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Erik Sahlin - Analytical Problem- Brevetoxin levels in Ocean

My analytical problem will be a focused on the effects of Red Tide in the oceans. Red tide is a major problem for ocean wildlife, since harmful toxins get released into the oceans from clusters of algae. The algae are composed of phytoplankton, single-celled plant-like organisms that release natural toxins into the ocean's water which ends up killing the marine life. "The most common type of toxin released from the algae is brevetoxin, which has been known to cause problems for humans and marine life. It can be harmful to humans because people who live near the ocean, spend a lot of time in the ocean, breathe in the toxin that gets released into the water vapor (1)." The marine life cycle suffers because the toxin affects all organisms from the small microscopic organisms to the larger fish that consume the smaller organisms (1)." Since the fish get contaminated with the toxin, the fishing communities that surround the ocean suffer because the fish can no longer be consumed or if people consume the fish they get sick.
The main problem I want to look at deals with the toxicity of the water. How toxic is brevetoxin and more importantly, what toxicity levels of brevetoxin would be harmful to the marine life? Is the toxicity level different for being harmful to humans? Are there any other toxins that can be blamed?
There will be other toxins that can be found in the ocean water samples but the most prevalent toxin found in the water will be brevetoxin, which is supported by multiple scientific experiments. The best place to gather water samples would be found where there are several species of fish washed up on the shore.
The analyte in my analytical problem is the brevetoxin or other lesser known toxins that are extremely harmful to the environment. The matrix is the ocean water and any other contaminants that are found in the water.

UV-Vis Absorption Spectrometry

Scientists primarily use multi-wavelength spectroscopy to measure brevetoxin levels in ocean water. "Multi-wavelength spectroscopy is a versatile, rapid and reliable tool that has immediate applications as a biosensor for the detection, identification and enumeration of pathogens. The sample information contained in a typical multiwavelength ultraviolet/visible (UV/vis) spectrum includes cell size, chemical composition and shape. This information is obtained from the spectroscopic analysis of a sample measured over a broad range of wavelengths (200 - 900 nm) with scattered light measured at one or many different angles (3)." Multi-wavelength spectroscopy just became the primary tool to measure various toxin levels in ocean water, including brevetoxin, within the last ten years.
The range of wavelengths that are primarily used in every experimental procedure is 200 to 900 nm. In the Forward, Tester, and Cohen journal, "the Karenia brevis and a local, non-toxic isolate of Prorocentrum minimum were grown in filtered Gulf Stream seawater diluted with distilled water to a salinity of 30, enriched with f/2 nutrients at 22 degrees Celsius(4)." The f/2 nutrients are added to the ocean water and keeps the original sample clear of any contaminants that may effect the results of the experiment. The temperature and specific salinity of the ocean water may vary depending on what the scientist wants to study. Pure ocean water with a certain salinity would be used as the control (blank) of the experiment and various concentrations would be used to obtain multiple absorbances. The exact values for the molar absorptivity or optical density were never found from my research.

Similar Analytical Problem

John Raia - Detection of Anionic Surfactants in Water -
His hypothesis is that anionic surfactants should be present in higher concentrations in areas that have been exposed to dispersant chemicals compared to other natural water sources with no known history of intentional contamination. The analyte is anionic molecules like sodium lauryl sulfate or sodium octanesulfonate, which can be found in an ocean water matrix.
Our analytical problems are similar because our samples will be taken from the same matrix, the ocean. Not only do we both use the ocean water as the matrix, but we both can use a version of UV-Visible Spectrometry in order to test our samples for our desired product, for John it's the sodium lauryl sulfate or sodium octanesulfonate and for me it would be brevetoxin.
Our analytical problems are different because one of the analytical methods that I will use for my experiment is a multiwavelength spectrometer. The multi-wavelength spectrum includes cell size, chemical composition and shape of the sample that I will be studying.

Chemical Structure and Standards

The brevetoxin standards can be found on the CalBioChem / EMD4Biosciences company webpage.

View image
Brevetoxin PbTx-2
Catalog Number - 79580-28-2
Quantity - 100 microliters
Purity - >95%
Price - $449

View image
Brevetoxin PbTx-3
Catalog # - 85079-48-7
Quantity - 100 micrograms
Purity - >95%
Price - $499

EMD4Biosciences. 203732 Brevetoxin PbTx-2, Ptychodiscus brevis. http://www.emdchemicals.com/life-science-research/brevetoxin-pbtx-2-ptychodiscus-brevis/EMD_BIO-203732/p_F8eb.s1LILsAAAEWlWEfVhTm?WFSimpleSearch_NameOrID=brevetoxin&BackButtonText=search+results. (Accessed Oct, 23rd
EMD4Biosciences. 203734 Brevetoxin PbTx-3, Ptychodiscus brevis. http://www.emdchemicals.com/life-science-research/brevetoxin-pbtx-3-ptychodiscus-brevis/EMD_BIO-203734/p_F8eb.s1LILsAAAEWlWEfVhTm?WFSimpleSearch_NameOrID=brevetoxin&BackButtonText=search+results. (Accessed Oct, 23rd)

Atomic and Mass Spectrometry

The ESI-MS/MS spectrum of Brevetoxin PbTx-2 in 0.002 M HCl in a methanol:water (4:1 ratio) solution is shown in the figure below (the graph on top)
View image

References for mass spec.
Hua, Yousheng. Cole, Richard B. Electrospray Ionization Tandem Mass Spectrometry for Structural Elucidation of Protonated Brevetoxins in Red Tide Algae. http://pubs.acs.org/doi/full/10.1021/ac990433o. (Accessed October, 26th)


1. Ramsdell, John. Science Daily. Aerosol Toxins from Red Tides May Cause Long-Term Health Threat. July 9, 2008. http://www.sciencedaily.com/releases/2008/07/080709110049.htm (Accessed Sept. 16th, 2011)
2. Anderson, Dr. Don. Harmful Algae. May 31, 2011. http://www.whoi.edu/redtide/ (Accessed Sept 16, 2011)
3. Mattley, Yvette D. Garcia-Rubio, Luis H. Multiwavelength Spectroscopy for the Detection, Identification and Quantification of Cells. Nov. 5th, 2000. http://www.marine.usf.edu/sapd/spiep00ym.pdf (Accessed Sept. 26th, 2011)
4. Cohen, Jonathan H. Tester, Patricia A. Forward Jr., Richard B. Oxford Journals: Journal of Plankton Research. Sublethal Effects of the Toxic Dinoflagellate Karenia Brevis on Marine Copepod Behavior. January 12th, 2007. http://plankt.oxfordjournals.org/content/29/3/301.full (Accessed Sept. 26th, 2011)

Blog 9- Chromatographic Techniques

1. Reverse Phase chromatography can be used while gas chromatography and HILIC probably could be used in some similar experiments. Gas chromatography would be more helpful if I were analyzing the Brevetoxin levels in the air composition from the ocean water. HILIC could probably be used but I don't think it would help to deal with more water in the process since the sample is already in ocean water. Ion exchange chromatography will not be used because my analyte is not an element and doesn't have ions present. Size exclusion chromatography is usually used for macromolecules and my analyte would not show good results from SEC. Affinity chromatography is mostly used for biochemistry experiments that relate antibodies to antigens, enzyme to substrate, or receptor to ligand and my analyte does not deal with those types of interactions. Chiral chromatography will not be used because my analyte does not have chiral centers.

2. My first choice of chromatography would be reverse phase liquid chromatography. Reverse phase liquid chromatography will be useful because since the stationary phase in non-polar and my analyte is polar, this will create a good separation. Reverse phase chromatography is almost always used for experiments that are analyzed by liquid chromatography .

3. The columns that will be used are "C18 "Aqua"column [3 μm, 125 A, 75 × 200 mm, (Phenomenex #003-4311-B0, Torrance, CA)] and C18 guard column (Phenomenex #AJO4287, Torrance, CA) (Yung, McDonald)." The stationary phase is C18 in a fully porous silica, with a particle size of 3 microliters and the pore size is 125 A. The column length is 30mm with an internal diameter of 2mm. The column temperature was set to 32 degrees Celsius. I could not find any value for the typical backpressure used in the column.

4. The mobile phase will be made up of purified water (solvent A) and methanol:1 mM acetate (solvent B). "The solvents were operated on the following time cycle: 20% B for 0-1 min, linear gradient to 90% B at 3 min, 90% B 3-10 min, 20% B for 10.1-12.5 min (Yung, McDonald)."

5. The detector that will be used is an MS/MS. The MS/MS will be used because since the different forms of Brevetoxin have molecular weights very similar to one another, the use of MS/MS will be able to separate the different types of Brevetoxin.

6. Not Available

(1) Yung Sung Cheng. McDonald, Jacob D. "Concentration and Particle Size of Airborne Toxic Algae (Brevetoxin) Derived from Ocean Red Tide Events." National Institutes of Health. May 15, 2005. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652738/ (Accessed November 1, 2011)
(2) Phenomenex. "C18 "Aqua"column [3 μm, 125 A, 75 × 200 mm, (Phenomenex #003-4311-B0) and C18 guard column (Phenomenex #AJO4287)" Phenomenex-DNV. Torrance, CA. 2011. http://www.phenomenex.com/Products/Part/00A-4311-B0 (Accessed November 9th, 2011)

Blog 11 - Capillary Electrophoresis Techniques

1. The CE techniques that would work for my analytical problem would be CZE and MEKC. The CZE technique will be able to separate the brevetoxins from the ocean water matrix but it would be hard to separate the different types of brevetoxin from each other. The cIEF technique would not be useful for my analytical problem because it separates by pI. The pI is the pH of a certain molecule where the molecule has no net charge. Since the brevetoxin molecule is neutral, it will not have a varying pH under different specific conditions. The CGE technique is not useful for my analytical problem because it is used to separate charged polymers like DNA and since my analyte is neutral this is not a good technique to use.

2. The MEKC technique would be the best technique to use for my analytical problem. Since the micelles form inside the capillary tube, it creates a hydrophobic interior inside the micelle and a hydrophilic buffer solution. This will separate all the components of the ocean water. The neutral brevetoxin molecules will also separate since they can travel through the hydrophobic micelle interiors and the hydrophobic buffer solution at different rates which makes them elute from the capillary at different times.

3. In the paper referenced below from Damian Shea, they used a 50 cm x 75 micrometer ID bare silica capillary(Polymicro Technologies, Phoenix, AZ). The buffer that was used was a 10mM sodium borate / 30mM SDS / 10% methanol buffer solution at a pH of 9.3. The experiment was run with an electric field of 30kV and at a temperature of 30 degrees Celsius +/- 0.3 degrees celsius. The MEKC experiment was also run with a UV detection at 214nm.

4. The detector used for Damian Shea's experiment was laser-induced fluorescence (LIF). The LIF used was a 25 mW He/Cd laser with an excitation at 354 nm and a fluorescence emission at 410 nm. The LIF is useful because it will yield a greater sensitivity than UV detection and better selectivity of the different types of brevetoxins.


Shea, Damian. "Analysis of Brevetoxins by Micellar Electrokinetic Capillary Chromatography and Laser-induced Fluorescence Detection." Department of Toxicology. North Carolina State University. April 14, 2005. http://onlinelibrary.wiley.com/doi/10.1002/elps.1150180216/pdf. (Accessed November 19th, 2011)

My post didn't seem to work.
The analytical problem that is similar to mine is Sara's. The Geochemical Mobilization of Arsenic in Groundwater. It is similar because we are both studying a certain chemical/toxin in water. It is different because her problem deals with groundwater from wells and mine deals with the ocean.


Blog 13? -1 pt

Good answers for blogs 9, 10, 11.

Blog 10

1. The best technique that I have found for my analytical problem is reverse-phase HPLC-MS/MS.

2. The technique will also be done by the following people:
Vinh Tran - Triclocarban in Human Urine.
Ian Ronningen- Pesticides and toxins in fragrances and natural flavors.
Chuxin Chen - Comparative Analysis of Arbutin and
Tranexamic Acid in Skin Whitening Products

I am entirely clear if you are spiking a standard onto filters in which you have captured algae or filters that have been used to enrich for the toxin. This part will need to be clarified in future posting. (-0.5 pt).

As part of your analytical problem will you need to analyze brevetoxin in other matrices (e.g. fish)?

Blog 8 - Procedure to prepare(clean) sample
1. The brevetoxin, PbTx-2 are spiked directly to filters at concentrations ranging from 100-1000ng.
2. Dry the sample at room temperature for about 60 minutes.
3. Cut the filters in half and fold one half and roll filter into 15mL polypropylene tube. (Place second half in freezer at -20 degrees Celsius)
4. Add 10 mL of analytical grade acetone (Fisher Scientific) to the tube and vortex from 20 seconds.
5. Sonicate the tube for 20 minutes.
6. Place tube on circular rotator (Roto-Torquie, low speed #10, Cole-Parmer instruments, Vernon Hills, IL) for 20 minutes.
7. Remove filter and 10 mL extract and evaporate under a gentle stream of nitrogen to ~100 microliters.
8. Vortex the ~100 microliters sample for 5 seconds to reconstitute the extract.
9. Recombine the sample with a 50:50 solution of methanol/purified water to a final volume of 200 microliters.

Yung Sung Cheng. Mcdonald, Jacob D. "Concentration and Particle Size of Airborne Toxic Algae (Brevetoxin) Derived from Ocean Red Tide Events." National Institutes of Health. May 15, 2005. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652738/ (Accessed November 1, 2011)

Blog 6. Good answers.
Blot 7. HPLC is only a separation technique. It also uses ESI four coupling to the mass analyzer. (-0.1 pt)

The two types of mass spectrometry that can be used to measure Brevetoxin PbTx-2 (mol wt 895.08 grams) and Brevetoxin PbTx-3 (mol wt 871.1 grams) are electrospray ionization (ESI-MS/MS) and liquid chromatography mass spec (LC-MS/MS).

The ESI will have an electrospray ionization and the type of mass analyzer is a Quattro II triple-quadrupole. The HPLC-ESI-MS/MS will have a liquid chromatography- electrospray ionization and the type of mass analyzer is a triple-quadrupole.

The ESI-MS/MS mass spectrum of Brevetoxin PbTx-2 in 0.002 M HCl in a methanol:water (4:1) solution is shown in the body of the analytical problem because I couldn't add the photo here.

1. Hua, Yousheng. Cole, Richard B. "Electrospray Ionization Tandem Mass Spectrometry for Structural Elucidation of Protonated Brevetoxins in Red Tide Algae." http://pubs.acs.org/doi/full/10.1021/ac990433o. (Accessed October, 26th)

2. Hua, Yousheng. Cole, Richard B. "High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry for the Determination of Brevetoxins in “Red Tide” Algae." http://pubs.acs.org/doi/pdf/10.1021/ac00107a010. (Accessed October, 26th)

Both answers for BLOGS 4 and 5 are correct. Your hypothesis has three sub-hypotheses. You may want to select one and focus your efforts.

The Brevetoxin that I'm studying in this analytical problem is fluorescent. Brevetoxin has a maximum excitation at about "354nm and a maximum emission at about 410nm. The solvent used to seperate the brevetoxins from the ocean water was a sodium borate/ sodium dodecyl sulfate buffer with a pH at 9.3. The solvent for the study was acetonitrile (1)."

The best instrument that could be used for my problem is fluorescence detector or in the case of this study a laser-induced fluorescence detector.

Shea, Damian. "Analysis of Brevetoxins by Micellar Electrokinetic Capillary Chromatography and Laser-induced Fluorescence Detection. Wiley Online Library. Wiley Online Library, 14 Apr. 2005. Web. 17 Oct. 2011. .

Hypothesis- The Brevetoxin levels in the ocean will be higher near the algal blooms than ocean water samples taken far from algal blooms. Very low concentrations of Brevetoxin will contaminate and kill fish and other marine life. The fish and other marine life that live or swim around the algal blooms will have a higher concentration of Brevetoxin in their blood and tissues.

Studies- 1) Measure the brevetoxin levels in ocean water samples near algal blooms. 2) Measure the brevetoxin levels in ocean water samples from shorelines that are close to the algal blooms. 3) Measure the brevetoxin levels in ocean water samples far away from the algal blooms. 4) Measure the brevetoxin levels in ocean water samples along shorelines far away from the algal blooms. 5) Measure the brevetoxin levels in ocean water samples along the shoreline where several fish have washed up on shore. 6) Measure the brevetoxin levels in ocean water samples off shorelines where several fish have washed up on shore. 7) Compare the brevetoxin levels in all of the ocean water samples. 8) Map out where brevetoxin/red tide blooms are or where they have been found recently. 9) Add varying levels of brevetoxin to ocean water samples with fish to test how the brevetoxin levels effect the fish.

An estimate of the brevetoxin levels in the ocean water samples will be between 0.25 ng/mL to 1.00 ng/mL. The standard ocean water sample will be zero or a value really close to zero.

Flewelling, Leanne, and Jerome Naar. "Brevetoxicosis: Red Tides and Marine Mammal Mortalities : Nature." Nature. Nature Publishing Group, 8 June 2005. Web. 14 Oct. 2011. .

BLOG 2 - Although your answer is related to the use of UV-VIS in the area of interest, it does not answer the question requested for this blog. Based on the molecular structure of brevetoxins you could have answered 5(a) or 5(b) (-0.5 pt).
I have made note that you could not post on Sara's entry. Talk with Chad to solve this issue. Once you have solved it, let him know so that he releases your grade. Thanks. Edgar

BLOG 3, Parts (b) and (c)? (-0.5 pt)

You need to define a hypothesis related to the problem that you are investigating. You may need to refine your analytical problem more to define your hypothesis (-0.2 pt)