Annex B-12, Appendix 1. Technical note on biological material sampling and sample handling for the analysis of persistent organic pollutants (PAHs, PCBs and OCPs) and metallic trace elements

1. General principles

Muscle tissue or liver of fish have to be dissected while they are in good condition. If biological tissue deteriorates, uncontrollable losses of determinands or cross-contamination from other deteriorating tissues and organs may occur. To avoid this, individual fish specimens must be dissected at sea if adequate conditions prevail on board, or be frozen immediately after collection and transported frozen to the laboratory, where they are dissected later.

If the option chosen is dissection on board the ship, two criteria must be met:

1. The work must be carried out by personnel capable of identifying and removing the desired organs according to the requirements of the investigations; and
2. There must be no risk of contamination from working surfaces or other equipment.

2. Tools and working area

Crushed pieces of glass or quartz knives, and scalpels made of stainless steel or titanium are suitable dissection instruments.

Colourless polyethylene tweezers are recommended as tools for holding tissues during the dissecton of biological tissue for metallic trace element analysis. Stainless steel tweezers are recommended if biological tissue is dissected for analysis of chlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and polunuclear aromatic hydrocarbons (PAHs).

After each sample has been prepared, including the samples of different organs from the same individual, the tools should be changed and cleaned.

The following procedures are recommended for cleaning tools used for preparing samples:

1) for analysis of metallic trace elements

• a) Wash in acetone or alcohol and high purity water.

• b) Wash in HNO₃ (p.a.) diluted (1+1) with high purity water. Tweezers and haemostates in diluted (1+6) acid.
• c) Rinse with high purity water.

2) for analysis of CBs and OCPs

• a) Wash in acetone or alcohol and rinse in high purity water.

The glass plate used during dissection should be cleaned in the same manner. The tools must be stored dust-free when not in use.

The dissection room should be kept clean and the air should be free from particles. If clean benches are not available on board the ship, the dissection of fish should be carried out in the land-based laboratory under conditions of maximum protection against contamination.

3. Fish muscle and liver samples dissection

For fish analysis, commercial catches can be used if fish transport to the laboratory does not take longer than 24 hours. The fish must be transported on ice. The dissection then takes place at the laboratory.

For analysis of fish muscle, the epidermis and subcutaneous tissue should be carefully removed from the fish. Samples should be taken under the red muscle layer. In order to ensure uniformity of samples, the right side dorso-lateral muscle should be taken as the sample. If possible, the entire right dorsal lateral filet should be used as a uniform sample, from which subsamples can be taken after homogenizing for replicate dry weight and contaminant determinations. If, however, the amount of material obtained by this procedure is too large to handle in practice, a specific portion of the dorsal musculature should be chosen for the sample. It is recommended that the portion of the muscle lying directly under the first dorsal fin should be utilised in this case. As both fat and water content vary significantly in the muscle tissue from the anterior to the caudal muscle of the fish (Oehlenschläger, 1994), it is important to obtain the same portion of the muscle tissue for each sample.

To sample liver tissue, the liver must be identified in the presence of other organs such as the digestive system or gonads (Harms and Kanisch, 2000). The appearance of the gonads will vary according to the sex of the fish and the season. After opening the body cavity with a scalpel, the connective tissue around the liver should be cut away and as much as possible of the liver is cut out in a single piece together with the gall bladder. The bile duct is then carefully clamped and the gall bladder dissected away from the liver.

When fish samples which have been frozen at sea are brought to the laboratory for analysis, they should be dissected as soon as the tissue has thawed sufficiently. The dissection of fish is easiest when the material, at least the surface layers of the muscle tissue, is half frozen. For dissection of other organs, the thawing must proceed further, but it is an advantage if, for example, the liver is still frozen. It must be noted that any loss of liquid or fat due to improper cutting or handling of the tissue makes the determinations of dry weight and fat content, and consequently the reported concentrations of determinands, less accurate.

After muscle preparations, the liver should be completely and carefully removed while still partly frozen to avoid water and fat loss. Immediately after removing it from the fish, the liver should be returned to the freezer so that it will be completely frozen prior to further handling. This is particularly important for cod liver.

4. Shellfish sampling

The blue mussel (Mytilus edulis) occurs in shallow waters along almost all coasts of the Baltic Sea. It is therefore suitable for monitoring in near shore waters. No distinction is made between M. edulis, M. gallopovincialis, and M. trossulus because the latter species fills a similar ecological niche. A sampling size range of 20–70 mm shell length is specified to ensure availability throughout the whole maritime area.

Two alternative sampling strategies can be used: sampling to minimise natural variability and length-stratified sampling. Only details of length-stratified sampling are described in this document, as this strategy is used in monitoring programmes for temporal trends of contaminants in biota.

For shellfish, the upper limit of shell length should be chosen in such a way that at least 20 mussels in the largest length interval can easily be found. The length stratification should be determined in such a way that it can be maintained over many years for the purposes of temporal trend monitoring. The length interval shall be at least 5 mm in size. The length range should be split into at least three length intervals (small, medium, and large) which are of equal size after log transformation.

Mussels are collected by a bottom grab and selected onboard. The number of specimens selected for analysis depends on their length, e.g. 80-100 individuals are necessary to suffice material within the length range 4-5 cm.

5. Storage of Fish and Mussel Samples

Material from single fish specimens should be packaged and stored individually.

• Samples for analysis of metallic trace elements can be stored in polyethylene, polypropylene, polystyrene or glass containers.

• Samples for analysis of CBs and OCPs should be packaged in precleaned aluminium foil or in precleaned glass containers.

Liver tissue can deteriorate rather rapidly at room temperature. Consequently, samples should be frozen as soon as possible after packaging. They can be frozen rapidly by immersion in liquid nitrogen or blast freezing, but both these techniques need care. Whatever system is used, freezing a large bulk of closely packed material must be avoided. The samples in the centre will take longer to cool and will therefore deteriorate more than those in the outer layer.

Once frozen, samples can be stored in a deep freezer at temperatures of -20^oC or below.

Frozen liver tissue should not be stored longer than six months, while lean muscle tissue can be stored up to two years. Each sample should be carefully and permanently labelled. The label should contain at least the sample's identification number, the type of tissue, and the date and location of sampling.

Mussels should be shucked live and opened with minimal tissue damage by detaching the adductor muscles from the interior of at least one valve. The soft tissues should be removed and homogenised as soon as possible, and frozen in glass jars at -20 °C until analysis. Mussel tissue for trace metal determination is homogenised and decomposed in a wet state while for persistent organic pollutants determination it is homogenised and water is removed by freeze-drying. Frozen liver tissue should not be stored longer than six months, while lean muscle tissue can be stored up to two years. Each sample should be carefully and permanently labelled. The label should contain at least the sample's identification number, the type of tissue, and the date and location of sampling.

References

Harms, U.; Kanisch, G. (2000): Intra-Individual Variability of levels of lead detected in the liver of Baltic cod (Gadus morhua). Mar. Poll. Bull., 40 / 8, 710-712

Oehlenschläger, J. 1994. Quality assurance during sampling onboard. In ICES/HELCOM Workshop on Quality Assurance of Chemical Analytical Procedures for the Baltic Monitoring Programme. Ed. by G. Topping and U. Harms. Baltic Sea Environment Proceedings No. 58: 82-84.

Last updated: 29.10.2012 (Annex number changed from Annex B 13 to Annex B 12)