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# Water Bucket Protocol

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The practical field guide to the Water Bucket Protocol (WBP). For theory and science see Methodology [Water Bucket Protocol](/biodiversity-credits.md).

#### **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](https://www.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**


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