total dissolved inorganic carbon (CT) Total alkalinity (AT) 3. Sampling and conservation procedures: Each sample requires 1.5 liter of seawater Samples must be taken just after those used to measure oxygen in order to avoid CO2 exchange. Ideally, all samples should be preserved at 4°C. 4. Outline of the method used: Samples for CT and AT analysis will be collected into 500 ml borosilicate glass screw cap bottles using standard sampling protocols (DOE 1994) and poisoned with saturated HgCl2 solution to prevent modification in TCO2 due to biological activity. TA and TCO2 will be measured simultaneously by a potentiometric titration system using the method of Edmond (1970) described in the DOE Handbook of Methods for CO2 Analysis (DOE, 1994). TA and TCO2 will be estimated using a non-linear least-squares approach similar to that used by Dickson (1981). The accuracy of the measurements will be monitored by routine analysis of Certified Reference Materials (CRMs provided by A.G. Dickson, Scripps Institution of Oceanography). 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory 6. Precision and detection limits The precision, evaluated using both CRMs and replicate analysis of surface and deep samples, is estimated to be ± 2 and ± 4 µmol.kg-1 for CTand AT, respectively. 7. References D.O.E., Handbook of methods for analysis of the various parameters of the carbon dioxide system in sea water; version 2, DICKSON, A.G. & GOYET, C., eds. ORNL/CDIAC-74, 1994. 2 DISSOLVED ORGANIC CARBON (DOC) 1. Details of Institute: Laboratoire Arago – UMR 7621 – BP44 66651 Banyuls sur Mer cedex

Person reporting results: Mireille Pujo-Pay Email: pujopay@obs-banyuls.fr 2. Parameter measured Dissolved organic Carbon (DOC) 3. Sampling and conservation procedures: Sampling in glass vials rinsed 3 times before collection of samples. Sub samples are filtered through pre-combusted glass fiber filters (2 Whatman GF/F) and collected in glass tubes. They are poisoned with orthophosphoric acid are immediately analysed on board.

4. Outline of the method used: Pre-treatment and conservation techniques: Instrumental details and analysis: These sub-samples, collected in glass tubes and poisoned with orthophosphoric acid are immediately analyzed on board by high temperature catalytic oxidation (HTCO) (Sugimura & Suzuki 1988, Cauwet, 1994, 1999) with a Shimadzu TOC-V analyzer. Carbon concentration is determined by automatic comparison with a four-points calibration curve performed with standards (generally 50 to 200 µM C) prepared by diluting a stock solution of Acetalinid in MilliQ water. Each value reported is the average of at least three injections. Analytical accuracy of measurements is better than 2 %. Deep Sargasso Sea reference water (47 µM C, ± 0.5 SE ; http://www.rsmas.miami.edu/groups/biogeochem/CRM.html) is injected before analyses and every 10-12 samples to insure stable operating conditions. 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory -Participation to numerous Intercalibration exercises (February 2000, September 2001 in USA, January 2002) organized by Prof. Jonathan Sharp, Graduate College of Marine Studies, University of Delaware, Lewes, USA and supported by grants from the National Science Foundation (NSF) -https://www.ocean.udel.edu/cms/jsharp/interest_definitions.html#interest2 -Use of Deep reference water for DOC analyses http://www.rsmas.miami.edu/groups/biogeochem/CRM.html) 6. Precision and detection limits Analytical accuracy of measurements is better than 2 %. 7. References Cauwet G. 1994. HTCO method for dissolved organic carbon analysis in seawater : influence of catalyst on blank estimation. Mar. Chem., 47 (1) : 55-64. Cauwet G., 1999. Determination of dissolved organic carbon (DOC) and nitrogen (DON) by high temperature combustion. In: K. Grashoff, K. Kremling and M. Ehrhard (Eds), Methods of seawater analysis, 3 rd edition. Wiley-VCH, Weinheim. pp. 407-420.

Sugimura Y, Suzuki Y., 1988. A high-temperature catalytic oxidation method for the determination of non-volatile dissolved organic carbon in seawater by direct injection of a liquid sample. Mar Chem 24:105-131. 3 DISSOLVED AND PARTICULATE ORGANIC N AND P

1. Details of Institute: LOB UMR6535 Person reporting results: Véronique Cornet-Barthaux Email: veronique.cornet-barthaux@univmed.fr 2. Parameter measured Dissolved Organic Nitrogen (DON), Dissolved Organic Phosphate (DOP), Particulate Nitrogen (PN), Particulate Phosphate (PP) 3. Sampling and conservation procedures: DON & DOP: 20 ml samples will be collected from the Niskin bottles and immediately stored in Teflon vials. PN & PP: 500-1000 ml samples will be collected from the Niskin bottles, filtered onto GF/F filters and immediately stored in Teflon vials. 4. Outline of the method used: Pre-treatment and conservation techniques: Instrumental details and analysis: Total nitrogen and Total phosphate (TN & TP) will be estimated on board on 20 ml samples of seawater, using the same high-temperature persulfate wet-oxidation as for PN & PP (Pujo-Pay & Raimbault, 1994). Nitrate (NO3) and orthophosphate (PO4) obtained will then be analysed using the classical procedures previously described. DON = NO3TN- NO3PN-NO3 DOP = PO4TP- PO4PP-PO4 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory 6. Precision and detection limits Precision and accuracy: DON DL: 200 nmol L-1 DOP DL: 20 nmol L-1 DON & DOP concentrations accuracy decrease with increasing depth, when NO3 and PO4 concentrations became the dominant components of the total dissolved nutrient pools. Detection limit for 1000 mL of sample processed PON DL: 10 nmol L-1 POP DL: 1 nmol L-1 7. References Pujo-Pay M, Raimbault P (1994) Improvement of the wet-oxidation procedure for simultaneous determination of particulate organic nitrogen and phosphorus collected on filters. Mar Ecol Prog Ser 105: 203-207.

4 PIGMENTS

1. Details of Institute: CNRS - Laboratoire d’Oceanographie de Villefranche sur mer (LOV) BP 08 – Quai de la Darse – 06238 Villefranche sur mer - France Person reporting results: Josephine Ras Email: jras@obs-vlfr.fr 2. Parameter measured Phytoplankton pigments 3. Sampling and conservation procedures: Vacuum filtration of 2 L of seawater on 25 mm diameter Whatman GF/F glass fiber filters. Filters are placed in cryotubes. Immediate storage in liquid nitrogen then at -80°C. 4. Outline of the method used: Pre-treatment and conservation techniques:

Extraction of pigments: Filters are placed in 3 mL methanol 100%. Incubation at -20°C for 30 minutes. Filter disruption using an ultrasonic probe. Incubation for another 30 minutes at -20°C. Clarification of the extracts: by vacuum filtration on Whatman 25 mm GF/F filters. Instrumental details and analysis: Analysis is carried out on an Agilent Technologies HPLC (High Performance Liquid Chromatography) system. This comprises a degasser, binary pump, automated sampler, including Peltier temperature control (set at 4°C) and a programmable autoinjector with sample preparation prior to injection, programmable column oven compartment, Diode Array Detector and Chemstation for LC software (A.09.03). According to a modified version of the method described by Van Heukelem and Tomas (2001), sample extracts are injected onto a Zorbax Eclipse XDB-C8 column (3 x 150 mm, 3.5µm) with a 0.55 mL.min-1 flow rate. Separation, within 24 minutes, is based on a linear gradient between a 70:30 methanol:TBAA (Tetrabutyl ammonium acetate) 28mM mixture and a 100% methanol solution. Retention times and absorption spectra determine peak identification, while the quantification is carried out using the peak areas and corresponding specific absorption coefficients. 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory Short-term quality control (during a sequence run) is monitored using the internal standard solution in order to verify retention time reproducibility, peak area precision (should be less than 1%) and instrument stability during the analytical sequence. The stability of the pressure signal is also monitored during the analyses. Long-term quality control is carried out using a mixed pigment standard supplied by DHI Water and Environment (Denmark). This standard is regularly injected, at least in triplicate, to monitor the quality of the column and the instrument performance.

Signs of deterioration of the column or instrumentation can therefore be rapidly detected. The calibration of the volumetric measuring devices (pipettes, syringes, etc) is carried out annually. All these data are used to provide a set of performance metrics which have been determined in the frame of three previous intercomparison exercises (SeaHARRE-1, 2 an 3). In this way, the objective is to evaluate and maintain the “state of the art” level of analysis at the LOV. 6. Precision and detection limits Precision: instrument precision :0.4% precision for natural samples: 2% Detection limits (based on a filtration volume of 2.8 L):

Detection limit, mg.m-3 Pigments

0.0004 Chlorophyll a

0.0007 Carotenoids

7. References Van Heukelem L. and C. S. Thomas 2001. Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments. Journal of Chromatography A 910: 31-49.

5 BACTERIA AND PICOPLANCTON BIOMASS 1. Details of Institute: Laboratoire Arago CNRS / Université Pierre et Marie Curie – Paris VI 66650 Banyuls-sur-mer France Person reporting results: Philippe Catala Email: philippe.catala@obs-banyuls.fr 2. Parameter measured Enumeration of heterotrophic and autotrophic prokaryotes and autotrophic pico-and nanoeukaryotes by Flow Cytometry 3. Sampling and conservation procedures: Sampling is done with Niskin bottles. Duplicate aliquots of 4,5 ml are formaldehyde fixed (2% final concentration), stored 15 min at room temperature, then frozen in liquid nitrogen. The samples are stored at -80°C until flow cytometric analysis. 4. Outline of the method used: Pre-treatment and conservation techniques: cf references Instrumental details and analysis: cf references

5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory cf references 6. Precision and detection limits cf references 7. References Ingrid Obernosterer, Urania Christaki, Dominique Lefèvre, Philippe Catala, France Van Wambeke and Philippe Lebaron (in press) Rapid bacterial mineralization of organic carbon produced during a phytoplankton bloom induced by natural iron fertilization in the Southern Ocean. DSRII KEOPS Special Issue Baldwin A. J., J. A. Moss, J. D. Pakulski, P. Catala, F. Joux, W. H. Jeffrey. 2005. Microbial diversity in a Pacific Ocean transect from the Arctic to Antarctic circles. Aquat Microb Ecol. November : 91-102 Obernosterer I., P. Catala, T. Reinthaler, G. J. Herndl and P. Lebaron. 2005. Enhanced heterotrophic activity in the surface microlayer of the Mediterranean Sea. Aquat Microb Ecol. June : 293-302 Agogué H., E. O. Casamayor, F. Joux, I. Obernosterer, C. Dupuy, F. Lantoine, P. Catala, M. G. Weinbauer, T. Reinthaler, G. J. Herndl and P. Lebaron. 2004. Comparison of samplers for the biological characterization of the sea surface microlayer. Limnology and Oceanography : Methods. 213–225 6 MICROPLANKTON BIOMASS 1. Details of Institute: Laboratoire d’Océanographie et de Biogéochimie, Centre d’Océanologie de Marseille, UMR 6535, CNRS - Université de la Méditerranée, 163 Avenue de Luminy, Case 901, F-13288 Marseille, France Person reporting results: Fernando Gómez Email: fernando.gomez@fitoplancton.com 2. Parameter measured Microplankton 3. Sampling and conservation procedures: For microplankton analysis, seawater samples were collected with Niskin bottles from 0 to 250 m depth (9 depths). Four hundred seawater samples (9 samples × 44 stations) were preserved with acidified Lugol’s solution and stored at 3-8ºC until the microscopical analysis. 4. Outline of the method used: Pre-treatment and conservation techniques: At the laboratory, A 500 ml aliquot of the sample was concentrated by sedimentation in glass cylinders. During six days of settlement, top 450 ml of the sample was progressively but slowly siphoned off with small-bore tubing. About 50 ml of the concentrate representing 500 ml whole water sample was used for settlement in a composite settling chamber.

Instrumental details and analysis: The entire chamber was scanned at 200× magnification under an Nikon inverted microscope equipped with a digital camera. The specimens were photographed at 400×, 600x or 1000x magnification. 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory Not required 6. Precision and detection limits This technique is suitable for plankton cells higher than 15 µm in one dimension. Diatoms, dinoflagellates, ciliates and nauplii were well preserved, while most of the nanoplankton (i.e. cryptophytes and coccolithophores) was lost due to the fixation treatment or the incomplete sedimentation. The organisms were identified to species level when it was possible. Some groups of phytoplankton, especially the small pennate diatoms or small athecate dinoflagellate were counted, but not initially identified at the species level during the routine microscopy analysis. Further analysis by using scanning electron microscopy will allow to identify the most conflictive taxa. 7. References Gómez, F. 2007. Trends on the distribution of ciliates in the open Pacific Ocean. Acta Oecologica 32, 188-202. Gómez, F. 2007. On the consortium of the tintinnid Eutintinnus and the diatom Chaetoceros in the Pacific Ocean. Marine Biology 151, 1899-1906. Gómez, F., Claustre H., Raimbault, P. & Souissi, S. 2007. Two High-Nutrient Low-Chlorophyll phytoplankton assemblages: the tropical central Pacific and the offshore Peru-Chile Current. Biogeosciences Discussions 4, 1535-1554. Gómez, F., Furuya, K. & Takeda, S. 2005. Distribution of the cyanobacterium Richelia intracellularis as an epiphyte of the diatom Chaetoceros compressus in the western Pacific Ocean. Journal of Plankton Research 27, 323-330. Gómez, F. & Gorsky, G. 2003. Microplankton annual cycles in the Bay of Villefranche, Ligurian Sea, NW Mediterranean Sea. Journal of Plankton Research 25, 323-339. Gómez, F. & Claustre, H. 2003. The genus Asterodinium (Dinophyceae) as a possible biological indicator of warming in the Western Mediterranean Sea. Journal of the Marine Biological Association of United Kingdom 83, 173-174. Gómez, F., Echevarria, F., Garcia, C.M., Prieto, L., Ruiz, J., Reul, A., Jimenez-Gomez, F., Varela. M. 2000. Microplankton distribution in the Strait of Gibraltar: coupling between organisms and hydrodynamic structures. Journal of Plankton Research 22, 603-617. 7 MEZOZOOPLANCTON 1. Details of Institute:

Station Marine d’Endoume, Laboratoire d’Océanographie et de Biogéochimie, Centre d’Océanologie de Marseille, CNRS UMR 6535, rue de la Batterie des Lions, 13007 Marseille, France Person reporting results: François Carlotti Email: francois.carlotti@univmed.fr 2. Parameter measured (one report for each parameter or class of parameters) Mesozooplankton (200 µm – 2000 µm) species composition, abundance and biomass Abundance (species, development stages): taxonomic description, ZOOSCAN Abundance (size classes): Laser Optical Plankton Counter Biomass: Dry weight, CNP contents 3. Sampling and conservation procedures: Seawater samples are collected with Niskin bottles Net twos 200-0m twice a day, every day for long stations, once during daylight and once at night. Formalin buffered seawater preservation Oven dried 4. Outline of the method used: Pre-treatment and conservation techniques: For biomass, samples will be collected on a pre-combusted pre-weighted filter. Filters will be dried on board in an oven for 3 days then put in a dessicator. Instrumental details and analysis: Utermöhl chambers for nauplii counts. Nikon inverted microscope connected to a digital camera. Scale for dry weight measurements CHN autoanalyser for stoichiometric measurements Dissecting microscope for mesozooplankton diversity 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory Not required 6. Precision and detection limits 7. References Carlotti, F., Thibault-Botha, D., Nowaczyk, A, Lefèvre D. (2007) Zooplankton community structure, biomass and role in carbon transformation during the second half of phytoplankton bloom on the Kerguelen shelf (January –February 2005) and comparison with the oceanic areas. Deep Sea Res. (in press) Thibault D., E.J.H. Head and P.A. Wheeler, 1999. Mesozooplankton in the Arctic Ocean in summer. Deep-Sea Research, 46: 1391-1415 8 MICROZOOPLANCTON 1. Details of Institute: Université du Littoral Côte d'Opale -MREN Laboratoire d'Océanologie et de Géosciences CNRS, UMR 8187 LOG 32 avenue Foch, 62930 Wimereux, Fr Person reporting results: Urania Christaki

Email: Urania.Christaki@ univ-littoral.fr 2. Parameter measured heterotrophic protists abundance and biomass 3. Sampling and conservation procedures: Niskin bottle, 2 % formol or 2 % lugorl v/v 4. Outline of the method used: Pre-treatment and conservation techniques: 5-10 °C Instrumental details and analysis: Microscopy (epifluoresece and inverted) 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory - 6. Precision and detection limits - 7. References

Utermöhl, H. (1958) Wur Vervollkommnung der quantitativen Phytoplqnkton - metodik. Mitt. Int. Ver. Theor. Angew. Limnol. p. 9 – 38.

Porter, K.G., Y.S., Feig (1980) The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr., 25, 943 – 948

THIRD PRIORITY PARAMETERS: 1 PRIMARY PRODUCTION 1. Details of Institute: 1. Hellenic Centre for Marine Research (HCMR), Institute of Oceanography 2. Laboratoire d’Océanographie et de Biogéochimie (LOB), Centre d’Océanologie de

Marseille Person reporting results: Stella Psarra1, Thierry Moutin2 Email: spsarra@her.hcmr.gr, thierry.moutin@univmed.fr 2. Parameter measured Primary production (PP) (mgC m-3 h-1, mgC-2 d-1) 3. Sampling and conservation procedures: A. In Situ measurements Samples are obtained with 12-l Niskin bottles with silicone rubber closures and tubing that has carefully been checked to avoid introducing toxic metals during sampling. Up to 12 depths of sampling will be chosen according to the « in vivo » fluorescence profiles. B. On board microcosm experiment Waters for the microcosm experiment will be taken at 5 m depth at 3 long duration station using the same 12-l Niskin bottles and sampling procedure. 4. Outline of the method used: Photosynthetic carbon fixation within BOUM project will be measured in situ in vertical profiles along a transmediterranean transect according to the experimental protocol recommended by France-JGOFS-P.F.O. (1991), as modified by Moutin et Raimbault (2002). At the same time, primary production will also be measured in a nutrient addition bioassay experiment on board according to Steeman- Nielsen (1952) as modified by Psarra et al. (2005). A. In situ measurements: Each sample (320-ml polycarbonate bottle, 3 light and one dark sample per depth) is collected before sunrise, inoculated with 250 µl of the 14C working solutiona just before sunrise, and then incubated in situ. After 24h, the samples are filtered on GF/F filters to measure net absorption (AN mgC m-3). Filters are immediately covered with 500 µl of HCl 0.5 M and stored for counting at the laboratory. Each day, 3 samples are filtered immediately after inoculation for to determination, and 250 µl of sample is taken at random from 3 bottles and stored with 250 µl of ethanolamine to determine the quantity of added tracer (Qi). At laboratory, samples are dried during 12 h at 60°C, 10 ml of ULTIMAGOLD-MV (Packard) is added to the filters. and dpm is counted. B. On board microcosm experiment: For the on board nutrient addition experiment subsamples of 100ml will be taken from the incubation carboys of 9L in polycarbonate bottles (3 light and one dark per carboy) every second day of the experiment, inoculated with 250 µl of the 14C working solutiona and incubated for 2-3h during midday. By the end of incubations, samples are immediately filtered on GF/F filters to measure gross carbon assimilation.

The same procedure as for in situ measurements will then be followed until counting in the laboratory. Pre-treatment and conservation techniques: All glassware and working materials will be acid cleaned with 0.5 M HCl and autoclaved where appropriate prior to the experiments. Special care to avoid contaminations during sampling will be taken by using gloves. Calculations Net absorption AN(Ti ;T) for dark and light bottles was calculated from :

AN(Ti ;T) (mgC m-3) = (dpm-dpm(to))/(dpm(Qi)*1280) * TCO2b

where Ti corresponds to the starting time of the incubation since dawn and T to the incubation duration. Primary production rates PP* (* = 24h from dawn-to-dawn) were obtained from: PP* (mgC m-3 j-1) = ANlight* - ANblack* = ANlight(Ti ;T) / τ(Ti ;T)

c - (ANblack(T) /T)*24 Integrated primary production IPP* (mmol m-2 d-1) has been calculated with trapezium method assuming (1) that subsurface (about 5 m) rates are identical to surface rates (not measured) and (2) that rates are zero at 20 m below the deepest sampled depth. Notes: a Working solution : 12.5 ml of NaH14CO3 (25 mCi, 50-60 mCi/mmole, Amersham CFA3) was added to a solution containing 0.09 g of Na2CO3 (Aldrich 20,442-0) per 300 ml of sterilized milliQ water. This solution was stored in sealed 15 ml glass flasks. b TCO2 = 2 106 µmolC m-3. c τ(Ti ;T) (conversion factor depending on the date and the latitudinal position) was determined according to Moutin et al. (1999). Applying this procedure allows to normalize primary production rates obtained from incubation duration ≤ 24h in a given region at a given date, to daily rates, thus allowing the comparison of data obtained from different experimental incubation durations.

Remark : for all the 24-h incubation duration, τ(Ti ;T) = 1. Instrumental details and analysis: After 24h of scintillation fluor addition dpm counts are obtained with a Packard Tri carb 2100 TR liquid scintillation analyser. 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory Intercalibration is repeated periodically between the persons involved in the measurements. 6. Precision and detection limits According to Steeman- Nielsen (1952) the detection limit for Primary production is 0.05 mgC m-3 h-1.

7. References JGOFS, Core measurements protocols : report of the core measurement working group. JGOFS report n°6, Joint Global Ocean Flux Study, SCOR (1988) 1-40. Moutin, T. and Raimbault, P., 2002. Primary production, carbon export and nutrients availability in western and eastern Mediterranean Sea in early summer 1996. J. Mar. Sys., 33-34: 273-288. Moutin, T., P. Raimbault & J.C. Poggiale. 1999. Production primaire dans les eaux de surface de la Méditerranée occidentale : Calcul de la production journalière. . C. R. Acad. Sci. Paris, Sciences de la vie. 322 : 651-659. Psarra, S., Zohary, T., Krom, M.D., Mantoura, R.F.C., Polychronaki, T., Stambler, N., Tanaka, T., Tselepides, A., Thingstad, T.F., 2005. Phytoplankton response to a Lagrangian phosphate addition in the Levantine Sea (Eastern Mediterranean). Deep Sea Research II, 52(22-23): 2944-2960 Steeman Nielsen, e. (1952). The use of radioactive carbon (14C) for measuring organic production in the sea. J. Cons. Perm. Int. Explor. Mer, 18:117-140 2 BACTERIAL PRODUCTION 1. Details of Institute: Laboratoire de Microbiologie, Géochimie et Ecologie Marines, LMGEM, UMR CNRS 6117, Case 901, Campus de Luminy, 13 288 Marseille Cedex 9, France Person reporting results: France Van Wambeke Email: france.van-wambeke@univmed.fr 2. Parameter measured (one report for each parameter or class of parameters) Bacterial production with 3H-leucine technique and treatment with centrifuge protocol. 3. Sampling and conservation procedures: Niskin from CTD rosette. Sea water collection in polycarbonate bottles. (total sea water, no pre-filtration). Processing of samples within 30 min after sea water collection. 4. Outline of the method used: Pre-treatment and conservation techniques: Storage of sea water before incubations with 3H leucine is not possible Instrumental details and analysis: Prepare 3 centrifuge tubes per layer1 Add in the blank 200 µl TCA 45 % Add 1,5 ml seawater sample per tube Add 100 µl of a mixture Hot-Cold leucine2 Incubate appropriate time periods3, in the dark4, at in situ temperature stop the 2 duplicates with addition of 200 µl TCA 45 % mix (vortex)

1 Always use the same Manufacturer .We use Safe-Lock 2ml Eppendorf tube. 2 [4,5-3H]-leucine, 150-180 Ci mmol-1, by Amersham-France, code TRK 636. Final concentrations 13nM unlabelled+ 6 nM radioactive is generally OK for surface Mediterranean seawater 3 In surface Med Sea 2 hours is generally OK 4 Recently effect of light has been shown to have incidence on the results. It should be checked.

storage in fridge possible at this stage, no longer than couple of days (optional: add 50 µl of a Bovine Serum Albumine solution (final concentration required 100 µg/ml), Mix, centrifuge 16 000g 10 minutes at 13°C)5 pump out the supernatant with a vacuum pump add 1,5 ml of TCA 5 % Mix, centrifuge 16 000 g 10 minutes a 13°C, pump out the supernatant (optional: add 1.5 ml 80% ethanol, mix, centrifuge again, pump out supernatant) add 1,5 ml scintillation liquid vortex after 5 minutes count on board or store in fridge for delayed counting at the laboratory computation of rates of leucine incorporation and bacterial carbon production as in Kirchman, 1993. 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory For each cruise are checked different points: - Linearity of the incorporation with time (checked with time kinetics) - Saturation of the label / isotopic dilution (checked with concentration kinetics) - Unspecific lipid labeling (comparison of the effect or not of an 80% ethanol rinse step) And optionally, if possible to manage onboard: - Cross comparison of methods (centrifuge/filtration, and effect of addition of BSA on the results of this comparison) - Effects of light vs dark incubations in euphotic layers - Estimation of empirical conversion factor using dilution experiments (filtration of sea water through 0.2 µm, inoculation with a 0.8 µm filtrate, incubation in the dark for a few days during which both bacterial abundances and bacterial production are regularly measured). 6. Precision and detection limits With both the filtration and the centrifugation technique, accuracy measurements are made when the signal in disintegration per minutes (dpm) is a least 2 times the blank value (generally it is much higher). For this, it is possible to increase the detection limits by slights modification of the protocol: - increasing the proportion of radioactive leucine in the mixture hot/cold leucine, (at the maximum only radioactive leucine is added) - increasing the volume of sample incubated (only for filtration technique) - increasing the incubation time (but still with respect of linearity and still if less than 10% of the label is used during the incubation time) For these reasons, the centrifuge technique (maximum volume incubated 1.5-2 ml) is generally limited to approx. 200m depth.

5 This step is used to add a co-precipitant molecule for helping precipitation and gives the advantage that the pellet is visible. However this step is avoided in most protocols, because in the original method it had been shown to have no effect on the results and to increase blank signal and variability between duplicates (Smith and Azam, 1992). However it should be checked in very oligotrophic environments particularly if both centrifuge and filtration technique will be used and compared (Van Wambeke et al 2002).

Reproducibility within duplicates is on average 10% 7. References Kirchman, D. L., 1993: Leucine incorporation as a measure of biomass production by heterotrophic bacteria. Handbook of methods in aquatic microbial ecology, P. F. Kemp, B. F. Sherr, E. B. Sherr, and J. J. Cole, Eds., Lewis, 509-512. Moran, X. A., Massana, R., and Gasol, J. M.: Light conditions affect the measurement of oceanic bacterial production via leucine uptake. Appl. Environ. Microbiol., 67, 3795-3801, 2001. Pace, M. P., del Giorgio, P., Fisher, D., Condon, R., and Malcom, H.: Estimates of bacterial production using the leucine incorporation method are influenced by differences in protein retention of microcentrifuge tubes. Limnology and Oceanography: Methods, 2, 55-61, 2004. Smith, D. C. and Azam, F.: A simple, economical method for measuring bacterial protein synthesis rates in sea water using 3H-Leucine. Marine Microbial Food Webs, 6, 107-114, 1992. Van Wambeke, F., Christaki, U., Giannakourou, A., Moutin, T., and Souvemerzoglou, K.: Longitudinal and vertical trends of bacterial limitation by phosphorus and carbon in the Mediterranean Sea. Microbial Ecology, 43, 119-133, 2002. 3 ZOOPLANKTON PRODUCTION 1. Details of Institute: Laboratoire d’Océanographie et de Biogéochimie, Centre d’Océanologie de Marseille, CNRS UMR 6535, Campus de Luminy, Case 901, Marseille, France Person reporting results: Delphine Thibault-Botha Email: delphine.botha@com.univmed.fr 2. Parameter measured (one report for each parameter or class of parameters) Metabolic rates (feeding, respiration, excretion and egg production) 3. Sampling and conservation procedures:

Long stations

- Water samples from chlorophyll maximum (~5L) - 0-200 m Bongo net tow, samples will be collected at night several times per long stations; daylight sampling can also be added.

Short stations

- Water samples from chlorophyll maximum (~5L) - 0-200 m Bongo net tow, samples will be collected if sampling preferably done at night. Liquid nitrogen preservation Formalin buffered seawater preservation 4. Outline of the method used: Pre-treatment and conservation techniques:

1)- Gut fluorescence technique: measurements of Chlorophyll contained in copepods by flurimetry 2)- Feeding rates: gut evacuation rates methods, and pigment-taxon specific grazing through incubation 3)- Respiration through oxygen depletion assessed by an oxygen probe 4)- egg production rates through incubation of gravid female Instrumental details and analysis: Fluorimeter Turner Design, HPLC YSI oxygen probe Dissecting microscope Fluorimeter Turner Design 5. Quality control / Quality assurance (QC/QA) routinely employed within the laboratory 6. Precision and detection limits 7. References Carlotti, F., D. Thibault-Botha, A. Nowaczyk, and D. Lefèvre, 2007. Zooplankton community structure and physiological rates on the Kerguelen shelf and oceanic areas: responses to the phytoplankton bloom in January –February 2005. Deep-Sea Research II Thibault D., S. Roy, C.S. Wong and J.K. Bishop, 1999. The downward flux of biogenic material in the NE Subarctic Pacific: Importance of algal sinking and mesozooplankton herbivory. Deep-Sea Research II, 46, (11-12): 2669-2697 Boyd P.W., N.D. Sherry, J.A. Berges, J.K. Bishop, S.E. Calvert, M.A. Charette, S.J. Giovanni, R. Goldblatt, P.J. Harrison, S.B. Moran, S. Roy, M. Soon, S. Strom, D. Thibault, K.L. Vergin, F.A. Whitney and C.S. Wong, 1999. Transformation of biogenic particulates from the pelagic to the deep ocean realm. Deep-Sea Research II, 46, (11-12): 2761-2791 Thibault D., E.J.H. Head and P.A. Wheeler, 1999. Mesozooplankton in the Arctic Ocean in summer. Deep-Sea Research, 46: 1391-1415 Thibault D., R. Gaudy and J. Le Fèvre, 1994. Zooplankton biomass, feeding and metabolism in a geostrophic frontal area (Almeria-Oran, Western Mediterranean). Significance to pelagic food webs. Journal of Marine Systems, 5: 297-311 Carlotti , F., Rey, C., Javanshir, A. & Nival, S.,1997. Laboratory studies on the rates of egg production and fecal pellet production for the copepod Centropages typicus: individual variability, effect of age and effect of temperature. J. Plankton Res. 19, 8, 1143-1165 Halsband, C., Nival, S., Carlotti, F., Hirche, H.J., 2001. Seasonnal cycle of egg production of two co-dominant copepods Centropages typicus and Temora stylifera in the north-western Mediterranenan Sea. J. Plankton Res. 23(6): 597-609 Rey-Rassat C, Irigoien X, Harris R, Head R, Carlotti F., 2002. Egg production rates of Calanus helgolandicus females reared in the laboratory: variability due to present and past feeding conditions. Marine Ecology Progress Series, 238:139-151