How to Sample Water with a GoFlo

Entering Seattle

Fur Seal visits the R/V Melville

Festive ending

As we continue into the Straight of De Fuca, halfway between the U.S. and Canada, the final batch of sampling is being taken for analysis. I'm looking at the mountains in the Olympic National Forest as the familiar music plays as on the fantail, mostly infamous songs from the movie, "National Lapoon's Christmas Vacation." We have a few more locations of interest as we pull into the port at the University of Washington campus on Friday. It's now time to begin the equally difficult work of packing all that has been brought on board which will take all of the next 2-3 days. The sun is out and the mood is that of elation. Science at sea is not easy work. From preparation and planning to tackling problems with equipment and limited resources at sea, these marine scientists have persevered through it all.

The research has been successful, and the learning has been overwhelming, as everyone is eager to teach and learn. These are lifelong learners unlike any I have ever witnessed firsthand. Science is knowledge, an understanding of mysteries one test at a time. As Dr. Wells said to me, "If we knew everything that we were doing, it wouldn't be called research." 

After this research cruise, we are one step closer to understanding the vast unknown of the ocean and the future of life as we know it. 

I am currently working on videos to add to this blog once I get home to sufficient bandwidth. I will be interviewing each principal investigator and report as I have on the other parts of this journey. The day by day work with many more pictures and information can be found on my friend and collegue, Denis Costello's blog, socalcostello.blogspot.com. Feel free to comment if there is more that you'd like to see or know about life and science at sea.

UW joins the science party

After several strenuous days of sampling and working in the lab, we are now in the home stretch with quite a few sites of interest in our path. In coordination with NOAA-PMEL, Rachel Van Giessen represents the University of Washington on this cruise. Her team of three is playing an important role in both the grand design of our research onboard and the collaboration between NOAA and UW. They are analyzing both dissolved oxygen (DO) and collecting samples for the labs back at the university to analyze for dissolved inorganic carbon (DIC). Rachel's team has been supporting in every way on board by deploying the CTD with me each day and helping wherever is needed. Now that we are off the coast of Washington, there are specific sites that NOAA is particularly interested in that are now our target sites. These sites have been chosen because of their value for comparing data in the same location over time. We have monitored the water chemistry for many years from these exact spots and are able to see the changes, year to year. Entering the Strait of Juan De Fuca, we will be increasing our workload to collect data all along the way.

Their work:

Rachel Vander Giessen, UW, titrating to find DO in a sample

Hannah and Kit work together to collect the water sample
and poison the sample to stop the biological processes.

Titration mobile station.
Notice the cloudy precipitate in the glass vial.

Rachel, Hannah and Kit work together to carefully collect samples from various depths without any contamination. They are the first to sample from the CTD and make sure that no air in the Niskin enters their specialized glass containers. Contamination from the atmosphere is their number one concern  and the reason that they follow such tight protocols. As soon as the sample is taken without a single bubble, they poison the sample to stop any biological processes inside the bottle, freezing the chemistry of the water for analysis. Otherwise, the organisms within would continue using up the nutrients and change the oxygen and carbon dioxide content of the water. By adding 1 mL of manganese chloride and another milliliter of a combination of sodium hydroxide and nitrogen iodide, they create a precipitate, clear evidence of a chemical change. The solid (precipitate) that is formed is directly proportional to the DO (dissolved oxygen) that they ultimately want to calculate on board. Within a five day window, they add 1 mL of diluted sulfuric acid which frees the iodine and turns the basic solution to the acidic side of the pH scale (under 7). They then add a starch solution (think potato) to use as an indicator to determine when the solution becomes perfectly neutral. Using this method that has been used since the late 1800's (1888 to be exact), they titrate with a device that allows them to add one thousandth of a milliliter of sodium thiosulfate at a time, looking carefully for when the solution becomes completely clear. At this point, they record the amount of sodium thiosulfate that was needed and input this number into a formula to find the dissolved oxygen content, accurately and without contamination.

Rachel works on the deck with the chemicals used for DO calculations.

These calculations and those for the DIC (dissolved inorganic carbon) will be used in other formulas all over the vessel. It's a tight-knit family here with everyone collaborating, adding to the big picture one titration at a time.

Meet the team: 

Rachel Vander Giessen, 33, has had a passion for the ocean as long as she's lived in Seattle. All of her life. After graduating from high school, her passion propelled her to apply for the Maritime Marine Academy in Seattle, like so many of the full-time crew on board the Melville, but a subpar math score on the entrance exam kept her from pursuing this dream. Ironically, her degree now is in physics. "All it took was a good professor," she explained, to change her mind and turn her on to the mathematical world around us. After a few years of crewing private vessels through the inside passage to Alaska, she finished her degree and began volunteering at UW. It was on another research cruise as a volunteer with Jan Newton (UW) to repair the Cha Ba buoy (one of our sites of interest) that she was offered a permanent job with the university that has led to her work here with us.

Rachel holds the clipboard and lets each person know when
to start sampling. This helps avoid confusion and contamination.

Hannah Glover sampling for DO.

Hannah Glover, 23, moved from her lifelong northeastern home to Seattle after graduating from Bowdine College in Brunswick, Maine and working for a while with the Maine geological survey. Hannah is a natural at sea and loves the work and the outdoors. Her move to Seattle quickly turned into an internship at a land trust making maps before joining the UW applied physics lab as a volunteer. With UW and Rachel, she helped deploy ORCA buoys; as with everything in science, ORCA is an acronym for oceanic remote chemical analyzer. When asked if she wanted to join this science excursion, Hannah did not hesitate to join.

"Kit" Kallista Angeloff

"Kit" Kallista Angeloff, 26, graduated from Rhode Island School of Design (RISD) which led to work as an illustrator for a science team. She created technical drawings for archaeological finds. It was in this field, that she kindled a passion for science and realized that she had a desire to know more, especially in the area of chemistry, geochemistry, and marine chemistry. Kit moved to Seattle and started her Ph.D. at South Seattle Community College. But, determined to know more, she emailed and called all over UW's campus to find opportunities to learn first-hand. Her persistence led to her contact with Rachel, and here she is, working side-by-side with best oceanographers in the world.

Kit stops the biological processes,
preserving the chemistry of the sample.

The formula to calculate DO from
the Winkler Method.

Each volume is known;
they use these values in their calculations.

Each glass container has an exact
known volume to six places after the zero.

Adding the "poison"

Precipitate formed.

Chuck and the Rad-van

Tweeted this ad because "Chuck and the Rad-Van" sounded like a band
that could be playing in our destination: Seattle!

As a junior grad student under Dr. Cochlan at San Francisco State University, Charles "Chuck" Wingert, 27, is earning his master's degree in marine biology with a rare opportunity to work with the brightest and best from Romberg Tiburon Center for Environmental Studies. Chuck's been tirelessly working onboard, and the major-majority of that time has been spent in a 8 foot by 20 foot container box that has been converted into a mobile science lab. This one in particular is called the "Rad-Van" because Chuck is working with radioactive materials inside. He has the proper certification and dons the appropriate safety gear as he handles the radioactive isotopes needed for his work.

 

Calibrating the light intensity before
departing San Francisco, no protection
needed at this point

All plants gain their energy for production of glucose from carbon dioxide (CO2) and sunlight. Phytoplankton are no different in this respect; they are autophototrophs--converting the sun's energy into food for themselves and for consumers. Carbon dioxide (CO2) is relevant to us because of oceanic acidification (OA). Increased CO2 production is leading to a decreased pH, an increased acidity by the production of carbonic acid. "It's all about the free positive H's" as Chuck and Chris have said to me, both graduate students at RTC-SFSU. Acids have a surplus of positive hydrogen ions, and as the ocean's pH lowers, the more "positive H's" are floating around. But, the question is: Does a lower pH affect the efficiency of the "plants" (phytoplankton) ability to make its own food? Our research team hypothesizes that a lower pH due to OA decreases the photosynthetic ability of phytoplankton, some more than others. This is one of the many questions being answered on this research cruise.

Remember that every group on board has a specific focus for their research, but they all collaborate, sharing data and using differing methods to compare data as well. For example, Brian Bill' s taxonomy of phytoplankton was supported by Julia Matheson's work with the flow cytometer. Andrew Schellenbach's FIRe work determined photosynthetic health, happy versus distressed cells; now, we look at Chuck Wingert's work in the Rad-Van, testing similarly for the cells photosynthetic health but with another method.

Photosynthetron with samples, notice
the brighter intensity light at the
bottom of the picture

Chuck is using an instrument called THE PHOTOSYNTHETRON, a name so futuristic it deserves all caps. Basically, the photosynethetron measures the rate of photosynthesis at each intensity of light, providing a nice standard curve, steadily increasing then flattening out. This machine with Star-Trekish name measures the amount of carbon uptake to make glucose from the varying light intensities. Some species of phytoplankton are low-light adapted while others are high-light adapted. This is the way that we can determine their efficiency to perform photosynthesis at differing light intensity levels.

Photosynthetron without
samples added; each of these
cells are tuned for a specific
light intensity. 

In order for the photosynthetron to measure carbon uptake, Chuck has to add a radioactive isotope of carbon, C-14. Every atom of carbon has 6 protons, and on the periodic table, carbon has an atomic mass of 12.011 amu. That means every atom of carbon has not only 6 protons but 6 neutrons as well, combining to make up the atomic mass. An isotope is when the neutrons differ from the stable atom of the element on the periodic table; therefore, C-14 is an isotope of carbon that has not 6 neutrons but 8.

6 protons plus 8 neutrons equals 14, C-14.

Fume hood takes up all gases from the reaction. 

After Chuck adds the radioactive isotope C-14, he allows the phytoplankton to incubate for 2 hours to take up the radioactive carbon, C-14.  Afterwards, he then moves the samples to the fume hood and adds 10% hydrochloric acid, HCl. The hydrochloric acid, HCl, helps remove any C-14 that is not taken up by the cells. Through a chemical  reaction between the C-14 and HCl, carbon dioxide is produced as a gas and taken up in the fume hood safely to the atmosphere. This degassing takes 12-24 hours. All the C-14 that is left is that which is taken up by the cells; the excess removed.

After degassing, he adds a special solution and places the samples into another machine, called a liquid scintillation counter. This device detects radioactive isotope, C-14 without detecting the stable carbon (C-12). The machine provides a printout of "disintegrations per minute" or DPM. The higher the DPM, the more C-14 present in the sample which means that it has a higher photosynthetic capacity. Chuck then uses this number in a formula to find the carbon uptake or the "rate of photosynthesis."

Liquid Scintillation Counter detects C-14.

Two photosynthetrons with samples for testing

Different species of phytoplankton all have the same general shape of the curve, increasing photosynthetic rates with higher light intensity but leveling off at capacity, but this curve varies with its values. In other words, some species will be better adapted than others for the chemistry of the ocean and the changes of the future. Our work here is particularly interested in which phytoplankton cells are best adapted for the higher acidity and lower available nutrients as well as the effect of higher acidity on photosynthetic health. 

Chuck is playing a huge role in this grand investigation. And, it continues long after this month of science at sea. In order for Chuck to truly see the photosynthetic abilities of each sample, he will need to input data for the formula to work. That data will come later as the group from University of Washington in conjunction with NOAA-PMEL (post upcoming) analyzes samples for DIC, dissolved inorganic carbon. It's all coordinated and connected--a beautiful picture of the ocean which we are studying.

Chuck Wingert and Chris Ikeda are both graduate students
 at RTC-SFSU under Dr. Cochlan.

Quick story from the bridge...

One the best things about being at sea is meeting new people and hearing the stories they have from life experiences. Sailors, especially for research vessels like the Melville, have been all over the world to some of the most exotic places. I have been amazed by their adventures, and I can understand why a young man or woman finds themselves with saltwater in their blood. This salty life, however, is reserved for those with an adventurous spirit and few ties to the mainland. With a wife and four children at home, I could never do what they do which is why I have enjoyed learning about their jobs and hearing of their adventures.

When I was writing my post about the bridge, I met all of the officers and the captain. Heather Galiher is the 2nd mate onboard, and she told be me about some of her favorite places that she's had the chance to visit in her young career. Heather told me about Papua New Guinea, and how she climbed into the mouth of an active volcano with the rest of her crew. She said that

the very next day that same volcano erupted! 

It's a pretty amazing story. But, it didn't make the impact that the pictures do.

Take a look at what Heather sent me a few days later to support the magnitude of her story.

Hiking up to the volcano

Eruption the next day

Heather Galiher unscathed, ready to board the R/V Melville.

The reason that I could never be a full time sailor.
left to right: Bryce, nearly 2, Olivia , 11, Jax, 3, Grady, 9

Canadians, eh?

left to right: Dr. Mark Wells (U. Maine), Trey Joyner,
Dr. Charlie Trick (Western U.), and Dr. Bill Cochlan (RTC-SFSU)

As a southern boy, I haven't had too many encounters with our friends north of the border. Now, I find myself talking hockey and comparing climates of our homes with three of the four principal investigators: Dr. Cochlan, Dr. Wells, and Dr. Trick. All three of these men of science hail from the colder regions of North America and are proud of it. Dr. Wells lives and works in the States at the University of Maine, and Dr. Cochlan lives and works in California at San Francisco State University. Dr. Charlie Trick, however, continues to live and work in the great Dominion of Canada at Western University in London, Ontario, but was raised in Ohio.

Dr. Charlie Trick proving that fun and science do mix!
This group has been incredible, and it stems from the leadership.

I will be writing more about Dr. Trick in the near future, so I will concentrate on the complimentary work that he and his team from Western U. are doing in our oceanic lab at sea. Julia Matheson, "PJ," as she's known onboard, is a graduate with her master's degree in biology. She has been brought on by Dr. Trick to be his research assistant because of her experience with a sophisticated machine, called a "flow cytometer." Julia quietly works all day from her station, donning heavy jackets in the cold lab. With every 2 mL sample, she uses the flow cytometer to determine the phytoplankton species and concentration. While I was interviewing her, she showed me that the sample in her hand had 19 cells of the raphidophyte, Heterosigma akashiwo a species known for forming harmful algal blooms ("red tides"), a fish killer. That means that the concentration of the sample contains 19 of these harmful cells per 100 mL of seawater. The PI's will analyze these results and the results of others to determine a possible conclusion, but they never jump to conclusions. Just like in my classes, they rely on the evidence to guide them, and just like my classes, they use multiple sources.

Julia Matheson, research assistant, Western University,
London, Ontario, will be spending 12 weeks after
our research at sea in Bermuda with BIOS, Bermuda
Institute of Ocean Sciences as an intern with more time at
sea on the R/V Atlantis.

Julia is basically determining the same thing as Brian and Kathryn (see previous post) but using laser technology rather than traditional light microscopy. Both parties are observing the assemblage of the type and concentration of the phytoplankton in each sample. Brian and Kathryn are using concentration techniques, then, looking under the microscope for the general abundance of the phytoplankton present. Julia is using the flow cytometer which automatically sorts the cells by size and chlorophyll fluorescence, essentially observing the same things as Brian and Kathryn. From these two sources, the PI's can be sure that they have an accurate view of the biodiversity of the ocean's primary producers. Julia's machine also allows her to breakdown each sample into high, medium, and low chlorophyll production for each cast and each depth sampled. This is additional information to be shared with the teams to assess the relative health of the planktonic community.

Speaking with Dr. Trick, I realized that the food chain communities are size-based assemblages. In other words, large planktonic cells are food for larger zooplankton. The larger the phytoplankton, the shorter the food web, making it more efficient energetically. If phytoplankton cells, due to oceanic acidification or other variables, are smaller in size, then the food chain is longer, less efficient, meaning that more sun and nutrients will be necessary to provide the same amount of food for fish and other predators at the top of the food chain. Therefore, it is important to find out the cell size and their relative photosynthetic contribution to the natural community. And, like the iPhone, there's an app for that; in fact, there are multiple tools available that our marine scientists utilize aboard the R/V Melville.

Andrew Schellenbach, senior at Western University with
Dr. Cochlan (RTC-SFSU) and Denis Costello and
Kathryn Ferguson in the background.

Andrew Schellenbach, our youngest science crew member at 20 years of age, is a senior at Western Univ., and hopes to use this experience to help guide him in the next chapter of life as he continues his scientific career. His job in this investigation has been to use two types of fluorometers, one approach uses a chemical, called DCMU to disrupt the flow of photosynthetic energy and then measure the fluoresence using a traditional fluorometer; and in another approach, rapid rates of light are used to saturate the photosystems of the planktonic cells. This latter type of fluorometer is called a FIRe. By firing light through the samples, FIRe is able to provide a measure of photosynthetic health--determining whether we have "healthy, happy cells or distressed, sad cells," according to Dr. Trick, co-PI. Andrew explained that FIRe uses a ratio from two different blasts of light, one short and intense, another longer, less intense. The light excites the photosynthetic cells until they are saturated. With each specific ray of light, FIRe records the cells' absorption of light until, no more light can be absorbed; it is saturated. Andrew's analysis using these two methods compliment another experiment onboard that goes even a step further. I will focus on Chuck and the "Radvan" later and show how it provides more evidence from yet another source to determine the health of the phytoplankton in our samples.

FIRe measures photosynthetic health from the ratio derived
from maximums that saturate the cell.

With every experiment, with every method, with every collaboration and with every conversation; together - they reveal a clearer picture. Like observing a diamond from different angles, using different instruments and different eyes, we not only have a better understanding but a greater appreciation for its beauty. Personally, that is what I'm observing here everyday at sea: Beautiful complexity revealed one test at a time.

Domoic Acid in the Phyto's

To say that I'm learning a lot would be a gross understatement. I'm drinking scientific knowledge from a firehose here. Every day and night, our conversations with the brightest and best scientists, experts in multiple disciplines from biology to chemistry to geology, supersede my limited knowledge of the marine world. Little by little, my understanding grows. How do you eat an elephant? One bite at a time. 

Dr. Cochlan (SFSU-RTC) with former student, Brian Bill (NOAA-NWFSC),
enriching seawater for a domoic acid experiment.

The way research cruises work is that the principal investigators collaborate ideas, plan, and write a grant proposal to fund a specific investigation. In our case, Dr. Bill Cochlan was selected to be the chief scientist by the group. They then sent a proposal to the National Science Foundation (NSF) that was accepted relatively quickly due to its uniqueness and importance. Normally, these proposals will take several drafts, but because of the urgency of field data to monitor effects of oceanic acidification (OA) on lipid quality and quantity on the only marine organisms responsible for their production--effecting the entirety of the marine food web, their proposal was accepted and funded on the first submission. After the proposal and budget is approved, Dr. Cochlan and his team not only begin the hard work of planning and testing for the target experiments on board but continue to reach out to other noted marine scientists to coordinate experiments that will compliment this target research. Therefore, many peer-reviewed publications of the past and the future will have repeating names as authorship: Dr. Wells (U. Maine), Dr. Trick (Western U.), Dr. Trainer (NOAA-NWFSC), Dr. Bidigare (U. Hawaii) and Dr. Cochlan (RTC-SFSU). Every group benefits independently from the use of the research vessel, gathering data from otherwise unaccessible waters without this great lab at sea, but they all benefit cooperatively by the sharing and comparing of data.

One of the complimentary groups that is adding to the overarching goal of this sea-faring excursion is NOAA (National Oceanic and Atmospheric Administration). Through the leadership of Dr. Vera Trainer, who I will be writing a post about specifically later, NOAA's Northwest Fisheries Science Center has partnered with Dr. Cochlan and San Francisco University's Romberg Tiburon Center for years to collaborate and present the most honest picture of the ocean's "pulse." As teachers, we strive for interdisciplinary, even integrated, connections, and here at sea, this is best example that I have seen. 

Kathryn Ferguson and Brian Bill hard at work. 

Brian Bill, the research associate for NOAA-NWFSC, has been working diligently day and night with the team and on his own, using equipment foreign to me, analyzing the assemblage and health of the phytoplankton communities throughout our sampling sites. His focus has been on methods that reveal the taxonomy of organisms, classifying the autophototrophs (photosynthesizing organisms) and analyzing the production of domoic acid by harmful species. The question that he and NOAA-NWFSC is trying to answer is: Do the toxin levels in phytoplankton increase with lower pH (higher acidity levels) of the ocean? 

Brian has been working for NOAA since a biology undergrad at UW (pronounced "u-dub" by those that go there) in Seattle. From the area, Tacoma, Washington, Brian continued his work with NOAA as he began and completed his master's degree in marine biology at SFSU with Dr. Cochlan. After his master's degree, his title became oceanographer with NOAA, a fantastic career that few chose and fewer attain.

Kathryn Ferguson, Hollings Scholar, FSU/NOAA-NWFSC

Kathryn Ferguson, 21, has been assisting Brian. She's an intern for the summer with NOAA and will be staying in Seattle to work. Kathryn, or "Sunshine," as she's known on board for her contagious smile, is a prestigious Hollings scholar from Florida State University. Originally from Maryland, she now makes Orlando her home (in the "Sunshine State"). 

Brian and Kathryn have been physically counting the "general abundance" of phytoplankton and identifying the types that are present with each water sample. They use a technique that concentrates the phytoplankton through a sieve allowing them to see as many as possible in a small volume of water. The most common genera in their samples so far have been: Chaetoceros and Pseudo-nitzschia. Both of these can be harmful species of diatoms. The former sometimes kills fish; and the latter produces the poison, domoic acid. 

Remember that in the food web it's all connected. Phytoplankton produce their own food as autophototrophs, but they also produce lipids (think Omega-3) and toxins, such as domoic acid. These cannot be produced by any other source. As zooplankton, such as krill, eat the phytoplankton, they gain the nutrients as well as the lipids and toxins. Then the predators eat the zooplankton and all that is within them. So, just like the omega-3 lipids that are transferred to us when we eat fish, the domoic acid is transferred and accumulates in our bodies, causing sickness and even death. ASP, amnesic shellfish poisoning, is named because of the attack on the nervous system that causes temporary memory loss. It's a scary thought because the fish and shellfish are unaffected; it's us consumers that pay the price.

Pseudo-nitzschia

Chaetoceros

Dr. Vera Trainer, Brian Bill and Kathryn Ferguson are playing a huge role in the determination of toxic content in the communities that we are analyzing. Finding these two phytoplankton types, Chaetoceros and Pseudo-nitzschia, in the greatest abundance in our samples is not a good sign. 

After finding these two harmful organisms in abundance, Brian goes to work analyzing the amount of domoic acid present in their cells. He uses a tool to detect toxic activity called ELISA (Enzyme Linked Immune Assay). He creates a standard curve with an expensive plate and machine that measures the optical density or color of the reaction on the special plate. Because this is a competition reaction, the more color, the less domoic acid. These plates are showing a lighter blue which means that the amount of domoic acid is high. 

NOAA-Northwest Fisheries Science Center works hard daily to analyze the waters, to communicate with fisheries, and to create public awareness programs for toxins such as domoic acid as well as harmful algae blooms which both may be increased by the lowering pH of the ocean. But, we don't know that yet, which is why we are here.

These are the samples before the diluted hydrochloric
acid (HCl) is added to each.

Brian uses this special pipette to add 0.1mL to each sample.

After the HCl (diluted hydrochloric acid) is added,
the samples change colors (indication of chemical change).
The lighter blue or clearer indicates high amounts of
domoic acid. This one is off the charts.

The plate goes into machine that reads the optical density of each sample.

Analysis of the plate gives Brian the standard curve.

Making a plate for domoic acid analysis

Standard curve used to determine
production of domoic acid.

For quick analysis of the general
abundance, a phytoplankton net tow is
used to make collections.