"Ecological use of marine phytoplankton pigments at the Antares-ChloroGIN time-series-stations" Workshop
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“Ecological use of marine phytoplankton
pigments at the Antares-ChloroGIN time-series-
stations” Workshop
Latin American Regional Project
EDIMAR, Margarita Island, Venezuela. 21 to 24 October 2013.
Report of the Workshop of the Latin American Regional Project 2013.
“Ecological use of marine phytoplankton pigments at the Antares-
ChloroGIN time-series-stations” Workshop
Latin American Regional Project - 2013
The workshop “Ecological use of marine phytoplankton pigments at the Antares-ChloroGIN
time-series-stations” (hereafter Margarita Workshop) was carried out in the framework of the second
phase of the Latin American Regional Project for the NANO Network (LA-NANO Project, http://www.nf-
pogo-alumni.org/about+NANO). The workshop took place from 21 to 24 October of 2013 and it was
hold at the Station of Marine Research of the La Salle Foundation for Natural Sciences in Margarita
Island, Venezuela.
The LA-NANO Project started in 2012, after an initiative of the Nippon Foundation and POGO
who gave financial and logistic support to former scholars to carry out research projects at a regional
scale. One of the achievements of the first phase of the project was to send more than 50 pigment
samples from 6 time series stations to be analyzed by HPLC at a the Ocean Ecology Laboratory, NASA-
Goddard Space Flight Center (Greenbelt, Meryland, USA).
Briefly, the workshop consisted of lectures, working sessions and group discussions among the
topics of phytoplankton pigments uses in oceanographic research and the possibilities of carrying out
HPLC analyses in Latin America. The main goals set for the workshop are stated below:
Offer a brief introduction to the High Performance Liquid Chromatography (HPLC) technique for the
analysis of phytoplankton pigments. Principles and comparison of methods. Applications in
oceanography; pigment indices, CHEMTAX, remote sensing of Phytoplankton Functional Types, etc.
Discuss and interpret the results of the 2012 NANO Regional Project for Latin America; including,
whenever possible, a comparison of pigment results obtained at each center and those provided by
NASA.
Discuss a first outline of a manuscript/report using this pigment information in conjunction with
satellite data. This manuscript will be a concrete output of the Latin American NANO Project, where
the NANO Alumni and young ANTARES participants will be co-authors; and the senior scientists
NANO-Friends will generously guide them.
Evaluate the technical and budgetary challenges of each ANTARES station to conduct HPLC pigment
analysis locally that meet NASA quality standards.
Design a strategic plan to ensure that pigment HPLC analyses are systematically carried out at all
ANTARES stations.
Gather the pigment samples collected at each of the participating stations during 2013 at EDIMAR in
order to be shipped to NASA for HPLC analysis in a single shipment.
The workshop counted with two special guests. On the one hand, Dr Suzanne Roy, from the
Institut des sciences de la mer de Rimouski, Université du Québec à Rimouski (Canada), offered lectures
on general topics regarding the diversity of phytoplankton pigments, CHEMTAX and the ecological use of
the pigment data in oceanographic research. On the other hand, Crystal Thomas, from the Ocean
Ecology Laboratory, NASA-Goddard Space Flight Center (USA), dictated a few classes on HLPC analysis,
quality control and HPLC methods. Both guests provided a valuable input as lectures and also by getting
involved during the discussions regarding practical issues concerning how to implement or improve
HPLC analysis in our Latin-American centers. The complete list of the participants is shown in the
appendix A; the workshop agenda is reproduced below.
Workshop Agenda
Monday 22 October
Morning
Welcome- Main goals of the workshop - Introduction of participants. Vivian Lutz – Jaimie Rojas.
Brief introduction to NANO. Guillermina Ruiz – Jaimie Rojas.
Lecture 1: Diversity of phytoplankton pigments (type of pigments, properties, roles). Suzanne Roy.
Lecture 2: Pigment indices, CHEMTAX. Suzanne Roy.
Afternoon
Lecture 3: Methods, starting from collection of samples, and overview of different techniques to measure
pigments. Crystal Thomas.
Brief presentations of the work being carried out at the participating time series stations, stating if they carry
out HPLC analysis, expectations of the use of pigments data to enhance their studies.
1. Antares – EPEA (Vivian - Guille)
2. Antares – Ubatuba (Natalia - Mayza)
3. Antares – Cartagena (Liseth)
4. Antares - Ensenada (Eduardo - Roberto)
5. Antares – IMARPE (Jesus)
6. Antares – CARIACO (Yrene - Jaimie)
Tuesday 22 October
Morning
Lecture 4: Applications of pigments information in oceanography (show a few examples). Suzanne Roy
Lecture 5: Use of pigments in conjunction with remote sensing of ocean color to study Phytoplankton
Functional Types. Suzanne Roy – Vivian Lutz
Break
Lecture 6: Components of high-quality HPLC pigment analysis. Crystal Thomas.
Lecture 7: Different methods; comparisons (inter-calibration exercises). Crystal Thomas
Afternoon
Brief overview of the methods used at the stations which are carrying out HPLC analysis.
- Ensenada – Roberto Millan
- EPEA – Vivian Lutz (for Nora Montoya)
- GSFC - Crystal Thomas
Discussion regarding the best protocol of sample collection (volume filtered, time of filtration, etc.) and
method of analysis (extraction solvent, disruption, etc.).
Recommendations.
Wednesday 23 October
Morning
Case example of a preliminary interpretation of the pigment data (provided by NASA) and discussion
considering ancillary data (phytoplankton taxonomy, temperature, mixing regime, etc.). Vivian Lutz.
In depth explanation of the information provided in the pigments spreadsheet of the NASA laboratory (data,
quality control, abbreviations, etc.). Crystal Thomas.
Each station works on a first interpretation of the pigments data obtained by NASA, evaluation of the
possible main groups of phytoplankton present; calculation of the pigment indices (Uitz et al. 2006).
Everybody, with inputs from Suzanne Roy, Roberto Millan Nuñez, Crystal Thomas, Vivian Lutz.
Afternoon
Each station works on a first interpretation of the pigments data obtained by NASA, evaluation of the
possible main groups of phytoplankton present; calculation of the pigment indices (Uitz et al. 2006).
Everybody, with inputs from Suzanne Roy, Roberto Millan Nuñez, Crystal Thomas, Vivian Lutz.
Possible use of satellite information corresponding to the different stations; date of sampling, different spatial
and temporal scales.. Eduardo Santamaría
Discussion.
Thursday 24 October
Outline of the table of contents of a report using these data.
Discussion about how to improve the capability to carry out pigment analysis in Latin-America.
- Which will be the minimum requirements involving sampling and analysis.
- Which type of instrument, supplies, calibration, maintenance and expert handling it requires.
- Concrete possibilities of the different participating centers to implement this and maintain it in the long run.
- Advantages and disadvantages of proposing a centralized laboratory in Latin-America to carry out
measurements for the Antares time-series.
- Possible sources of funding towards this aim.
Sample collection and storage Recommendations
It is important to be careful on the collection, filtration and preservation of the samples.
The filtration volume was discussed. It was agreed
(comments from Crystal Thomas, Suzanne Roy and other
experts in the group) that the volume to filter should be
enough to reach visible color on the filter (i.e., as much as 3
liters in oligotrophic waters, and as little as 100 ml or less in
eutrophic waters). On the other hand, the total time of
filtration is also important, it should not exceed 30-40
minutes; even if sometimes in oligotrophic waters the filter
will not get too colored. The possibility of using two filters
was also discussed, especially in waters with a high
concentration of sediments; since in these cases the filter
gets clogged fast, but without much phytoplankton in it. On
the other hand, Crystal commented that when there are two
filters for a single sample, she needs to make the extract with
the double of volume of solvent; hence there is not too much
to gain in the final concentration of pigments (since the
sample was diluted).
It was reminded that the pressure of filtration should not exceed 5 PSI (< 35 kPa) to avoid sucking up
small cells. There was also a discussion about the use of positive filtration; the group from Ensenada
showed pictures and explained the advantages of this system.
It was suggested that it is advisable to remove any copepods visible on the filter (taken them from
the antennas with a twisters) or if possible when they are floating in the filtration funnel.
Do not let the filters dry on the filter support; after wrapping them, put them immediately in the
liquid nitrogen. It was advised that whenever possible keep the Dewar beneath the filtration bench,
or even have a small one for that purpose. In the case of not having this possibility, an alternative
would be to put the filters immediately inside a cooler with cryo-gel-packs (-23 ºC). The possibility of
getting a small liquid nitrogen producing machine was commented.
Extra care should be put on the labels. Use adequate markers that do not wash away with the liquid
nitrogen and solvents. Write the labels, either directly on the aluminum foil or on stickers, before
wrapping the samples (i.e., the surface is not wet and avoids smear of the ink, and there is no risk of
piercing the aluminum and contaminate the sample). Stickers should be of a quality that will not
detach; it was commented the use of a kind of ‘hypoallergenic medical adhesive tape’ with good
results. Another possibility is using two stickers per sample (one inside the folding apart from the
one outside).
Strategy to obtain quality phytoplankton pigments information at the Antares stations
1) The question whether it was important to get information on pigment composition or just on
chlorophyll a concentration was discussed. All the participants expressed their interest in counting with
pigment composition to be used in different studies:
To help identify phytoplankton groups (small cells) in conjunction with microscopy information, and
hence help to:
o detect changes associated to climate and anthropogenic sources
- e.g., to monitor the richness and diversity of the phytoplankton community and the
impacts of changes produced by El Niño and La Niña events.
o evaluate the impacts of such changes in the productivity.
o understand the seasonal dynamics of the phytoplankton communities.
To be used in conjunction with satellite information:
o To have accurate estimates of Chla for ocean colour match-ups.
o To develop new satellite applications (e.g., PFT models, HABs detection) in conjunction with
microscopy and bio-optical data.
2) The current situation regarding carrying out these analyses at each station was commented:
Ensenada (Mexico): They are running HPLC analysis within their own laboratory.
Ubatuba (Brazil): They do not have an HPLC, the only data they have so far is that analyzed by NASA.
They have however some possibilities to work in collaboration with other laboratories within other
Brazilian universities and they will make efforts to carry out pigment’s analysis in Brazil as soon as
possible.
Cartagena (Colombia): They are in the process of buying an HPLC which may take a year to be
achieved. They have also considered the budget to hire and train personal to run the analysis, as
well as, to acquire the required standards, solvents and other consumables for the analysis.
IMARPE (Peru): There is an HPLC system at another laboratory in IMARPE and they have already
made an agreement to start analyzing pigments samples from their time-series. There is budget for
training and maintenance of high quality analysis.
EPEA (Argentina): They have an HPLC system, and they have already analyzed some samples from
the time series at INIDEP.
3) The way to try to achieve the best quality data was discussed.
Attention especially for the stations that are buying HPLC instruments: do not be concerned only
with the instrument required, but also the standards and solvents which are expensive and most
importantly the training of the personal that will be responsible to run the analysis, as well as to
process and analyze the data.
Regarding the use of a common protocol and ‘best-practice’ method to analyze pigments by HPLC, it
was suggested that:
o It is crucial to make a good calibration of any system used.
o To know the strengths and short-comes of the method used.
o According to previous inter-comparison exercises most methods give comparably good
results of the primary pigments (Chla, and others present in relative high concentrations).
o Pigments data ran at each laboratory may also be compared to algal culture mix prepared
by DHI which may be bought with the standards and are not expensive ($20 USD) to check if
the results are consistent.
It will be important in a near future to carry out an inter-
comparison of pigments results by HPLC obtained at the
different Antares stations.
Also to follow up on the fluorometric Chla inter-comparison
(from the one performed in 2006).
Regarding intercomparisons, it was suggested that a first step
will be comparing the pigments data obtained at each station
with that provided by the NASA laboratory. Even more,
Crystal Thomas offered to analyze all the samples received
from this second-phase LA-NANO project by the classic
fluorometric technique as well as by HPLC.
The groups from the Peruvian and Colombian stations, who
will be starting soon to analyze their own samples, should
check for training opportunities for the HPLC analysis.
Throughout the whole workshop, the necessity to
compliment pigment data with the identification of
phytoplankton species using microscopy was stated.
Tasks from the workshop
Homework for each station
Deadline to submit to SeaBASS the pigment data already analyzed by NASA: End of November.
Interpret the pigments data provided by NASA. Look at the results, check the Tables with
pigment composition in the different algae groups; write a brief description of the possible
phytoplankton groups present in your samples. Send these brief descriptions to Roberto Millan
and Suzanne Roy by end of December.
Calculate the pigment indices PPC/Tpig; PSC/Tpig; PPC/PSC; Uitz et al (2006) indices.
Analyze this data together with microscopy phytoplankton information. Compare whether there
are coincidences or discrepancies.
If possible, try to adjust Uitz indices.
Interpret this phytoplankton diversity information with ancillary oceanographic information.
Eduardo Santamaría will write a description of the satellite information, match-ups.
End of January each group will send these parts to Guille and Jaimie. They will draft a
manuscript.
Outline LA-NANO manuscript
A preliminar outline of the manuscript was agreed among the participants which is the following:
1. Index of phytoplankton biomass [TChla]
a. HPLC
b. Fluorometer or spectrophotometer
c. Satellite (different sensors, different scales)
2. Main phytoplankton groups present
a. Description of the main possible groups found according to pigment composition in the
samples.
b. Pigments indices (Uitz)
c. Microscopy information
d. Compare both, and readjust indices
3. Photoacclimation status of phytoplankton
a. Indices PPC/Tpig, PSC/ Tpig; PPC/PSC
b. Microscopy information
A description of phytoplankton richness (biomass and diversity) at the different NANO stations
will be made. This information will be interpreted in relationship with the biogeochemical provinces
(physical, chemical environment), and seasons to which they belong. An analysis of the phytoplankton
groups and photoacclimation according to the physical conditions with season and depth (MLD, Zeu) will
be made. A match-up TChla in situ vs satellite will be made. Other satellite applications will be explored.
Future Directions
The future direction of a LA-NANO project will be discussed in December during the NANO
Meeting in Berlin (Germany).
A new advisor and coordinator for the project should be selected. This issue was discussed
within the participants from the stations and was commonly agreed that ‘Ensenada’ (Mexico)
could lead the next stage of the LA-NANO; they have three NANO alumni/instructors (Roberto
Millan-Nuñez, Eduardo Santamaria del Angel, Adriana Gonzalez-Silvera).
It will be good to try to include other stations (e.g. from Chile, Ecuador and south of Argentina)
in the next stage of the LA-NANO.
A project was recently approved for the Antares network by the Inter-American Institute for
Global Change Research CNR-3094 “Assessment of Marine Ecosystem Services at the Latin-
American Antares Time-Series Network”. It will be interesting to make a link with the next LA-
NANO project.
Further Outcomes from the Workshop
A total of 63 samples from the different participants were gather at EDIMAR. Fortunately all the
samples arrived to Venezuela at the appropriate temperature but once again we confirmed that
transporting samples refrigerated in dry ice overseas is a difficult task. Customs regulations are quite
strict; some of us were required to drop the dry ice or even to make holes in the polystyrene boxes,
which put at risk the integrity of the samples. It was recommended during the workshop to acquire cry-
gel packs that can be chilled at -23°C to avoid future problems.
Antares samples
An ad-hoc ANTARES Meeting was held among
the present members, where future work directions
were discussed. Topics such as the website
maintenance, satellite images provision and the
possibility of incorporating new time series stations to
the Antares network were discussed, among others.
Suzanne Roy kindly brought a copy of the book
“Phytoplankton Pigments: Characterization,
Chemotaxonomy and Applications in Oceanography”
Ad hoc Antares meeting.
(Cambridge Environmental Chemistry Series), of which
she is one of the editors, which was drawn among those participants who didn´t have a copy at their
laboratories. Notwithstanding, contact with Ed Urban from SCOR was made, who promised to send
copies to the remaining groups.
Time for recreation was also stipulated during the workshop. During one of the afternoon
breaks, hosts Dr. Ramón Varela and Dr Yrena Astor showed us around the oceanology museum “Benigno
Roman”, museum housing the biggest algae collection from the Caribbean (among many other
interesting collections). On Thuesday 24, after finishing the last session, we went onto a mini-tour
around the isle and have dinner in town for a final toast. It goes without saying that all these activities
facilitate the strengthening of the relationships between NANO, Antares members and the special
guests as well.
Finally, it is worth mentioning that the Margarita Workshop served as a medium for the consolidation of a group work dynamics within the NANO and ANTARES communities, which promise to yield profitable outcomes for the whole region. Moreover, both Crystal Thomas and Suzanne Roy perceived and acknowledged the commitment of our group to find the way to overcome logistics and budgetary restrictions in order to get high quality data.
Appendix – List of participants Special guests: Suzanne Roy – Institut des sciences de la mer de Rimouski (Canada) Crystal Thomas – NASA Goddard Ocean Biology Processing Group (USA) NANO – Antares: Guillermina Ruiz – Antares – EPEA (INIDEP, Argentina) Vivian Lutz – Antares – EPEA (INIDEP, Argentina) Natalia Ruddorf – Antares – Ubatuba (INPE, Brazil) Mayza Pompeu – Antares – Ubatuba (USP, Brazil) Liseth Arregoces – Antares – Cartagena (CIOH, Colombia) Eduardo Santamaria – Antares - Ensenada (UABC, México) Roberto Millan – Antares – Ensenada (UABC, México) Jesus Ledesma – Antares – IMARPE (IMARPE, Peru) Rafael Rasse – (Instituto Venezolano de Investigaciones Científicas (IVIC)) Rene Ayala – (SURADI Suministros; CARIACO Caracas, Venezuela) Yrene Astor – Antares – CARIACO (EDIMAR, Venezuela) Jaimie Rojas – Antares – CARIACO (EDIMAR, Venezuela) Ramón Varela – Antares - CARIACO – (Venezuela) Luis Troccoli - Antares- CARIACO – (Venezuela) Laurencia Guzman- Antares- CARIACO – (Venezuela) Luis Medina - Antares- CARIACO – (Venezuela) Alberto Rosales - Antares- CARIACO – (Venezuela)
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