*2.3. Chemical Analyses*

Total element concentrations in the original MIBA and the solid residues after cultivation were analyzed at accredited laboratories (Eurofins, Luxembourg, and Synlab, Munich, Germany) by melting with LiBO2 followed by dissolution in HNO3 according to ASTM D3682: 2013 and ASTM D4503: 2008, or with HNO3/HCl/HF according to SS-EN 13656: 2003. Final analysis of the solutions was performed using ICP-SFMS (Inductively Coupled Plasma-Sector Field Mass Spectrometry) according to SS-EN ISO 17294-2: 2016 and EPA method 200.8: 1994. A total content analysis of the elements in the soil before and after cultivation was carried out at an accredited laboratory (Eurofins, Luxembourg) by dissolution in aqua regia according to SS-ISO 11466, followed by analysis with ICP-SFMS (SS-EN ISO 17294-1, 2 (mod) or EPA method 200.8 (mod)) or ICP- AES (Inductively coupled plasma atomic emission spectroscopy) (SS-EN ISO 11885 (mod) and EPA method 200.7 (mod)). The contents in the original MIBA and reference soil were analyzed in triplicates, while the residues after harvest, due to limited sample volume, were analyzed as singlicates.

The total organic carbon (TOC) and acid-neutralizing capacity (ANC) of the MIBA were analyzed at an accredited laboratory (Eurofins, Luxembourg) in duplicate, using SS-EN 13137: 2001 and EN 14429: 2015, respectively. The particle size distribution in MIBA was carried out as singlicate (30 g) at the Chalmers University of Technology using EN 933-1. The particle size distribution and humus content in soil were analyzed in triplicates using SS ISO-11277 (2009), while TOC was analyzed using SS-EN 15936: 2012 metodappl. A/SS-EN 13137: 2001 m and pH using DIN ISO 10390: 2005–12, all in triplicates and at accredited laboratories (Eurofins, Luxembourg). The bulk density of the soil was analyzed in triplicate (internal method at the accredited laboratory Eurofins, Luxembourg), while the corresponding value for MIBA was calculated from data in [30,31].

A 5-step sequential extraction scheme based on [32] was performed by an accredited laboratory (Synlab, Munich, Germany) on the original MIBA (single sample) to study the potential mobility and operational binding forms of selected elements [33]. The method uses 1 M NaOAc, pH 5, to extract easily adsorbed and exchangeable metals and carbonates, followed by Na4P2O7 to extract metals weakly bound to organic matter, 0.25 M NH2OH·HCl in 0.1 M HCl in 60 ◦C to extract metals bound to amorphic iron and manganese oxides, 1.0 M NH2OH·HCl in 25% CH3COOH in 90 ◦C to extract metals bound to crystalline iron oxides, and finally KClO3 in 12 M HCl 4 M HNO3 in 90 ◦C to extract metals bound to the stable organic forms and sulfides. The leachates were analyzed according to SS-EN ISO 17294-1,2 (mod)/EPA-method 200.8 (mod) and SS 028150-2.

To evaluate potential leaching, SS-EN-12457-3 was performed on representative original MIBA samples. The test was carried out in duplicate. The pH of the leachates was measured according to SS-EN ISO 10523: 2012. The electrical conductivity (EC) of the leachates was measured according to SS-EN 27888-1 at a liquid-to-solid (L/S) ratio of 2. All analyses were conducted at accredited laboratories (Eurofins, Luxembourg, and Synlab, Munich, Germany).

The elemental contents of the harvested plants (above- and below-ground biomass) were analyzed after microwave digestion with HNO3/H2O2 using ICP-SFMS according to SS-EN ISO 17294-2: 2016 and EPA method 200.8: 1994. The analyses were done at an accredited laboratory (Synlab, Munich, Germany) in duplicate.

#### **3. Result and Discussion**

#### *3.1. Characterisation of Original MIBA*

Biomass ashes are known to act as a fertilizer and promote the growth of trees when spread in woods or used in agricultural processes [34,35]. The Swedish Forest Agency has issued recommendations on the total content of different elements in biomass ashes to be reintroduced to a forest [36]. A comparison between those values and the contents of the MIBA samples used in this study showed that of the macronutrients (Ca, K, P, Mg), Ca and Mg were present in sufficient concentrations, whereas the K and P contents were too low (Table 1). This shows that there is potential for plants to grow in MIBA and that growth could be promoted by adding NPK fertilizer. However, the amounts of the potentially toxic metals Cr, Cu, Ni, and Pb all exceeded the recommended levels, and it is well known that this can inhibit growth, especially of the roots; see, e.g., [37] and references therein. On the other hand, their mobility in water is low (Table 1) and sequential extraction has shown that of these elements, Pb was the only one with a high proportion present in exchangeable and adsorbed forms (Figure 1). In addition to Pb, Zn, Cd, and Ca are also highly mobile and were present in more than 50% of the exchangeable fraction. Chromium and Ni were mainly present in stable forms (steps 4 and 5), and are unlikely to be as available for phytoextraction. For most elements, the presence in step 2 (labile organic forms) was <10%, which was expected, as the TOC in MIBA is low (Table 1). The predicted mobility of Cu is low, and >70% was present in the sulfide fraction (step 5). The consistency of the results from the total elemental analyses for the other elements was acceptable between the methods. The mobility of anions, such as chloride and sulfate, as well as ECsw, were noteworthy (Table 1), as high salinity is known to affect plant growth negatively [38].

**Figure 1.** Sequential extraction distribution for original MIBA. Step 1: easily adsorbed and exchangeable metals and carbonates, Step 2: metals weakly bound to organic matter, Step 3: metals bound to amorphic iron and manganese oxides, Step 4: metals bound to crystalline iron oxides, and Step 5: metals bound to stable organic forms and sulfides.

#### *3.2. Plant Growth and Biomass*

The plant height growth was initially similar for all sunflower plants (Figure 2a). However, after a few weeks, the rate decreased in the MIBA boxes, and after a few more weeks, the MIBA plants started to lose their leaves and dried out. The addition of fertilizer had only a minor effect on growth, which could have been due to the presence of potentially toxic elements like Cu, Zn, and Cl that may inhibit nutrient uptake and affect plant growth negatively [37–39]. Additionally, sunflower roots are likely to be too weak to efficiently penetrate the compact MIBA and therefore don't absorb enough water and nutrients (Table 1).

Rapeseed showed a different pattern. Initially, the plants in the MIBA with fertilizer (RF) grew slower than the reference plants (Rref) (Figure 2b). However, after a few weeks, the rate increased and at harvest, the heights of the Rref and RF plants were almost the same. However, the height variation was larger for RF than for Rref, implying that cultivation in original MIBA is likely to be more diverse. Contrary to the sunflowers, the addition of fertilizer was crucial. After only a few weeks, the plants without nutrient addition (RwoF) already had a slower growth rate, and after 8 weeks, their growth stopped completely (Figure 2b). The leaves of these plants were purplish, which is a known effect of

P deficiency; see, e.g., [40]. The P content was 5 times higher in MIBA than in soil, but was not in available forms for rapeseed (Table 1). Shortage of P is more common in alkaline soils with low TOC and high Fe, and in plants with weak roots, which describes the conditions in the MIBA boxes without fertilizer (Tables 1 and 2). No difference in the growing capacity trends was observed for rapeseed plants planted at longer or shorter distances from each other, indicating that many plants can be cultivated in a limited space, giving a higher potential phytoextraction capacity.

**Figure 2.** Average growth in cm for (**a**) sunflowers and (**b**) rapeseed cultivated in MIBA or reference soil (Sref and Rref) over 20 weeks. Error bars indicate variations in height.

The main fraction of the biomass was found in the above-ground biomass irrespective of the cultivation conditions or plant type (Table 2). However, the above-ground biomass was significantly higher for the control plants, Sref, than for the sunflowers grown in MIBA, which was due to the fact that the MIBA plants dried out and stopped growing. Both the heights and the distances between the plants in Rref and RF were similar, but the total biomass was approximately 3 times larger in the control plants, Rref (Figure 2b and Table 2). The ocular inspection confirmed that both the above- and under-ground biomass of the RF

plants was weaker than that of the Rref plants. This indicates that although the taproots of rapeseed developed better than those of the sunflowers, their growth was still restricted in MIBA. There are likely several reasons for this, and except for the higher bulk density of MIBA (Table 1), which can inhibit root growth [41], another major difference was the particle size distribution (Figure 3). MIBA contains a higher fraction of larger particles, which is well known to give lower water- and nutrient-holding capacities. At harvest, it was noted that the reference soil contained more water than the MIBA samples, despite the same amount of water having percolated through all cultivation boxes. This was also noted during the growing season, when the top layer of the MIBA appeared slightly drier than the reference soil. MIBA is known to be a draining material, and much of the added water probably percolated faster through the cultivation boxes and collected in the lower parts, where the roots were unable to reach it, while the water in the reference boxes was more evenly distributed. Additionally, the organic matter (TOC) in MIBA was low (Table 1), which further affects this. Mixing the MIBA with organic matter would likely give a better material for cultivation, as the presence of organic matter not only improves the nutrientand water-holding capacities but also provides better access to nutrients [42]. The fraction of smaller particles will increase, and organic matter can also protect plants from absorbing too much salt [42]. The latter might be of high importance in MIBA (Table 1).

**Table 2.** Above- and under-ground biomass yield [g] for sunflowers and rapeseed cultivated in MIBA or reference soil (Sref and Rref) with SD in brackets.


1This includes the whole plant, due to insufficient biomass for division into parts.

**Figure 3.** Cumulative particle size distribution curves for original reference soil and MIBA.

Rosenkranz et al. added compost in their study, but unfortunately, they did not include any cultivation in reference soil [25]. The mass above ground per rapeseed plant (*Brassica n.*) in their study was, however, consistent with the Rref in this study, showing the effect of the addition of organic matter to MIBA. However, the higher contents of especially Cu, Pb, and Zn in the conventional MIBA used here are also likely to have contributed to the inferior growth rate [37]. Cultivation of rapeseed in sandy soil contaminated with

Zn and Pb in amounts corresponding to those in the MIBA used here resulted in 30% less above-ground biomass, indicating that the contamination level and the density of the cultivation material both have an impact [43].

#### *3.3. Metal Accumulation in Plants*

Although most of the biomass was present in the parts above ground, the highest metal concentrations were generally found in the roots for both plant types (Table 3). This was particularly true for minor and trace elements such as Cu, Pb, and Zn in RF (Table 3). Accumulation of Pb and Zn in rapeseed roots has previously been reported and suggested to be caused by the formation of low mobility compounds between the heavy metals and organic substances, as the metals enter the plasma in the roots [29,43]. For RwoF, the distribution could not be evaluated due to insufficient biomass. The distribution trend was weaker for the sunflowers, likely due to the limited germination of the MIBA plants. Earlier research has indicated that the Zn content is highest in the leaves of the sunflower [18]. However, this was not found in the present study, neither in the sunflowers planted in MIBA nor in those planted in the reference soil (Table 3). The metal contents were generally higher in the rapeseed plants grown in MIBA than in the sunflowers (Table 3). For instance, the Zn content in the below-ground biomass was about 10 times higher for the rapeseed plants than for the sunflowers (Table 3). This is probably because the root systems of rapeseed are more suitable for cultivation in MIBA than those of sunflowers, as discussed above, although the effects of potential water and nutrient shortages are also likely to influence this.

When calculating the bioaccumulation coefficients (BAC), i.e., the concentration of metal in plant parts above ground divided by the metal concentration in original MIBA, it is obvious that none of the used plant types should be classified as hyperaccumulators for any of the elements studied, as BAC < 1. On the other hand, BAC might not be optimal for evaluating the extraction efficiency in this material, as MIBA is more complex and generally contains higher concentrations of metals than polluted soils. However, the results indicate that for most elements, the higher the pollutant content in the solid material, the higher the content in the plants. The Zn concentration was approximately 5 times higher in the above-ground sunflower parts cultivated in MIBA than in the reference soil, even though the growth was limited (Table 3). This was even more distinct for rapeseed, where the Zn concentrations detected in the RwF roots were almost 20 times higher than those in the Rref roots (Table 3). The highest enrichments compared to the reference cultivation were found for Cu (50 times), and for Sn and Pb (25 times). Marchiol et al. reported that the Zn content in rapeseed tissues cultivated in polluted soil substrate was 100 times higher than in reference soil, while the enrichment for elements like Cu and Pb was one order of magnitude higher [43]. This indicates that if the cultivation properties of MIBA are improved by, e.g., the addition of organic matter [42], the extraction efficiency in rapeseed probably will increase.

#### *3.4. Metal Contents in MIBA after Harvest*

Although the enrichment of several elements in the plants was significant, the observed decreases in the MIBA samples were limited (Figure 4). This was also confirmed by the low BACs, as calculated above. Except for elements that to a large extent form water-soluble compounds (Ca, K, Na, and S), i.e., are washed out during watering, no noteworthy changes in the MIBA composition were identified. Naturally, this was partly due to the limited growth, and better cultivation conditions would most likely result in higher extraction rates. Regardless, cultivation in MIBA should primarily be seen as a way to recover unutilized metals, rather than as a way to efficiently remediate the material, as this would take many years due to the complex contamination situation [22]. However, phytoextraction would likely decrease metal mobility, consequently enabling further utilization alternatives, even though metal compounds would still be present.


1 Whole plant due to limited biomass.

**Table 3.** Total amounts in parts above ground and roots from sunflower and rapeseed cultivated in MIBA, with or without fertilizer, and in reference soil. All values

**Figure 4.** Contents (mg/kg DS) of selected elements in MIBA before and after cultivation of sunflowers and rapeseed.

#### *3.5. Potential for Recovery of Zn and Other Metals from MIBA*

From the results, it is obvious that the direct cultivation of sunflowers and rapeseed in conventional MIBA is not straightforward. However, although both growth and metal reduction were limited, a glimpse of the potential for using plants grown in MIBA as a metal-enriched biofuel is shown. Plants from phytoextraction could be incinerated in WtE plants, and the metals could be recovered from the fly ash, as discussed in the Introduction. Calculations assuming 6% of fly ash mass after incineration of rapeseed plants [44] provide ash with interesting quantities of Co, Cu, Pb, and Zn, for which the estimated amounts are comparable to workable ores (Table 4) [45]. Additionally, Co has been identified by the EC as a critical raw material [1]. However, for this to be interesting on a larger scale, metal enrichment in these plants must increase. In addition to improving important cultivation parameters of MIBA, such as the particle size distribution and the content of organic matter, the choice of more suitable plants for phytoextraction in this kind of material is urgent. As the metal content in MIBA is high, it would probably be more efficient to use perennial plants, as one cultivation season is too short to reach efficient extraction. For instance, plants in the salix family could be an alternative, as they are known to grow in highly polluted soils in the climate of northern Europe and have a high capacity for extracting Zn and other metals [20–22]. In a study using Salix caprea, Zn was enriched in the leaves, while Co, Cu, and Ni had mainly accumulated in the roots [20]. By collecting the leaves regularly, Zn can be recovered continuously, whereas metals mainly enriched in the roots can only be recovered when the whole plant is harvested. An additional advantage with plants in the salix family is their high biomass; this makes them suitable for incineration, which makes them interesting from an energy perspective as well, as they may be capable of displacing fossil fuels.


