Water Bucket Protocol

How to execute the Water Bucket Protocol (WBP) in the field.

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The practical field guide to the Water Bucket Protocol (WBP). For theory and science see Methodology Water Bucket Protocol.

Materials

• Four locally-sourced buckets, sterilized
• 10% bleach solution (for sterilization); clean water (for rinsing)
• ~4 L of filtered water for filling
• A sterilized, twice-rinsed 1 L sample bottle
• Gloves and face masks
• SimplexDNA filtration kit (simplexdna.com)
• KoboToolbox or equivalent for field data capture

Bucket coloring should follow a consistent scheme: light colors to attract pollinators, dark or terrain colors to attract wood-dwelling insects. Bucket material, shape, and color may vary across experiments but must be held constant within a single sampling effort and matched to the objectives of data collection.

Procedure

1. Sterilize. Clean each bucket with a 10% bleach solution and rinse twice with water. To avoid cross-contamination, sterilize on-site in the field or seal buckets during transport from the disinfection site.
2. Place. Position the four buckets around the centroid of the plot, approximately 10 m apart. Stabilize so they cannot fall over: brace with sticks on sloping ground; weight with rocks where grazing animals are present. Maintain consistent placement across fields (always in the open, or always near bushes) — comparability between sites matters more than any single placement choice.
3. Fill. Add approximately 1 L of filtered water to each bucket. Place a stick taken from the surrounding area into the water so that any insects falling in can escape; we want their eDNA, not the insects themselves.
4. Expose for 72 hours. Leave undisturbed.
5. Observe. Return after 72 hours and record for each bucket: whether it rained during the exposure period, the fill level, and the presence of any living or dead organisms. Photograph each bucket.
6. Composite. Wearing gloves and a face mask, use the sterilized 1 L bottle to measure one litre from each bucket and combine all four into one of the remaining buckets at the site.
7. Filter and store. Filter the composite sample using the SimplexDNA kit. The kit operates manually (syringe-driven), is intuitive in the field, and stores the filter in a buffer that preserves DNA at room temperature — a significant practical advantage where refrigeration is unavailable. Filterable volume varies with debris load; the filter clogs progressively and may require considerable force toward the end of filtration.

Figure X. Water bucket protocol, colored buckets