Processing Whole Fish Samples

This page outlines the steps for preparing whole fish composite homogenate samples for analysis. Wear powderless, disposable gloves when handling fish for tissue processing, changing gloves between each sample.

1. Inspect individual fish.

Unwrap and inspect fish to ensure that they have been properly preserved during shipment.
Discard any specimen deemed unsuitable for processing (e.g., thawed, severely damaged, mutilated fish, or fish with skin lacerations, wounds or anomalies that expose muscle tissue or viscera) and note on the sample processing record.
Frozen fish specimens should be thawed only to the point where it becomes possible to make an incision into the flesh (U.S. EPA, 1991d). Grinding and homogenization is easier when fish are not completely thawed.

2. Weigh individual fish.

Weigh the wet weight (mass) of each individual fish sample to the nearest gram using a balance. Record mass on the sample processing record. Check balance calibration at the beginning and end of each weighing session and after every 20 weighings in a weighing session.
Weigh fish on a foil-lined balance tray or decontaminated stainless steel bowl. Replace the foil lining after weighing each individual fish for individual samples or after the last fish in a group for a composite sample to prevent cross contamination between samples or follow decontamination procedures for cleaning the bowl between samples.
As the fish thaws, keep all liquid as part of the sample.

3. Remove otoliths, scales, spines or maxillary bone for age determination (optional).

Age provides a good indication of the duration of exposure to pollutants.
Age may be determined by a fisheries biologist using otoliths, scales, spines or in some cases, maxillary bones.
Scaleless fish (catfish, etc.) - clip pectoral fin spines.
For Lake Trout, studies found that thin sections of maxillary bone (upper jaw) are easier to read and just as accurate as otoliths (Retherford, 2014 and Wellenkamp and He, 2015).
Store the otoliths, scales, spines, or maxillary bones in small envelopes or plastic bags labeled with the tissue specimen identification number. Note the removal of otoliths, scales, spines, or maxillary bones from each fish on the sample processing record.

4. Determine the sex of each fish (optional).

Fish sex should be determined by a knowledgeable fisheries biologist.
Make an incision on the ventral surface of the body from a point immediately in front of the anus toward the head to a point immediately posterior to the pelvic fins. If necessary, make a second incision on the left side of the fish from the initial point of the first incision toward the dorsal fin.
Fold the resulting flap back to observe the gonads. Ovaries range from granular in texture (in immature stages) to fully developed eggs, and their color can vary depending on the species (e.g., white, golden brown, pink, orange, or red). Testes appear uniform in texture and are typically white or cream in color. (Fish Necropsy Manual)
Record the sex of each fish on the sample processing record.

5. Note morphological abnormalities (e.g. deformities, eroded fins, lesions, and tumors)

Assessment of gross morphological abnormalities in finfish can be conducted in the field or during initial inspection at the processing laboratory and should be noted on the field record form.
When documenting morphological abnormalities, consult resources such as Sinderman (1983) or Smith et al. (2002).

6. Wash the outside of the fish with distilled water (or another form of contaminant-free water).

Place on a clean cutting board for cutting into pieces, if necessary, prior to homogenization.
Control cross-contamination between composites by following the decontamination procedures described in the Equipment subsection before processing each new composite of fish. Rinse the cutting board and knife with distilled water (or another form of contaminant-free water) before processing each fish within a composite.

7. Homogenize individual whole fish.

Grind and homogenize individual fish prior to analysis to ensure even distribution of contaminants throughout tissue samples and to facilitate extraction and digestion of samples.
Use a stainless-steel grinder, high-speed blender, or homogenizer to grind and homogenize fish. Large fish may be cut into smaller cubes with high-quality stainless steel or titanium knives prior to homogenization. Parts of the blender or homogenizer used to grind the tissue (i.e., blades, probes) should be made of tantalum or titanium rather than stainless steel.
For best results, grind and homogenize when the biological tissue is still partially frozen. Additionally, chilling the grinder/homogenizer briefly with a few chips of dry ice will reduce the tendency of the tissue to stick to the grinder. These techniques will make grinding easier (Stober, 1991).
Grind the sample into a stainless steel or glass bowl until the tissues appear to be homogeneous. Repeat as necessary until the tissue is a uniform color and finely ground texture. Do not let the homogenate heat up in the process.
Whole fish (especially skin) can be difficult to homogenize completely. No chunks of tissue or skin should remain in the sample homogenate because these may not be extracted or digested efficiently and could bias the analytical results.
Note the preparation specifics for each individual homogenate on the sample processing record.
Individual homogenates may be either processed further to prepare composite homogenates or frozen separately and stored at ≤-20 °C.

8. Prepare a composite homogenate.

If analyzing composite whole fish, combine individuals making sure to thoroughly mix the tissue.
It is essential that the mass of the composite homogenate yields adequate mass to perform all targeted analyses. mass requirements vary based on:
- The number of target chemicals that will be analyzed
- The amount of tissue required for analyses
Sample size requirements may vary among laboratories and the analytical methods used. Consult with the analytical laboratory supervisor to determine the actual weights of homogenates required to analyze for all selected target analytes.
Label containers with aliquot identification number, sample weight (to the nearest 0.1g), and date aliquots were prepared.
Weigh aliquots of the homogenate to meet the analytical laboratory needs for each particular target analyte and store the analytical aliquots (as described in each method analysis) prior to shipment to the laboratory.
Freeze the remainder of each composite homogenate (i.e., tissue in excess of that needed for analysis) at ≤-20 °C to archive it. Record the designation "Archive" and the preparation date on the sample label. Record the location of the archived samples on the sample processing record under "Notes." Archived samples provide a valuable backup if samples are lost in transit or there are analytical problems.