Metal mining technical guidance: revised guidance for sample sorting, chapter 1
Sample Processing Methods
The sample processing steps recommended for all EEM benthic invertebrate sample processing are schematically represented in Figures 1-3. These are illustrative examples which would be applicable to most samples with decision points based on type and amount of organic and inorganic material. If sufficient numbers of organisms are encountered in a given fraction then a subsampling method can be considered (Figure 2 and 3, Section 3). Each laboratory should outline sample-specific sorting and subsampling procedures and provide full documentation of the methods used.
Preservation quality check and initial assessment of samples
Immediately upon return from the field, a quality check should be performed on the samples to ensure that the preservative has effectively penetrated the entire sample (Figure 1). This is especially important if there is a large amount of fine material in the samples. At this time, an initial assessment of the samples can be conducted to help determine the most efficient and cost effective method of sample processing. This assessment would include volume and type of debris material in the sample and if possible an assessment of the quantity of invertebrates in the samples. However, until sorting commences it is sometimes difficult to determine rough numbers of organisms in a sample. If sample size is small to moderate, sorting of the entire sample may be the simplest and most cost efficient method of sample processing. If large numbers of organisms are present then subsampling may be warranted (see section 3.1 for determining when to subsample).
Figure 1: Summary of general sample processing protocols
Washing, Elutriation and Storing of Samples
If samples have large amounts of inorganic matter (silt and sand), which was not removed in the field by bucket swirling and pouring the floating organic matter onto a sieve (Needham and Needham 1962), this procedure should be done in the laboratory as it can reduce sorting time by as much as 50% (Rosillon 1987, Ciboroski 1991). Furthermore, this elutriation process minimizes the adverse effects large amounts of inorganic matter may have on invertebrates (damaged specimens are more difficult to identify), or the subsampling process (easier distribution on sieves or trays). Before discarding any inorganic sample portions, a thorough visual inspection should be conducted to detect any cased or shelled invertebrates (e.g., rock encased caddisflies) that may be retained in the inorganic portion.
Prior to microscopic sorting of samples in the laboratory, sample preservative should be thoroughly rinsed from the organic matter. Sorting samples directly from a 10 % formalin solution is unnecessary and unwise from a health and safety perspective. The preserved sample can be placed into a sieve (freshwater = 500 µm) and the preservative and residual fine debris rinsed from the sample. Disposal of used preservatives must be performed in accordance to provincial hazardous waste regulations (e.g., BC Special Waste Regulations, Alberta Waste Control Regulation, Ontario Regulation # 347 etc.). While flushing the samples, care should be taken not to damage the organisms with too much water pressure. Samples can be sorted in water immediately, providing they are transferred back to preservative at the end of the workday (i.e., after 8 hours). Alternatively, samples may be transferred to 80% ethanol after the washing process, prior to sorting and taxonomic work provided that the sample has been thoroughly exposed to 10% formalin for a minimum of 72 hours. After sorting or identification, most freshwater macro-invertebrates should be stored in a solution of 70 to 80% ethanol, and 5% glycerin in vials or jars with air-tight lids. (Marine samples should be transferred to 70-80% ethanol within 3 months to protect the samples from breakage as the organisms become brittle.) If screw-cap lids are used, they should be sealed with wax film, and will require periodic checking (once or twice annually) to replace evaporative losses. Since preservatives can have a variety of effects on the lengths and weights of invertebrates (Howmineer 1972, Leuven et al. 1985), it is essential that the exact preservation protocol for all steps be drafted a priori and that it be consistently applied between all phases, areas, replicate stations and habitats.
Fractionation and Sorting of Samples
For samples, which have large amounts or pieces of organic matter, the samples can be divided in the laboratory into appropriate size fractions to expedite the sorting process (Figure 2). The most commonly used fractions are coarse (> 1.00 mm) and fine (500 µm - 1.00 mm), which correspond to the divisions used to define coarse and fine particulate organic matter (CPOM and FPOM, respectively). If there are very large pieces of organic material or large invertebrates it is sometimes beneficial to separate these from the rest of the sample with a 4.00-mm sieve. All fractions should then be sorted and if warranted, by large numbers of organisms, the fractions can be subsampled independently (see Section 3.0). Note that for samples with large amounts of clumping algae, fractionation may not be beneficial. Careful note taking is recommended for these more complex sorting procedures so that densities are calculated accurately. After the initial washing and fractionation of samples, the invertebrates should be sorted from the debris by trained technicians on a gridded tray or petri dish under a dissecting microscope at 10X to 20X magnifications.
Various additional techniques exist which are designed to speed up the manual sorting required for benthic invertebrate samples. These techniques include flotation of organisms in high-density solutions (e.g. magnesium sulphate, sodium chloride, calcium chloride, sugar, D-mannitol) or bubbling to trap animals in the surface film and to promote separation from denser sediment particles. Heavier organisms such as molluscs will not float and must be removed directly with forceps. Also, various stains such as rose bengal or phloxine B at a concentration of 100 mg/l can be used to make the organisms easier to see. A cautionary note is required, however, as the efficiency of many of these techniques differ between taxa (Rosillon 1987). If these techniques are used, then the QA/QC performed on these samples should be designed to detect any differential sorting efficiency of taxa caused by the staining procedures.
Figure 2: Sample processing with sieve fractionation
Figure 3: Sample processing without sieve fractionation
QA/QC for sample sorting
The basic processing of benthic invertebrate samples involves the time-consuming removal of organisms from large amounts of debris. Inevitably, processing errors occur during this sorting phase regardless of how diligent the processor and must be estimated (e.g. Kreis 1986, 1989). Thus, the first QA/QC component of benthic invertebrate sample processing is the requirement to assess this sorting efficiency (i.e., the proportion of total organisms extracted from the sample upon sorting). High sorting efficiencies will ensure that endpoint calculations are reasonably reliable and without bias between samples. The recommendation for assessing sorting efficiency for the PP and MM EEM programs is that at least 10% of all samples from each study be resorted and any organisms found on the second sort be enumerated. The criteria for an acceptable sort is that > 90% of the total number of organisms are recovered during the initial sort. If > 10% of the total number is found during the resort, then all the samples within that particular group of samples requires resorting. The factors which should be considered when determining similar groups of samples include:
- sampling area
- habitat class
- individual sorters
The QA/QC guidelines apply independently to each sample fraction and group of samples sorted. A further criterion which would require a resort is if an entire group of benthic invertebrates were not extracted from the debris (e.g., ostracods were not recognized and sorted), even if the missed organism constituted < 10 % of the total numbers. Unsorted and sorted fractions are to be retained until taxonomy and sorting efficiency are confirmed and the data are reviewed by the regional EEM coordinator. The sorting efficiencies obtained on each set of samples should be calculated as below and reported for all studies. Note, that if the sorting efficiency is acceptable (>90%), the "resorted" organisms are left out of any further analysis as they are not part of the complete sorting process.
Determination of sorting efficiency
Sorting Efficiency = [1- (# in QA/QC re-sort/(# sorted originally + # in QA/QC re-sort)]*100
Example:
- 37 invertebrates found in the QA/QC re-sort
- 393 invertebrates were recovered in the original sort process
- the sorting efficiency is calculated as 91.4% :
-% Sorting Efficiency = [1- (37/(393 + 37))]*100
If the efficiency is less than 90% for any one group of samples, all samples within that group of samples should be completely re-sorted.
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