Question

Researchers conducted an experiment to measure the rate of crude engine oil degradation by the soil bacterium *Pseudomonas putida* in the presence of varying concentrations of a chemical surfactant (Surfactant X). Five culture flasks were prepared with identical volumes ($100\text{ mL}$) of sterile mineral salts growth medium and equal amounts ($2.0\text{ g}$) of crude oil:

- Flask 1: Growth medium + engine oil (no bacteria, no Surfactant X)
- Flask 2: Growth medium + engine oil + $1.0\text{ mL}$ bacterial culture (no Surfactant X)
- Flask 3: Growth medium + engine oil + $1.0\text{ mL}$ bacterial culture + $0.5\text{ g}$ Surfactant X
- Flask 4: Growth medium + engine oil + $1.0\text{ mL}$ bacterial culture + $1.0\text{ g}$ Surfactant X
- Flask 5: Growth medium + engine oil + $1.0\text{ mL}$ bacterial culture + $2.0\text{ g}$ Surfactant X

All flasks were incubated at $30^\circ\text{C}$ for 14 days, after which the total percentage of degraded oil was measured.

Which flask was included to establish the baseline rate of non-biological (abiotic) oil breakdown in the absence of bacterial activity?