Per- and Polyfluorinated Alkyl Substances

Environmental DNA (eDNA) is quickly gaining attention as a tool for ecological surveying. It is gaining popularity due to its accuracy, compared to intrusive traditional methods. Below is a selection of Frequently Asked Questions on this topic, complementing Bureau Veritas’ technical webinar. Questions related to this method are either focused on applicability or analytical considerations.

Applicability of eDNA

1. How does eDNA work and what are the advantages of eDNA testing?

eDNA testing is cost effective, more accurate than traditional methods, has less risk of pathogen transfer, and is less invasive to species (especially at-risk species) and ecosystems. The eDNA tests can be performed for multiple species using a single sample and it provides early detection of invasive species. This molecular biology method detects DNA of target species through quantitative polymerase chain reaction (qPCR) using the TaqMan^® Assay.

2. Can the presence of specific fish species be confirmed through the eFish eDNA assay?

Our current eFish eDNA assay tests for the presence of a group of 12 fish species: Sockeye salmon, Pink salmon, Chum salmon, Arctic Grayling, Cutthroat trout, Rainbow trout/Steelhead, Chinook salmon, Coho salmon, Atlantic Salmon, Dolly Varden, Round Whitefish, and Slimy Sculpin. It does not identify a specific species. The test can be reported as “detected” of any one specie or as “not detected” result.

3. Can DNA from a species of fish that no longer lives in a watercourse, remain in the habitat for some time and bias the results?

Generally, eDNA persists in the environment for a short time. The duration is dependent on environmental conditions but is quantified, in standard conditions, between 4-58 days. So within this time frame, it is still possible to determine the presence of fish species that may no longer be present in the watercourse

4. What is the applicability of the method to terrestrial, or semi-aquatic species? Is a fully aquatic life stage mandatory for using the method?

Suspension of eDNA in water allows for transport (active or through osmotic diffusion) through the system (transport distances are variable and depend on output rates, flow, etc.).  When suspended and transported in water, eDNA can be easily captured, extracted and tested for the focal taxa. eDNA methods can also be applied to terrestrial habitats, but advantages conferred by aquatic systems are lacking. Hence, design considerations are even more critical and false negatives are more likely. eDNA has just been applied successfully on Sharp-tailed snake (Contia tenuis). So it can be done but it’s not the ‘norm’.

5. How are hybridization issues addressed, for example, between rainbow and cutthroat trout, or subspecies?

eDNA detects mitochondrial DNA. Thus, only the maternal lineage would be detected and subspecies cannot be identified.

6. Does eDNA settle in lakes? Should samples be collected further down the water column?

Research is ongoing regarding distribution and persistence of eDNA in environments.  However, at this time, there is evidence suggesting that most eDNA is found close to the area the animal can be found, likely because of degradation.  Thus, an understanding of the ecology of the organism is important for determining where to best collect samples.

Analytical Considerations for eDNA

7. What volume of water is required per sample?

The ideal volume required is 1L of water which is to be filtered through a cellulose nitrate filter (45µm pore size). This increases detection probabilities, however in some cases this volume may not be possible due to filter clogging. The minimum volume to be passed through the filter is 70ml.

8. What type of containers are required? Does Bureau Veritas provide these? What is the hold time and should samples be submitted on ice at <10C?

The intent is to eventually provide sampling kits, including required containers and filters but these are not supplied by Bureau Veritas at this time. It is not necessary to submit preserved filters on ice, and samples should be filtered prior to submission to maximize the potential DNA recovery and reduce the amount of degradation. Water samples should be collected in 1L Nalgene bottles. If the water sample is to be filtered in the field, insert the rolled-up filter in a 2mL tube, preserved with ethanol or silica and then transport to the lab.

9. How quickly should samples be submitted to the laboratory after filtration? How long do preserved samples last?

It depends on the contents of the water sample and what remains on the filter. Filtering the water sample and preserving the filter within 24 hours of sample collection is the recommended approach. Preserved filters should be sent to the laboratory to have eDNA recovered as soon as possible. There is no definite or standard time frame because the degradation rate will be related to properties of the microbial community of the sample. The ability to detect eDNA from target species does demonstrably decline over time from stored filter samples. Extracted eDNA is retained in a buffered solution and it is more stable than eDNA on the filter.

10. What is the detection limit? i.e how many fish need to be present in a stream to shed enough DNA for a positive result?

Detection of eDNA is not related as closely to abundance as it is to proximity.  It is possible to detect eDNA from one animal if enough cells were shed recently and very close to the sampling site.  That being said, if sampling did not occur close enough to these organisms, it is possible that no DNA would be present in the sample, and would not be detected.

11. How many species are available for the eDNA assay? Is this library collaborative across the country?

Each eDNA assay (other than eFish) has been designed for a specific species. Maxxam currently has 14 validated eDNA assays, and we are working to validate more species (as presented in the webinar). These validations are aligned with 13 additional assays developed at the University of Victoria, BC. There is no collaborative library across the country.

12. Are there any challenges with collecting, transporting and preserving samples when working at remote sites? Or alternatively, how do samples need to be handled and how long can it take between collection and lab processing?

There are considerations around these aspects that are easily accommodated by sound sample design. The standards to sample collection, preservation and submission for laboratory analysis are documented in the Protocol prepared for the British Columbia Ministry of Environment. 

13. Does Bureau Veritas use externally developed assays for species not covered by the current method?

Bureau Veritas’ focus is not on developing new assays. Typically, we use developed assays that are revalidated in-house and applied to commercial testing. However, if we are provided with a developed assay, revalidation of applicability for species and commercial testing can be done. This ensures that the assays have been designed properly and they do not detect closely related species or sympatric species.

For more information, Contact Bureau Veritas today.