*3.3. Phytotoxic Activity Estimation of the EOs*

The extracted EOs were tested for their phytotoxicity against two noxious weeds *Dactyloctenium aegyptium* and *Bidens pilosa*. The seeds of *D. aegyptium* were collected from cultivated fields near the Mediterranean coast, at Gamasa City, northern Egypt (31◦27 03.9 N 31◦27 44.8 E), while the seeds of *B. pilosa* were collected from a garden in Mansoura University campus, Mansoura, Egypt (31◦02 40.2 N 31◦21 18.4 E). The homogenous and ripe seeds were selected, sterilized with 0.3% sodium hypochlorite, rinsed with distilled and sterilized water, dried, and stored in sterilized vials.

The phytotoxicity experiments were conducted in vitro following the methodology described by Abd El-Gawad et al. [38]. In brief, 20 seeds of the weed were transferred to a

Petri plate lined with Whatman No. 1 filter paper wetted with 4 mL of each concentration of the EOs (25, 50, 75, and 100 μL L<sup>−</sup>1). Different concentrations of the EOs were prepared using 1% Tween® 80 (Sigma-Aldrich, Darmstadt, Germany) as an emulsifier. The plates were sealed with Parafilm® tape and incubated in a growth chamber adjusted with a temperature of 25 ◦C and light/dark cycle of 12/12 h. Besides, Tween® 80 was used as a control treatment. After seven days of incubation, the germinated seeds were counted and the length of shoots and roots of the seedlings were measured. The inhibition of germination and seedling growth were calculated based on the following equation:

$$\text{Inhibtion }\%=100 \times \frac{(\text{Length/Number}\_{\text{Control}} - \text{Length/Number}\_{\text{Treatment}})}{(\text{Length/Number}\_{\text{Control}})}$$

The IC50 (the concentration of the EO required to reduce the germination or growth by 50%) was calculated using MS-Excel.
