11Fish Oils

R. G. Ackman

Canadian Institute of Fisheries Technology, Dalhousie University

Halifax, Nova Scotia, Canada

1. INTRODUCTION

At one time ‘‘fish oils’’ were low-cost industrial materials for the paint and lino-

leum industries. After World War II (WWII), these industries switched to chemicals

and plastics, and therefore, much information in older books became obsolete by

1960. Hydrogenation of fats to produce margarines and shortenings, starting about

1900, led to improved oil refining and better quality and included whale oils when

these animals were still plentiful. Two factors have recently impacted negatively on

large-scale and continued use of marine oils in our food supply.

Because of one of the earliest media-stimulated public health panics in the

late 1970s, that of the erucic acid (22:1) of rapeseed and mustard oils, alleged to

damage hearts, food use of partially hydrogenated fish oils in that form petered out

because of their content of both natural and artifact 22:1 isomeric fatty acids, as

described in Barlow and Stansby (1). As millions of healthy Germans and Poles

had thrived on rapeseed, and the fish-eating Scandinavians were universally healthy,

this fear was based on scanty evidence. The major result of this scare against

sources of very long-chain monoethylenic fatty acids was acceleration of develop-

ment of low-erucic-acid rapeseed oils such as canola (2). The desirable physical

properties of partially hydrogenated fish oils in some margarines and in shortening

for baking purposes continued but depended primarily on conversion of most of the

Bailey’s Industrial Oil and Fat Products, Sixth Edition, Six Volume Set.Edited by Fereidoon Shahidi. Copyright # 2005 John Wiley & Sons, Inc.

279

natural cis-ethylenic bonds of the fatty acids to trans-ethylenic bond configurations,

rather than to saturated fatty acids (3). Recently, a media storm against trans-acids

followed initial scientific research on the health aspects of small changes in serum

cholesterol, despite long-term human exposure (4), and again partially hydroge-

nated fish oil use was condemned. Despite the adverse image, the baking industry

was conservative and this usage persisted in technically advanced countries such as

Denmark until recently (5). Dairy products and ruminant meat products continue to

include ‘‘natural’’ trans-acids (6).

In a positive turn of fate, by 1980, the observations of Dyerberg and Bang (7) on

the excellent cardiac health of the Eskimo population of Greenland, who had a high

intake of dietary omega-3 (n-3) fatty acids from seal and fish fats, were also noted

in the media. This publication in 1979 created an astounding public interest in fish

oils as nutrition supplements, usually taken in capsules. In 1985, another positive

media bombshell exploded when a long-term study in Zutphen in the Netherlands

showed that a large male population group had reduced their cardiovascular mor-

tality rate by eating fish regularly (8). Obviously, fish omega-3 (n-3) fatty acids

were beneficial.

By 1994, the United Kingdom had followed up on such reports and released an

official government medical report recommending that people eat fish at least twice

a week, one meal being of fatty fish (9). Belatedly the American Heart Association

released a ‘‘Scientific Statement’’ in 2002, with a similar recommendation (10).

Through this exciting period the dietary intake of fish in western society had

declined as other modern foods were easier to produce, store, and distribute. Fish

were also beginning to be more scarce as fisheries were overexploited and fish

populations diminished or were even wiped out.

In this review, only fish ‘‘oils’’ can be considered. The fat in edible parts of fish

ranges from about 16% down to 0.7%, the latter being almost exclusively the basic

muscle phospholipids (Figure 1). These lipids are an excellent source of the highly

unsaturated C20 and C22 omega-3 fatty acids of medical interest, and the content of

omega-3 fatty acids is stable at roughly 0.5-g/100-g muscle. However, it is the mus-

cle triacylglycerols that are variable for quantity and quality. This subject is conve-

niently reviewed for the food industry in one medium-length paper (11) in a journal

available in most major libraries, being a part of a whole issue (No. 4) on seafood.

A chapter on marine fatty acids in a reference book associating all types of fatty

acids with specific health problems could also be useful (12). As for the actual

health benefits of the omega-3 fatty acids, a special supplement issue of the

American Journal of Clinical Nutrition (13) is also highly recommended as it draws

together the thoughts and facts from several dozen distinguished authors active in

the field of omega-3 fatty acids.

It must be noted that the promotional bandwagon for fish oils and seafood

omega-3 fatty acids has been neatly hijacked by those oilseed companies offering

a vegetable oil omega-3 fatty acid, alpha-linolenic acid (18:3n-3). Only about

10% of this fatty acid in the diet survives immediate catabolism and is needed

for ‘‘essential’’ roles in the skin and elsewhere (14). Whether enough survives to

be elongated and to enrich brain and retinal tissues and thus to perform the vital

280 FISH OILS

functions of the fish DHA or docosahexaenoic acid (22:6n-3) and EPA or eicosa-

pentaenoic acid (20:5n-3) is not known, but the distinction by chain lengths should

always be made. The term ‘‘conditionally dispensable’’ coined by one expert (15) is

perhaps suitable for 18:3n-3 at this time and in this context of recommending mar-

ine oils rich in long-chain n-3 fatty acids. Only human trials can be trusted to be

definitive, and modest doses of vegetable (C18) or fish (C20 þ C22) fatty acids for

12 weeks, although informative (16), cannot accurately predict the life expectancy

H2C

HC

H2C

OC

OC

O

O

O C

O

H2C

HC

H2C

OC

OC

O

O C

O

H2

H2C

HC

H2C

OC

OC

O

O C

O

O

O

O CH2

CH2

NH3C CH3

CH3

Triacyglycerols

Diacyl-1-glyceryl ether

Phosphatidyl Choline

(+)(−)

Glycerol-Based Fish Lipid Classes

Figure 1. Three classes of lipids found in fish bodies and sharing in common glycerol fatty acid

linkages.

INTRODUCTION 281

and good health desired by the public. These trials are often linked to modifications

in diet, for example, reduction in intake of n-6 fatty acids. Critical reexamination of

published results is highly desirable and useful (17, 18).

Throughout the last 250 years, cod liver oil has steadily maintained a health role

well known to be inclusive of vitamins A and D. Our aging population is increas-

ingly faced with osteoporosis and mobilization of calcium intake to strengthen

bones, now a high priority in the adult population that requires Vitamin D, whereas

formerly the benefit target was rapidly growing children. Cod liver oil remains a

nutritional supplement staple for vitamins, but it also contains omega-3 fatty acids

(19). However, it is a specific staple outside the scope of this book except for a brief

mention on oil production and refining.

2. WHY DO WE STILL HAVE FISH OILS?

Fish oils are a byproduct of the production of fish meal. Both commodities are sub-

ject to price fluctuations that are only indirectly related. The meal has been histori-

cally used for terrestrial animal feedstuffs. The production process can be

condensed to apply to two basic functions: take fish or fish waste, cook it, and

squeeze it (Figure 2). Subsequent steps are varied for each commodity, but they

essentially are well described by Young (20), whereas the desirable properties of

fish meal are concisely described by Bimbo and Crowther (21). Similar summaries,

especially on sources and production, are provided by various chapter authors in a

book on pelagic fish (22), because most fish caught for meal and oil are pelagic

(migratory) and subject to seasonal exploitation. The principal competition to

fish meal is soybean meal and other oilseed meals (23).

Fish

FishMeal

Crude Fish Oil

Grind and Cook

Press

Centrifuge

Fish Oil Manufacture

Oil + Stickwater Press Cake

H2O

H2O

EvaporatorSolids

Drying

Figure 2. A simplified diagram of the fish meal manufacturing process. Shading denotes the

principle products.

282 FISH OILS

The gloomy chronology in Section 1 for food and health aspects of fish oils

since WWII would have ruined most basic industries, but as a byproduct of fish

meal production, that of oil has continued. The human (and pet) nutritional

supplement markets would absorb only a small amount of the total world pro-

duction, which has remained at approximately 1.2–1.4 million tons for the last

decade (23).

Aquaculture has provided part of the solution, which is particularly true for sal-

monids, and to trout farmed in freshwater, a very large amount of the former indus-

trial grade (unrefined) production of fish oil goes to Atlantic salmon (Salmo salar)

production in places as far away from their origin in the North Atlantic as New

Zealand and Chile. Other species are now coming into aquaculture production,

usually higher priced fish such as Atlantic halibut, grouper, sea bass, tilapia, and

so on. Fish oil is not just a cheap fat, as most of the fish will grow on diets of

fish meal plus other fats, but around 1–2% of the total dietary fatty acids should

be the long-chain polyunsaturated fatty acids (20:5n-3 and 22:6n-3) that are basic

to the functional membranes of new cells when combined with the protein of the

fish meal. Fish meal usually contains approximately 8–10% of fat, and it is often a

good source of this minimum requirement of omega-3 fatty acids. Excessive fat in

fish diets is thought to spare protein degradation for energy, which results in more

growth, and the 20% of total dietary fat for the salmon diets common a decade ago

is now frequently replaced with a total of 26–28%, the so-called high-energy diets.

Peru has been a major producer of industrial grade fish oil for the aquaculture

industry, but the anchovy fished there fails to return to the coast occasionally for

Pacific Ocean and climate reasons (an EI Nino event), and reduced production

can drive the world price of fish oil to above $500 (US)/tons compared with

more normal prices of $250–$400/ton (23).

The new factor in the fish oil industry is as a built-in supplement in the human

diet instead of a separate food additive. It is in the form (usually) of highly refined

fish oil added in a microencapsulated format. The oils used must be highly refined

to meet Food and Drug Administration (FDA) standards, especially in the United

States, where menhaden oil was the first oil approved for this purpose. There, the

intake of the oil must not exceed 3 g/day of the two fatty acids 20:5n-3 and 22:6n-3

in a designated list of typical foodstuffs. Although ethyl esters and concentrates of

oils and esters will eventually be approved, it is almost certain that the starting

materials will be those fish oils essentially very low in 20:1 and 22:1. As will be

discussed below, these oils typically have 20:5n-3 > 22:6n-3, i.e., EPA > DHA in

the popular terminology. The initial omega-3 publicity in the 1980s favored EPA

for superior blood vessel function, but gradually this public image has changed

to favor DHA as superior for heart muscle function and neural problems. Infant

nutrition is yet another popular field for debate on omega-3 fatty acids, and it

should be noted that the Martek Biosciences Corp., Columbia, nw, was the first

to develop algal (i.e., vegetarian) sources of 22:6n-3 and 20:4n-6 (arachidonic

acid) as preformed long-chain fatty acids representative of human breast milk

when added to enriched infant formulas. Revenues for this company were forecast

to exceed $100 million in 2003.

WHY DO WE STILL HAVE FISH OILS 283

3. FISH OIL FATTY ACIDS AND GAS-LIQUIDCHROMATOGRAPHY

Fish oils mostly contain triacylglycerols of fatty acids (Figure 1), recovered by

fairly simple technology (Figure 2) from the whole bodies of fish, often from spe-

cies considered inedible in contemporary Western society. In fact, many exceptions

to these generalizations exist, because in addition to the triacylglycerols, there can

be wax esters (1/2 fatty alcohols), diacylglyceryl ethers (2 fatty acids, Figure 1),

cholesterol and cholesteryl esters (Figure 3), and even the hydrocarbon squalene

(Figure 4). Also, the marine mammals are to be considered. The depot fats of

baleen whales and seals yield oils similar to the above general description of fish

oil including fatty acid composition, but the triacylglycerols differ in fatty acid

molecular arrangements and warrant a separate discussion. The depot fats of the

toothed whales can include wax esters (sperm whales), or even triacylglycerols

and wax esters incorporating short-chain fatty acids such as isovaleric (the dolphins

and similar small species). These topics are covered elsewhere (24). Marine inver-

tebrates also will have to be excluded from this discussion as there is no large-scale

or industrial use of their triacylglycerols. ‘‘Krill’’ oil produced from small Antarctic

crustacea such as Euphausia superba is now offered in small amounts for the nutri-

tional health product market, but the investment prospects are daunting (25). The

two volumes of Marine Biogenic Lipids, Fats and Oils offer lipid class and lipid

C

O

HO

Free Fatty Acid

HO OC

O

R

Cholesterol and/or Ester

H3C (CH2)x CH2

O C

O

(CH2)x CH3

Wax Ester

Fish Oil Miscellaneous Natural Lipids

Figure 3. Lipids that may be found in marine oils. Free fatty acids are artifacts of postmortem

processes in fish bodies and may range up to 5% or even 10% of oils. Cholesterol and its esters

are usually in the 0.5–1.0% range, but wax esters may be major oil components in oils from

certain fish.

284 FISH OILS

component composition information on these topics from resource and biochemical

points of view (24).

Although traditional quality factors such as iodine value, free fatty acids

(Figure 3) and unsaponifiable content (Figures 3 and 4) are still important in trading

fish oils, the fatty acid composition determined by gas-liquid chromatography

(GLC) of methyl esters is now frequently required. Figure 5 is a typical example

of what is desirable, and it may help to explain the fatty acids to be expected. This

analysis is on a special class of liquid phases based on polyglycols (e.g., SUPEL-

COWAX-10, DURA-WAX, Stabilwax, Omegawax-320, etc.), which limits even

chain length overlaps to two C24 fatty acids and 22:6n-3. These chain lengths

are marked accordingly.

Figure 6 is an extensive list of the fatty acids typical of the triacylglycerols of

fish oils and includes one baleen whale oil. These were all of interest to industrial

and food fats and oils companies two decades ago (1), but even later were of limited

interest in human nutrition (26). For practical purposes, marine oils can be defined

by 12 fatty acids that add to about 90% of the array of peaks in Figure 5 and deter-

mine the properties of a given oil. These are (in a shorthand giving the chain-length,

HO

CH3 CH3 CH3 CH3

Phytol

CH3 CH3 CH3 CH3

Phytane

CH3 CH3 CH3 CH3

Pristane

CH3

CH3 CH3 CH3

CH3 CH3 CH3

CH3

Squalene

Neutral Fish Oil Compound

Figure 4. Phytol and phytol-related hydrocarbons, pristane and phytane, are associated and

may be found in some fish oils. Phytanic, pristanic, and 4,8,12-trimethyltridecanoic acids are

common fish oil components derived from phytol. Squalene is usually of animal origin and a

feature of some shark liver oils.

FISH OIL FATTY ACIDS AND GAS-LIQUID CHROMATOGRAPHY 285

number of cis-methylene-interrupted ethylenic bonds, and position of the ethylenic

bond nearest the methyl end of the chain):

14:0 16:1n-7 18:2n-616:0 18:1n-9 18:3n-318:0 20:1 18:4n-3

22:1 20:5n-322:6n-3

After considering the many analyses of fish oils available, the author concluded that

there was only one ‘‘basic’’ fatty acid composition of fish oils from coldwater or

from northern latitudes (27). Generally this is typified by menhaden oil, a species

that feeds exclusively by filtering phytoplankton out of the ocean water in the Gulf

of Mexico or in the Atlantic Ocean off the east coast of the United States or the

Figure 5. Analysis of menhaden (fish) oil on an Omegawax-320 capillary column. Equipment:

Varian 3400 GLC, splitless injection helium carrier gas. Initial temperature 69 �C for 1.4 min;

ramp to 170 �C at 50 �C/min; hold for 8 min at 170 �C; ramp to 220 �C at 3 �C/min; hold 15 min;

total time 43 min. Note FAME chain lengths below baseline. Peaks identified are as follows:

1 ¼ 14:0; 2 ¼ 15:0;3 ¼ 16:0; 4 ¼ 16:1n-7; 5 ¼ 16:16:2n-4; 6 ¼ 16:3n-4; 7 ¼ 16:4n-1; 8 ¼ 18:0;

9 ¼ 18:1n-9; 10 ¼ 18:1n-7; 11 ¼ 18:2n-6; 12 ¼ 18:3n-3; (followed by 18:3n-1); 13 ¼ 18:4n-3

(followed by 18:4n-1); 14 ¼ 20:0; 15 ¼ 20:1n-9 (followed by 20:1n-7); 16 ¼ 20:4n-6; 17 ¼ 20:4n-

3; 18 ¼ 20:5n-3; 19 ¼ 22:1 group; 20 ¼ 21:5n-3; 21 ¼ 22:5n-3; 22 ¼ 22:6n-3 (with 24:0 pre-

ceding and 24:1n-9 following). Analysis time, 30 min. Reproduced by permission of Anal.

Chem.Acta.

286 FISH OILS

anchovy oil from Peru (Figure 6), which also feeds close to the plant base of the

food chain.

Menhaden oil has a fatty acid composition that provides a good example of the

‘‘basic’’ marine fish oil fatty acid system. For example, it is characterized (Figure 6)

by low values of 20:1 and particularly 22:1. The origin of these two fatty acids has

been discussed in reviews of freshwater lipids (28) as well as of marine lipids (29,

30). In principle the extension by one acetate unit of the plentiful 18:1n-9 and

18:1n-7 will give 20:1n-9 and 20:1n-7, and a second step leads to some 22:1n-9

and 22:1n-7. Generally the process stops there, and only small amounts of these

are found relative to 22:1n-13 and especially 22:1n-11. In addition to a little

24:0, a more obvious peak for 24:1 is found, which accounts for that in Figure 6.

The marine 24:1n-9 includes nervonic acid, functional in various organs of animals,

and may accumulate from food fatty acids. Other isomers are possible. The 22:1

peak is actually resolved by efficient open-tubular GLC into four distinct peaks

(Figure 7), of which the major peak is primarily 22:1n-11 but also includes some

22:1n-13, followed by 22:1n-9, 22:1n-7, and 22:1n-5. Similarly the 20:1 array

(Figure 7) includes a frontal shoulder of 20:1n-11, sometimes difficult to see on

the dominant 20:1n-9 peak, which is followed by 20:1n-7 and 20:1n-5 and some

other peaks of non-methylene-interrupted dienoic (NMID) acids that might occur

as discussed below.

The origin of the unexpected 22:1n-13 and 22:1n-11 fatty acid isomers (Table 1)

is now not a mystery (29, 30). The pioneering work of many scientists some dec-

ades ago on marine lipids led to those of a group of tiny marine crustaceans called

copepods. They found that one major lipid class in several species known to be

important as food for fish in the North Atlantic was wax esters (31). In fact, lipids

of a copepod sample examined in Halifax contained 61.2% wax esters and only

31.6% triacylglycerols (29). The distribution of ethylenic bond positions in the

copepod lipid wax ester fatty alcohols is compared with the alcohols recovered

from the body depot fats of several regional fish species known to feed directly

on copepods in Table 1. It is believed that the copepod fatty alcohols are converted

directly by the fish to the corresponding fatty acids, which accounts for the very

high proportion of 22:1 usually observed in fish feeding at this trophic level

(Table 1). However, it is fair to point out that the copepod fatty acids of both the

triacylglycerols and the wax esters had modest contents of the same isomers (29).

The dominance of the unusual 22:1 wax ester isomer n-11 in the copepod is not

readily explained, but it may be based on physical properties such as melting point,

specific gravity, and so on.

It will be noticed that in most tables of fish oil, fatty acid compositions 20:1

and 22:1 are simply listed as such and do not include isomer details. They are

perfectly acceptable in diets for aquaculture fish (32). As reviewed earlier, their

presence in fish oils did contribute to false alarms about 22:1 fatty acids and alleged

heart damage in animals consuming rapeseed oil or fish oils, the latter usually

being in partially hydrogenated form for margarines and shortening. This was inde-

pendent of the much more recent possibilities of trans-fatty acids of any origin

leading to cholesterol and atheroma problems in the human population (33, 34).

FISH OIL FATTY ACIDS AND GAS-LIQUID CHROMATOGRAPHY 287

HerringNorthSea

Anchovy

Fatty acid composition of six samples of fish oil and one whale oil in commercial trade. Unpublished datareproduced by courtesy of W. Schokker and H. Boerma, Unilever Research, Vlaardingen.

PeruWhaleAntarctic

PilchardSouthAfrica

Sardine MenhadenU.S.APortugal

Pilchard?

12 0.10 0.10 0.20 0.20 0.10 0.15 0.1014 6.10 7.45 7.45 7.75 6.70 7.30 7.3014:1 0.15 − 0.75 0.15 − − −16:Branched 0.40 0.40 0.40 0.40 0.50 0.45 0.5515:0 0.40 0.60 0.65 0.40 0.75 0.65 0.6016:0 10.75 17.45 13.40 15.65 17.80 19.00 15.6016:1 7.30 9.00 10.50 8.50 6.00 9.05 9.0016:2 ω 7 0.20 0.20 0.20 0.55 0.40 0.50 0.40

16:2 ω 4 0.40 1.00 0.65 1.45 0.65 1.25 1.55

16:3 ω 4 6.70 2.05 0.10 2.00 0.40 1.45 1.70

16:3 ω 3 − − 0.20 − 0.20 0.20 0.15

16:4 ω 4 0.10 − − − 0.10 0.15 0.20

16:4 ω 1 1.20 2.45 0.95 3.20 1.60 2.30 2.6017:Br 0.30 0.35 0.25 0.25 0.20 0.20 0.1517:0 0.35 0.55 0.95 0.80 0.80 0.90 0.8517:1 0.30 − 0.25 − 0.30 − −18:Br 0.80 0.70 1.10 0.60 0.60 0.45 1.0018:0 1.40 4.00 2.70 3.65 3.60 4.20 3.4518:1 10.30 11.55 27.60 9.25 13.00 13.20 10.4018:2 ω 9 tr. 0.10 0.10 0.15 0.15 0.30 0.20

18:2 ω 6 0.95 1.20 1.90 0.80 1.20 1.30 1.30

18:2 ω 4 0.10 0.60 0.20 0.50 0.30 0.40 0.50

18:3 ω 6 0.05 0.30 0.20 0.35 0.20 0.25 0.3018:3 tr. 0.20 0.10 0.30 0.10 0.30 0.2018:3 ω 3 2.00 0.75 0.85 0.45 1.00 1.30 0.65

18:4 ω 3 3.15 3.05 1.05 2.05 3.15 2.75 2.6518:4 0.15 0.20 0.20 0.15 0.10 0.15 0.2019:Br − 0.10 − − 0.20 0.20 −19:0 0.20 0.10 0.60 0.20 0.40 0.40 0.1019:1 0.10 − 0.40 − − − −20:0 0.10 0.30 0.20 0.60 0.40 0.35 0.3020:1 13.40 1.55 6.75 2.50 4.30 2.00 1.4520:2 ω 9 − 0.30 0.10 0.40 0.15 0.45 0.15

20:2 ω 6 0.15 0.35 0.15 0.25 0.20 0.35 0.30

20:3 ω 6 0.10 0.10 0.20 0.30 0.10 0.15 0.20

20:3 ω 3 0.30 1.10 0.60 1.35 0.85 0.80 1.00

20:4 ω 6 tr. 0.10 0.25 0.10 0.10 0.15 0.15

20:4 ω 3 0.75 0.70 1.30 0.70 1.05 1.35 0.80

20:5 ω 3 7.45 17.00 4.70 19.30 11.00 11.00 18.3021:0 0.10 tr. 0.05 0.15 0.10 0.05 0.1021:5 ω 2 0.25 0.70 0.15 0.90 0.50 0.60 0.9022:0 0.05 0.05 0.10 0.20 0.20 0.20 0.1522:1 21.25 1.15 2.40 3.10 3.80 0.55 1.5522:2 0.20 0.10 0.05 0.05 0.10 0.20 0.1022:3 ω 3 − 0.15 0.25 0.15 0.15 0.15 0.20

22:4 ω 3 0.25 0.55 0.20 0.40 0.70 0.50 0.60

22:5 ω 3 0.75 1.60 2.40 2.35 1.30 1.90 1.80

22:6 ω 3 6.75 8.75 5.70 6.45 13.00 9.10 9.6023:0 0.10 0.05 0.05 0.10 0.10 0.10 0.1524:0 0.15 0.05 tr. 0.15 0.10 0.15 0.1024:1 0.75 0.50 0.30 0.50 0.60 0.35 0.70Wijs I.V. 135.7 181.0 121.7 182.0 169.7 162.1 189.4GLC- I.V. 138.0 181.5 122.1 183.9 170.0 161.5 190.7

Figure 6. Reproduction of a personal communication to R. G. Ackman from scientists of

Unilever Research, Vlaardingen, the Netherlands, as published in (1). Reproduced by

permission of Academic Press.

288 FISH OILS

As of the time of writing (mid-2003), the FDA has announced that trans-acid

contents of foods will soon be required on food composition labels. Fish oils,

now omega-3 nutritional supplements in some foods, are essentially excluded

from such considerations because they contain almost exclusively cis-ethylenic

bonds.

Figure 7. Part of gas-liquid chromatographic analysis of methyl esters of cod liver oil on an

Omegawax-320 column, 0 :32 � 30 � 0 :25. Temperature 160 �C for 8 min, 3 �C/min to 220 �C,

hold. Peaks identified are as follows: 1 ¼ 18:4n-3; 2 ¼ 18:4n-1, unmarked probably 18:5n-3;

3 ¼ 20:0; 4 ¼ 20:1n-9 with frontal shoulder of 20:1n-11 after peak 3; 5 ¼ 20:1n-7; 6 ¼ 20:1n-5;

unknown; 7 ¼ 20:2n-6; unknown; 8 ¼ 20:3n-6; 9 ¼ 20:4n-6; 10 ¼ 20:3n-3; 11 ¼ 20:4n-3;

12 ¼ 20:5n-3; small peak for 22:0; 13 - 22:1n-13 þ 22:1n ¼ 11; 14 ¼ 22:1n-9; 15 ¼ 22:1n-7;

16 ¼ 22:1n-5 (Ackman, unpublished).

FISH OIL FATTY ACIDS AND GAS-LIQUID CHROMATOGRAPHY 289

4. SATURATED, ISOMERIC MONOENOIC, AND UNUSUALFATTY ACIDS

The ‘‘basic’’ fish oil (27) also included a generous amount of saturated fatty acids.

As can be seen from Figure 5 and Table 1, the saturated fatty acids are dominated

by the 16:0 (palmitic acid), usually accompanied by about half as much or less of

14:0 (myristic acid) and much less of 18:0 (stearic acid). Usually the saturated fatty

acid totals are at least 20%, especially as the odd chain (15:0, 17:0) and methyl-

branched (iso, anteiso, pristanic, phytanic) fatty acids (compare Figure 4) are satu-

rated and will total around 2–3%. An unsaturated peak that is often observed is

17:1n-8, which is roughly equal to 17:0. The details of these peaks are discussed

in other publications, but those researchers attempting modern open-tubular gas

chromatography analyses should be aware of their presence and influence on

peak identification and quantitation. As can be seen from Figure 6, there is an

TABLE 1. Principal n-3 Fatty Acids, Saturated, and Monoethylenic Fatty Acid Isomers

(w/w%) in Triacylglycerols and Wax Esters of Copepods and Commercial Oils of Pelagic

Species of North Atlantic Fish Likely to be Consuming Copepods.

Commercial Oils

Copepod Capelin Mackerel Herring

Triacylglycerol Wax Esters Total Acids Wax Esters Total Acids Total Acids

14:0 19.84 38.42 7.85 5.23 7.81 8.77

16:0 28.98 11.15 8.81 8.36 15.93 14.84

18:0 1.04 0.35 0.72 1.03 1.73 0.97

16:1n-9 0.05 0.35 0.03 1.58 0.29 ND

16:1n-7 8.89 12.33 15.42 18.16 8.20 7.22

16:1n-5 0.73 1.04 0.73 0.12 0.54 0.52

18:1n-9 3.14 3.46 4.40 5.95 8.61 12.27

18:1n-7 0.91 0.56 3.43 1.69 3.78 3.66

18:1n-5 0.41 0.15 0.62 0.65 0.54 0.64

18:2n-6 0.97 0.83 0.78 0.86 1.28 0.78

18:3n-3 1.08 1.00 0.20 0.36 0.99 0.39

18:4n-3 3.23 5.63 1.36 1.87 2.47 0.93

20:1n-11 0.33 0.36 1.20 0.46 0.24 0.50

20:1n-9 4.12 4.37 14.53 9.34 10.59 14.37

20:1n-7 0.34 0.55 1.84 0.92 1.13 0.94

20:1n-5 0.02 0.01 0.23 0.10 0.09 0.19

20:4n-6 0.29 0.19 0.29 0.60 0.36 0.24

20:5n-3 8.38 5.81 9.35 22.38 7.84 2.85

22:1n-11(13) 5.16 4.59 17.45 5.90 12.74 20.92

22:1n-9 0.34 0.65 1.70 0.92 1.00 1.36

22:1n-7 0.11 0.11 0.42 0.19 0.19 0.33

22:5n-3 0.60 0.23 0.60 0.74 0.57 0.37

22:6n-3 4.90 0.64 2.70 5.32 7.66 2.70

24:1 0.49 0.24 0.59 0.13 0.69 0.52

ND ¼ not detected.

From Ratnayake and Ackman (30).

290 FISH OILS

inverse relation among classes of fatty acids, so oils rich in 20:1 and 22:1 generally

have lower levels of saturated fatty acids.

The peaks for C14 monounsaturated fatty acids follow 14:0 and are usually a

jumble of peaks for 14:1 isomers, mixed up with those for iso and anteiso 15:0

and the isoprenoid 4,8,12-trimethyltridecanoic acid, followed reasonably clearly

by that for 15:0 (Table 2). Little interest exists in these details, and the only obvious

next peak before 16:0 should be iso-16:0, and sometimes another isoprenoid acid,

pristanic or 2,6,10,14-tetramethylpentadecanoic, is found just ahead of 16:0.

TABLE 2. Fatty Acid Composition (w/w%), with Relative Retention Times, for Japanese

Sardine Oil (36), Compared with That of Triacylglycerols of Cultured Cells of the Marine

Diatom Phaeodactylum tricornutum (39).

Peak No. Fatty Acid RRT Sardine P. tricornutum

1 10:0 0.057 0.02 –

2 12:0 0.119 0.11 0.4

3 iso-13:0 0.144 – –

4 13:0 0.166 0.02 1.1

5 iso-14:0 0.208 – –

6 14:0 0.245 7.25 6.9

7 14:1 (n-9) 0.260 0.25 –

8 14:1 (n-7) 0.275 0.06 –

9 14:1 (n-5) 0.287 0.07 –

10 iso-15:0 0.296 0.15 –

11 anteiso-15:0 0.313 0.08 –

12 15:0 0.348 0.31 2.0

13 iso-16:0 0.421 – –

14 anteiso-16:0 0.437 0.08 –

15 16:0 0.502 19.42 21.2

16 16:1 (n-11) 0.529 0.84 –

17 16:1 (n-9) 0.541 – 4.4

18 16:1 (n-7) 0.564 8.64 23.0

19 iso-17:0 0.600 0.07 –

20 anteiso-17:0 0.631 0.32 –

21 16:2 (n-7) 0.643 0.05 –

22 16:2 (n-4) 0.686 1.23 3.4

23 17:0 0.704 0.33 –

24 17:1 (n-10) 0.732 0.52 –

25 16:3 (n-4) 0.762 1.57 3.3

26 17:1 (n-8) 0.785 0.13 0.4

27 17:1 (n-6) 0.830 0.05 –

28 iso-18:0 0.844 0.09 –

29 anteiso-18:0 0.875 0.03 –

30 16:4 (n-1) 0.892 2.55 0.2

31 18:0 1.000 2.62 2.3

32 18:1 (n-13) 1.041 0.11 –

33 18:1 (n-9) 1.110 6.38 6.5

34 18:1 (n-7) 1.131 3.04 0.8

35 18:1 (n-5) 1.169 0.23 1.2

(Continued)

SATURATED, ISOMERIC MONOENOIC, AND UNUSUAL FATTY ACIDS 291

The C16 polyunsaturated fatty acids are often confusing because two series can

coexist and overlap in GLC analyses, basically the familiar n-3 and n-6 series with

16:2n-6, 16:3n-3, and 16:4n-3. Superimposed on these acids are members of an n-1,

n-4, and n-7 series. The latter are well documented (35), and they may be prominent

in fish lipids where algal fatty acids are deposited more or less directly, for example,

in Japanese sardine oil (Table 2). All should be included in extensive tabulations of

marine fatty acids, and their methyl ester peak relative retention times for the low-

polarity GLC phase SILAR-5CP (36) are included in Table 2, which will clarify

their positions after 16:1n-7 for similar GLC columns, but on higher polarity

GLC columns they will overlap with C18 fatty acids. In default of GC-MS, older

techniques of plotting or separation factors may help in identifications (37, 38),

although these require an isothermal GLC analysis. To illustrate how the n-1, n-

4, and n-7 fatty acids can be transferred to fish oils, the fatty acids in the triacylgly-

cerols of a well-known unicellular alga, Phaeodactylum tricornutum, are included

in Table 2, because it is a basic diatom in that geographic location (39). It may be

noted that 16:4n-1 is a minor fatty acid presumably because very little 18:4n-3

exists. The 16:4n-1 would be generated in algae by the enzymatic process that pro-

duces 18:4n-3, but acting on a C16 chain length instead of a C18 chain length.

36 iso-19:0 1.222 – –

37 18:2 (n-6) 1.291 0.85 2.5

38 18:3 (n-6) 1.371 0.47 0.3

39 19:0 1.415 0.14 –

40 19:1 (n-8) 1.515 0.11 –

41 18:3 (n-3) 1.575 0.40 1.3

42 18:4 (n-3) 1.730 2.06 0.2

43 18:4 (n-1) 1.768 0.22 –

44 20:0 1.995 0.11 0.1

45 20:1 (n-11) 2.142 2.37 –

46 20:1 (n-9) 2.176 1.33 –

47 20:1 (n-7) 2.235 – –

48 20:2,5,11 2.403 – –

49 20:2 (n-6) 2.783 – 0.4

50 20:4 (n-6) 2.928 1.26 0.3

51 20:4 (n-3) 3.396 0.74 0.4

52 20:5 (n-3) 3.608 17.01 8.7

53 22:1 (n-11 þ 13) 4.205 2.09 –

54 22:1 (n-9) 4.288 0.27 –

55 22:2,7,11 4.902 – –

56 22:2,7,13 4.985 – –

57 21:5 (n-3) 5.158 0.60 –

58 22:5 (n-3) 7.150 2.17 0.9

59 22:6 (n-3) 7.510 10.09 2.3

60 24:1 (n-9) 8.408 1.09 –

TABLE 2. (Continued )

Peak No. Fatty Acid RRT Sardine P. tricornutum

292 FISH OILS

Other sources must provide the amount obvious in the sardine oil, and similar or

lesser contents are common in other fish oils.

Curiously, in fish oils, 18:4n-1 is frequently clearly visible after the peak for

18:4n-3 (No. 12) in Figure 5 and is usually about one-quarter of its size. Another

unusual fatty acid of algal origin is 18:5n-3 (40). It is known from GLC analyses of

both phytoplankton and zooplankton lipids. In Figure 7, it is probably the small

sharp peak between peak 2 (18:4n-1) and peak 3 (20:0), approximately as for the

moderately polar SILAR-5CP liquid phase of (40). All unusual fatty acids should

be neatly handled by the mammalian body degradation process and even by those

in fish, and they are seldom reported in fish oils. Incidentally, peak 3 is wider than

expected, a characteristic of methyl esters of saturated fatty acids in such analy-

ses. Of the other unusual fatty acids in fish oils, the 21:5n-3 (20 in Figure 5) is

common and well documented (41). It is of interest if 23:0 (tricosanoic acid methyl

ester) is used as an internal standard in GLC (42). The latter may coincide with

21:5n-3, and the Omegawax-320 was a slight modification of SUPELCOWAX-10

to avoid this problem. On the other hand, the NMID referred to earlier (both C20

and C22) appear in mollusc lipids, possibly in physical imitation of the polyunsa-

turated fatty acids of membranes (43), and they are not apt to be observed in indus-

trial pelagic fish oils. On tropical reefs or among fish-consuming molluscks, they

should be considered as likely if the normal resolution among the later-eluting

C20 and C22 monoethylenic isomers (n-7, n-5) is obscured on polyglycol capillary

columns.

5. POLYUNSATURATED FATTY ACIDS

The C18 polyunsaturated fatty acids include the familiar terrestrial fatty acids

18:2n-6 and 18:3n-3, along with some 18:3n-6, but in most marine fish oils, these

are �2% of each (Figure 6, Table 1). The amount of 18:4n-3 is in the 2–4% range.

Among the C20 polyunsaturates, there will be found very small proportions of

20:2n-6, 20:3n-6, 20:3n-3, and 20:4n-3. The 20:4n-6 (arachidonic acid) peak is gen-

erally the same size as these except in tropical fish lipids, where it can be more

important. The latter are, however, not usually commercial fish oil sources.

Although of nutritional importance in mammals, 20:4n-6 is usually grossly over-

shadowed by the C20 and C22 omega-3 polyunsaturated fatty acids in marine

lipids. One reason for avoiding higher polarity gas-liquid chromatographic columns

is that with the chain length overlap, this usually puts 22:1 and 20:4n-6 in juxtapo-

sition or coincidence.

The polyunsaturated fatty acids were intimately associated with early (ca. 1960)

attempts to lower serum cholesterol with varying degrees of success (44). In that

particular study, with ethyl ester concentrates given to provide up to 4 g/day n-3

PUFA, a side effect of ‘‘an increased feeling of well-being coupled with improved

cerebration’’ was reported only for the group receiving omega-3 fatty acids. About

the same time, up to 26 mL/day of seal oil with a content of about 5 g of omega-3

fatty acids was fed for cholesterol-lowering effect without ill effects (45). Today

POLYUNSATURATED FATTY ACIDS 293

this simplistic dietary approach to one cardiac risk factor, cholesterol, is no longer

acceptable in some circles because of the multiplicity of risk factors for ‘‘heart dis-

ease’’ (46). Essentially this recent review rules out arachidonic acid (20:4n-6) as

having little effect, and it considers EPA (20:5n-3) as being more effective than

DHA (22:6n-3) in the lowering of serum triacylglycerols, another independent

risk factor (47). Another study also suggested that EPA was more effective than

DHA in lowering blood pressure (48).

Tables 1 and 2 and Figures 5 and 6 show a third long-chain omega-3 fatty acid,

7,10,13,16,19-docosapentaenoic acid (or 22:5n-3), one of two DPA isomers. The

other is the n-6 isomer, 22:5n-6, eluting just before 22:5n-3 on GLC. In Figure 5,

the 22:5n-3 is peak 21 and a 22:4n-3 may be present after peak 20. In 1996, two

papers (49,50) gave this 22:5n-3 fatty acid considerable potency in respect to

endothelial cells. Considering that the related 20:5n-3 was originally considered

to keep the blood vessel walls elastic through providing a prostaglandin (51),

this could well be a beneficial role for EPA and even DPA in circulation separate

from the heart, but research on the relative roles of the three omega-3 fatty acids in

the aortic endothelium seems to have fallen into abeyance, perhaps because they are

often said to be freely interconverted (52). This statement is not necessarily true,

but the conversion of EPA to n-3 DPA and the reverse process, by one acetate

unit, is generally accepted. The critical step, the conversion of 22:5n-3 to 22:6n-

3, is not so easy, involving elongation to 24:6n-3 and peroxisomal shortening to

22:6n-3, and some biochemists now think that exogenous supplies of DHA are

the preferable route to increasing the amount available in the body, especially in

late pregnancy and lactation. This digression into the biochemistry of the logical

beneficiaries of fish oils, humans, cannot cover the maternal/infant problems dis-

cussed at length by various authors in Ref. 13, a topic not without controversy

(53, 54).

More emphasis has been placed on the loss of heart function taking place

through arryhthmia (55), and it is possible that DHA is the more functional ome-

ga-3 fatty acid in the heart muscle. Unfortunately, the available fish oils divide into

the menhaden/anchovy type, richer in EPA, an omnivorous group such as cod with

EPA� DHA, and the tuna oils, both body and orbital, with 20–25% DHA. In fact,

the first research on the beneficial aspects of omega-3 fatty acids in arrhythmia was

conducted in Australia (56), conveniently near an Asian source of tuna oil rich in

DHA, which lead to misunderstandings over a role for this omega-3 fatty acid in

particular for preventing arrhythmia.

The health and welfare interest in long-chain omega-3 fatty acids inevitably

raises the question of ‘‘where do they come from’’ and ‘‘are they safe.’’ The latter

question applies to oils and will be addressed in a production and quality section,

but in reality, most fatty acids are of plant origin and perfectly safe.

The phytoplankton produce toxins dangerous even to fish, usually observed as

‘‘red tides’’ (57). These are fish kills in the oceans and near the shores, and fish

oils are not made from fish found dead. A different issue is shellfish toxins where

the digestive assimilation of unstable algal toxins does not kill the host (58), but fish

oils are not made from such filter-feeding animals.

294 FISH OILS

Each new discovery of unusual fatty acids in marine organisms leads to concerns

that eventually dissipate. An example is the furanoid fatty acids that were once well

documented as common in most oils (26). More recent papers and books on omega-

3 fatty acids simply ignore the subject.

Figure 8 is a simplification of much work by many scientists over decades. The

phytoplankton in the ocean produce all fatty acids necessary for fish oil and with

somewhat similar compositions in places as remote as Australia and Scotland. Spe-

cific differences exist among them, but broadly the fatty acid patterns are related to

colors familiar to people who see the macrophytes growing on the edges of the sea:

red, green, and brown. However, even the accumulation of the amount of triacyl-

glycerols is controlled by the available nutrients, especially nitrogen, and light

intensity (59). The 22:6n-3 is not universal in phytoplankton, but it is found all

over the world in benthic algae (60) or phytoplankton (61,62). For unknown rea-

sons, fatty acid phytoplanton biosynthesis often stops at 20:5n-3, which was pre-

sumably the reason for the original (ca. 1980) ratio of 18:12 for 20:5n-3 and

22:6n-3 in oil from the filter-feeding menhaden fish (Figure 5), repeated on hun-

dreds of labels for bottles of fish oil capsules, and repeated again in most concen-

trates prepared from it or from anchovy oil (Fig. 6). Actually the production of

18:2n-6 and 18:3n-3 is limited in a total lipid context for brown and red macrophyte

EPA > DHA Phytoplankton

TGEPA > DHA

PLDHA > EPA

18:2n-6 18:3n-3

ShellfishCrustacea

ShellfishCrustacea

Fish

Energy

Diet

DHA

Neurological systems?Retina?Cell membrane?

EPAcirculatory

system

TXA3 PGI3

Prostaglandin Group 3

Human Seals Seabirds

Origin of Marine Fatty Acids

Figure 8. Origin of unsaturated fatty acids in phytoplankton, followed by discrimination

by invertebrates leading to accumulation of EPA and DHA in oils and eventually in higher

animals.

POLYUNSATURATED FATTY ACIDS 295

algae, but it is more common in the green, Ulva pertusa being a familiar and much

studied example (60).

As already remarked, various invertebrates feed on the phytoplankton, and smal-

ler carnivorous fish feed on those vegetarian species as well as on carnivorous inter-

mediate invertebrates. Available 22:6n-3, with its neural and visual implications, is

probably conserved at these lower levels and is vital in fish muscle phospholipids

(30–40% of fatty acids). In oils, 18:4n-3 and 18:4n-1 are preserved as shown in Fig-

ures 5 and 6. Recent progress in the lipid biochemistry of fish shows that the rain-

bow trout can perform biosynthesis of 22:6n-3 (DHA) from 18:3n-3 (alpha-

linolenic acid). Only a small part of that provided is converted to DHA (63), and

surprisingly this was substantially converted by the pyloric cecum as well as by the

liver (64), which raises an interesting point about adaption of fish biochemistry to

circumstances. The marine fish ingest preformed EPA and DHA, but there was a

curious change in diet for North American freshwater fish such as the rainbow trout.

The recent glaciations should have wiped out the resident invertebrates, and after

salmonids returned from the ocean, recolonization of food species for freshwater

fish would have been based on insect life introduced from Central America. Thus,

deprived of an excess of marine long-chain omega-3 fatty acids, adaption to elongate

the available insect C18 fatty acids would have been necessary once the returning

salmonids penetrated waters remote from the ocean. A similar situation for insect

lipids and fatty acids is known to come from Britain (65). These sources could pro-

vide EPA but not DHA for freshwater fish. Freshwater fish, although beneficial in

most respects among our sources of both n-3 and n-6 fatty acids (12,66,67), are not

apt to produce large volumes of fish oils of distinctive character. An exception is the

U.S. farmed catfish industry, which is subject to an excess of n-6 dietary fatty acids

from local aquaculture diets. The production of the visceral oil has been described,

in 2003 in JAOCS but with confusion in published fatty acids. The northern lakes of

Canada support fisheries with more potential for accumulating longer-chain omega-

3 fatty acids in the oils (67) or muscle. In four oils, the longer chain omega-3 fatty

acids (including 18:4n-3) totaled 9.6%, 13.3%, 17:0%, and 18:6% of total fatty

acids. The corresponding totals for the longer chain n-6 fatty acids arachidonic

acid (20:4n-6), 22:4n-6, and 22:5n-6 were 3.3%, 2.5%, 3.7%, and 4.2%, mostly

of 20:4n-6. Thus, a generally higher level of n-6 fatty acids over marine oils

is found, but overall a favorable balance exists between preformed C20 and C22

n-3 and n-6 longer chain PUFA contents in the oil. The fatty acids of the edible

muscle, widely eaten locally and also exported, are similarly skewed in favor of

omega-3 fatty acids, but with the additional n-6 fatty acids already mentioned

(R.G. Ackman, unpublished).

6. FISH OIL PRODUCTION AND QUALITY

A paramount concern in maintaining oil (and meal) quality is speedy processing

after catch. As 100 tons or more could be involved, chilling is not always practical,

but fish pumps can transfer the catch quickly from boats to the processing plant.

296 FISH OILS

Even there delays must be avoided. Preferably no more than 24–30 hours should

elapse, depending on temperature before fish reduction. The fish enzymes, both

of muscle tissue and of digestive tissue, and those of gut bacteria, combine to break

down protein. The oil degradation is basically from lipolytic enzymes, but some of

the oil-soluble free fatty acids may come from partially digested food and phospho-

lipids and not necessarily from triacylglycerols. The free fatty acids, abbreviated to

FFA, are one of the oldest and simplest guides to fish oil quality. In one detailed

review (68), it is pointed out that the oil stored in fat cells (adipocytes) illustrated

for salmon by Zhou et al. (69) will be set free by 50 �C, although cooking with

steam usually reaches 95 �C. Separation of the oil is achieved with presses and

with very expensive and very efficient centrifuges. Once cooled, the oils are stable,

provided no protein particulates are carried over from the first separations of oil. A

second ‘‘polishing’’ centrifuge can handle this matter. The oil should be cooled

before storage in clean, dry tanks. These tanks should be filled as full as possible

and provided with provision for drainage of any sediments and water (foots).

Complex flow diagrams are provided by various authors (20,23,26,70). They are

complex because the thermodynamics, mainly for water removal, dictate costs.

References 20, 23, 26, and 70 provide additional detailed diagrams for those inter-

ested. Subsequent technology leading to consumer products is summarized in

Figure 9.

The basic crude fish oils are exemplified by the quality details of Table 3. Ranges

are given because these are specifications for crude oils, produced at the level of

over a million tons per year. The first six properties are traditional wet chemistry

assays and the American Oil Chemists’ Society (AOCS) Official and Tentative

Crude Fish Oil

Neutralization

Bleaching

WinterizationMolecularDitillation

Finished OilProduct

Vacuum DistilledFish Oil

Packaging, Microencapsulation,Soft Gelatin Capsules, Bottles

Deodorizer

EsterFatty Acid

ConcentratePurified Fatty Acid

Purified EsterMono-Diglyceride

Production

Figure 9. Production of pharmaceutical-grade fish oil or nutritional supplements. Reproduced

from (70) by permission of the American Oil Chemist’s Society.

FISH OIL PRODUCTION AND QUALITY 297

Methods, Champaign, IL, or the Association of Official Analytical Chemists

(AOAC), Gaithersburg, MD, are the usual sources for North America to access

recognized, standardized, and detailed analytical methods. Traditionally, even dif-

ferent staff members in one laboratory can get somewhat different results. Techni-

cal spectral methods are now becoming useful, but the search for some exact and

rapid replacement fish oil technology goes on. Some problems are discussed in

Appendix 1, courtesy of Codex Alimentarius. A problem peculiar to the peroxide

value, the anisidine number, and hence the totox value is that it is a moving target.

Figure 10 shows that oxidation of highly unsaturated fatty acids proceeds, but at the

TABLE 3. Crude Fish Oil Quality Guidelines and Physical

Characteristics.

Quality Guidelines

Moisture and impurities, % usual basis 0.5 up to 1% maximum

Free fatty acids, % oleic range 1–7% but usually 2–5%

Peroxide value, meq/kg 3–20

Anisidine number 4–60

Totox value 10–60

Iodine value

Capelin 95–160

Herring 115–160

Menhaden 120–200

Sardine 160–200

Anchovy 180–200

Jack mackerel 160–190

Sand Eel 150–190

Color, Gardner scale up to 14

Iron, ppm 0.5–7.0

Copper, ppm less than 0.3

Phosphorus, ppm5-100

Physical characteristics

Specific heat, cal/g 0.5–0.55

Heat of fusion, cal/g about 54

Caloric value, cal/g about 9,500

Slip melting point, �C 10–15

Flash point, �CAs triglycerides about 360

As fatty acid about 220

Boiling point, �C greater than 250

Specific gravity

At 15 �C about 0.92

At 30 �C about 0.91

At 45 �C about 0.90

Viscosity, cp

At 20 �C 60–90

At 50 �C 20–30

At 90 �C about 10

From A. P. Bimbo (70).

298 FISH OILS

same time degradation of the peroxides can also proceed, with degradation to alde-

hydes producing the familiar ‘‘fishy’’ flavor of both oils and fish muscle lipids

undergoing development of rancidity. Many candidate molecules are offered for

consideration (26), some being unstable themselves. The acids produced can be

volatile, and one ending to a peroxide free radical’s career can be to lead to poly-

merization, either within a triacylglycerol or between triacylglycerols. The splitting

of an oxidized fatty acid chain can take place anywhere, but one-half of the pro-

duct(s) will still be attached to the glycerol molecule. Thus, removal of free volatile

aldehydes, for example, reduces the aroma from rancidity, but after their removal,

refining can leave the anisidine value for the remaining glycerol-bound aldehydes

as a real number of 5 or more. Addition of antioxidants depends on the value of the

raw material and of the final product, so it is not likely to be added to crude fish oils.

These already include the natural antioxidant benefit of natural alpha-tocopherol.

Sometimes cheaper vegetable oil deodorization mixtures of tocopherols may be

added after refining because the alpha-tocopherol may be lost in that step. The pro-

ducts of oxidation of oils protected by mixed tocopherols may then differ some-

what, but they have recently been studied in detail (71).

As already mentioned, the iodine value has been largely replaced by exact fatty

acid composition from gas-liquid chromatography of the methyl esters of the single

fatty acids. In addition to the AOCS Ce 1b-89 and AOAC 991.39 methods,

European Pharmacopeia 4 method 01/02/1352 includes their standards for ‘‘ome-

ga-3 acidorum triglycerida’’ and the GLC analysis for EPA and DHA in triacylgly-

cerols and an associated method for ethyl ester products (see below), as does the

Voluntary Monograph (October 2002) on Omega-3, DHA, EPA and DHA þ EPA

of the Council for Responsible Nutrition of Washington. This body also gives

TIME

PEROXIDES ACIDS

POLYMERSALDEHYDESN

UM

ER

ICA

L V

ALU

ES

Figure 10. Time relationship among peroxides and their degradation products after oxidation of

marine oils. No quantitative relationship is implied. Described in text as a ‘‘moving target’’ for

analytical methods.

FISH OIL PRODUCTION AND QUALITY 299

recommended maxima for heavy metals, dioxins and PCBs, and wet chemistry

value maxima (Table 4).

New regulations require new technology, which is especially true for food use.

Bleaching with activated clays has been a long-established practice to remove

chlorophyll green or a brownish tint acquired from heating oils in the presence

of other materials, and the objective of a clear yellow oil is usually possible.

Recently, the purification target has been extended to removal of organochlorine

materials, polyaromatic hydrocarbons, and most recently dioxins in particular. Acti-

vated carbon is recommended for dioxins, typically at 0.5–1.5% carbon for 20–45

minutes at 80–100 �C. Some operators add this to the bleaching earth to reduce

handling steps. Various associated matters, such as disposal of the oil-coated

clay, develop that should be considered, and all are dealt with in a recent conference

report (72).

Occasionally, a highly purified fish oil is required for research purposes. Such

oils can be prepared by large-scale chromatography (73) for use in studying oxida-

tion products. The recent paper based on such technology is instructive in illustrat-

ing the variety of products produced from oxidation of even purified fish oil (71).

Modern technology is beginning to investigate such materials in situ (74).

For crude fish oil, deodorization has changed considerably, with new technology

designed to reduce temperature and/or exposure times. In 1990, a review chapter in

a book (75) described classic batch tray technology with gravity cascade transfer of

the oil and steam sparging to carry away volatiles. This process was satisfactory for

hydrogenated fish oils, but thermal damage to one of the highly unsaturated fatty

acids of vegetable oil had been recognized nearly 20 years earlier (76). A critical

temperature of about 185 �C was observed, and at 230 �C, severe isomerization of

ethylenic bonds (cis to trans) was observed in 18:3n-3 with prolonged heating.

These isomers were also found in retail oil samples and so were produced by

TABLE 4. Council for Responsible Nutrition Quality Standards for Nutraceutical Grade

Fish Oils in the United States.

CRN Quality Standards for Nutraceutical Grade Fish Oils

Measures of Oxidation

Peroxide Value (PV), meq/kg 5 Max

Anisidine Value (AV) 20 Max

TOTOX ((2 � PV) þAV) 26 Max

Purity

Dioxins (PCDDs, PCDFs) 2 pg/g WHO-TEQ Max

PCB’s <0.09 mg/kg (ppm)

Lead <0.10 mg/kg (ppm)

Cadmium <0.10 mg/kg (ppm)

Mercury <0.10 mg/kg (ppm)

Arsenic <0.10 mg/kg (ppm)

Omega 3 Fatty Acids Expressed on a weight/weight basis (mg/g)

Acid Value 3 mg KOH/g Max

300 FISH OILS

standard deodorizers of the type mentioned and illustrated in a chapter in a book on

fish oils (75). A thin-film design, the Campro unit is designed for a very short resi-

dence time, and with oil transfer as a thin film by gravity and by high-velocity

sparge steam, and it is described as suitable for fish oil refining at 210 �C. In

fact, this is satisfactory for fish oils based on recent experience with this unit for

seal oil (private communication). Designs for throughputs of up to 300 tons/day

are available. The damage done to fish oil fatty acids at 220 �C was investigated

in depth with open-tubular gas-liquid chromatography (77), and it illustrated the

problems of prolonged heat exposure (Figure 11). This case is a time-temperature

relationship, so operating flexibility is desirable.

A true molecular still ejects a low-molecular-weight molecule from a less vola-

tile liquid surface. If a very good vacuum exists (� 10�3 mm), this molecule may

bounce around with a few residual air molecules but eventually will either fall back

or attach to a cold condenser. Sometimes this is described as short-path distillation,

which is not usually an efficient separation process as shown by a comparison of

removal of DDT from cod liver oil and concurrent losses of Vitamin A (78). The

term molecular still has now become attached to units of a different design applied

to fish oils, for example, from the Pope Company of Menomonee Falls, WI, or the

Pfaudler company of Rochester, NY. The Pope model of molecular still illustrated

(75, 79) shows this wiped-wall concept. There is an outer cylindrical shell with heat

applied on the outside. An interior centered post is actually a cold condenser, and

wiper blades rotate continuously against the inner walls of the outer shell as the oil

is fed in at the top, spreading the oil as a film of <1 mm thickness. The constant

agitation of the film moves fresh volatile oil materials to the surface, and they pass

into the evacuated space. The desired vacuum is sufficient to cause the volatiles,

especially organochlorine materials (MW � 358), but even cholesterol (MW

386), to pass over to the condenser at moderate (ca. 200 �C) temperatures. Also

removed are squalene (MW 410) and most of the volatile fatty acid oxidation pro-

ducts such as 2,4-decadienal (MW 152) and other lower molecular weight mole-

cules contributing to objectionable flavors (73,74). Unfortunately, the natural

alpha-tocopherol present (MW 430) may suffer the same fate. The antioxidant

value of it and of squalene (80) may be lost. That these materials are effectively

removed from triacylglycerol oils with molecular weights in the 800–1000 range

is a result of the repeated turnover and mixing of the oil during its descent of several

meters in very large units. The low viscosity at high temperatures helps refining by

such units, and very large surface areas permit flowthroughs of tons of fish oil per

hour.

As desirable products have similar molecular weights (ethyl docosahexaenoate

is 356, and ethyl eicosapentaenoate is 330), it is clear that this type of fish oil pro-

duct cleanup is best done at the oil stage to avoid losses at the same time as con-

taminant removal, which is carried out as described above. However, dimers and/or

other polymeric and/or colored materials may be left behind if wiped wall equip-

ment is used in its capacity of a simple and inefficient short-path still for the ethyl

esters only. This process will produce a water-white distillate product. Regrettably,

it is not an efficient way to separate ethyl-EPA from ethyl-DHA.

FISH OIL PRODUCTION AND QUALITY 301

Figure 11. The GLC C20 region of a menhaden omega-3 PUFA concentrate (ethyl ester): (a)

before and (b) after heat treatment at 220 �C, and (c) the 20:5 region of an artifact concentrate

isolated by AgNO3 column chromatography. Peaks A–E refer to artifacts formed after heat

treatment. Analysis on a SUPELCOWAX-10 fused-silica capillary column operated isothermally

at 195 �C. Note that components B–E fall into the region where several 22:1 isomers may be

found (cf. Figs. 5 and 7). From (77).

302 FISH OILS

7. CONCENTRATES OF FISH OIL OMEGA-3 PRODUCTS

Table 5 shows the marketing and label strategies for some current marine omega-3

products sold in Halifax, Canada. Obviously samples No. 1 and No. 6 are natural

oils. Perhaps No. 5 is simply winterized oil, a process demonstrated in Table 6 for

menhaden oil, which follows from the tendency for DHA to be in the 2-position of

fish oil triacylglycerols (81). EPA is reputed to be somewhat less specific. In the

absence of 20:1 and 22:1, the outer 1- and 3-positions may, in some molecules, pre-

sent two saturated fatty acids from 14:0, 16:0, and 18:0 in one triacylglycerol mole-

cule, which leads to the stearine composition of Table 6.

Simple biochemical rules are made more complex by other factors, and in fish

oil triacylglycerols, the 20:1 and 22:1 fatty acids of a high melting point confuse the

issue (82). The traditional enzymatic approach to fatty acid distribution will soon

be replaced by nondestructive instrumental methods, particularly nuclear magnetic

resonance (NMR). It can distinguish the proportion of DHA between the 1,3- and

2-positions (74) and otherwise provide the details shown in Table 7 for a few oils,

which shows verification of the method through an international exchange (83). A

TABLE 5. Some Recent Omega-3 Product Retail Labels in Canada.

Label Content in mg Capsule size or liquid intake

No Description EPA DHA (1000 mg basis)

1 Wild Sockeye (Total 90) 1000

2 Wild Harvested Pacific Salmon 180 111 1000

3 O3mega 400 200 1065

4 Natural Sea* 775 500 1500

(517) (333) (1000)

5 Holista Premium Fish Oil** 180 120 1000

6 Omega Gold (liquid) 900 (180) 600 (120) 5000 (1000)

*Other (undefined) 225 mg.

**Each capsule is said to be equivalent in omega-3 content to 2.5 oz (70 g) portion of cooked salmon.

TABLE 6. Fatty Acid Composition (w/w%) of Menhaden Oil with

Olein and Stearine Fractions.

Menhaden Oil Olein Fraction Stearine Fraction

14:0 9 8 11

16:0 21 18 31

16:1 11 12 9

18:0 3 3 5

18:1 12 12 10

20:1 2 2 2

20:5 14 15 11

22:5 2 2 1

22:6 10 11 7

From A. P. Bimbo (26).

CONCENTRATES OF FISH OIL OMEGA-3 PRODUCTS 303

recent enzymatic examination of tuna oil (EPA 6.69%, DHA 26.4%) showed 8.7%

EPA and 56.3% DHA in the 2-position (84).

Retail product No. 1 of Table 5 is likely to be simply salmon waste oil, the fish

name conferring an elite status. Our research (Ackman, unpublished) suggests that

many ‘‘salmon oil’’ encapsulated oils are unrelated to any salmon oil in fatty acid

composition. In 1989, our analysis showed many products of this type to be exag-

gerated as to omega-3 fatty acid content (85), and a more recent European survey in

1998 gave comparable results and reported on quality (86).

In the menhaden oil winterization of Table 6, the increase in polyunsaturated

fatty acids is modest in the olein fraction, and the commercial objective may

have been the stearine fraction, 50% richer in saturated acids of commercial inter-

est. As already remarked for polyunsaturated fatty acids, 18/12 (in implied percents) or

180/120 (in mg/g) were obtained as triacylglycerols from menhaden and/or anchovy oils

with minimal trouble and technology. Retail product No. 2 is also a product like No. 5.

Winterization also would prevent either capsules or oil turning cloudy if refrigerated.

Strangely, hardly anybody challenges label claims as to chemical nature, although the

word oil may be carelessly used because concentrates are almost always ethyl esters, but

numbers should always be expressed in the free acid form.

Among the various laboratory procedures used for studying fatty acids, concen-

trations by chromatography on silver nitrate impregnated silica gel, or equivalent

(87), are too expensive to scale up; although effective for fish oils (88), they and

mercuric adducts (89) would not be acceptable for health and safety reasons.

Before 1980, there was a now forgotten industrial technology for concentrating

fish and vegetable oils called the Solexol process. It can be described as the first

large-scale use of supercritical gases, in this case, propane. A report with much con-

venient detail based on iodine values was published in 1949, but as a historical

record (90), because several large plants were closed during the war and the prime

TABLE 7. Comparison of DHA Content from the Interlaboratory 1H NMR Analysis

Between Japan and Norway Together with GC Data. Ethylene Glycol Dimethyl Ether was

Used as Internal Standard (1H NMR Analysis with 30s Pulse Repetition Time).

DHA Proportion Proportion (mol%)

DHA Content (mg/g) (mol%) n-3 Fatty Acids

Sample Oil Data Norway Japan GC Norway Japan GC Norway Japan GC

No. 1 Average 248.17 276.1 267.6 27.03 27.5 24.5 32.05 32.1 33.6

Bonito CV 0.64 1.33 1.67 0.78 0.46 2.49 0.08 0 2.03

No. 2 Average 123.79 122.6 123.4 11.93 12 11 22.56 22.2 21.6

Tuna CV 4.01 1.38 0.73 2.98 0.43 1.71 0.85 0.4 1.35

No. 3 Average 214.51 208.3 215.7 20.65 20.7 20.2 29.08 28.8 29.9

Tuna CV 2.72 0.91 0.2 0.66 2.02 1.43 0.05 0.73 0.89

No. 4 Average 217.97 206.7 215.4 21.18 20.8 19.7 32.22 31.8 32.1

Tuna CV 3.84 0.07 0.72 1.59 0.47 1.35 0.23 0.34 1.07

No. 5 Average 111.07 111.1 106 10.65 10.8 10.2 28.03 28.1 28.6

Salmon CV 3.27 0.95 1.47 0.88 0.32 1.15 0.32 0.33 0.74

From S. Wada (74).

304 FISH OILS

markets such as paints, linoleum, and oil cloth disappeared after the war. Two dec-

ades later supercritical carbon dioxide was to be the panacea in this field, but early

promises, including fish oil fractionation, were seldom realized (91, 92).

Much research was conducted to prepare concentrates when the U.S. National

Marine Fisheries Service provided funds for exploring most existing technologies

for concentrating fish oil fatty acids. This exploration would provide omega-3 con-

centrates for the medical research sponsored by the National Institutes of Health

and included supercritical fractionation of esters. Unlike commercial research,

results were available to all interested parties. A book chapter by authors located

in the Charleston, Seattle, and Gloucester NMFS laboratories, and in the Hormel

Institute, Austin, Minnesota (93), provides detail on all methods considered for frac-

tionating methyl or ethyl esters, because menhaden oil was clearly intractable. For

supercritical CO2, the differences in factors such as the influence of chain length

(i.e., molecular weight) and unsaturation, including explanations of results achieved

by others, are helpful in explaining why this fractionation process has not been

commercially developed. The final total enrichment process is described below.

Urea complexing was demonstrated for fractionation of fatty acids of a marine oil

(as methyl esters) as early as 1963 (94), and laboratory-scale tests in Halifax, Canada

(95) were followed by further tests on a scale of 40 kg of crude oil. At that time, 50%

omega-3 ethyl esters was considered a good possible result and doubling the omega-3

content made the product more acceptable on a retail basis by 1989 (79, 85).

A flow chart, courtesy of H. Breivik of Norway, is provided as Figure 12, the

work of Norsk Hydro, Porsgrunn, Norway, and dating to 1990. The molecular

weight of ethyl myristate (14:0, 256), palmitate (16:0, 284), palmitoleate (16:1,

282), and even oleate (18:1, 310) are sufficiently different from those of the ethyl

esters of C20 and C22 polyunsaturated acids (330 and 356) to allow removal of

most of the shorter chain fatty acids by short-path distillation. From Figures 5

and 6, it can be seen that 30–50% of the ethyl esters in question are available to

be distilled off, with 18:4n-3 as the omega-3 fatty acid of interest that may be lost.

By eliminating these fatty acids as a first step with the simple operation of short-path

distillation, the subsequent urea complexing step is much more economically carried

out, which led to the very successful Provnova Biocare product of 85% EPA þ DHA

as ethyl esters. Under the trade name EPAX, a variety of triacylglycerol and ethyl

ester products are now offered by this firm, with different proportions of EPA and

DHA, which is evidence of further process development since 1990.

At about the same time, the Charleston Laboratory of the U.S. National Marine

Fisheries Service prepared menhaden oil omega-3 fatty acid ethyl ester concen-

trates on a large scale for participants in projects funded by the U.S. National Insti-

tutes of Health. Their flow chart. Figure 13, combined development work also

carried out at the Seattle and Gloucester Laboratories, much of it recorded in the

book by M.E. Stansby (26). The final process adopted includes urea complexing of

ethyl esters in a total of seven stages, viz:

I. Vacuum deodorization

II. Transesterification

CONCENTRATES OF FISH OIL OMEGA-3 PRODUCTS 305

III. Urea adduction

IV. Film evaporation

V. Short-path distillation

VI. Supercritical fluid fractionation

VII. High-performance liquid chromatography

Although subsequently closed down only because of the cessation of the joint pro-

ject, the demonstration of what could be done, even if uneconomic, created wide

Fish Oil30.800 kg

Ethyl ester29800 kg

Ethyl ester (EPA + DHA = 50%)9575 kg

Ethyl ester (EPA + DHA = 84%)3140 kg

Further purification

2 step moleculardistillation

Urea precipitationUrea16150 kg

Isolated adduct18890 kg

Precut: 16510 kg

Residue: 3245 kg

MASS BALANCEPART OF EARLY SCALE-UP EXPERIMENT

PRIOR TO LATER MODIFICATIONS

Figure 12. Development stage of Norsk Hydro Research Center Porsgrun scheme for

production of ethyl ester concentrates of marine oil fatty acids, ca. 1990. Courtesy of H. Breivik.

306 FISH OILS

interest in employing concentrates in clinical trials of omega-3 fatty acids. The pro-

duct standards that were set were, for the time, remarkably high (Table 8).

Investigations carried out for the NMFS Charleston process indicated that chain

length (i.e., molecular size) was a dominant factor observed in research on super-

critical CO2 separations of ethyl esters of marine oil fatty acids (93). The initial

IIIII

IV

VI VII

V

I

REFINED MENHADEN OIL

OMEGA - 3 CONCENTRATES

PURE EPA AND DHA

DN2

ED

AN2

N2

P2P2

P2

P1P1

P1

N2

N2

O

N2

U R

C

C g

Figure 13. Production of biomedical test materials in the Charleston Laboratory of the U.S.

National Marine Fisheries Service for a joint NMFS-NIH project. Code numbers for steps are

explained in text. Small print identities are N2 ¼ nitrogen atmosphere, A ¼ alkali, E ¼ ethanol,

D ¼ distill, U ¼ urea, R ¼ reflux, C ¼ cold water, and S ¼ steam. From (96).

CONCENTRATES OF FISH OIL OMEGA-3 PRODUCTS 307

promise of supercritical fluid extraction (SFE) for actual recovery of lipids from

natural samples is a separate issue from oil fractionation, but a few references estab-

lish the difficulties faced with use of cosolvents, water removal, and other factors

not applicable to oil or ester fractionation (97–99). Thanks to many pioneers and

recent commercial stimulus, supercritical fluid fractionation has the potential for

concentrating ethyl esters of fish oil fatty acids.

Gradually, health benefits have attracted financial interest and a market role for

omega-3 concentrates was seen in U.S. foods. A leader in this field was Roche Vita-

mins, Inc., of Parsippany, NJ, soon to be acquired by the DSM company of the

TABLE 8. Quality Specifications for Fish Oil Derived n-3 Ethyl Esters

to be Shipped from Charleston Laboratory.

Test Material

Analysis Type n-3 Conc EPA DHA

Esters, % >90 >95 >95

EPA, mg/g >400 >900 <50

DHA, mg/g >200 <50 >900

Total n-3, mg/g >700 >950 >950

Free Fatty Acids, % <0.2 <0.2 <0.2

Trans Acids, % <5 <5 <5

Cholesterol, mg/g <5.0 <0.1 <0.1

Peroxide Value, meq/kg <10.0 <5.0 <5.0

Iodine Value, g I/100g >320 * *

Anisidine Value <80 * *

Antioxidant Content

a-tocopherol, mg/g 0.5–5.0 ** **

g-tocopherol, mg/g 0.5–5.0 ** **

TBHQ, mg/g 0.1–0.2 ** **

Moisture, ug/g <500 <500 <500

Residual urea, ug/g <20 <20 <20

PCB, ug/g <0.5 <0.5 <0.5

Total DDT, ug/g <0.5 <0.5 <0.5

Trace Metals, ug/g

Arsenic <1.0 <1.0 <1.0

Cadmium <1.0 <1.0 <1.0

Lead <1.0 <1.0 <1.0

Mercury <1.0 <1.0 <1.0

Selenium <1.0 <1.0 <1.0

Sensory Attributes:

Odor (TIO) <6.0 * *

Flavor (TIF) <6.0 * *

Other:

Specific Gravity 0.89 ** **

Solidification Range *** *** ***

*not applicable.

**not enough material to conduct these analyses routinely.

***Esters are a liquid at 5 �C or higher.

Reproduced from (96).

308 FISH OILS

Netherlands. The basic product was ‘‘ROPUFA ‘75’ n-3 EE’’ with specifications as

set forth in Table 9. Information on this ethyl ester product clearly shows a GLC

profile with traces of C16 fatty acids, traces of 18:0 and 18:1, a little of each of

20:4n-6 and of 20:1 and 22:1, and the ubiquitous 21:5n-3 and 22:5n-3.

Many students have received degrees in recent years for exploring enrichment of

marine oils (or other oils) by selective hydrolysis of triacylglycerols, or ester inter-

changes between esters and natural oils by enzymes. These explorations tend to be

somewhat theoretical (100), but they can be effective, although impractical, for

example, a 100-hour reaction time (101). Having a starting material rich in the

desired product fatty acid (DHA) helped in one case (102), but the complexity of

these proposed processes requires a separate article.

There is no real reason for concentrations of mixed omega-3 fatty acid ethyl esters

to be provided when the estimated need in oral supplement form is approximately 1 g

per day for adults. This supplement can be provided by several capsules of any sui-

table oil taken with meals. However, the food industry requirement is for oils that are

microencapsulated powders. These powders have to be provided with stable, yet

digestible, shells and in a suitable powder format to be unnoticed in the food and

yet carry a significant proportion of mass as the marine oil or omega-3 concentrates.

A ‘‘capacity’’ of about 50–50% oil is acceptable, and a target of 70–80% content is

being sought. Eventually, concentrates will boost delivery of EPA and DHA.

8. THE OTHER OILS

Aside from cod liver oil, no mention has been made of other fish liver oils, although

at one time, there was production of vitamins A and D from the liver oil of a Pacific

dogfish Squalus acanthius. This industry collapsed when synthetic vitamins were

introduced. The Atlantic spiny dogfish is essentially the same fish, and the liver

oils from both coasts have been compared (103). The distinguishing feature of

TABLE 9. Product Data for ROPUFA ‘75’ n-3 EE.

Product identification for refined ethyl esters of fish oil, containing eicosapentaenoic acid ethyl

ester and docosahexaenoic acid ethyl ester

Specifications EPA content (gas chromatography): min 42%

Appearance: yellowish liquid EPA content (weight as ethyl ester): min 380

Acid value: max. 3 KOH/g mg/g

Peroxide value: max. 10.0 mEq/kg DHA content (gas chromatography): min 22%

Anisidine value: max. 20 DHA content (weight as ethyl ester): min 200

Oligomers: max. 2% mg/g

Conjugated dienes: max. 1.5% Total content of n-3 PUFAs (gas

Iron: max. 1 ppm chromatography): min 75%

Copper: max. 0.1 ppm Total content of n-3 PUFAs (weight as ethyl ester):

min. 720 mg/g

Arsenic: max. 0.1 ppm

Courtesy of Roche Vitamins Inc., Parsippany, NJ.

THE OTHER OILS 309

this shark liver oil is the content of diacyl glyceryl ethers (DAGE, Figure 1). The

limited survey showed that the Pacific oil was the richer in DAGE (41% vs. 18% in

Atlantic liver oil), but the C20 and C22 omega-3 fatty acids quantitatively were

similar (103). In a further examination of the liver oil from Atlantic dogfish, the

ability of Atlantic salmon to digest was excellent (104). The oil, therefore, has

value for aquaculture feeds.

Squalene (Figure 4) is also sold in capsules in health supplement stores. Shark (and

related elasmobranch) livers do not necessarily have this hydrocarbon in any more

traces than those found in other species of fish. It depends on the exact species. A

survey of New Zealand shark resources (Table 10) shows what may be expected

(105). This report also explores liver oil recovery and processing. Unfortunately, local

fishing in underdeveloped countries, often for shark fins only, has destroyed a large

part of the resource. Squalene can exaggerate the iodine value of such oils (106),

but it is easily measured by GLC after hydrogenation of methyl esters carefully pre-

pared from the whole liver oil (107). Curiously, a small anadromous fish, ooligan or

eulachon, spawning in the Fraser and other Pacific coast rivers has considerable

(�19%) squalene in its body fats (108, 109). The eulachon Thaleichthys pacificus

was long recognized by local aborigines as a source of inedible but useful oil, but

although the fish is fatty, it is edible and is fished to some extent for that reason. As

olive oil is a potential source of squalene if it is needed, the slaughter of sharks for this

hydrocarbon for any purported health benefits is to be deplored.

Wax esters are another useful marine lipid class, which are now historical when

derived from the heads of sperm whales. Although various marine invertebrates

contain wax esters (110), there is an unexploited resource in relatively small fish

called myctophids. These fish can be caught by modern fishery technology as

was shown in South Africa some decades ago, but the use of any oil and meal pro-

duced would have to be carefully considered. The biosynthesis of their wax esters

has recently been resolved (111) and reviews most questions on that topic that were

TABLE 10. Liver Oil and Squalene Analysis of Dogfish from the Continental Slope

of New Zealand.

Oil Yield Squalene Yield

Total No. (g/100g Liver) (g/100g Oil)

No. of of Dogfish

Species Sex Samples Sampled Mean Range Mean Range

Shovelnose Dogfish Male 4 40 87.7 86–89 65.7 64–69

Shovelnose Dogfish Female 11 110 86.4 83–90 52.7 45–59

Shovelnose Dogfish Juvenile 3 30 89.2 86–90 58.4 57–60

Baxter’s Dogfish All 2 20 77.7 – 48.9 44–54

Seal Shark All 2 15 84.5 81–88 72.6 72–73

Leafscale Gulper Shark All 3 22 81.5 74–86 62.9 61–64

Plunket’s Shark All 1 2 84.2 – 1.4 –

Owston’s Dogfish Male 2 20 80.6 – 67.7 66–70

Owston’s Dogfish Female 2 16 – – 44.2 66–70

Portugese Dogfish All 1 5 81.4 – 44 41–47

From G. Summers et al. (105).

310 FISH OILS

left unanswered. Analysis of the whole lipids (18%) of a North Atlantic fish, the

barracudina Paralepis notolepis rissoi krøyeri, showed this to contain 85% wax

esters (112). The fatty acids of the wax esters differed from those of sperm body

or head wax esters, but the alcohols from barracudina showed a remarkable simi-

larity to those of sperm head wax.

Algae in theory and in practice can produce oils that are triacylglycerols

(39, 113). The DHA-rich oils commercially produced by the Martek Biosciences

Corp. are approved for use in infant formulas. They have also been shown to pos-

sess the favorable clinical attributes of fish oil DHA in healthy adults, whether

alone (114) or combined with arachidonic acid (20:4n-6) of fungal origin (115).

Products of these two sources of refined fatty acids show no particular resistance

to oxidation, compared with fish oils, when finally purified of natural components

that might not be allowed in products for human consumption (116). A competing

source of DHA (Nutrinova Inc., Somerset, NJ) advertises its products as ‘‘from

vegetarian source.’’ As noted earlier, algae can also be good sources of EPA

(117), which reopens the question posed earlier. Are the EPA and DHA of fish

oils all or mostly from the fatty acids originally supplied by phytoplankton?

9. CONCLUSION

In a decade, the ‘‘advanced’’ lipid analytical technology that defines DHA as a nat-

ural fatty acid of marine oils from a deep-sea shark described around 1994 (118)

has been surpassed by nondestructive NMR measurement of DHA in situ (Table 7).

Thus, advanced analytical technology is supporting with new developments the

benefits from marine oil omega-3 fatty acids in our daily lives.

APPENDIX 1. CODEX ALIMENTARIUS EXPLANATORYMATERIALS RELATING TO FISH OIL QUALITY TERMINOLOGY

Quality Guidelines and Potential Problem Areas or Disadvantages of Various Parameters.

Codex Specif. CAC/RS

Quality Unit Disadvantage or Potential Problem Area 19-1981 Rev. 1 1989.

Color Dark-colored oils may be crude and contain

contaminants normally removed by refining

or the color might indicate overheating during

refining.

No Standard

Iodine Value IV varies with the species of fish. In general, IV

is a measure of the unsaturation in oils. High IV

oils are generally more susceptable to oxidation.

No Standard

Acid Value1 High acid value crude oils might indicate that

poor-quality fish were processed or the oil

deteriorated in storage.

0.6 mg KOH/g fat max

refined oils

4 mg KOH/g fat max

virgin oils

4 mg KOH/g fat max cold

pressed oils

CODEX ALIMENTARIUS EXPLANATORY MATERIALS RELATING TO FISH 311

Peroxide Value Peroxide value is the primary measure of

rancidity (Oxidation) in an oil or fat. It reflects

recent oxidation.

10 meq/kg fat max virgin

and cold pressed oils

5 meq/kg fat max other

oils

p Anisidine

Number

The Anisidine Number also measures products

of oxidation; however, it reflects oxidation that

has taken place in the past.

No Standard

Totox Value A relationship between peroxide value and

anisidine number that is used to measure the

rancidity level of fats and oils. It is defined as

(2 � PV) þ AN. It reflects total oxidation to date.

No Standard

Moisture Considered an impurity. High levels of moisture

in an oil can lead to deterioration in storage.

0.20% max

Soap Soap can be formed when moisture is present

in the crude oil and reacts with the free fatty

acids and a catalyst (alkali ion), or it can result

from incomplete removal of soap from washed

refined oil.

0.005% max

Insoluble

Impurities

These are substances including traces of

protein, dirt, rust, and other materials that tend

to precipitate out of the oil during storage.

Depending on the substance, they can reduce

the stability of the oil.

0.05% max

Unsaponif. Matter These are composed of sterols, hydrocarbons,

glyceryl ethers, and fatty alcohols. There may

also be traces of pigments, vitamins, and

oxidized oil. Unsaponifiables vary with the

species of fish.

No Standard

Organochlorine,

Organophos-

phorous

Pesticides and

other Chlorinated

Hydrocarbons

Numerous compounds come under this group.

Generally, the pesticide content of the oil

reflects the environmental conditions in the

area where the fish are caught. The level of

these compounds in the oil must be within the

regulatory limits of the locality involved.

No Standard

Total Cholesterol Cholesterol is a major part of the unsaponifiable

fraction of fish oils. Generally it is not removed

except by vacuum stripping of the oil.

No Standard

Iron Iron is considered a pro-oxidant in fish oil and

is removed by degumming and refining.

1.5 mg/kg max refined oil

5 mg/kg max virgin oil

5 mg/kg max cold

pressed oil

Copper Copper is considered a pro-oxidant in fish oil

and is removed by degumming and refining.

0.1 mg/kg max refined oil

0.4 mg/kg max virgin oil

0.4 mg/kg max cold

pressed oil

Quality Guidelines and Potential Problem Areas or Disadvantages of Various Parameters.

Codex Specif. CAC/RS

Quality Unit Disadvantage or Potential Problem Area 19-1981 Rev. 1 1989.

312 FISH OILS

REFERENCES

1. S. M. Barlow and M. E. Stansby, eds., Nutritional Evaluation of Long-Chain Fatty Acids

in Fish Oil, Academic Press, London, 1982.

2. R. G. Ackman, Fatty Acids in Newer Fats and Oils, in Y. H. Hui, ed., Bailey’s Industrial

Oil and Fat Products, 5th ed., Vol. 1, Edible Oil and Fat Products: General Applications,

Wiley, New York, 1996, p. 427.

3. J-L. Sebedio, M. F. Langman, C. A. Eaton, and R. G. Ackman, J. Amer. Oil Chem. Soc.,

58, 41 (1981).

4. J-L. Sebedio and W. W. Christie, eds., Trans Fatty Acids in Human Nutrition, The Oily

Press, Dundee, (1998).

5. T. Leth, A. Bysted, K. Hansen, and L. Ovesen, J. Amer. Oil Chem. Soc., 80, 475 (2003).

6. R. G. Ackman, Natural Trans Fatty Acids, in Ref. 4, pp. 303–311.

7. J. Dyerberg and H. O. Bang, Lancet, (2), 433 (1979).

8. D. Kromhaut, E. B. Bosschieter, and C. De Lezenne Coulander, New Eng. J. Med., 312,

1205 (1985).

9. Committee on Medical Aspects of Food Policy, Department of Health, ‘‘Nutritional

Aspects of Cardiovascular Disease. Report of the Cardiovascular Review Group,’’ No.

46, HMSO, London, (1994).

Arsenic A heavy metal, naturally ocurring in sea

water. It is removed by the refining process.

0.1 mg/kg mx

Lead A heavy metal removed by the refining process. 0.1 mg/kg max

Mercury A heavy metal removed by the refining process. No Standard

Selenium A heavy metal removed by the refining process. No Standard

Cadmium A heavy metal removed by the refining process. No Standard

Hygiene Microbiological contamination by enterobacteria,

salmonella, coliforms, or E. coli would be an

indication of the sanitary conditions under

which the oil was manufactured.

CAC/RCP 1-1969, Rev. 2

1985 limits.

Oil Soluble

Vitamins

Normally part of the unsaponifiable fraction of

the oil. High Vitamin A and/or D would indicate

that the oil is a liver oil rather than a body oil.

No Standard

1Acid value is defined as two times the free fatty acid content of the oil. CAC defines edible fats and oils as

foodstuffs composed of glycerides of fatty acids. They are of vegetable, animal, or marine origin. They may

contain small amounts of other lipids such as phosphatides, unsaponifiables, or free fatty acids naturally

present in the fat or oil. CAC defines virgin fats and oils as edible fats and oils obtained without altering the oil,

by mechanical procedures, and the application of heat only. They may be purified by washing with water,

settling, filtering, and centrifuging only. CAC defines cold-pressed fats and oils as edible vegetable fats and oils

obtained, without altering the oil, by mechanical procedures without the application of heat. They may have

been purified by washing with water, settling, filtering, and centrifuging only.

Quality Guidelines and Potential Problem Areas or Disadvantages of Various Parameters.

Codex Specif. CAC/RS

Quality Unit Disadvantage or Potential Problem Area 19-1981 Rev. 1 1989.

REFERENCES 313

10. P. M. Kris-Etherton, W. S. Harris, and L. J. Appel, for the Nutrition Committee, AHA

Scientific Statement, Circulation, 106, 2747 (2002).

11. R. G. Ackman, Food Revs. Intern., 6, 617 (1990).

12. R. G. Ackman, Fatty Acids in Fish and Shellfish, in C. K. Chow, ed., Fatty Acids in Foods

and their Health Implications, 2nd ed., Marcel Dekker, New York, 2000, pp. 169–184.

13. W. E. Connor and A. Bendich, eds., Amer. J. Clin. Nutr., 71(suppl) (1), (2000).

14. A. J. Sinclair, N. M. Attar-Bashi, and D. Li, Lipids, 37 1113, (2002).

15. S. C. Cunnane, Can. J. Physiol. Pharmacol., 74, 629 (1996).

16. F. Thies, E. A. Miles, G. Nebe-von-Caron, J. R. Powell, T. L. Hurst, E. A. Newsholme,

and P. C. Calder, Lipids, 36, 1183 (2001).

17. R. Marchioli, F. Barzi, E. Bomba, et al., Circulation, 105, 1897 (2002).

18. A. Leaf, Circulation, 105, 1874–1875 (2002).

19. S. A. Reed, J. Sci. Food Agric., 15, 399 (1964).

20. F. V. K. Young, The Production and Use of Fish Oils, in Ref. 1, pp. 1–23.

21. A. P. Bimbo and J. B. Crowther, J. Amer. Oil Chem. Soc., 69, 221 (1992).

22. J. R. Burt, R. Hardy, and K. J. Whittle, eds., Pelagic Fish. The Resource and its

Exploitation. Fishing News Books, Blackwell Sci. Pubs., Oxford, United Kingdom,

1992.

23. A. P. Bimbo, Fish Meal and Oil, in R. E. Martin, E. P. Carter, G. J. Flick, Jr., and L. M.

Davis, eds., Marine and Freshwater Products Handbook, Technomic Pub. Co. Inc.,

Lancaster, Pennsylvania, 2000, pp. 541–581.

24. R. C. Ackman, ed., Marine Biogenic Lipids, Vols. 1 and 2, CRC Press, Boca Raton,

Florida, 1989.

25. J. K. McElvoy, A techno-economic model for designing large factory vessels and its

application to the Antarctic krill-finfish fishery, in Ref. 22, pp. 94–114.

26. M. E. Stansby, ed., Fish Oils in Nutrition, An avi Book, Van Nostrand Reinhold,

New York, 1990.

27. R. G. Ackman, W. M. N. Ratnayake, and B. Olsson, J. Amer. Oil. Chem. Soc., 65, 136

(1988).

28. R. G. Ackman, J-L. Sebedio, and M. I. P. Kovacs, Mar. Chem., 9, 157 (1980).

29. W. N. Ratnayake and R. G. Ackman, Lipids, 14, 795 (1979).

30. W. N. Ratnayake and R. G. Ackman, Lipids, 14, 804 (1979).

31. R. F. Lee and J. S. Patton, Alcohol and Waxes, in Ref. 24, Vol. 1, pp. 73–102.

32. D. Menoyo, C. J. Lopez-Bote, J. M. Bautista, et al., Aquat. Liv. Resour, 15, 217 (2002).

33. R. N. Lemaitre, I. B. King, and T. E. Raghunathan, et al., Circulation, 105, 697 (2002).

34. A. M. Katz, Circulation, 105, 669 (2002).

35. R. G. Ackman, Fatty Acids, in Ref. 24, Vol. 1, pp. 103–137.

36. Y. Ando, T. Ota, and T. Takagi, Yukagaku, Proc. Vol. 2, Sess. Lect. Sci. Pres. ISF-JOCS

World Congress, 1989, pp. 1263–1268.

37. R. G. Ackman, Anal. Chim. Acta, 465, 175 (2002).

38. R. G. Ackman, INFORM, 5, 1119 (1994).

39. Y. Itabashi, N. Harada, T. Ota, and K. Matsunaga., Yukagaku, 45, 271 (1996).

40. P. Mayzaud, C. A. Eaton, and R. G. Ackman, Lipids, 11, 858 (1976).

314 FISH OILS

41. P. Mayzaud and R. G. Ackman, Lipids, 13, 24 (1978).

42. J. D. Joseph and R. G. Ackman, J. Assoc. Off. Anal. Chem. Int., 75, 488 (1992).

43. R. G. Ackman and S. N. Hooper, Comp. Biochem. Physiol., 46B, 153 (1973).

44. H. E. Worne and L. W. Smith, Amer. J. Med. Sci., 710–721 (1959).

45. C. M. Harlow, L. Steeves, and A. Myrden, Nova Scotia, Med. Bull., 37, 127 (1958).

46. E. J. Schaefer, Amer. J. Clin. Nutr., 75, 191–212 (2002).

47. P. Cullen, Amer. J. Cardiol, 86, 943 (2000).

48. K. H. Bønaa, K. S. Bjerve, B. Straume, I. T. Gram, and D. Thelle, New Eng. J. Med., 322,

795 (1990).

49. T. Kanayasu-Toyoda, I. Morita, and S. Murota, Prostaglandins Leukotri. Essent. Fatty

Acids, 54, 319 (1996).

50. C. Benistant, F. Achard, S. Ben Slama, and M. Lagarde, Prostaglandins Leukotri. Essent.

Fatty Acids, 55, 287 (1996).

51. W. S. Harris, J. Lipid Res., 30, 785 (1989).

52. F. Achard, C. Benistant, and M. Lagarde, Biochim. Biophys. Acta., 1255, 260 (1995).

53. J. D. Carver, Amer. J. Clin. Nutr., 77(suppl.), 1550S (2003).

54. S. M. Innis and S. L. Elias, Amer. J. Clin. Nutr., 77, 473 (2003).

55. A. Leaf, J. X. Kang, Y-F. Xiao, and G. E. Billman, Circulation, 107, 2646 (2003).

56. J. S. Charnock, Prog. Lip. Res., 33, 355 (1994).

57. T. Helm, Dangerous Sea Creatures, Funk and Wagnall, New York, 1976.

58. W. M. Indrasena and T. A. Gill. J. Food Sci., 65, 948 (2000).

59. P. Pohl and H. Wagner, Z. Naturforsch., 27, 53 (1972).

60. K. Hayashi, S. Kida, K. Kato, and M. Yamada, Bull. Jap. Soc. Sci. Fish., 40, 609 (1974).

61. R. G. Ackman, C. S. Tocher, and J. McLachlan, J. Fish. Res. Bd. Can, 25, 1603 (1968).

62. S. M. Budge, C. C. Parrish, and C. H. McKenzie, Mar. Chem., 76, 285 (2001).

63. M. V. Bell, J. R. Dick, and A. E. A. Porter, Lipids, 36, 1153 (2001).

64. M. V. Bell, J. R. Dick, and A. E. A. Porter, Lipids, 38, 39 (2003).

65. C. Ghioni, J. G. Ben, and J. R. Sargent, Comp. Biochem. Physiol., 1143, 161 (1996).

66. T. Ota and M. Yamada, Bull. Fac. Fish. Hokkaido University, 26, 277 (1975).

67. R. G. Ackman, C. A. Eaton, E. G. Bligh, and A. W. Lantz., J. Fish Res. Bd. Can., 24,

1219 (1967).

68. W. Schmidtsdorff, Fish meal and fish oil—not only by-products, in A. Ruiter, ed., Fish

and Fishery Products, CAB International, Wallingford, 1995, pp. 346–376.

69. S. Zhou, R. G. Ackman, and C. Morrison, Can. J. Fish. Aquat. Sci., 53, 326 (1996).

70. A. P. Bimbo. INFORM, 9, 473 (1998).

71. E. Kulas, E. Olsen, and R. G. Ackman, Eur. J. Lipid. Sci. Technol., 104, 520 (2002).

72. K. Berger, INFORM, 14, 307 (2003).

73. E. Kulas, R. G. Ackman, J. Amer. Oil Chem. Soc., 78, 361 (2001).

74. S. Wada, J. Oleo Sci., 50, 329 (2001).

75. A. P. Bimbo, Production of Fish Oil, in Ref. 26, pp. 181–225.

76. R. G. Ackman, S. N. Hooper, and D. L. Hooper, J. Amer. Oil. Chem. Soc., 51, 42

(1974).

REFERENCES 315

77. R. C. Wijesundera, W. M. N. Ratnayake, and R. G. Ackman, J. Amer. Oil Chem. Soc., 66,

1822 (1989).

78. K. Julshamn, L. Karlsen, and O. R. Braekkan, Fiskeridirektoratets Skrifter, Ser. Tek.

Undersøkelser, Vol. 5, No. 15, Directorate of Fisheries Bergen, 1973.

79. R. G. Ackman, Chemistry and Industry, 7, 139 (1988).

80. M. A. Dessi, M. Deiana, B. W. Day, A. Rosa, S. Banni, and F. P. Corongiu., Eur. J. Lipid.

Sci. Technol., 104, 506 (2002).

81. G. Hølmer, Triglycerides, in Ref. 24, Vol. 1, pp. 139–173.

82. R. Ackman, V. R. Orozco, and W. M. N. Ratnayake, Fat. Sci. Technol., 93, 447 (1991).

83. T. Igarishi, M. Ausand, Y. Hirata, I. S. Gribbestad, S. Wada, and M. Nonaka, J. Amer. Oil

Chem. Soc., 77, 737 (2000).

84. M. Cansell, F. Nacka, and N. Combe, Lipids, 38, 554 (2002).

85. R. G. Ackman, W. M. N. Ratnayake, and E. J. Macpherson, J. Amer. Oil. Chem. Soc., 66,

1162 (1989).

86. V. K. S. Shukla and E. G. Perkins, INFORM, 9, 955 (1998).

87. W. W. Christie, Silver-ion high-performance liquid chromatography, in J. L. Sebedio and

E. G. Perkins, eds., New Trends in Lipid and Lipoprotein Analyses, AOCS Press,

Campaign, Illinois, 1995, pp. 59–80.

88. J. L. Guil-Guerrero and E-H. Belarbi, J. Amer. Oil Chem. Soc., 78, 477 (2001).

89. J-L. Sebedio and R. G. Ackman, Lipids, 16, 461 (1981).

90. H. J. Passino, Ind. Eng. Chem., 41, 280 (1949).

91. V. J. Krukonis, Processing with supercritical fluids, overview and applications, in B. A.

Charpentier and M. R. Sevenants, eds., Supercritical Fluid Extraction and Chromato-

graphy, Techniques and Applications, Amer. Chem. Soc., Washington, D. C., 1988, pp.

26–43.

92. S. S. H. Rizvi, R. R. Chao, and Y. J. Liaw, Concentration of omega-3 fatty acids from fish

oil using supercritical carbon dioxide, in B. A. Charpentier and M. R. Sevenants, eds.,

Supercritical Flyid Extractioned, 1988, pp. 89–108.

93. V. F. Stout, W. B. Nilsson, J. Krzynowek, and H. Schlenk, Fractionation of fish oils and

their fatty acids, in Ref. 26, pp. 73–119.

94. R. G. Ackman, R. D. Burgher, and P. M. Jangaard, Can. J. Biochem. Physiol., 41, 1627

(1963).

95. W. M. N. Ratnayake, B. Olsson, D. Matthews, and R. G. Ackman, Fat. Sci. Technol., 90,

381 (1988).

96. Jeanne D. Joseph, Ed. 1989. Biomedical Test Materials Program: Production Methods

and Safety Manual. NOAA Technical Memorandum NMFS-SEFC-234, 120 p.

97. J. Via, L. T. Taylor, and F. K. Schweighardt, Anal. Chem., 66, 1459 (1994).

98. S. L. Taylor, F. J. Eller, and J. W. King, Food Res. Intern., 30, 365 (1997).

99. T. H. Walker, H. D. Cochran, and G. J. Hulbert, J. Amer. Oil. Chem. Soc., 76, 595 (1999).

100. M. Linder, J. Fanni, and M. Parmentier, OCL, 8, 73 (2001).

101. G. P. McNeill, R. G. Ackman, and S. R. Moore, J. Amer. Oil Chem. Soc., 73, 1403 (1996).

102. Y. Shimada, K. Maruyama, A. Sugihara, S. Moriyama, and Y. Tominaga, J. Amer. Oil.

Chem. Soc., 74, 1441 (1997).

316 FISH OILS

103. S-J. Kang, M. C. A. Timmins, and R. G. Ackman, J. Amer. Oil. Chem. Soc., 75, 1667

(1998).

104. S-J. Kang, S. P. Lall, and R. G. Ackman, Lipids, 32, 19 (1997).

105. G. Summers, R. Wong, and L. Eyres, ‘‘Handling and processing shark livers for the

recovery of squalene and diacyl glyceryl ethers,’’ DSIR Crop Research Report No. 1,

Christchurch, New Zealand, 1990.

106. R. G. Ackman, E. Macpherson, and A. Timmins. J. Amer. Oil. Chem. Soc., 75, 1223

(1998).

107. R. G. Ackman, E. J. Macpherson, and A. Timmins, J. Amer. Oil. Chem. Soc., 77, 831

(2000).

108. R. G. Ackman, R. F. Addison, and C. A. Eaton, Nature, 220, 1033 (1968).

109. H. V. Kuhnlein, F. Yeboah, M. Sedgemore, S. Sedgemore, and M. Chan, J. Food Compos.

Anal., 9, 18 (1996).

110. J. Joseph, Distribution and composition of lipids in marine invertebrates, in Ref. 24, Vol.

2, pp. 49–143.

111. H-S. Seo, Y. Endo, M. Moku, K. Kawaguchi, and K. Fujimoto, Lipids, 36, 389 (2001).

112. R. Ackman, S. N. Hooper, S. Epstein, and M. Kelleher, J. Amer. Oil Chem. Soc., 49, 378

(1972).

113. P. W. Behrens and D. J. Kyle, J. Food Lipids, 3, 259 (1996).

114. J. A. Conquer and B. J. Holub, J. Nutr., 126, 3032 (1996).

115. S. M. Innis and J. W. Hansen, Amer. J. Clin. Nutr., 64, 159 (1996).

116. E. N. Frankel, T. Satue-Gracia, A. S. Meyer, and J. B. German, J. Agric. Food Chem., 50,

2094 (2002).

117. L. Poisson, M. Devos, G. Pancreac’h, and F. Ergan, OCL, 9, 92 (2002).

118. C. G. Bordier, N. Sellier, A. P. Faucault, and F. Le Goffic, Chromatographia, 39, 329

(1994).

REFERENCES 317