W-1 WATER FIELD SAMPLING

Field sampling of eDNA water samples involves two people. One person serves as the ‘sampler’ and the other person serves as a ‘helper’. The helper can look up details in these instructions when needed, keep track of samples, handle objects that are contamination risks, serve as a second set of eyes for potential contamination, and ensure safety of the sampler in potentially hazardous field conditions.

Find two suitable sampling locations within the water body representing different ecological systems, if possible.

At each sampling location, soil samples should be taken from two points, one at the edge of the waterhole and the second one meter in from the edge. Sediment collection at each sampling location should be done in triplicate.

Because ‘the edge’ of the water body may change due to precipitation changes, the edge should be defined at each month's sampling.

If the water body has a "drop off" (embankment of more than 20 cm), the soil and water samples should be taken where the water "meets" the soil. Aim to collect at locations where finer sediment is present, if possible.

At each sampling location, the helper opens the bag containing the vials to have one open and ready for the sampler. The helper will open and close the bags containing the collection materials. This is to prevent potential contamination of samples.

The Sampler takes the appropriately labeled water dipper (for example, SL1-edge would be the dipper head for sampling location 1 at the edge) from the bag and opens the corresponding Whirl-Pak® Bag which can be placed in a bucket for easier manipulation.

Check the depth of the water. If the depth of the water is greater than 1 m, the water column should be gently mixed using the water dipper without disturbing bottom sediment.

Once the water column is homogenized, collect water filling the dipper, and empty the sample into the Whirl-Pak® Bag until the 250 mL sample is collected (3/4 of a 532 mL Whirl-Pak® Bag).

Once the water is collected, measure pH of the sample by emerging litmus paper.

Close the Whirl-Pak® Bag and dry it using paper towels.

Label the Whirl-Pak® Bag with a Sharpie or water-proof marker.

Place the Whirl-Pak® Bags into the cooler box filled with ice for storage.

Proceed with REDI-NET SOP S-1 Soil Collection at the same sampling location.

Repeat the water and soil sampling 3 times per sampling location point.

Repeat steps 8-15 at the one meter mark at the same sampling location.

Clean boots thoroughly between sampling location sites.

Repeat steps 7-17 at the second sampling location until all the samples are collected.

When all samples for a collection site are taken, bring the samples back to the laboratory facility for further processing according to REDI-NET SOP W-2 Water Processing (filtration should be done within 24h 0m 0s).

Keep freshly collected water in labeled whirl-pak bags at 4°C cold chain (cooler box) until return to laboratory. Water samples can be stored at 4°C for up to 1 week.

Once at the laboratory, it is optimal to filter the water samples and freeze the filter disk in separate, labeled ziplock bags as soon as possible. If water samples can not be processed on the same day of collection, whole sample bags should be frozen at -80°C. If -80°C storage is unavailable, water bags/ filter disks should be stored at -20°C until processing. Water samples can be stored at -20°C for up to 1 month and at -80°C for longer term storage.