1. Lipid Determination in Aquatic Samples
7
1993; Parrish et aI., 1992a), algae (Dembitsky and Rozentsvet, 1996; Napolitano,
1994), sponges (Dembitsky et aI., 1994a), crustaceans (Butler, 1994; Dembitsky
et ai., 1994b; Wainman et ai., 1993; Arts et ai., 1992), and fish (Verreth et aI.,
1994). In the past 2 years, there have also been studies of fatty acids in algae
(Napolitano et aI., 1995; Napolitano, 1994), sponges (Dembitsky et aI., 1994a),
crustacea (Dembitsky et ai., 1994b; Rezanka and Dembitsky, 1994), and fish
(Andrade et ai., 1995; Fodor et aI., 1995; Harrell and Woods, 1995; Rahman et aI.,
1995). Other solvent extractable compounds have also been investigated recently
in freshwater samples including hydrocarbons (Yunker et ai., 1994; Meyers and
Eadie, 1993; Parrish et ai., 1992a), alcohols (Yunker et aI., 1994; Meyers and
Eadie, 1993), and organochlorine contaminants (Renaud et aI., 1995a,b). These
studies suggest that there are nearly as many different ways of determining lipids
in aquatic samples as there are laboratories that routinely perform these measurements. The purpose of this chapter is to indicate procedures that we have used
successfully over the past several years for a large number and a wide variety of
aquatic samples.
1.2. Results and Discussion
1.2.1. Sampling and Storage
As soon as a sample is taken, the lipid content and composition begin to change.
These changes are catalyzed by enzymes, heat, oxygen, and light. Thus, samples
should be kept as cold as possible and processed as soon as possible. Sampling of
tissues or filtration of seston should be performed as soon as it can be done
cleanly. To avoid contamination, it is important to avoid contact with any oils or
grease at this stage. After processing, the tissue sample or filter should be immediately placed in a clean glass vial containing chloroform. At this point, it is
important to avoid contact of the solvent with plastic or rubber to prevent contamination. Samples are handled with metal forceps or glass pipettes, and they
must be stored in vials that have previously been washed with good-quality
chloroform (e.g., high-performance liquid chromatography [HPLC] grade) and
that have caps with Teflon liners. After immersing the sample in the chloroform,
the airspace above the chloroform should be flushed with a clean, relatively
unreactive gas (e.g., nitrogen). The vial is then capped, and Teflon tape is wrapped
around the cap. Under these conditions, the sample can be stored in a freezer
« - 20°C) for months without appreciable alteration in the lipid content or
composition (Sasaki and Capuzzo, 1984).
After analysis, the integrity of the sample can be assessed from the free fatty
acid content (Parrish, 1988) or the free fatty acid plus alcohol content (Parrish et
aI., 1995). Ideally, fresh samples should be analyzed for comparison with stored
ones so that normal levels of these hydrolysis products can be determined. However, an interesting paper suggests that free fatty acid levels in marine diatoms
should, in fact, be close to zero and that problems are encountered at the sample
7
1993; Parrish et aI., 1992a), algae (Dembitsky and Rozentsvet, 1996; Napolitano,
1994), sponges (Dembitsky et aI., 1994a), crustaceans (Butler, 1994; Dembitsky
et ai., 1994b; Wainman et ai., 1993; Arts et ai., 1992), and fish (Verreth et aI.,
1994). In the past 2 years, there have also been studies of fatty acids in algae
(Napolitano et aI., 1995; Napolitano, 1994), sponges (Dembitsky et aI., 1994a),
crustacea (Dembitsky et ai., 1994b; Rezanka and Dembitsky, 1994), and fish
(Andrade et ai., 1995; Fodor et aI., 1995; Harrell and Woods, 1995; Rahman et aI.,
1995). Other solvent extractable compounds have also been investigated recently
in freshwater samples including hydrocarbons (Yunker et ai., 1994; Meyers and
Eadie, 1993; Parrish et ai., 1992a), alcohols (Yunker et aI., 1994; Meyers and
Eadie, 1993), and organochlorine contaminants (Renaud et aI., 1995a,b). These
studies suggest that there are nearly as many different ways of determining lipids
in aquatic samples as there are laboratories that routinely perform these measurements. The purpose of this chapter is to indicate procedures that we have used
successfully over the past several years for a large number and a wide variety of
aquatic samples.
1.2. Results and Discussion
1.2.1. Sampling and Storage
As soon as a sample is taken, the lipid content and composition begin to change.
These changes are catalyzed by enzymes, heat, oxygen, and light. Thus, samples
should be kept as cold as possible and processed as soon as possible. Sampling of
tissues or filtration of seston should be performed as soon as it can be done
cleanly. To avoid contamination, it is important to avoid contact with any oils or
grease at this stage. After processing, the tissue sample or filter should be immediately placed in a clean glass vial containing chloroform. At this point, it is
important to avoid contact of the solvent with plastic or rubber to prevent contamination. Samples are handled with metal forceps or glass pipettes, and they
must be stored in vials that have previously been washed with good-quality
chloroform (e.g., high-performance liquid chromatography [HPLC] grade) and
that have caps with Teflon liners. After immersing the sample in the chloroform,
the airspace above the chloroform should be flushed with a clean, relatively
unreactive gas (e.g., nitrogen). The vial is then capped, and Teflon tape is wrapped
around the cap. Under these conditions, the sample can be stored in a freezer
« - 20°C) for months without appreciable alteration in the lipid content or
composition (Sasaki and Capuzzo, 1984).
After analysis, the integrity of the sample can be assessed from the free fatty
acid content (Parrish, 1988) or the free fatty acid plus alcohol content (Parrish et
aI., 1995). Ideally, fresh samples should be analyzed for comparison with stored
ones so that normal levels of these hydrolysis products can be determined. However, an interesting paper suggests that free fatty acid levels in marine diatoms
should, in fact, be close to zero and that problems are encountered at the sample