*2.2. Cultivation Experiments*

Suitable plant types were chosen based on a review of the literature, with the prerequisites that the selected plants should be able to: grow on open land in a Nordic climate, obtain significant relative biomass within one cultivation season, and have a proven affinity for Zn. Further, the plants should preferably have a dense structure, to ensure good incineration properties in recovery processes. Sunflowers (*Helianthus annuus*) are known to be suitable for phytoremediation of Zn, but other cruciferous plants, such as rapeseed (*Brassica napus*), have also been shown to be appropriate [17,18,25,28,29]. Additionally, as rapeseed and sunflowers both have variations of taproots, these plants were chosen for the cultivation experiments.

The experiments were performed using six cultivation boxes of about 1 m<sup>2</sup> each. The boxes were prepared in several layers, starting with a plastic covering on the inside, to prevent uncontrolled leaching. Thereafter, an approximately 15-cm thick layer of leca (Light Expanded Clay Aggregate) spheres was placed in the bottom, followed by a ground cloth. Finally, about 30 cm of MIBA or reference soil (control cultivation) was placed on top of the ground cloth in each box. In total, 6 boxes were prepared for the cultivation experiments, 4 cultivation experiments in MIBA and 2 control cultivation experiments in reference soil:


Seeds of both sunflowers and rapeseed were supplied by the agriculture cooperative Lantmännen. The sunflowers were pre-cultivated in reference soil in a greenhouse and were about 20 cm high when planted in the boxes, while the rapeseed plants were grown directly in the boxes. To improve the growing conditions, small holes, about 2–4 centimeters deep, were made in the MIBA boxes. The holes were filled with reference soil and the plants and seeds were grown inside the holes. Eight holes, i.e., eight plants, were made in each sunflower box, while twelve holes were used for the rapeseed. Several rapeseed plants were planted in each hole. Rapeseed was also planted directly in MIBA. Fertilizer was added one week after planting, in amounts according to the recommendation from the producer. All boxes were placed in similar sun and wind conditions. Watering using ordinary tap water, management, and growth checks (ocular and height measurement) were carried out regularly during the growing season (end of April to the middle of September). Some excess water was collected in the bottom of the boxes, where it could not reach the roots. This water was not returned to the plants. Approximately 6 weeks after planting, the rapeseed plants were thinned out in some of the planting groups. This was done to study whether or not having a large number of rapeseed plants within a small area would harm their growth.

**Table 1.** Contents in original reference soil, MIBA and the fertilizer used. Additionally, leaching (L/S 10) from MIBA according to SS-EN-12357-3 is shown. All amounts are provided in mg/kg DS except pH, TOC, ANC, EC, and bulk density.


1 Not possible to calculate due to a single sample or parameter not analyzed; 2 Not analyzed; 3 Re-calculated from a maximum of 12 mg Cd/kg P; 4 L/S 2; 5 Re-calculated from a natural moisture content of 10%.

> After 20 weeks, the plants were gently harvested manually using small spades. The plants, and any reference soil or MIBA on or close to their roots, were collected and dried at room temperature for 1 week. Due to the low growth rate, small sub-samples were collected from several cultivation spots in each box, then mixed into one MIBA sample per box. For consistency, only one sample from each reference box was collected according to

the same methodology. The dried plants were separated into below-ground biomass, i.e., roots and above-ground biomass, before further analysis. The only exception was RwoF, as the biomass from this box was too small to allow separation into different plant fractions.
