Environmental DNA

The application of eDNA testing is particularly advantageous for aquatic and semi-aquatic species as DNA shed from the target organism is transported through the aquatic environment, improving the ability to detect the target species.

eDNA testing enables more informed decision-making about biodiversity and ecosystem health due to its enhanced sensitivity over conventional methods, which are time and labour intensive, subject to observer bias and limited in ability to confirm distribution for cryptic species or those with low population abundance.

Advantages of environmental DNA Testing

eDNA testing has several advantages when compared to conventional survey methods:

• Improved sensitivity – eDNA detects target species that are cryptic or present at low density with improved power of detection over conventional surveying.
• Time savings – reduced field time for sampling compared to conventional surveys.
• Cost effective – eDNA assays for multiple target species can be tested from a single collected sample.
• Less invasive – no need for trapping/electrofishing and reduced impact to sensitive habitats, including less risk of pathogen transfer.
• Permit and license not required – water sampling for eDNA with no requirement to trap, handle or even observe the target species.
• Reduced observer bias – simple environmental sam-pling reduces error associated with observer experience or variation in surveying efforts.
• Improved field safety – field sampling can occur during daylight hours and better weather conditions.
• Accurate – properly designed eDNA tests are specific to the target species not requiring a qualified expert in the field to identify the species.
• Retroactive testing – properly archived samples can be tested at a future date for additional species of interest.
• Expanded window of surveying – eDNA sampling can be completed outside of conventional restraints, e.g., window of amphibian calling.

environmental DNA Testing Protocol

Several methodologies for eDNA detection exist. Most methods utilize quantitative polymerase chain reaction (qPCR), also known as real-time PCR, to detect the presence of a target species. qPCR is a highly sensitive and specific DNA analysis that allows for detection of low quantities of DNA through cycles of PCR that exponentially generate many copies that are visualized by the lab instrument with a fluorescent reporter dye.

Given the nature of eDNA being low quantity, subject to variables in the environment and having a varied rate of release from the organism, eDNA assay results are qualitative (detect/non-detect) as opposed to quantitative.

Bureau Veritas has licensed the IntegritE-DNA^TM method from UVic Industry Partnerships, a technique developed by Dr. Caren Helbing [1] (Figure 1). The first step of this protocol is sample collection, filtration and preservation, assuring best eDNA recovery and conservation. The second step is the eDNA extraction from the sample collected. This protocol incorporates a quality check on the extracted eDNA as a third step in the form of an IntegritE-DNA™ qPCR assay to test for amplifiable eDNA extracted from the filtered water sample, but also absence of inhibitors. This is a critical step in preventing false negative eDNA results.

Figure 1: eDNA protocol at Bureau Veritas

1. Sample Collection, Filtration & Preservation
Image	For the best eDNA recovery, sample filtration and preservation (silica or 95 to 100% molecular grade ethanol) should take place within 24 hours of sample collection.
2. eDNA Extraction
Image	One quarter of the filter is extracted for eDNA testing. The remainder filter can be archived for future eDNA testing for the same species or new suspected species at site under study.
3. eDNA Quality Check
Image	IntegritE-DNA™ QC check for DNA quality is completed prior to testing for the target species. Only eDNA samples that successfully amplify IntegritE-DNA™ are carried forward for testing the target species. This minimizes the risk of false negatives due to poor DNA quality or qPCR inhibition.
4. Target Species qPCR Assay
Image	eDNA test for the target species. Some assays (e.g. eFish) test for multiple potential species (see table 1). Eight technical replicates are tested and reported for statistical confidence in detecting the target.

The fourth step of this protocol is a species-specific eTarget qPCR assay and confirms the detection of target species DNA. Eight replicates are analyzed for each sample to provide sufficient statistical power to confirm detection or no detection of the target species. Positive controls are incorporated into this method to ensure testing performs as expected.

The volume of extracted eDNA is sufficient for four to five eTarget qPCR assays should multiple species detection from the same eDNA extract be required. This reduces overall cost by not requiring additional sampling, filtering, DNA extraction and integrity check if additional species are to be tested.

Sample Collection

Considering the variety of target species and their diverse habitat, there is substantial variation in methods for sample collection, filtration, preservation and processing (Figure 2).

Figure 2: Sample collection workflow

1. Water Collection	2. Water Filtration	3. Filter Preparation	4. Preservation in Silica
Image	Image	Image	Image

To maximize detection rates, considerable planning and preparation of the study design is key to a successful “in the field” collection protocol and expert consultation is advised.

Prior to any such consultation, we recommend a review of the detailed discussion presented in “Environmental DNA Protocol for Freshwater Aquatic Ecosystems, Version 2.2” [2].

eDNA preservation of the collected sample is important to ensure successful testing at the laboratory. As a rule, water samples should be filtered, preserved and stored in either silica or molecular grade ethanol (95 – 100 % molecular grade ethanol), or silica within 24 hours of collection (and prior to submission to the lab). Once preserved and stored, DNA in filters should remain viable for 6 months, or longer if stored in a non-self-defrosting freezer at -20ºC or below. Lab extracted eDNA (in a buffered solution) is most stable and will remain viable for years under these same freezer storage conditions.

Considerations for Your environmental DNA Study

To ensure effective application of eDNA testing, the following must be considered:

Field Sampling Design

• When to sample.
• Where to sample.
• Number of sample replicates – temporally and spatially.

Sample Filtration & Preservation

• Water sample should be filtered as soon as possible and within 24 hours of collection.
• Preserve filter in 95 - 100 % molecular grade ethanol or silica immediately following sample filtration.

eDNA Assay

• Specificity – eDNA assay validation needs to include verification that only the target species is detected.
• Lab protocol includes checks and controls to assess for DNA degradation and assay inhibition.
• Number of test replicates in the lab ensures sufficient statistical power (Bureau Veritas tests 8 replicates per sample).

Available environmental DNA Assays

Currently available eDNA assays are presented in Table 1. All of the listed assays are species-specific except the eFish assay. The eFish assay is a general test that identifies eDNA from 19 fish species (Table 1) but cannot distinguish between these fish species.

Table 1: Available eDNA Assays

	eDNA TEST	SPECIES	COMMON NAME
Fish	ANRO	Anguilla rostrata	American Eel
Fish	CAAU	Carassius auratus	Goldfish
Fish	CACA	Catostomus catostomus	Salish Sucker
Fish	ESLU	Esox lucius	Northern pike
Fish	ESNI	Esox niger	Chain pickerel
Fish	HYPR	Hypomesus pretiosus	Surf Smelt
Fish	MIDO	Micropterus dolomieu	Smallmouth Bass
Fish	MISA	Micropterus salmoides	Largemouth bass
Fish	ONCL	Oncorhynchus clarkii	Cutthroat trout
Fish	ONGO	Oncorhynchus gorbuscha	Pink Salmon
Fish	ONKI	Oncorhynchus kisutch	Coho salmon
Fish	ONMY	Oncorhynchus mykiss	Rainbow trout (Steelhead trout)
Fish	ONNE	Oncorhynchus nerka	Sockeye salmon
Fish	ONTS	Oncorhynchus tshawytscha	Chinook salmon
Fish	RHUM	Rhinichthys umatilla	Umatilla Dace
Fish	SACO	Salvelinus confluentus	Bull trout
Fish	THAR	Thymallus arcticus	Arctic grayling
Fish	THPA	Thaleichthys pacificus	Eulachon
Fish	eFish*	Anguilla rostrata (ANRO)	American eel
Fish	eFish*	Cottus cognatus (COCO)	Slimy sculpin
Fish	eFish*	Esox lucius (ESLU)	Northern pike
Fish	eFish*	Gasterosteus aculeatus	Three-spined stickleback
Fish	eFish*	Micropterus dolomieu (MIDO)	Smallmouth bass
Fish	eFish*	Micropterus salmoides (MISA)	Largemouth bass
Fish	eFish*	Oncorhynchus clarkii (ONCL)	Cutthroat trout
Fish	eFish*	Oncorhynchus gorbuscha (ONGO)	Pink salmon
Fish	eFish*	Oncorhynchus keta (ONKE)	Chum salmon
Fish	eFish*	Oncorhynchus kisutch (ONKI)	Coho salmon
Fish	eFish*	Oncorhynchus mykiss (ONMY)	Rainbow trout
Fish	eFish*	Oncorhynchus nerka (ONNE)	Sockeye salmon
Fish	eFish*	Oncorhynchus tshawytscha (ONTS)	Chinook salmon
Fish	eFish*	Prosopium cylindraceum (PRCY)	Round whitefish
Fish	eFish*	Salvelinus confluentus (SACO)	Bull trout
Fish	eFish*	Salvelinus malma (SAMA)	Dolly varden
Fish	eFish*	Salmo salar (SASA)	Atlantic salmon
Fish	eFish*	Thymallus arcticus (THAR)	Arctic grayling
Fish	eFish*	Thaleichthys pacificus (THPA)	Eulachon
Frog	ANBO	Anaxyrus (Bufo) boreas	Western toad
Frog	ASMO	Ascaphus montanus	Rocky mountain tailed frog
Frog	ASTR	Ascaphus truei	Pacific (Coastal) tailed frog
Frog	LICA	Lithobates (Rana) catesbeiana	North American bullfrog
Frog	LIPI	Lithobates (Rana) pipiens	Northern leopard frog
Frog	RAAU	Rana aurora	Northern red-legged frog
Frog	RACA	Rana cascadae	Cascades frog
Frog	RALU	Rana luteiventris	Columbia spotted frog
Frog	RAPR	Rana pretiosa	Oregon spotted frog
Frog	SPIN	Spea intermontana	Great Basin spadefoot
Other	AMMV (AMTI)	Ambystoma mavortium	Blotched tiger salamander
Other	AMMV (AMTI)	Ambystoma tigrinum	Tiger salamander
Other	COTE	Contia tenuis	Sharp-tailed Snake
Other	GLIN	Glyptemys insculpta	Wood turtle
Other	SOBE	Sorex bendirii	Pacific water shrew

*The eFish assay is designed to be non-specific. It may detect eDNA from other fish species in addition or instead of the specific species listed here, which the assay has been validated for. It is important to note that in effort to design the eDNA assay to detect presence of any fish species, the assay also detects presence of some amphibian species.

environmental DNA Assay Development

To date, Bureau Veritas’ approach to eDNA assay development for commercial applications has been in-house (re)validation of previously published/documented methods. This ensures assay transferability for laboratory-controlled and field-derived samples regarding two crucial parameters: sensitivity (reliable detection criteria) and specificity (no cross-reaction, especially for closely related or sympatric species). Through client feedback, Bureau Veritas will continue to expand the suite of commercially available eDNA assays presented in Table 1.

It should be noted, however, that the above approach leaves an important, unanswered question. What do we do for target species where no eDNA assay has been developed?

Future availability of DNA sequence data for additional species will undoubtedly allow for primer and probe design. However, a prerequisite to functional assay design, which is the availability of tissue samples (or total DNA) from target species (and closely-related species or sympatric species), presents substantial difficulty for low density/rare species and could be prohibitive for endangered species. Bureau Veritas intends to establish third party, collaborative agreements that will facilitate the development of novel eDNA assays for target species of interest.

Limitations of environmental DNA Testing

eDNA testing has limitations that should be considered when designing the field survey and interpreting laboratory results:

• Binary results - eDNA testing provides a detected or not detected result. Results cannot be extrapolated to determine abundance or density of the target species.
• Limits to geographical information – since eDNA is transported in an aquatic environment, it cannot provide exact detail on the proximity of the organism or duration in the environment.
• eDNA does not confirm presence of a living organism, but rather detection of DNA from the target species.
• Source of DNA (e.g., gametes vs. skin) is not revealed by eDNA.
• Results are limited to detect or not detect - the age, gender, size, or reproductive status of the organism is not revealed.
• Subject to environmental variability for movement of DNA (water flow rate) and stability (water temperature, pH, salinity, exposure to microbes and ultraviolet light, which degrade DNA). Typically, eDNA can be detected 7 to 21 days after being released to the environment.

Moving Forward

eDNA testing is quickly becoming a reliable and robust technique that complements traditional environmental monitoring. It should be recognized that traditional survey techniques still have select advantages to eDNA testing, the most relevant of which may be their ability to distinguish live species and the different life stages of those species. Further, standards for eDNA testing across laboratories are not yet established but need to be carefully considered to create maximum utility of this evolving technique.

Bureau Veritas is proud to be the first laboratory in Canada accredited for eDNA testing by the Standards Council of Canada to rigorous ISO 17025 standards. We are committed to working closely with the eDNA community to develop consensus-based standards that will enable better interpretations of eDNA results.

References

[1] Veldhoen N, Hobbs J, Ikonomou G, Hii M, Lesperance M, and Helbing CC. Implementation of Novel Design Features for qPCR-Based eDNA Assessment. PLoS One. 2016; 11(11): e0164907. Published 2016 Nov 1. DOI:10.1371/journal.pone.0164907

[2] Jared Hobbs and Caren Goldberg. Environmental DNA Protocol for Freshwater Aquatic Ecosystems (Version 2.2). Prepared for: BC Ministry of Environment. November 2017