Fish Sample Types

Fish sample type describes whether the sample is composed of tissue from an individual specimen or is a composite of tissue from multiple specimens, and whether the fillet, plug or whole body is to be analyzed. A description of the sample type should be documented for each target species in the project work plan and the field record form. Program managers must select and document the sample type that best aligns with their study goals and the target audience for any advisories that are developed.

• Individual Samples
• Composite Samples
• Fish Fillet, Fish Plug, and Whole Fish Samples
• Skin-On vs Skin-Off Fish Samples

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Individual Samples

Collecting individual samples for target fish species provides a direct measure of the range and variability of contaminant levels in fish populations. A typical sample size is 10 individuals of each species, but this number may vary depending on the size and characteristics of the waterbody. A sample size as small as 5 individuals can be considered if it’s not possible to catch as many as 10.

Information on maximum contaminant concentrations in individual fish is useful for evaluating human health risks. Estimates of the variability of contaminant levels among individual fish can be used to ensure that studies meet desired statistical objectives. 

Analyzing individual fish incurs additional analytical laboratory costs, particularly when one considers that a number of individual sample analyses are required to achieve measurements of a reasonable statistical power. A power analysis can be performed to determine the minimum number of fish to be collected and analyzed to detect a specified minimum significant difference in chemical residues over space or time. If the sample size is too small, the measurements will not have the precision needed to provide reliable detection of the difference in chemical residues over time. If the sample size is too large, resources will be wasted because too many fish were collected and analyzed. 

Any remaining sample tissues could be archived to perform additional individual analyses (e.g., of other contaminants) without incurring more sampling costs.

Composite Samples

Composite samples may be necessary to secure enough mass for analysis and can be a cost-effective means of estimating average chemical concentrations in the tissues of target species. For example, the analytical laboratory costs for 3 composite samples of 5 specimens each is less than the cost of the analysis of 10 individual fish of the same species.

Once a base year of composite data is collected, the measured population variance can inform future study design. With a power analysis, the program manager can determine the most appropriate number of composite samples that would allow adequate determination of mean contaminant concentrations. Three composite samples may provide sufficient data for a monitoring program; however, some jurisdictions may analyze as many as 10 composite samples for each species.

The Analytical Methods and Fish Tissue Mass Table can be used to determine whether individual or composite sampling is appropriate for the target species.

Composite samples provide an average concentration. If maximum and minimum levels of contamination in individual specimens are necessary to meet monitoring objectives, then individual samples should be collected and analyzed, instead of or in addition to composite samples.

Individual specimens within a composite should be:

• Only one species. Accurate taxonomic identification is essential. Different species can have different contaminant bioaccumulation rates.
• Legal-size, or at least be of consumable size, if no legal harvest requirements are in effect.
• Live and intact.
• Similar in size so that the smallest individual in a composite is no less than 75% of the total length of the longest individual. Size is used as a surrogate for age, which provides some estimate of the total time the individual has been at risk of exposure. The primary target size range ideally should include the larger individuals harvested at each sampling site. In this way, fish exposure time is maximized, and it may maximize the chances of detecting high levels of chemical contamination in the single composite sample collected for each target species. If this ideal condition cannot be met, the field sampling team should retain individuals of similar length that fall within a secondary (smaller) target size range. The use of sizes of fish exceeding the size range recommended for compositing may introduce more variability. If the size range within each composite is broadened, the variability within the composite may increase. Overall inferences made from composites of different size ranges will have increased variability associated with them (e.g., wider confidence intervals). Greater size range may suggest the need for three size classes, rather than two.
• Collected at the same time (i.e., collected as close to the same time as possible but no more than 1 week apart). The timely collection of samples is important so that temporal changes in contaminant concentrations that are associated with the reproductive cycle of the target species are minimized. Individual fish may have to be frozen until all individuals for a composite sample are available for delivery to the designated laboratory.
• Collected in sufficient numbers (3-5 fish) to provide a sufficient mass of edible tissue (or whole-body tissue if warranted by the study design) for the analysis of target contaminants. Having an equal number of fish in each composite sample leads to a simpler data analysis (a weighted average is necessary when composite samples have different numbers of fish); however, it is more important to have similarly sized fish than an equal number of fish in a composite sample (Hoetig and Olsen, 1997). There may be occasion when as many as 10-20 individuals may be necessary depending on the species (e.g., small fish like smelt) and the number of analyses to be performed.

Table 1. Comparing individual fish sample collection to composite fish sample collection - advantages and disadvantages

Sample Type	Advantages	Disadvantages
Individual Samples – fish analyzed individually	Enables the development of size-specific fish consumption advisories Provides information on levels of chemical contamination in individual and degree of variability  Enables calculation of confidence limits about the mean	Requires more fish to be collected for size class analyses (a concern where fish populations are low or endangered) More funding to perform analyses if number of individual samples is greater than number of composite samples
Composite Samples – homogeneous mixtures of samples from 2 or more individuals of the same species collected at a particular site and analyzed as a single sample	Provides a cost-effective means of estimating average chemical concentrations in the tissue of target species Compensates for individual variability separate from size Ensures adequate sample mass for analysis of multiple chemicals	Loses maximum and minimum levels of contaminant concentration values for individuals  Requires multiple fish of similar size for each composite sample

Fish Fillet, Fish Plug and Whole Fish Samples

The sample type should reflect the typical fish preparation and consumption practices of the target audience. A critical factor when selecting sample type is maintaining consistency over time so studies are comparable over multiple years.

Most of the general population consumes the fish fillet; therefore, the fish fillet sample type is the most common in monitoring programs. EPA recommends using fillets with the skin on for most scenarios. When mercury is the contaminant of concern, skin-off fillets are recommended as described in the next section.

An alternative to the fish fillet is the collection of a fish plug if the contaminant of concern is mercury or selenium. Fish plugs are a presumably non-lethal biopsy of the meaty section (anterior dorsal area) of a live fish, a type of “mini fillet”. EPA conducted a study to address the data gap in the scientific literature for comparing fish plug sampling and harvesting fish for fillet sampling (Stahl et al., 2021). Both the mercury and selenium phases of the study “showed that there were no statistically significant differences between fillet plug and homogenized fillet results at the community level; however, a selenium plug monitoring alternative must employ a sufficiently sensitive analytical method and consider total solids.”

Another option for the sample collection is the whole fish. This option is appropriate if the target audience generally consumes the whole fish or parts other than the fillet.

Consumption advisories for specific fish body parts may be necessary to inform target audiences of how they can reduce their exposure to contaminants. For example, the New Jersey Department of Environmental Protection recommends that consumers “do not eat the heads, guts or liver, because PCBs usually concentrate in those body parts. Also, avoid consumption of any reproductive parts such as eggs (roe).”

Table 2. Advantages and disadvantages of fish fillet, fish plug and whole fish samples

Sample Type	Advantages	Disadvantages
Fish Fillet – muscle tissue of the fish	Provides sufficient tissue mass to analyze multiple contaminants, and the corresponding quality control (QC) samples for each analytical method, and for archive Allows tissue preparation in a controlled laboratory setting Identifies risks for commonly consumed portion of the fish	Requires the fish to be sacrificed May underrepresent the risk to populations who eat fillet tissue plus other portions of the fish (or the whole fish) due to potential risks associated with organs or other portions of fish not being captured in the fillet analysis Increases shipping costs compared to fish plugs due to the need to ship whole fish before it is prepared into fillet samples Requires laboratory preparation and ample space for freezer storage, leading to increased costs
Fish Plug – biopsy of the fillet	Allows fish to remain alive (although survival rates are generally unknown) if the plug is taken in the field Decreases shipping costs due to lighter samples if the plug is taken in the field Identifies risks for commonly consumed portion of the fish No laboratory preparation required before sample analysis Less laboratory freezer space required for storage	Provides insufficient tissue for analysis of multiple contaminants, QC samples, or for repeating analyses if necessary Increases vulnerability to sample contamination due to tissue preparation in the field (but not if the plug is taken in the lab) Increases possibility of moisture loss in long-term frozen storage which increases the contaminant concentration by reducing the wet weight of the sample May result in under reporting of risk for people who eat other parts of the fish
Whole Fish	Provides sufficient tissue mass to analyze multiple contaminants, and the corresponding QC samples for each analytical method, and for archive Allows tissue preparation in a controlled laboratory setting Assesses the risk more accurately to populations who eat fillet tissue plus other portions of the fish (or the whole fish) since different fish parts may have different levels of contaminants May be more applicable to smaller species of fish	Requires the fish to be sacrificed Whole fish have increased concentrations of PCBs and PFAS, compared to fillet samples (Fair et al. 2019 and Levanduski et al. 2024). An advisory could overestimate the risk for people who only consume fish fillet. Increases shipping costs due to whole fish shipping Requires laboratory preparation and ample space for freezer storage, leading to increased costs

Skin-On vs Skin-Off Fish Samples

The choice between skin-on or skin-off fish fillet samples should be based on the target contaminants, and the consumption practices of the target audience.

Analyzing skin-on fillets with the belly-flap is more representative for assessing potential exposure to PCBs, dioxin/furans, or organochlorine pesticides since these contaminants concentrate in the fatty tissue. It also accounts for the fact that some consumers may not trim the more contaminated fatty tissues from the edible muscle fillet tissue.

Analyzing skin-off fillets for solely assessing mercury contamination in fish is more representative of potential exposure since mercury concentrates in fillet muscle tissue (Gutenmann and Lisk, 1990). Analysis of skin-off fillets may also be more appropriate for some scaleless fish species such as catfish since consumers typically remove their skin before cooking them.

Table 3. Advantages and disadvantages of skin-on versus skin-off fillets

Sample Type	Advantages	Disadvantages
Skin-on Fillets - Samples are analyzed with the skin intact (most studies include removal of the scales, but leave the skin on)	Considers consumers who eat the fillet and skin Involves a more representative approach for assessing many organic contaminants (e.g., PCBs) which bioaccumulate in fatty tissue	May result in a lower mercury concentration per gram of skin-on fillet than per gram of skin-off fillet (Gutenmann and Lisk, 1990), which may result in less protective advisories
Skin-off Fillets - Samples are analyzed with the skin removed	Aligns with the consumption habits of the general population Considered to be appropriate for some target species such as catfish and other scaleless finfish species because the skin is typically removed by consumers Involves a more representative approach for assessing mercury contamination, which accumulates in fillet tissue	Does not account for the consumers who eat the fillet and skin May result in a lower organic contaminant concentration per gram of skin-off fillet than per gram of skin-on fillet, which may result in less protective advisories