Temperature Change Influences the Viscosity Flow Rate of Saturated Fatty Acids in Dietary Supplementation
Carlos Vega
Salt Lake Community College
Author Note
This paper has been created for Elementary Bio-organic chemistry 1120, section 500, taught by Professor Alvarez.
Abstract
The purpose of this analysis project is to identify essential fatty acids found in dietary supplementations in Utah. Marketed products that contain less than the equal advertised claim disables consumers, patients, clinical and medical researchers. The first phase of this project obtained different products, varying in cost and brand availability. This research also created a replicable method of producing these types of results. The final phase tested the viscosity of these samples with the change of temperature and examined the data while comparing known melting temperatures of DHA, EPA, and ALA. By identifying the percent of viscosity change, relative to each sample, 60% of the products tested failed to meet the label claim. The ratios used by manufacturers also associated with cost and ratios of DHA and EPA. This research will allow for more significant consideration for consumers, researchers and doctors on the change of the dietary supplementation market.
Keywords: omega, dietary supplementation, fish oil production, fish oil industries
Temperature Change Influences the Viscosity Flow Rate of Saturated Fatty Acids in Dietary Supplementation
Omega found in dietary supplements in the United States contain low quantities of EPA and ALA in the United States. A research study found that “70%” of the “47 commercial” dietary supplementation, made from “fish, krill, and algal oil” failed to meet the label claims of the EPA and ALA contents in the product (Kleiner, Cladis, & Santerre, 2015). That is true for other supplementations products from South Africa, where less than “89%” of the products out of “45” meet the label claims (Albert et al., 2015). Nonetheless, not all studies conducted are reasonable. In a different study, a research team found similar results in New Zealand and Australia, later being cited by reviewers for a “concern” and bias allegations, when the alleged research published a follow-up study that sponsored specific brand products(Nichols, Dogan, & Sinclair, 2016). Further review in New Zealand and Southern Africa confirmed the results marketing claims not meeting (Albert et al., 2015; Opperman, Marais de, & Spinnler Benade, 2011). Overall, most researchers concluded that omega containing supplements contained less than the advertised label, adding that agencies were unable to or were not enforcing laws against manufacturers. It seems then, that, the quality and production of these omega containing dietary supplementations could be improved in all countries using the assistance of the public, by conducting research and warning others.
In this report, the methods to measure the physical characteristics of EPF’s (polyunsaturated fatty acids) are derived using the use the viscosity of EPFs, using temperature as a variable and time for measurement, these provided semi-qualitative results of EPFs, based on melting points, comprehensive safety and replicability of testing. In researching past and current test methodologies, little results appeared. Past lab work conducted on the characteristics of EPF’s has provided consistent results.
Brief History
The history of omegas began in the 1800’s, in the area of vitamin discovery, commencing the scientific community to ponder the function of oils and fats. In 1929 with initial experiments, rats feed fats increased weight compared to rats fed a regular diet. Professor George Oswald Burr and Lafayette B. Mendel later examined their findings and discovered that rats feed fats soluble in either did not lose weight (Spector & Kim, 2015). The reason that rats weight remained challenged the scientific community in general. Both the researchers found that these fats must be essential to a diet, but others found his work inconclusive.
Throughout the next four decades, the scientific community came to little agreement on the effects of Omega Fatty Acids in Humans until the most durable case appeared. In 1982 Ralph Halman and colleagues reported of a six-year-old patient who had a 300cm of intestine removed. The patient’s treatment lead to the parental nutritional emulsion. The patient’s neurological symptoms alleviated when ALA 6.9% increase in the emulsion (Spector & Kim, 2015). The six-year-old patient case remains a vital credible case in the effects of EPFs in Human nutrition.
As a result of other notable cases where results persist with reexaminations throughout the decades. A very known case includes the famous Greenland Eskimo case. Dyerberg and H. O. Bang in Aalborg reported of the populations of Eskimos of high blood levels of EPA and DHA, with unusually low levels of heart attacks (Spector & Kim, 2015). Followed studies in 1972 found inconclusive data, studies conducted in 2002 found benefits of relations when Eskimo populations consumed traditional diets (Eilat-Adar et al., 2009). In 2010 a research team proposed that EPA’s and DHA’s have correlations with health, later reviewed as “inconclusive” and “further investigation” (Singh, Arora, Singh, & Khosla, 2016).
Fish Oil Production
Fish oil production methods have changed throughout little within the past fifty years. With “practical” costs in the 1970’s increasing, the “conventional” methods were not applicable for animal consumption (Pigott & Fisheries, 1967). These practices consist of high pressure cooking to yield large portions of low-fat oil from fish.
These methods standards are from the United States Department of Interior book, production of fish oil. The steps involved in manufacturing consist of pressing operations, where oil is pressed out into a raw liquid. The oils are then “separated” from solids by centrifuging, settling or both, the last step is “purification” where water and heat are used to separate “violate and water-soluble impurities” (Pigott & Fisheries, 1967).
Today the manufacturing has changed. According to a release from the United States Environmental Protection, fish oil industries use steam instead of pressing methods. The steam “ruptures” the cell membranes burst “releasing fish oil” (“AP 42, Fifth Edition, Volume I Chapter 9: Food and Agricultural Industries,”). Different manufacturers also further refine the process, where some manufactures steam the oil and doing so removes toxic elements found in fish oil (Anonymous, 2018).
General Information
With the use of manufacturing, Omega dietary supplementation assists in the nutrition needs of consumers. In the natural state, food consisting high level of omegas consist of seafood such as halibut, salmon, herring, mackerel, oysters, tuna, and sardines. In some products, common foods fortified with omegas are eggs, margarine and milk. Other products such as grains, nuts, and fresh produce also contain high levels of omega (Supplements, 2018). These products consist of the many different types of EPA’s as well.
Omegas consist of 3 forms of N-3’s, alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) The main difference between these EPF’s is the location of the double bond, number of double bonds and the number of carbons in the chain. These differences change the function of the EPFs.
N-3s belong to a family of PUFAs (polyunsaturated fatty acids), this family contain conjugated and the other types of PUFAs. EFAs are distinct structures. They are chains that very long, linking together carbons and covered by hydrogens (see figure 1).
When there are not enough hydrogens, the molecule forms the double bond(s), making the EFA curl in molecular structure. These oils also will form into thick substances, at considerably lowered temperatures. The familiar sources in the kitchen that have this type of oil include Olive oil, Canola Oil, and Fish Oil (see figure 2).
When hydrogens cover the long carbon molecule, then it is considered to be saturated. This saturated molecule is called saturated fat and is common in thick oils at room temperatures such as margarine and the lard, such as the lard from bacon, when cooled to room temperature. The following chart are known melting points (see table 1).
There most recognized test for saturation or unsaturation is known as the bromine test. This tests for how many hydrogens are missing from these molecules is called the bromide water test, where bromide is added to a solution to bromide, if bromide disappears when added to the solution, then the molecule is saturated. When added, bromine is unable to fit into the module. That makes the solution darken. It is without to say, bromine itself is carcinogenic and hazardous. All of the properties described make specific fatty acids unique, each containing chemical properties, and some have physiological functions, playing an essential and crucial role in the chemistry of life.
Methods or Methods and Materials
Sample
The test in this study consisted of seven samples. All samples contained a label claim and purchased through local shopping stores. All samples remain kept at room temperature with no sunlight and unpackaged until the day of experimentation.
• Sample #1 is produced using essential oils and filtration, with the claimed label of 600mg of omegas, 324mg EPA, 216mg DHA. The ingredients include Alaska Walleye Pollock, Pacific Whiting essential oils and Mixed Tocopherols. Costs per soft-gel are $0.07.
• Sample #2 is produced by molecular distillation, with the known label of 130mg of omegas, 430mg of EPA, 390mg of DHA and in triglyceride form. The ingredients include anchovy, tocopherols, rosemary extract and ascorbic palmitate. Average cost per soft-gel is $0.85.
• Sample #3 Production method is not labeled, with the claimed label of 720mg of omegas, 360mg of EPA, 240mg of DHA. The ingredients include sardine, mackerel, sodium alginate, ammonium hydroxide, medium chain triglycerides, tocopherols, stearic acid, sunflower oil. Costs per soft-gel are $0.13.
• Sample #4 Production method is not labeled, with the claimed label of 600mg of omegas, 400mg of EPA and 200mg of DHA. The ingredients include anchovy, sardine, and tocopherols. Costs per soft-gel are $0.10.
• Sample #5 Production method is not labeled, with the claimed label of 90mg of omegas, 50mg of EPA and 24mg of DHA. The ingredients include Krill, gelatin, glycerin, water, sorbitol. Costs per soft-gel are $0.66.
• Sample #6 Production method is not labeled, with the claimed label of 300mg of omegas, 700mg of unknown oil concentrate. The ingredients include anchovy, gelatin, glycerin, tocopherols. Costs per soft-gel are $0.05.
• Sample #7 is produced by purification, with the known label of 50mg of GLA and 369mg of LA. The ingredients include evening primrose oil (Seed). The cost per soft-gel is $0.55.
Design
The determination of the compensation in each sample is calculated using the lowest temperature reached by the sample. Each sample in each test interval is tested three times. The number of milliseconds of drips is averaged by the minimum (lowest) drip rate number for each sample and multiplied by 70%. If the sample drip rate is not measurable, then if the sample is soft, soft is equivalent to 80% viscosity; then else if wax, the wax is the equivalence to 90% viscosity, then if froze, froze is the equivalence to 100% viscosity, else sample is noted and skipped from data. Test set interval is -1C l unless the temperature is -15C. If the temperature is -15C, then Test set interval is -5C.
The temperature is lowered by -1C intervals until reaching 5C. The sample is cooled for at least 5 minutes. Temperature is checked with the second thermostat. If the temperature reaches -15C, then the intervals are changed to -5C intervals.
Viscosity is measured by minimum drip rate number in the experiment unless the sample reaches a soft, waxy or frozen consistency. The viscosity was determined by analyzation of video recordings of 30 fps using video editing software. An iPad Pro 9.7” is used to record using default settings. Final cut pro is used to edit the video and is overlaid with a time counter video. This video is freeze framed after each sample is analyzed. The video is cut after the 3 second mark. No additional manipulation of the video is used. Titles are placed on samples that are too viscous to stir. The freeze frame setting is set to 3 seconds in settings menu. All data is placed on charts using excel. A line chart for each sample.
Materials
• 22qt. Insulated Styrofoam cooler (for dry ice)
• Video camera (30 fps)
• Styrofoam board
• 4 feet by 1 foot
• Snap-off utility knife
• Two glass panes (11×8 inches)
• Aluminum tape
• General masking tape
• Electrical tape
• 12v digital thermostat temperature control switch sensor module with the minimum temperature ranges of -50 to 110c
• 10 feet of electrical wiring (use caution, follow switch guidelines)
• Eight pounds of dry ice (use caution)
• Clear vials with lids
80mm cooling fan (insignias worked fine, until reaching -30 ℃)
• Wire strippers
• Probe thermometer
• Digital thermometer
• Alcohol 90% isopropyl alcohol
• 110v to 12v dc 5a 60w regulated switching power supply
A 22qt. Styrofoam cooler box was used for this experiment. A square hole was cut on the long side of the box, the size of the hole is 10×7 inches or less (See figure 1). One glass pane was placed in outside of the container and taped in place using scotch tape. The second pane was placed in the inside of the box, covering the hole. This created an air pocket that acted as an insulator. Scotch tape was used to seal and hold the windows into place (See figure 2, 3, 4).
A bottom Styrofoam plate was placed 2 inches above the bottom, supported by thin strips of Styrofoam. This play fit snuggled next to the two long walls of the box. After experimentation, three layers of scotch tape were added to the plate with one final layer of aluminum. The bottom plate has gaps on the sides to allow air circulation. A wall was taped to the inside of the box, 2 inches away from the side. The wall contains a fan that allows the movement of circulation. The digital was set to turn on and off at when the temperature increased by – / + 0.2 degrees C.
A second inside lid was created on top of the cooler with holes for tube access. The original lid can cover and seal if needed (see figures 4 and 5).
Different types of Styrofoam boxes were used for this experiment. I found that the 2” thick wall sided Styrofoam hold temperate better than other types of Styrofoam. Aluminum tape transfers heat quickly and is not an ideal product for sealing purposes, for this project.
Procedure
A large dry ice piece is broken by a hammer and placed, under the Styrofoam plate. The inside lid is on, and tubes are placed through the holes created. The new scotch tape is used to cover the inside lid. Thermometers are inside a control specimen of omega 3, and both the thermostat and digital thermometer are placed in a solution of alcohol 90% isopropyl alcohol inside the box. The power was turned on.
After a couple of minutes, the thermostat began switching on and off, repeating fast, indicating that temperatures are stabilized for the alcohol solution. Using the probe thermometers, the samples were checked. Then all samples were recorded using a video camera and tested. A plastic straw is used to carry the sample from the vial into the straw. The straw is plugged by one finger and is placed a distance away from the bottom of the container. The straw is unplugged, and the sample drops back into the container. This process is repeated and recorded until the samples no longer can drip from the straw. The viscosity as described in the scoring section were used to describe viscosity.
Scoring
Each sample is repeated three times to obtain an average. If the sample is unable to drip, then the sample is stirred to check for one of three consistencies. Soft consistency is resembled cold honey and is unable to drip. Wax consistency resembles shortening or butter; it is soft enough to mold. Froze is the consistency of ice; a toothpick will be unable to penetrate the surface.
The time range for each sample begins one millisecond before the drop and one millisecond after the first drop. A plastic straw is used to carry the sample from the vial into the straw. The straw is plugged by one finger and is placed a distance away from the bottom of the container. The straw is unplugged, and the sample drops back into the container.
Results
In a study containing 7 different omega marketed samples, containing fish oil, krill oil, flaxseed oil and evening primrose oil are tested for EPA, DHA, ALA or GLA. In this test, 57% of these samples failed to meet the label claim. 5 of 7 samples, 71% contained EPA and 3 of those samples, 43% contained DHA. 4 of the 7 samples, 57 % of the samples contained ALA. 4 of the 6, 66% of the samples contained GLA. 2 of the 5 samples, 40% of the samples failed to meet or contain DHA. 3 of the 5, 60% samples failed to meet or contain EPA. 1 additional sample failed to contain DHA, this sample claimed of containing varied Omegas. Sample #1 failed to contain either Linoleic Acid and Linolenic Acid. This determines that 40% of the products tested
In further analysis, using negative and positive chart line flow rates indicate that all of the samples may contain Palmitoleic acid 100% of all samples, Nonmelodic acid at 50% of the samples, 40% Docosahexaenoic acid of all samples and 20% of Docosahexaenoic acid of all samples.
In further prospective analysis of costs and ratios of DHA and EPA, the cost average correlate with the ratio of claimed EPA and DHA. 2 samples that are less than $0.10 contain EPA and typically consists of a ½ or 2/3 ratio of EPA and DHA respectively. 2 samples cost greater than $0.10 per capsule and claim greater levels of EPA and DHA of 13/43 and 12/25 respectively. The samples show that greater ratio of DHA are greater cost and contain less than 75% of DHA. While 2/3 ratios of costs lower than $0.10 per capsule contain more EPA and DHA overall.
As a review. 60% of the samples contained a form of Linolenic acid, in either gamma or alpha form. 75% of the products contained Linolenic acid in GLA form. 20% of the products contained Linolenic acid in GLA form. 50 % of those samples contained Linoleic or Nonmelodic acid. 100% of these samples contained Palmitoleic acid. 40% of these samples contained Docosahexaenoic acid. 20% of these samples contained Arachidonic acid.

Discussion
In the dietary supplementations studied, A total of 60% of the products samples failed to meet the dietary claim of contain both EPA and DHA. 3 of the 5 products, 60%, failed to meet the dietary label claims of containing EPA, while 2 out of the 5, 40% failed to meet the label claim of docosahexaenoic acid. 1 product only EPA as marketed for containing “various Omegas”. 1 additional product failed to contain DHA, while meeting the label claim of containing GLA.
Costs and advertising are also briefly analyzed, determining that most of these products may contribute to marketing schemes. In two of the highest costing samples, the ratio of EPA to DHA mismatched the 2:3 ratios, while the costs of 2:3 are $0.7-$0.10 per capsule, these samples contained EPA and DHA.
This finding is concerning and calls for additional follow-up research and analysis review to determine the effectiveness of product label claims found in most common supper markets. It is also essential to educate the public and medical communities of the product label claims.
The application of these findings affects many ongoing claims, research studies, the FDA and the general public. Many studies report of little of effectiveness of omega-3 supplementation when physicians, cross links of those patients taking low or no dosage of the studied supplementation may play an essential outcome of the true nature and effectiveness other studies have found. This may also reduce the constraints the FDA has placed in the market for dietary oils, known as common food source dietary supplements. This overall will have a larger impact on patients.
Literture Cited
Albert, B. B., Derraik, J. G., Cameron-Smith, D., Hofman, P. L., Tumanov, S., Villas-Boas, S. G., . . . Cutfield, W. S. (2015). Fish oil supplements in New Zealand are highly oxidised and do not meet label content of n-3 PUFA. Sci Rep, 5, 7928. doi:10.1038/srep07928
Anonymous (Producer). (2018). Anonymous.
. AP 42, Fifth Edition, Volume I Chapter 9: Food and Agricultural Industries. In (Vol. AP 42, Fifth Edition, Volume I pp. 1-7). 1995: U.S. Environmental Protection Agency. Retrieved from https://www3.epa.gov/ttn/chief/ap42/ch09/final/c9s13-1.pdf.
Eilat-Adar, S., Mete, M., Nobmann, E. D., Xu, J., Fabsitz, R. R., Ebbesson, S. O., & Howard, B. V. (2009). Dietary patterns are linked to cardiovascular risk factors but not to inflammatory markers in Alaska Eskimos. J Nutr, 139(12), 2322-2328. doi:10.3945/jn.109.110387
Kleiner, A. C., Cladis, D. P., & Santerre, C. R. (2015). A comparison of actual versus stated label amounts of EPA and DHA in commercial omega-3 dietary supplements in the United States. J Sci Food Agric, 95(6), 1260-1267. doi:10.1002/jsfa.6816
Nichols, P. D., Dogan, L., & Sinclair, A. (2016). Australian and New Zealand Fish Oil Products in 2016 Meet Label Omega-3 Claims and Are Not Oxidized. Nutrients, 8(11), 703. doi:10.3390/nu8110703
Opperman, M., Marais de, W., & Spinnler Benade, A. J. (2011). Analysis of omega-3 fatty acid content of South African fish oil supplements. Cardiovasc J Afr, 22(6), 324-329. doi:10.5830/CVJA-2010-080
Pigott, G. M., & Fisheries, U. S. B. o. C. (1967). Production of fish oil: U.S. Dept. of the Interior, Fish and Wildlife Service, Bureau of Commercial Fisheries.
Singh, S., Arora, R. R., Singh, M., & Khosla, S. (2016). Eicosapentaenoic Acid Versus Docosahexaenoic Acid as Options for Vascular Risk Prevention: A Fish Story. Am J Ther, 23(3), e905-910. doi:10.1097/MJT.0000000000000165
Spector, A. A., & Kim, H. Y. (2015). Discovery of essential fatty acids. J Lipid Res, 56(1), 11-21. doi:10.1194/jlr.R055095
Supplements, O. o. D. (2018). Omega-3 Fatty Acids Fact Sheet for Health Professionals. Retrieved from https://ods.od.nih.gov/factsheets/Omega3FattyAcids-HealthProfessional/

Appendices

Abstract 2
Introduction 3
Brief History 4
Fish Oil Production 5
General Information 5
Methods or Methods and Materials 7
Sample 7
Design 8
Materials 9
Procedure 10
Scoring 11
Results 11
Discussion 13
Literture Cited 14
Appendices 16
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Figures 25
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Figures
Table 1 Melting Points of saturated fatty acids.
Name of Acid Number of Carbons Number of Double Bonds Melting Point ℃
Oleic 18 1 4
Linoleic 18 2 -5
Linolenic 18 3 -11
Arachidonic 20 4 -50

Table 2 All statistical data
TEMP
in ℃ Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7
1 1728000 5184000 7776000 1728000 Wax 34560000 8640000
0 1728000 6048000 17280000 17280000 Wax 001 25920000
-1 1728000 7776000 1000 1728000 Wax 17280000 8640000
-2 2592000 7776000 2000 1728000 Soft 002 8640000
-3 2592000 7776000 6000 2592000 Soft 003 8640000
-4 2592000 8640000 4000 2592000 Soft 006 51840000
-5 2592000 69120000 Wax 2592000 Soft Wax 17280000
-7 2592000 17280000 Wax 1728000 Soft Wax 43200000
-8 1728000 43200000 Soft 2592000 Soft Wax 001
-9 2592000 001 Soft 2592000 Soft Wax 001
-10 1728000 86400000 Soft 2592000 Soft Soft 43200000
-13 3456000 001 Soft 1728000 Froze Soft 60480000
-14 3456000 002 Soft 2592000 Froze Soft 003
-17 1728000 002 Soft 2592000 Froze Soft Wax
-18 2592000 001 Soft 2592000 Froze Soft Soft
-30 5184000 Wax Froze 2592000 Froze Soft Soft
-43 Soft Soft Froze 3456000 Froze Soft Soft
-50 Soft Soft Froze 4320000 Froze Soft Soft
-51 Soft Soft Froze 4320000 Froze Soft Soft
Note: This is the raw data is calculated in milliseconds.

Table 3 Data Calculations
Temp Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7
1 23% 4% 32% 4% 0% 70% 3%
0 23% 5% 70% 40% 0% 0% 10%
-1 23% 6% 0% 4% 0% 35% 3%
-2 35% 6% 0% 4% 50% 0% 3%
-3 35% 6% 0% 6% 50% 0% 3%
-4 35% 7% 0% 6% 50% 0% 20%
-5 35% 56% 80% 6% 50% 80% 7%
-7 35% 14% 80% 4% 50% 80% 16%
-8 23% 35% 90% 6% 50% 80% 0%
-9 35% 0% 90% 6% 50% 80% 0%
-10 23% 70% 90% 6% 50% 90% 16%
-13 47% 0% 90% 4% 100% 90% 23%
-14 47% 0% 90% 6% 100% 90% 0%
-17 23% 0% 90% 6% 100% 90% 80%
-18 35% 0% 90% 6% 100% 90% 90%
-30 70% 80% 100% 6% 100% 90% 90%
-43 90% 90% 100% 8% 100% 90% 90%
-50 90% 90% 100% 10% 100% 90% 90%
-51 90% 90% 100% 10% 100% 90% 90%

Table 4 Results of All Samples
Sample Linoleic Acid
(MP of -5℃) Linolenic
(MP of -11℃) Arachidonic
(MP of -50℃) Other 1 Other 2 Other 3
#1 Negative Positive Negative -1, -5 -17, -30
#2 Positive Positive Negative -7, -9 -18, -30
#3 Positive Negative Negative 1, -1 -7, -8 -18, -30
#4 Negative Negative Positive 1, -1 -50 < X #5 Positive Positive Negative 0, -2 -50 < X #6 Positive Positive Negative 0, -1 -50 < X #7 Positive Positive Negative 1, -1 -50 < X Table 5 Changes based on other oils Lipid Numbers TEMP °C Common Name #1 #2 #3 #4 #5 #6 #7 C16:1 -1.0 Palmitoleic acid + – + + + + – C6:0 -3.4 Hexanoic acid – – – – NA – + C18:2 -5.0 Linoleic acid – + + – NA + + C18:2 -5.0 Nonmelodic acid – + + – NA + + C18:3 -11.0 γ-Linolenic acid + + – – + + – C18:3 -17.0 α-Linolenic acid + – – – – – + C22:6 -44.0 Docosahexaenoic acid – + + + – – – C20:4 -50.0 Arachidonic acid – – – + – – – C20:5 -54.0 Eicosapentaenoic acid NA NA – NA – NA NA   Table 6 EPA and DHA label claims EPA Claim EPA Result DHA Claim DHA Result GLA Claim GLA Result LA Claim LA Result Cost DHA/EPA Ratio Sample 1 430 ➕ 130 ➖ None ➕ None ➖ 0.85 13/43 Sample 5 50 ➖ 24 ➖ None ➕ None NA 0.66 12/25 Sample 3 360 ➖ 240 ➕ None ➖ None ➕ 0.13 2/3 Sample 4 400 ➕ 200 ➕ None ➖ None ➖ 0.10 1/2 Sample 2 324 ➕ 216 ➕ None ➕ None ➕ 0.07 2/3 Sample 6 Various ➕ Various ➖ None ➕ None ➕ 0.05 NA Sample 7 None ➕ None ➖ 50MG ➖ 369mg ➕ 0.55 NA Table 7. Sample #1 Table 8. Sample #2 Table 9. Sample #3 Table 10. Sample #4 Table 11. Sample #5 Table 12. Sample #6 Table 13. Sample #7 Figures 1. Eicosapentaenoic acid 2 behenic acid   3 Styrofoam Box Freezer 3. Styrofoam Box Without Windows   4. Styrofoam Box with Windows 5 Top view of Styrofoam box. 6 Top view of Styrofoam box. 7 Top view of Styrofoam box with legs. 8 Top view of Styrofoam box. 9 Top view of Styrofoam box. 10 Top view of Styrofoam box.