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Biomonitoring requires thorough evaluation of methods used to detect impairment. Using a data set of 202 reference sites and 66 test sites from the Fraser River, British Columbia, Canada, we analyzed the effects of assemblage (benthic macroinvertebrates and periphyton) and reference site classification (ecoregion, stream order, null models, and biotic groups) on two bioassessment methods (BEAST (BEnthic Assessment of SedimenT) and RIVPACS (River InVertebrate Prediction And Classification Scheme)). Although largely undisturbed, the Fraser River is affected in some areas by logging, mining, agriculture, pulp mill effluent, and urban land use. Overall performance was evaluated using the harmonic mean of precision, accuracy, and two measures of sensitivity. Invertebrates and periphyton were equally accurate and precise, but invertebrates were more sensitive. Biotic groups were the least accurate and precise classification, but also the most sensitive and had the greatest overall performance. BEAST was slightly less accurate and precise than RIVPACS, equally sensitive to simulated disturbance, and more sensitive to real disturbance. Assessments with higher sensitivity frequently had lower accuracy, indicating a possible trade-off among these aspects of performance.

Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93–99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity.

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