at-risk amphibians study - govtogetherbc · at-risk amphibians study i preface ... at-risk...

At-Risk Amphibians Study

APRIL 2015

George Massey Tunnel Replacement Project

AT-RISK AMPHIBIANS STUDY

i

Preface

Technical studies were initiated in 2014 to support both ongoing Project planning and future

permitting and approval requirements. Since the technical studies were initiated prior to

finalizing the scope of the proposed Project, the scope of some studies consider physical

activities and spatial areas that are beyond the scope of the Project assessed under the B.C.

Environmental Assessment Act. The results of future studies will be presented in the

Environmental Assessment Certificate Application that will be prepared for the Project.



ii

Executive Summary

The B.C. Ministry of Transportation and Infrastructure (Ministry) is proposing the George

Massey Tunnel Replacement Project (Project) to meet regional, provincial, and national

transportation management goals. The proposed Project involves replacing the George Massey

Tunnel (Tunnel) with a new bridge spanning the Fraser River South Arm and Deas Island,

decommissioning the Tunnel, and improving Highway 99 between Bridgeport Road in Richmond

and Highway 91 in Delta.

To support project planning and future permitting and approval requirements, the Ministry

initiated studies to document existing conditions of environmental and socio-economic

components that could potentially be affected by the Project. An at-risk amphibians study was

conducted and was focused on the collection and collation of information to further understand

and document existing conditions in the study area for amphibian species at risk.

Northern red-legged frog (red-legged frog) was identified as a key at-risk amphibian species that

has the potential to occur in South Richmond and North Delta, specifically in watercourses

within and adjacent to the Highway 99 right-of-way. The spatial scope of this study extends

along the Highway 99 corridor from immediately north of the Highway 91 interchange in

Richmond and continues south to the interchange at King George Boulevard.

Red-legged frog is Blue-listed by the B.C. Conservation Data Centre, and is considered a

species of Special Concern by the Committee on the Status of Endangered Wildlife in Canada.

It is also on Schedule 1 of the federal Species at Risk Act. Study of the potential presence of

this species was therefore selected as a means of assessing the use of the Project Area by at-

risk amphibians.

Using environmental DNA (eDNA) methods, a survey was conducted in May 2014 to assess the

presence of red-legged frog DNA in watercourses within and adjacent to the Highway 99 right-

of-way. Presence of red-legged frog eDNA within a sampled aquatic feature was interpreted as

an indication that the sample area was being used by this species, either at or shortly preceding

the time of sample collection.

Red-legged frog DNA was detected at two of the 15 aquatic features sampled: one near the

intersection of Highway 99 and Highway 91, and the other closer to the intersection of Highway

99 and Highway 10. This suggests the potential use of aquatic features in the vicinity of the

Project by at-risk amphibians.



iii

Abbreviations and Acronyms

Term Definition

µS/cm microsiemens (unit of conductivity) per centimetre

CDC Conservation Data Centre

COSEWIC Committee on the Status of Endangered Wildlife in Canada

DNA deoxyribonucleic acid

eDNA environmental deoxyribonucleic acid

FLNR Ministry of Forests, Lands and Natural Resource Operations

Ministry Ministry of Transportation and Infrastructure

PCR polymerase chain reaction

qPCR quantitative polymerase chain reaction

SARA Species at Risk Act

SC Special Concern

Tunnel George Massey Tunnel

UTM Universal Transverse Mercator



iv

Glossary

Term Definition

Blue List; Blue-listed The B.C. CDC designation for species considered to be of special concern (formerly vulnerable) in British Columbia.

critical habitat

The habitat that is necessary for the survival or recovery of a listed wildlife species and that is identified as the species’ critical habitat in the recovery strategy or in an action plan for the species. SARA S.C. 2002, c. 29

element A term used by the B.C. CDC to describe a wildlife species or subspecies at risk.

extirpated Species that no longer exist in the wild in B.C. but occur elsewhere.

Highway 99 corridor The right-of-way owned by the Province of B.C. for Highway 99 from the Peace Arch border crossing in Surrey to the Oak Street Bridge in Richmond.

Project Area The Project footprint plus the Project disturbance area.

Project disturbance area

All lands and lands under water, except the Project footprint, which are subject to disturbance during Project construction and required for maintenance activities during Project operation.

Project footprint The land and water surface occupied by Project facilities and structures.

Red List; Red-listed The B.C. CDC designation for species considered extirpated, endangered, or threatened.

taxon A group of one or more populations of an organism or organisms to form a unit.



v

TABLE OF CONTENTS

Preface .......................................................................................................................................... i

Executive Summary .................................................................................................................... ii

Abbreviations and Acronyms ................................................................................................... iii

Glossary ...................................................................................................................................... iv

1.0 Introduction ..................................................................................................................... 1

1.1 Study Background ................................................................................................. 1

1.2 Study Components and Major Objectives ............................................................. 1

2.0 Review of Available Literature and Data ....................................................................... 2

3.0 Methods ........................................................................................................................... 4

3.1 Spatial Scope ........................................................................................................ 4

3.2 Temporal Scope .................................................................................................... 4

3.3 Study Methods ...................................................................................................... 4

3.3.1 Background of Environmental DNA Method ........................................... 4

3.3.2 Habitat Assessment ................................................................................ 5

3.3.3 Sample Collection ................................................................................... 5

3.3.4 Sample Filtration ..................................................................................... 6

3.3.5 Laboratory Analysis ................................................................................ 6

4.0 Results ............................................................................................................................. 8

4.1 Incidental Observations ......................................................................................... 9

5.0 Discussion ..................................................................................................................... 10

5.1 Key Findings ........................................................................................................ 10

5.2 Data Gaps and Limitations .................................................................................. 10

6.0 Closure ........................................................................................................................... 12

7.0 Statement of Limitations .............................................................................................. 13

8.0 References ..................................................................................................................... 14



vi

LIST OF TABLES

Table 1 At-Risk Amphibians Study Components and Major Objectives ............................. 1

Table 2 Amphibian Species with Potential to Occur in the Project Area ............................ 2

Table 3 Results of qPCR Analysis for Red-legged Frog DNA in the Study Area ............... 8

Table 4 Habitat and Water Quality Data for Positive Detection Sites ................................. 9

LIST OF APPENDICES

Appendix A Figures

Appendix B Red-legged Frog Tissue Sample Data for Assay Development

Appendic C Complete Sample Data and Results of qPCR Analysis



1

1.0 Introduction

This report presents the objectives, methods, and findings of the at-risk amphibians study

undertaken to support project planning as well as future permitting and approval requirements

for the George Massey Tunnel Replacement Project (Project).

1.1 Study Background

The British Columbia Ministry of Transportation and Infrastructure (Ministry) is proposing the

Project to meet regional and provincial transportation management goals. To support project

planning and future environmental permitting and approval, the Ministry initiated studies in early

2014 to understand and document existing conditions of environmental components that could

potentially be affected by the Project.

Because the studies were planned prior to the Project scope being finalized, a broader spatial

area was considered to accommodate potential refinements in the Project design. This broader

spatial scope was established based on a general understanding that the Project would involve

modifications of the Highway 99 corridor, including replacement of the George Massey Tunnel

(Tunnel) with a clear-span bridge, removal of all or part of the Tunnel, and replacement or

upgrade of interchanges and widening of the highway as required.

1.2 Study Components and Major Objectives

Key components and major objectives of the at-risk amphibians study, and a brief overview of

the each study component are provided in Table 1.

Table 1 At-Risk Amphibians Study Components and Major Objectives

Component Major Objective Brief Overview

Review of literature and available data

Determine the potential for presence of at-risk amphibians in the study area

A review of the B.C. Conservation Data Centre (CDC) Species and Ecosystems Explorer database to compare known geographic range and habitat requirements of at-risk amphibian species to habitat available in the study area.

eDNA sampling and analysis

Determine whether at-risk species with a potential to occur in the study area are likely to be present in watercourses in and adjacent to the Project Area.

Based on a review of literature and available data, northern red-legged frog (Rana aurora) was identified as a key at-risk amphibian species that has the potential to occur in the study area.

Environmental deoxyribonucleic acid (eDNA) was used to assess the presence of red-legged frog DNA in Project area aquatic features potentially used by this species for breeding or other requisite life history stages.



2

2.0 Review of Available Literature and Data

To determine the potential for presence of at-risk amphibians in the study area, a search of the

B.C. Conservation Data Centre (CDC) Species and Ecosystems Explorer database was

conducted. This online database includes wildlife species considered to be at-risk by the CDC

(i.e., Red- or Blue-listed), or by the Committee on the Status of Endangered Wildlife in Canada

(COSEWIC), or under schedule 1 of the federal Species at Risk Act (SARA), SC 2002, c. 29.

The species list generated from the CDC database was refined by comparing each species’

known geographic range and habitat requirements to the available habitat in the study area.

Five amphibian species are anticipated to potentially occur within the study area (Table 2).

Table 2 Amphibian Species with Potential to Occur in the Project Area

Scientific Name Common Name SARA Schedule1 COSEWIC2 B.C. List3

Ambystoma gracile Northwestern salamander

Not at risk (1999)

Yellow

Lithobates catesbeianus American bullfrog Exotic

L. clamitans Green frog Exotic

Pseudacris regilla Northern Pacific treefrog

Yellow

Rana aurora Red-legged frog SC (2005) SC (2004) Blue

Notes: 1

SARA listing: SC = Special Concern 2 COSEWIC: SC = Special Concern

3 B.C. List: Blue = Special Concern; Yellow = Secure; Exotic = introduced, non-native species

not listed

Pacific treefrog, red-legged frog, and northwestern salamander are native to the Lower

Mainland. American bullfrog and green frog are introduced species. American bullfrog, green

frog, Pacific treefrog, and northwestern salamander have been observed previously in or near

the Project area (Corporation of Delta 2003). Numerous bullfrog tadpoles, several northwestern

salamanders, and one adult red-legged frog were observed incidentally during the Freshwater

Fish and Fish Habitat technical study for the Project. The red-legged frog was caught in a

minnow trap in the roadside ditch on the north side of the Highway 99 right-of-way, east of the

King George Boulevard interchange (Appendix A, Figure 1d) on April 22, 2014. The frog was

released into suitable habitat nearby. Subsequently, a second adult red-legged frog was

observed approximately one kilometre north of the Highway 99 centreline, between Highway 91

and Highway 1A, during the field program for the Conspicuous Raptor and Great Blue Heron

Study.



3

Of the species with confirmed or potential presence in the Project Area, red-legged frog is a key

species that is considered at-risk by federal and provincial regulators, and therefore is the focal

species in this report. Red-legged frog is considered a species of Special Concern by the CDC

and COSEWIC, and is on SARA Schedule 1. The regulatory recognition of the status of this

species implies that the species may become threatened or endangered as a result of a

combination of biological characteristics and identified threats.



4

3.0 Methods

3.1 Spatial Scope

The study area includes all aquatic features (e.g., streams, wetlands, sloughs, and ditches)

within 100 m of the highway right-of-way (Appendix A, Figures 1a through 1d), if the features

were determined to be potentially suitable for use by red-legged frog (see Section 3.3.2 for

description of habitat suitability methodology). Onsite habitat suitability assessments were

conducted to identify aquatic features for eDNA sampling. The regional context of the study

area includes adjacent areas within the Lower Mainland (south Richmond and North Delta).

3.2 Temporal Scope

This study intended to capture existing conditions for at-risk amphibians in the study area. Field

sampling was conducted in May 2014 to coincide with the timing window of March to late

July/early August (Maxcy 2004) for red-legged frog tadpoles using aquatic habitat features at

breeding sites.

3.3 Study Methods

The study team of biologists identified approximately 30 watercourses in the study area having

potential to support red-legged frog breeding. During the fieldwork on May 28 and 30, 2014, this

list was refined to 14 watercourses, based on two factors:

1. Expert-based assessment of habitat features present at each site (see Section 3.3.2)

2. Persistence of standing water at the time of eDNA sampling

3.3.1 Background of Environmental DNA Method

Environmental DNA is any trace fragment of DNA that is released by an organism into the

environment (Biggs et al. 2014). Recently, the detection of aquatic vertebrate species using

eDNA from a variety of freshwater systems has been confirmed as an effective and reliable

survey method for the species tested (Ficetola et al. 2008, Goldberg et al. 2011, Thomsen et al.

2012). This method requires collection of water samples from habitats potentially inhabited by

the species of interest, with subsequent ex situ analysis of the sample for the presence of DNA

from the target taxon. Analysis of eDNA is currently being applied to a diverse suite of

freshwater habitats, including streams and wetlands (Goldberg et al. 2011).



5

Testing for the presence of a species’ DNA using a quantitative polymerase chain reaction

(qPCR) assay requires development of a set of species-specific primers that target a small

section of the mitochondrial DNA cytochrome b gene (Goldberg et al. 2011). Development of

this species-specific primer for red-legged frog was required to facilitate use of eDNA methods.

The biological assay was developed using egg masses collected under a provincial Wildlife Act

RSBC 1996, c. 488 permit from sites in the Lower Mainland and from several confirmed

breeding sites near Port Hardy, on Vancouver Island (see Appendix B for collection details).

3.3.2 Habitat Assessment

Upon arrival at each site, biologists assessed the suitability of the site for sampling based on the

following criteria:

Presence of standing fresh water deeper than 0.5 m

Permanent or ephemeral status of water feature

Presence of emergent vegetation suitable for egg mass attachment

Connectivity to other water features having suitable habitat for red-legged frog

Proximity to CDC element occurrences of red-legged frog (MFLNRO 2014)

If a site was determined to be potentially suitable for supporting red-legged frog breeding, water

samples were collected.

3.3.3 Sample Collection

At each site, biologists collected triplicate 250 mL water samples using polypropylene bottles.

Using an indelible marker, bottles were labelled with the site name, collection time and date,

and name of collector. To prevent contamination from boots or other gear, biologists did not

enter the water during sampling. Biologists wore clean, sterile nitrile gloves to triple-rinse the

sample bottles with site water, and each bottle was filled with water from the surface of the

feature.

Immediately after sample collection, a YSI-brand water quality meter was used to collect water

chemistry data to facilitate the calculation of detection probabilities. Using a GIS capable tablet

(Apple iPad Air), biologists marked site coordinates (in UTM) and collected water quality data.

Water chemistry parameters collected in the field included water temperature (oC), pH,

conductivity (µS/cm), and dissolved oxygen (mg/L).



6

Because DNA in samples can be degraded if exposed to elevated temperatures and ultraviolet

rays from sunlight (Pang and Cheung 2007), during sample collection in the field, sample bottles

were stored in an insulated cooler with ice packs to prevent DNA degradation prior to off-site

filtration and preservation.

One sample (site 16) was collected at the location of the red-legged frog incidentally captured in

a minnow trap (Appendix A, Figure 1d). The sample from this site was used as a probable

positive control sample (i.e., suspected presence of target DNA in the sample) to confirm the

eDNA test efficacy for red-legged frog.

3.3.4 Sample Filtration

Samples were processed following a modified version of a standard eDNA protocol (Goldberg

and Strickler 2013). Samples were stored in a refrigerator set to 4oC during holding for filtering.

Samples were processed within 24 hours of collection to prevent degradation of DNA. Samples

were processed in the same order that they were collected. Each sample was poured into a

250 mL sterile polypropylene filter funnel containing a 0.45 µm pore diameter cellulose nitrate

membrane. Filtering through the membrane was facilitated using a 115-volt alternating current

Masterflex Economy variable-speed drive motor to create a vacuum. On completion of filtration,

the filter was removed, using sterile nitrile gloves. To remove the cellulose membrane from the

funnel, tweezers sterilized in a 50% bleach solution were used. The membrane was placed into

a 2 mL sterile polypropylene cryogenic vial and filled the vial with 100% molecular-grade

ethanol. Vials were labelled with the collection date and site number, and placed inside labelled

Whirl-Pak ® storage bags for shipping.

One 250 mL control sample of distilled water was processed for each site using the same

filtration protocol to serve as a contamination test of both the filtration and laboratory analysis

processes. Preserved membranes were shipped to Dr. Caren Goldberg at Washington State

University for subsequent extraction and analysis; distilled water control samples were not

identified to the lab prior to analysis.

3.3.5 Laboratory Analysis

Sixty-one preserved filter samples from 14 sites (see Appendix A for site locations) were

extracted in a laboratory dedicated to the analysis of low-quantity DNA sources using a

Qiashredder/DNeasy protocol (Goldberg et al. 2011). Each extract was run in triplicate using a

species-specific qPCR assay that includes positive and negative controls in each plate as well

as an internal control to detect PCR inhibition. When PCR inhibition was detected, samples

were run through a One-Step PCR Inhibitor Removal kit column (Zymo Research) and



7

reanalyzed. When triplicate wells did not test consistently (i.e., one or two samples tested

positive), the sample was rerun to confirm the result. The assay was designed to not

cross-amplify with other sympatric amphibian species and was validated against DNA from

western toad (Anaxyrus boreas), northern leopard frog (Lithobates pipiens), wood frog

(L. sylvaticus), American bullfrog, green frog, and Pacific treefrog.



8

4.0 Results

Table 3 presents the results of the qPCR analysis. The targeted DNA sequence was recovered

in four (seven per cent) of 61 samples, representing two (14%) of the 14 sampled sites. As

expected, none of the 14 control samples yielded positive results (i.e., no DNA was detected).

Complete sample data and results are provided in Appendix C.

Table 3 Results of qPCR Analysis for Red-legged Frog DNA in the Study Area

Site ID Result of qPCR1

2 Negative / Positive2

3 Negative

4 Negative

5 Negative

6 Negative

7 Negative

8 Negative

9 Negative

10 Negative

12 Negative

13 Negative

14 Negative

15 Negative

16 Positive

Notes: 1 Results were the same for all three sample replicates unless otherwise indicated

2 Two of three replicates tested negative, and the third replicate was confirmed positive

The two positive detection sites (2 and 16) are at opposite ends of the study area (Appendix A,

Figure 1a). The habitat characteristics and water quality data for these two positive locations

are summarized in Table 4.



9

Table 4 Habitat and Water Quality Data for Positive Detection Sites

Parameter

Site 2

Highway 91 Interchange with Highway 99

Site 16

King George interchange with Highway 99

Habitat Characteristics

Presence of standing fresh water Yes No

Permanent fresh water Yes Yes

Presence of emergent vegetation Yes No

Connectivity to other water features with suitable habitat

No Yes

Proximity: Within 1 km of CDC observation record for red-legged frog

No No

Water Quality Data

Temperature (oC) 11.7 12.5

pH 7.27 7.90

Conductivity (µS/cm) 876 1358

Dissolved oxygen (mg/L) 1.61 6.62

4.1 Incidental Observations

During sampling on May 28, 2014, green frogs were heard calling near Serpentine Fen Wildlife

Management Area. Unidentified frog species were observed jumping into ditches at sample

sites 6, 7, 14 and 15. A pair of wood ducks (Aix sponsa) was observed swimming in a slough

near site 13. One red-tailed hawk with an active nest was observed at site 7. There were no

other incidental observations of wildlife during the eDNA sampling.



10

5.0 Discussion

5.1 Key Findings

The qPCR analysis did not detect red-legged frog DNA in the majority (86%) of aquatic

features tested within the study area. This suggests that extant occurrences of red-legged frog

are limited within the study area. All estimated suitable water features within the study area

were assessed by qualified environmental professionals, and all aquatic features with

anticipated potential for occurrence were subsequently tested using eDNA methods described

in Section 3.3.1. Detection of red-legged frog DNA at two of the sampling locations, however,

suggests the use of aquatic features within the study area by at-risk amphibians.

The positive detection of red-legged frog DNA at site 16 (probable positive control) provides

validation that the assay was effective at detecting eDNA at a site having recently confirmed

red-legged frog activity. The one positive replicate from site 2 (a roadside ditch just north of Exit

23A on Highway 99) indicates that red-legged frog is present in watercourses at the north end of

the study area. However, the lack of positive detections of red-legged frog DNA in other aquatic

features in close proximity to this area (e.g., sites 3 through 6) suggests low densities and

sporadic occurrence of the species in this area. Given this species’ preference for emergent

vegetation and covered or shaded habitat (Zevit et al. 2012), the lack of positive detections of

red-legged frog DNA in aquatic features adjacent to these positive detections is likely due, at

least in part, to the lack of suitable red-legged frog habitat in this area.

Small sample size (i.e., four positive test results and 37 negative test results) prevented the

calculation of detection probabilities using water chemistry data. There was no evidence that

false positives were generated in the control samples, indicating that the procedures were

effective at eliminating potential sources of contamination during collection, filtration, and

extraction methods.

5.2 Data Gaps and Limitations

Methods used to detect at-risk amphibian species in aquatic habitats (e.g., egg mass surveys,

acoustic surveys) are prone to type 1 (false positive) and type 2 (false negative) errors.

Environmental DNA is also subject to these potential errors; however, evidence suggests much

greater survey efficacy can be obtained using eDNA methods (Ficetola et al. 2008, Goldberg et

al. 2011, Thomsen et al. 2012). Positive detection during qPCR analysis of the target taxon

DNA in a sample indicates that a specimen of the species was recently (i.e., within seven to

21 days) in the tested water feature (Pilliod et al. 2013). A positive detection does not rule out

the possibility that the source of the DNA was contaminated gear (e.g., minnow traps or waders)

associated with other sampling programs. However, the biologists conducting the Freshwater

Fish and Fish Habitat study (Hemmera 2014) for the Project in the same watercourses that were



11

sampled for red-legged frog DNA followed the Interim Hygiene Protocols for Amphibian Field

Staff and Researchers (B.C. MOE 2008), which most likely eliminated contamination from fish

sampling gear as a source of false-positive detections.

Positive detection of eDNA does not convey information about the life stage of the source

organism. Using current eDNA methods, it is not possible to distinguish between the DNA of

eggs, tadpoles, metamorphs, and adults. For the purposes of this study, however, the confirmed

presence of any life stage of red-legged frog in Project watercourses yields useful information

by confirming use of the aquatic features in the study area by red-legged frog for any or all life

history requirements.

Negative qPCR results indicate that the DNA of the target species was not detected in the

sample. A negative result should not be used to conclusively reject potential for occurrence and

therefore should not be used to conclusively suggest that the target taxon is absent from a site

(Pilliod et al. 2013). Negative results can arise for two reasons: the species is truly absent from

the site during or immediately (seven to 21 days) preceding the time of sample collection; or, the

method simply failed to detect the DNA of the species despite its presence (i.e., a false

negative). False negatives, however, are unlikely for this study given that eDNA sampling was

conducted during the peak tadpole maturation period, when the highest amounts of red-legged

frog biological material are present in the sampling media. Furthermore, sampling in triplicate

reduces the potential for false negatives by increasing detection probabilities (Waits and

Paetkau 2005).

High levels of compounds such as tannins and other suspended particles in the water have the

potential to affect results at both filtration and analysis stages. These compounds can clog the

filter membrane, resulting in less than the desired 250 mL volume being filtered through each

membrane. To mitigate this possibility, the highest possible volume of water from each set of

samples was filtered (see Appendix C). During the qPCR analysis, some of the compounds

co-extracted with the DNA prevented the original amplification of the PCR. Although further

purification removed the compounds, the sensitivity (i.e., ability to detect DNA) of the assay may

have been affected by this process. For samples that require this extra processing, there is an

estimated 25% reduction in the concentration of eDNA in a sample (C. Goldberg. pers.com).

This can lead to a reduced eDNA detection probability and thus the potential for a Type 2 error

(false negative) is increased, particularly for samples that contain very low levels of

environmental DNA from the target taxa. Statistical quantification of decreased detection

probability is challenged by sample size; however, despite this limitation, eDNA methods are still

regarded as highly effective for detection of species with low densities, patchy distributions, and

cryptic life history traits. In the case of this study, analysis of triplicate samples from each site

allowed for sufficient material to be analyzed to provide confidence that DNA from the target

taxa was detected if present.



12

6.0 Closure

Major authors and reviewers of this technical data report are listed below.

Report prepared by: Elizabeth Vincer, MRM, A.Ag. Ecologist Report peer reviewed by: Paul McElligott, PhD, R.P.Bio. Senior Ecologist

Jared Hobbs, M.Sc., R.P.Bio. Senior Ecologist

This document represents an electronic version of the original hard copy document, sealed, signed and dated by Jared Hobbs, M.Sc., R.P.Bio. and retained on file. The content of the electronically transmitted document can be confirmed by referring to the original hard copy and file. This document is provided in electronic format for convenience only. Hemmera Envirochem Inc. shall not be liable in any way for errors or omissions in any electronic version of its report document.

Mami

Text Box

ORIGINAL SIGNED AND STAMPED



13

7.0 Statement of Limitations

This report has been prepared for the sole benefit of the B.C. Ministry of Transportation and

Infrastructure to describe existing conditions of at-risk amphibians within a specific study area.

This report is based on field studies and desktop studies, and the data presented herein

represent at-risk amphibian conditions at the time field observation and desktop studies were

undertaken.



14

8.0 References

Biggs, J., N. Ewald, A. Valentini, C. Gaboriaud, R. Griffiths, J. Foster, J. Wilkinson, A. Arnett, P.

Williams, and F. Dunn. 2014. Analytical and methodological development for improved

surveillance of the great crested newt. Freshwater Habitats Trust, Oxford.

British Columbia Ministry of Environment (B.C. MOE). 2008. Interim hygiene protocols for

amphibian field staff and researchers. Standard Operating Procedures: Hygiene

Protocols for Amphibian Fieldwork, Ecosystems Branch, B.C. MOE, Vancouver, B.C.

British Columbia Ministry of Forests, Land and Natural Resource Operations (MFLNRO). 2014.

iMap BC Version 2.0. Online Database created by the MFLNRO. Available at

http://webmaps.gov.bc.ca/imfx/imf.jsp?site=imapbc. Accessed May 2014.

Corporation of Delta. 2003. Delta watersheds fish and amphibian distributions. Available at

http://www.corp.delta.bc.ca/assets/Environment/PDF/fish_amphib_watersheds_distributi

ons.pdf. Accessed April 2014.

Ficetola, G. F., C. Miaud, F. Pompanon, and P. Taberlet. 2008. Species detection using

environmental DNA from water samples. Biology Letters 4: 423–425.

Goldberg, C., D. Pilliod, R. Arkle, and L. Waits. 2011. Molecular detection of vertebrates in

stream water: a demonstration using Rocky Mountain tailed frogs and Idaho giant

salamanders. PloS one, 6: e22746.

Goldberg, C., and K. Strickler. 2013. eDNA protocol sample collection with cellulose nitrate

filters. University of Idaho.

Hemmera. 2015. George Massey Tunnel Replacement Project: Freshwater Fish and Fish

Habitat Baseline Study Technical Data Report. Prepared for the British Columbia

Ministry of Transportation and Infrastructure, Vancouver, B.C.

Maxcy, K. A. 2004. Red-legged frog. Pages 79–90 in. Accounts and measures for managing

identified wildlife Coast Forest Region. Ministry of Water, Land and Air Protection

(MWLAP), Victoria, B.C. Available at:

http://www.env.gov.bc.ca/wld/frpa/iwms/accounts.html#fourth_. Accessed April 2014.

Pang, B. C., and B. K. K. Cheung. 2007. One-step generation of degraded DNA by UV

irradiation. Analytical Biochemistry 360:163–165.



15

Pilliod, D., C. Goldberg, M. Laramie, and L. P. Waits. 2013. Application of environmental DNA

for inventory and monitoring of aquatic species: U.S. Geological Survey Fact Sheet

2012-3146. Available at http://pubs.usgs.gov/fs/2012/3146/. Accessed April 2014.

Thomsen, P. F., J. Kielgast, L. L. Iversen, C. Wiuf, M. Rasmussen, M. T. Gilbert, L. Orlando,

and E. Willerslev. 2012. Monitoring endangered freshwater biodiversity using

environmental DNA. Molecular Ecology 21: 2562–2573.

Waits, L. P., and D. Paetkau. 2005. Noninvasive genetic sampling tools for wildlife biologists—

Review of applications and recommendations for accurate data collection. Journal of

Wildlife Management 69: 1419–1433.

Zevit, P., B. Matsuda, J. Malt, International Forest Products (Interfor), and Capacity Forestry

(CapFor). 2012. BC’s coast region: Species & ecosystems of conservation concern

northern red-legged frog (Rana aurora). Available at

http://ibis.geog.ubc.ca/biodiversity/factsheets/pdf/Rana_aurora.pdf. Accessed April 2014.

APPENDIX A

Figures

Path:

O:\!2

17-29

9\285

\077\0

3\mxd

\Wild

life\TD

R\Fig

1a_2

85_0

77_0

3_TD

R_At

RiskA

mphib

iansO

vervi

ew_1

6061

6_FIN

AL.m

xd

Legend

±1:100,000

0 1 2 3 4 5Kilometres

GEORGE MASSEY TUNNEL REPLACEMENT PROJECT

AT-RISK AMPHIBIANS OVERVIEW

Figure 1a 15/04/2015

SOURCESParks and Protected Lands, First Nations Reserves from GeoBC, United States basemap data courtesy of USGS. Burns Bog courtesy of The Corporation of Delta and based on the Metro Vancouver Burns Bog Ecological Conservancy Area Management Plan - May 2007, all other data courtesy of Canvec - GeoGratis.

_̂ Red-legged frog sighting

_̂ Red-legged frog in minnow trap

Red-Legged Frog eDNA Sample Location(eDNA present)Red-Legged Frog eDNA Sample Location(No eDNA present)CDC records of red-legged frog observations 1First Nation ReserveMunicipal BoundariesBurns Bog Ecological Conservancy AreaWaterbodyCanada - U.S. BorderHighwayArterial/Collector Road

_̂

_̂

GeorgeMasseyTunnel

Burns Bog

PEACE ARCH PARK

MUSQUEAMI.R. 4

Fraser R iver North Arm

River Road

River Road62

bStre

et

BoundaryBay

Deas IslandRegional Park

Deas

Sloug

h

Dyke Road

RichmondNature Park

Annacis Channel

Fraser River South Arm

UV1Bridgeport Road

No 5

Road

Blundell Road

Steveston Highway

Westminster Highway

SEMIAHMOO

UV17

Delta

Richmond

Surrey

TsawwassenFirst Nation

Vancouver

White Rock

BurnabyNew

Westminster

003 (2014)

002 (2014)

004 (2014)005 (2014)

006 (2014)

007 (2014)

008 (2014)

009 (2014)010 (2014)

012 (2014) 013 (2014)

014 (2014)

015 (2014)016 (2014)

GMT1 (2015)

GMT3 (2015)GMT4 (2015)

GMT5 (2015)

2 (2016) UV91

UV99

UV17

UV17A

UV17

UV10 UV99

UV91

UV10UV99

UV17

Area EnlargedRichmond

DeltaSurrey

Tsawwassen First Nation

GeorgeMasseyTunnel

VancouverBurnaby

Langley

Maple Ridge

CoquitlamPitt

Meadows

WhiteRock Canada

U.S.AWashington

Boundary Bay

0 5 10Kilometres

1Variation in polygon size represents differences in mapping accuracy associated with source data for each B.C. CDC element occurrence record

Figure 1b

Figure 1c Figure 1d

Path:

O:\!2

17-29

9\285

\077\0

3\mxd

\Wild

life\TD

R\Fig

1b-d_

285_

077_

03_T

DR_A

mphib

ian-S

heets

_160

616_

FINAL

.mxd

Legend

±1:54,000

0 0.5 1 1.5 2Kilometres


AT-RISK AMPHIBIANS

Figure 1a 15/04/2015


Red-Legged Frog eDNA Sample Location(eDNA present)Red-Legged Frog eDNA Sample Location(No eDNA present)CDC records of red-legged frog observations 1Municipal BoundariesFirst Nation ReserveBurns Bog Ecological Conservancy AreaWaterbodyCanada - U.S. BorderHighwayArterial/Collector Road

GeorgeMasseyTunnel

Burns Bog

Fraser River North Arm

River Road

62bS

tr eet

Deas IslandRegional Park

DeasSlo

ugh

Dyke Road

RichmondNature Park


Bridgeport Road

No 5

Road

Blundell Road

Steveston Highway

Westminster Highway

Richmond

Vancouver

Burnaby

003 (2014)

GMT1 (2015)

GMT3 (2015)

GMT4 (2015)

GMT5 (2015)

002 (2014)

004 (2014)005 (2014)

006 (2014)

007 (2014)

008 (2014)

2 (2016) UV91

UV99

UV17

Richmond

DeltaSurrey


GeorgeMasseyTunnel

VancouverBurnaby

Langley

Maple Ridge

CoquitlamPitt

Meadows

WhiteRock Canada

U.S.AWashington

Boundary Bay

0 5 10Kilometres


Figure 1b

Figure 1c Figure 1d

Path:

O:\!2

17-29

9\285

\077\0

3\mxd

\Wild

life\TD

R\Fig

1b-d_

285_

077_

03_T

DR_A

mphib

ian-S

heets

_160

616_

FINAL

.mxd

Legend

±1:44,000



AT-RISK AMPHIBIANS

Figure 1c 15/04/2015


Red-Legged Frog eDNA Sample Location(eDNA present)Red-Legged Frog eDNA Sample Location(No eDNA present)CDC records of red-legged frog observations 1Municipal BoundariesFirst Nation ReserveBurns Bog Ecological Conservancy AreaWaterbodyCanada - U.S. BorderHighwayArterial/Collector Road

_̂

Burns Bog

River Road

BoundaryBay


Delta

Surrey

GMT4 (2015)

GMT5 (2015)

009 (2014)010 (2014)

012 (2014) 013 (2014)

UV17

UV10 UV99

UV91

Richmond

DeltaSurrey


GeorgeMasseyTunnel

VancouverBurnaby

Langley

Maple Ridge

CoquitlamPitt

Meadows

WhiteRock Canada

U.S.AWashington

Boundary Bay

0 5 10Kilometres


Figure 1b

Figure 1c Figure 1d

Path:

O:\!2

17-29

9\285

\077\0

3\mxd

\Wild

life\TD

R\Fig

1b-d_

285_

077_

03_T

DR_A

mphib

ian-S

heets

_160

616_

FINAL

.mxd

Legend

±1:54,000



AT-RISK AMPHIBIANS

Figure 1d 15/04/2015


_̂ Red-legged frog sighting

_̂ Red-legged frog in minnow trap

Red-Legged Frog eDNA Sample Location(eDNA present)Red-Legged Frog eDNA Sample Location(No eDNA present)CDC records of red-legged frog observations 1Municipal BoundariesFirst Nation ReserveBurns Bog Ecological Conservancy AreaWaterbodyCanada - U.S. BorderHighway

_̂

_̂

BoundaryBay

WhiteRock

012 (2014) 013 (2014)

014 (2014)

015 (2014)

016 (2014)

017 (2014)

RAAU April16 Incidental (2014)

UV10

UV99

Richmond

DeltaSurrey


GeorgeMasseyTunnel

VancouverBurnaby

Langley

Maple Ridge

CoquitlamPitt

Meadows

WhiteRock Canada

U.S.AWashington

Boundary Bay

0 5 10Kilometres


Figure 1b

Figure 1c Figure 1d

APPENDIX B

Red-legged Frog Tissue Sample Data

for Assay Development


AT-RISK AMPHIBIANS STUDY – Appendix B

Appendix B - 1

Figure B1 Red-Legged Frog Tissue Sample Data for Assay Development

General Location UTM Zone Easting Northing Collection Date Collector Name Type of Specimen

South Fraser Perimeter Road 10 513992 5451282 15-Apr-2014 Jared Hobbs1

Kathy Paige2 Eggs


Kathy Paige2 Eggs


Kathy Paige2 Eggs

Port Hardy - Site 1 9 570278 5624088 18-Apr-2014 Chris Chutter1 Eggs









Notes: 1 Hemmera

2 B.C. Ministry of Forests, Lands and Natural Resource Operations (MFLNRO)

APPENDIX C

Complete Sample Data and

Results of qPCR Analysis


AT-RISK AMPHIBIANS STUDY – Appendix C

Appendix C - 1

Figure C1 Complete Sample Data and Results of qPCR Analysis

Site ID

Sample ID

Collector Initials1

Collection Date

Filter Date Initials of Filter Staff

Sample Contents

Sample Volume

(mL)

Result of qPCR

Notes

2 002A EV, AV 31-May-14 31-May EV Site water 200 Negative

2 002B EV, AV 31-May-14 31-May EV Site water 200 Negative

2 002C EV, AV 31-May-14 31-May EV Site water 200 Positive Inconsistent, but confirmed with 3 runs

2 002D EV, AV 31-May-14 31-May EV Distilled water 250 Negative


3 003B EV, AV 31-May-14 30-May EV Distilled water 250 Negative

3 003C EV, AV 31-May-14 30-May EV Site water 200 Negative

3 003D EV, AV 31-May-14 30-May EV Site water 200 Negative

4 004A EV, AV 31-May-14 31-May EV Distilled water 250 Negative



4 004D EV, AV 31-May-14 31-May EV Site water 200 Negative






Appendix C - 2

Site ID

Sample ID

Collector Initials1

Collection Date


Sample Contents

Sample Volume

(mL)

Result of qPCR

Notes

5 005D EV, AV 31-May-14 31-May EV Distilled water 200 Negative

6 006A EV, AV 31-May-14 31-May EV Site water 200 Negative Originally inhibited

6 006B EV, AV 31-May-14 31-May EV Site water 200 Negative Originally inhibited

6 006C EV, AV 31-May-14 31-May EV Distilled water 250 Negative

6 006D EV, AV 31-May-14 31-May EV Site water 200 Negative Originally inhibited

7 007A EV, JH 28-May-14 28-May-14 EV Site water 250 Negative

7 007B EV, JH 28-May-14 28-May-14 EV Distilled water 250 Negative

7 007C EV, JH 28-May-14 28-May-14 EV Site water 250 Negative

7 007D EV, JH 28-May-14 28-May-14 EV Site water 250 Negative

8 008A EV, AV 30-May-14 30-May-14 EV Distilled water 250 Negative

8 008B EV, AV 30-May-14 30-May-14 EV Site water 200 Negative

8 008C EV, AV 30-May-14 30-May-14 EV Site water 200 Negative

8 008D EV, AV 30-May-14 30-May-14 EV Site water 200 Negative


9 009B EV, JH 28-May-14 29-May-14 EV Site water 50 Negative




Appendix C - 3

Site ID

Sample ID

Collector Initials1

Collection Date


Sample Contents

Sample Volume

(mL)

Result of qPCR

Notes

9 009D EV, JH 28-May-14 29-May-14 EV Distilled water 250 Negative

9 009E EV, JH 28-May-14 29-May-14 EV Site water 50 Negative

10 010A EV, JH 28-May-14 29-May-14 EV Site water 100 Negative Originally inhibited

10 010B EV, JH 28-May-14 29-May-14 EV Site water 100 Negative Originally inhibited

10 010C EV, JH 28-May-14 29-May-14 EV Distilled water 250 Negative


10 010E EV, JH 28-May-14 29-May-14 EV Site water 100 Negative Originally inhibited

12 012A EV, AV 30-May-14 30-May-14 EV Distilled water 250 Negative

12 012B EV, AV 30-May-14 30-May-14 EV Site water 100 Negative

12 012C EV, AV 30-May-14 30-May-14 EV Site water 100 Negative

12 012D EV, AV 30-May-14 30-May-14 EV Site water 100 Negative

12 012E EV, AV 30-May-14 30-May-14 EV Site water 100 Negative




13 013D EV, JH 28-May-14 28-May-14 EV Distilled water 250 Negative



Appendix C - 4

Site ID

Sample ID

Collector Initials1

Collection Date


Sample Contents

Sample Volume

(mL)

Result of qPCR

Notes



14 014C EV, JH 28-May-14 29-May-14 EV Distilled water 250 Negative



15 015B EV, JH 28-May-14 28-May-14 EV Distilled water 250 Negative



15 015E EV, JH 28-May-14 28-May-14 EV Site water 150 Negative

15 015F EV, JH 28-May-14 28-May-14 EV Site water 150 Negative

16 016A EV, JH 28-May-14 28-May-14 EV Distilled water 250 Negative

16 016B EV, JH 28-May-14 28-May-14 EV Site water 200 Positive

16 016C EV, JH 28-May-14 28-May-14 EV Site water 200 Positive

16 016D EV, JH 28-May-14 28-May-14 EV Site water 200 Positive

Notes:

1 EV = Elizabeth Vincer; AV = Andrew Venning; JH = Jared Hobbs

Cells with grey shading indicate samples with positive qPCR results

at-risk amphibians study - govtogetherbc · at-risk amphibians study i preface ... at-risk...

Documents