WAITS_eDNA_STATEMENT_OF_WORKv4_esmbewFinal.pdf

STATEMENT OF WORK

Environmental DNA (eDNA) Sample Collection and digital PCR Analysis

National Exposure Research Lab (NERL) System Exposure Division (SED) and Ecosystem Integrity Branch (EIB) Cincinnati, OH

Purpose:

This statement of work is for the collection of and molecular analysis of environmental DNA (eDNA) from environmental samples to produce species occupancy data to fulfill requirements of the United States Environmental Protection Agency (USEPA) Chemical Safety for Sustainability (CSS) Research Program, Ecological Modeling Subtask, 18.04.02.

Background:

The rapid advance of molecular techniques for taxonomic identification of macro-biota based on their DNA present in environmental water samples may offer a sustainable solution to the limitations traditional bio-monitoring. Living organisms constantly shed DNA into the environment. This free DNA, or environmental DNA (eDNA), can be obtained from environmental samples (e.g., water, soil). Because each organism’s DNA contains a unique genetic code, eDNA can be used for precise taxonomic identification.

United States Environmental Protection Agency scientists are developing contemporary species distribution maps based on environmental DNA (eDNA) surveillance within the Sacramento River watershed, CA, and contributing waters. In order to estimate temporal population trends, these contemporary eDNA based species maps will be evaluated relative to historical and contemporary eDNA based species distribution data as well as to contemporary and historical physical catch data obtained from rotary screw traps (RST) of the same region previously generated for California State and Federal agencies. Critical to this research is knowledge of juvenile Chinook salmonid out-migration population trends over the last decade that has been determined using the RST monitoring station at Caswell Memorial State Park (Caswell) on the lower Stanislaus River, CA.

Task

United States Environmental Protection Agency scientists will leverage eDNA surveillance methodology that has been used historically, and is currently being used, to monitor fish populations in the Sacramento watershed area, American River system and the Yolo Bypass; the focal research areas of our Chemical Safety for Sustainability (CSS) Research Program, Ecological Modeling Subtask, 18.04.02.

In order to maintain data consistency, integrity, and compatibility with: A) historical environmental DNA data from within the Sacramento Watershed area, and B) environmental DNA data currently being generated from within the Sacramento Watershed area in collaboration with the California Department of Fish and Wildlife, all tasks outlined within this statement of work must be performed by the same contractor, using the same molecular analysis equipment (the actual instrumentation), that has and continues to provide said services described in A) and B) above. Furthermore, due to the vulnerable nature of the samples to be collected and the time sensitive nature of the environmental DNA assays requested by this Statement of Work, all tasks must be performed by the same vendor, from sample collection up-to-and-including molecular assays, in order to maintain sample and data integrity.

TASKS:

Table 2 in this SOW specifies 43 sites that will be used to perform this study. The specific site will be obtained from this list and provided to the contractor by United States Environmental Protection Agency scientists (see Table 2 of SOW).

1. Collect and filter 1.2 liters of water through a USEPA approved Sterivex filter (EPA#

90260-ITA-001)1. If the filter clogs before 1.2 liters is filtered due to water turbidity, additional approved filters may be used (up to 6) to achieve the target volume of 1.2 liters. From sites specified by United States Environmental Protection Agency scientists (Table 2), the vendor will collect and filter two water samples and one sediment sample.

The filtrate captured on the filters will be retained, the eluate will be discarded. Each site will be sampled on two different occasions.

2. Collect and filter a second water sample (1.5 Liters) through a 1.5 micron filter (Whatman 1827-047; Glass Microfiber Binder Free Grade 934-AH circles, 47 mm)1. If the filter clogs before 1.5 liters is filtered due to water turbidity, additional approved filters may be used (up to 2) to achieve the target volume of 1.5 liters. After Filtration the filter will be placed in a sterile 5ml screw-top centrifuge tube and frozen at -20’C or below until it will be packed on dry ice and mailed to USEPA, Cincinnati, via overnight-air. Samples shall be mailed to the following address within one month of collection.

Samples shall be received by USEPA staff within 36 hours from when they were shipped:

Eric Waits, Ph.D.

Environmental Genetics U.S. Environmental Protection Agency

Systems Exposure Division Ecosystem Integrity Branch

26 W. Martin Luther King Dr.

Mail Stop 587

Cincinnati, OH 45268

3. Collect a sediment sample (40 grams) in a sterile screw-top 50ml centrifuge tube and freeze at -20’C or below until it will be packed on dry ice and mailed to U. S. EPA Systems Exposure Division Ecosystem Integrity Branch c/o Eric Waits 26 W. Martin Luther King Drive Mail stop 587 Cincinnati, OH 45268 via overnight-air.

Samples shall be mailed to the aforementioned address above within one month of collection.

Samples shall be received by USEPA staff within 36 hours from when they were shipped.

4. The vendor will extract environmental DNA (eDNA) from one of the said filters using protocols (MoBio sterivex power water DNA extraction) consistent with those used to generate historical and contemporary eDNA data within our study area.

5. The vendor will perform Molecular assays interrogating said extracted environmental DNA using protocols and instrumentation (Fluidigm digital PCR) consistent with those used to generate historical and contemporary eDNA data within our study area. Each assay will include four target species as specified by United States Environmental Protection Agency scientists from among the assays listed in TABLE 1. Resultant raw and analyzed dPCR (Fluidigm) data shall be sent electronically to Eric Waits at Waits.Eric@EPA.gov in a standard spreadsheet format (MS excel) within one week of when the assay was performed.

TABLE 1: Species specific molecular assays.

Common name Latin name Brook Trout Salvelinus fontinalis Bull Trout Salvelinus confluentus Chinook Salmon Oncorhynchus tshawytscha Delta Smelt Hypomesus transpacificus Giant Garter Snake Thamnophis gigas Green Sturgeon Acipenser medirostris Inland Silverside Menidia beryllina Largemouth Bass Micropterus salmoides Longfin Smelt Spirinchus thaleichthys Oregon Spotted Frog Rana pretiosa Rainbow trout/Steelhead Oncorhynchus mykiss Sacramento Pikeminnow Ptychocheilus grandis Sacramento Splittail Pogonichthys macrolepidotus

Smallmouth Bass Micropterus dolomieu Striped Bass Morone saxatilis Threadfin Shad Dorosoma petenense White Sturgeon Acipenser transmontanus

Location

The services requested within this SOW will be performed in the Sacramento River watershed, at the sites specified in Table 2 below. Many of these sites include restoration monitoring sites as well as a rotary screw trap (RST) at Caswell on the lower Stanislaus River, CA.

TABLE 2: Specific sites by coordinates (Latitude/Longitude) to be sampled by the contractor.

mailto:Waits.Eric@EPA.gov

Site Number Latitude Longitude FULL SITE ID

1 37.8304 -121.3117 South Delta 2

2 37.956 -121.3475 South Delta 4

3 37.955 -121.347 South Delta 5

4 38.0555 -121.459 South Delta 16

5 38.046 -121.499 South Delta 17

6 38.113 -121.498 East Delta 1

7 38.1275 -121.5795 East Delta 2

8 38.2265 -121.4915 East Delta 3

9 38.238 -121.5175 East Delta 4

10 38.383 -121.52 Upper Sacremento River 6

11 38.478 -121.544 Upper Sacremento River 7

12 38.532 -121.528 Upper Sacremento River 8

13 38.597 -121.508 Sacremento Valley 1

14 38.672 -121.625 Sacremento Valley 4

15 38.05 -121.701 Lower San Joaquin River 5

16 38.058 -121.788 Confluence 1

17 38.043 -121.889 Confluence 4

18 38.04 -122.273 E San Pablo Bay 1

19 38.0125 -122.364 E San Pablo Bay 2

20 38.6363 -121.2222 Nimbus Basin

21 38.6344 -121.2292 Upper Sailor Bar

22 38.6344 -121.2442 Lower Sailor Bar

23 38.6371 -121.2512 Upper Sunrise

24 38.6377 -121.256 Upper Sunrise Side Channel

25 38.6217 -121.2854 Sacramento Bar

26 38.5909 -121.3306 River Bend

27 37.787 -120.7439 Buttonbush

28 37.7805 -120.8381 Rodden Road

29 37.516 -120.392 Merced River Ranch

30 37.5188 -120.4179 Henderson Park

31 37.5173 -120.3813 MID

32 39.1977 -121.4724 Hallwood

33 39.2304 -121.2787 Yuba Canyon

34 38.601 -121.482 Sacremento Valley 2

35 38.599 -121.543 Sacremento Valley 3

36 38.056 -121.7857 Confluence 2

37 38.9425 -121.5724 Bear River 1

38 38.9743 -121.5351 Bear River 2

39 38.9783 -121.5167 Bear River 3

40 38.9998 -121.4066 Bear River 4

41 39.0378 -121.3384 Bear River 5

42 39.0415 -121.3329 Bear River 6

37.7021 -121.179 RST monitoring station at

Caswell on the lower Stanislaus River

The contractor will repeat Tasks 1 through 5 during a single month specified by USEPA scientists that falls within the period of performance of this contract.

Period of performance

18 months from date of award

Deliverable

For each water sample collected, extracted, and analyzed, the contractor will deliver raw and analyzed dPCR (Fluidigm) data electronically to USEPA Eric Waits at Waits.Eric@EPA.gov.

The contractor will ship environmental water samples and sediment samples that were collected for archival and additional molecular analysis packed with Dry Ice to U. S. EPA Systems Exposure Division Ecosystem Integrity Branch c/o Eric Waits 26 W. Martin Luther King Drive Mail stop 587 Cincinnati, OH 45268.

mailto:Waits.Eric@EPA.gov