The modified setup of the Phytotoxkit™ allowed to assess the importance of the time factor on the results of this bioassay. The graphs clearly show that the results after 3 and 6 days of testing do not match. Differing results obtained from the same treatment after 3 and 6 days (testing the same combination of a plant species and the same proportion of soil amendment) can be caused by many factors. The phytotoxicity test has not been designed for long-term testing [
17]. Testing plates contain only a limited amount of air in the upper compartment and the air becomes exhausted, the conditions for the growth and development of a plant can deteriorate. Even though after 72 h the exchange of the air in the upper compartment was facilitated (during opening the cover of the plate to moisten the roots), the soil air could not be exchanged because the soil was saturated up to reaching the maximum water holding capacity at the beginning of the test. A moistened filter paper can be another factor affecting plant growth because it can start degrading with the time placed in the permanently wet conditions. The initial seed germination is sustained by accumulated reserves in the seed coat which suggests that testing that lasts longer may correspond more accurately with a real state of a contaminated area because plants already need to take up substances and nutrients present in the soil. Increased proportions of soil amendments do not display significantly better effect on seed germination or the root growth than the recommended proportion 3%
w/
w [
25]. A widely held assumption that a recovery action is necessary immediately after a fire is erroneous and a given action can even worsen the ecological stress experienced by the fire-affected area [
26]. The main post-fire management priority is to boost the ability of the fire-affected area to recover naturally and primarily to abandon such activities that can further damage or slow down the recovery of original plant species [
5,
26].
3.1. Results of the Phytotoxicity Test
Figure 3 shows the effect of diatomite application in increasing proportions. Diatomite is a naturally occurring sedimentary rock consisting of fossilized remains of diatoms which are single-celled golden-brown algae. Diatomite is mainly made of silicon dioxide and is highly absorbent thanks to a high porosity and a big surface area [
27]. It was demonstrated in studies by Angin et al. [
28] and by Aksakal et al. [
27] that it enhances nutrient and moisture retention in light textured soils and prevents crust and large aggregate formation [
28].
Figure 3 indicates that the less inhibitory effect on root growth was shown by the burnt soil with no added amendment in 3-day testing. This finding applies to all tested amendments (diatomite, biochar, compost). Increasing proportions of diatomite in 3 days of testing had an inhibitory effect on root growth of
Sinapis alba L. A proportion of 15%
w/
w of diatomite stimulated the root growth of
Sinapis alba L. in the 6 days of testing. Unamended burnt soil had the highest stimulation effect on the root growth of
Sinapis alba L. and it even exceeded the conditions provided by OECD soil in the 6-day testing.
Above mentioned differences in phytotoxicity have not been statistically significant. The effect of individual tested amendments on reducing the phytotoxicity of fire-affected burnt soil or in OECD soil (
Figure 3,
Figure 4,
Figure 5 and
Figure 6) was not proven. This was confirmed by the results of Tukey’s HSD test and F-test (
p < 0.05). The reciprocal comparison of biochar and diatomite (
Table 4) shows no mutual significant difference in the effect that biochar and diatomite had on the soil phytotoxicity during the testing with OECD soil (
Figure 3 and
Figure 5).
The properties of burnt soil did not improve after the addition of diatomite and on the contrary the
Figure 4 shows an inhibition effect on
Sinapis alba L. root growth. Proportion of 6%
w/
w of diatomite proved to be less inhibitory in 6-day testing just like the proportion of 15%
w/
w of diatomite than in 3-day testing. A decreasing trend in toxicity can be observed with time. This trend has not been statistically significant.
Figure 5 displays effects of addition of increasing proportions of biochar to burnt soil compared to OECD soil. Biochar is produced through thermochemical conversion of different kinds of biomass (plant- or animal-based) under oxygen-limited conditions. Recent studies promote biochar as a soil conditioner enhancing plant growth and crop yield; however, long-term effects are not known yet because available findings have been obtained only during the course of recent years [
29]. It is noticeable in
Figure 5 that all proportions of biochar played a negative, inhibitory effect on root growth in 3 days of testing; however, statistically significant differences have not been confirmed. Results after 6 days of testing changed and 3%
w/
w and 9%
w/
w proportions stimulated root growth slightly but less than unamended burnt soil. Similar findings of biochar application resulting in inhibition of plant growth were confirmed in several studies where the inhibition effect was explained by the fact that biochar decreases the availability of nutrients in unfertilized soils owing to stronger adsorption [
30].
The efficacy of biochar addition with the aim to improve burnt soil properties was not confirmed and all added proportions of biochar affected the root growth negatively. The inhibitory effect after 6 days is lower in the case of 3%
w/
w and 9%
w/
w proportion than after 3-day testing (
Figure 6). But this decrease is not statistically significant. The changes in the phytotoxicity in the treatments with increased proportion of biochar (6%
w/
w, 9%
w/
w, and 15%
w/
w) are noteworthy. It is generally known [
31] that biochar increases phytotoxicity because it contains polycyclic aromatic hydrocarbons. By contrast, values for treatments with the proportion of 9%
w/
w and 15%
w/
w shows decrease in spite of not being statistically significant. Adsorption of phytotoxic substances by the soil is increased after biochar application [
32]. This subsequently reduces biologically available concentrations of these substances and lowers the soil phytotoxicity.
Table 4 and
Table 5 provide a comparison of effects of diatomite and biochar on phytotoxicity in the test setup applied to the OECD soil (
Table 4) and to the burnt soil (
Table 5). There was a difference in the effect on soil phytotoxicity after application of diatomite and biochar regarding both soils—the OECD and the fire-affected burnt soil. Diatomite showed higher values of growth stimulation on indicator plant (
Sinapis alba L.) than in the case of biochar. On the other hand, these differences were not significant in either of the soils. It was only proven that after the application of diatomite and biochar in proportion higher than 3%
w/
w to the burnt soil, the phytotoxicity of the amended soil was lower than the phytotoxicity of the 100% burnt soil (100% BS after 3 days (
Table 5)).
The trends regarding the soil toxicity after 3 and 6 days of testing are contrary for
Lepidium sativum L. and
Sinapis alba L. Three days of exposure of burnt soil and burnt soil supplemented with compost had a less inhibitory effect on root growth of
Lepidium sativum L. than it had after 6 days of exposure (
Figure 7) which matches with the research conducted by Radziemska et al. [
25] where
Lepidium sativum L. proved to be more sensitive to soil contamination than
Sinapis alba L. Although differences between 3- and 6-day testing were detected, they were not significant for any of the indicator plants (
Sinapis alba L. or
Lepidium sativum L.). The significance of differences in phytotoxicity after 3- and 6-day test was excluded based on the pair T-test and no significant difference was discovered.
The efficacy of compost application to burnt soil increases with time according to
Figure 8. The lowest inhibition effect on root growth was seen with the proportion of 3%
w/
w and 15%
w/
w of compost in 3-day testing but this effect was insignificant. The potential positive effect of compost is based on its composition, i.e., the content of a large amount of stable organic substances [
33]. When comparing the effect of compost and biochar on the phytotoxicity (test setup with OECD soil) (
Table 6), it was found out that despite of the positive effect of compost no significant difference was confirmed.
Guerrero et al. [
33] carried out a one-year study investigating the impact of compost application to burnt soils in the Mediterranean. The importance of reclamation of fire-affected soils lies in minimising erosion and recovering the chemical quality of the soil thanks to the addition of the organic amendment which can be supplied by compost [
33]. There is a big difference between compost and biochar as biochar contains mainly C-substances without a presence of larger quantities of organic matter [
32].
Conditions for
Lepidium Sativum L. growth in burnt soil enriched by different proportions of biochar, when compared to optimum conditions that OECD soil should provide, are more inhibitory after 6 days of exposure than after 3 days of exposure (
Figure 9). Differences between 3- and 6-day test were confirmed by pair T-test which detected the only significant difference within the whole experiment (
Table 6). Despite the measured values indicate a positive effect on reducing the soil phytotoxicity compared to the effect of biochar, this effect has not been statistically proven in either test setup with OECD soil (
Table 6) or burnt soil (
Table 7). The positive effect of compost on reducing the soil phytotoxicity was expected because of its physicochemical properties [
34] which are the result of the composting process and the properties of input materials [
35,
36,
37]. Compost is, thanks to its sorption capacity which is similar to the one of the soil sorption complex, able to bond positively charged particles (heavy metals and other compounds) which can cause soil phytotoxicity [
34].
However, when evaluating the efficacy of biochar addition on burnt soil properties, the findings are opposite (
Figure 10) and a longer exposure promotes root growth more than the shorter one and the proportion of 3%
w/
w of biochar stimulates the root growth of
Lepidium Sativum L. after 6 days of exposure. This finding could not be proven statistically because of the high variance of measured values. Various studies on biochar application to contaminated soils brought contradictory findings. While some show positive effects on crop yield others report decrease in plant growth. The explanation lies in different reaction of biochar application in fertilized and unfertilized soils. Its ability to increase nutrient retention plays a positive role in fertilized soils while in unfertilized soils it reduces its availability for the uptake by plants [
29].
3.2. Results of Pot Experiment
The aboveground biomass yield from individual treatments are shown in
Figure 11. The tested plant species were chosen for their common usage in aided phytomanagement and their ability to immobilize PTEs [
25]. Biomass yield among individual plant species differ.t.
Festuca rubra L. biomass is much finer than
Lolium perenne L. biomass which is naturally more robust.
Brassica juncea L. is an annual herb used as a leaf vegetable or oil crop. Measured values of aboveground biomass yield can be evaluated from several points of view: (a) a factor of the effect of soil amendment application (fertilization); (b) a factor of the effect of the plant species. Stated factors were analyzed with one-way and two-way ANOVA (
Figure 11,
Appendix A,
Figure A1).
The measured values show evident effect of individual soil amendments and type of soil (unburnt/burnt;
Appendix B;
Table A1). The lowest values of aboveground biomass yield were detected in the treatments with 100% unburnt soil (control) and with burnt soil amended with 3%
w/
w of biochar (
Figure 11). A positive effect of a synergic application of biochar because of elimination of its potential phytotoxicity was confirmed by Yu et al. (2012) [
35]. The highest average values of aboveground biomass yield were achieved in the treatment with 3%
w/
w of bentonite followed by treatment with compost and diatomite. These data are statistically significant (
Figure 11) and indicate an important effect of bentonite and diatomite application into the burnt soil with respect to fire-affected soil recultivation.
The effect of individual plant species on the biomass yield was analyzed with one-way ANOVA in combination with post hoc Tukey′s HSD test (
Figure 12). The results of the statistical analysis confirm that the effect of plant species is partial because from the point of view of average values of aboveground biomass yields differences were found out only between
Festuca rubra L. and a
Lolium perenne L. with
Brassica juncea L. Therefore, it is obvious that application of individual soil amendments to the burnt soil and sensibility of the plants on these amendments were more important than the plant species.
The most favorable treatments for root growth differed for each tested plant species.
Lolium perenne L. root system benefited from bentonite and biochar application,
Festuca rubra L. roots grew best in unamended burnt soil and in the treatment with diatomite.
Brassica juncea L. created the biggest root system in treatment with diatomite as well (
Figure 13;
Appendix A:
Figure A2;
Appendix B:
Table A2).
The effect of amendment application to the burnt soil on pH is shown in
Table 8. The application of amendments in the proportion of 3%
w/
w affected the pH only slightly which can be related to the low amendment proportion. Biochar and bentonite decreased the pH of the burnt soil most. The application of biochar with the aim to decrease the pH of alkaline soils was investigated in a study carried out by Liu and Zhang [
38] where a decreasing pH trend was detected with increasing biochar application rates. The pH of treatments enriched with diatomite and compost differed from the pH of the unamended burnt very little. Low pH alterations induced by the chosen amendment application proportion suggest that the plants reacted on other soil modifications evoked by the amendments rather than on the altered pH.
Yields of under-ground biomass (roots) correspond with the yields of aboveground biomass even though the differences between individual treatments were lower (
Figure 14). The highest values were detected in the treatment with bentonite and diatomite. Average values of root yield in the treatment with compost were lower than in the unburnt and burnt soil where on average exceeded 0.02 g for individual plant species. An unexpected value was seen in the treatment with biochar where the total average value was the third highest.
Equally as in the case of aboveground biomass the effect of plant species was probably secondary which emerges from the measured values (
Figure 13 and
Figure 14) and from the absence of considerable deviations within individual treatments (
Figure 13;
Appendix B;
Table A2). Average values from all the treatments were significantly highest with
Lolium perenne L. and
Brassica juncea L., and lowest with
Festuca rubra L. Studies employing a pot experiment to investigate the reclamation of fire-affected soils with the application of organic amendment (poultry manure) were carried out by Castro et al. [
39] and Villar et al. [
40]. These studies focused on determining the lowest effective and the optimum poultry manure dose while Villar et al. [
40] evaluated the difference between efficiency of poultry manure and inorganic fertilizer (NPK) application to burnt soil. Based on the findings by Villar et al. [
40], restoration of physical and biological soil properties seems to be more relevant for
Lolium perenne L. growth than improvement of chemical properties induced by inorganic fertilizer. Based on long-term research, it can be stated that ecotoxicological diagnosis is an effective research tool [
41] and important for the selection of plant species which have higher chances to tolerate the unfavorable conditions [
42].