The Usability of Sorbents in Restoring Enzymatic Activity in Soils Polluted with Petroleum-Derived Products

Due to their ability to adsorb or absorb chemical pollutants, including organic compounds, sorbents are increasingly used in the reclamation of soils subjected to their pressure, which results from their high potential in eliminating xenobiotics. The precise optimization of the reclamation process is required, focused primarily on restoring the condition of the soil. This research are essential for seeking materials sufficiently potent to accelerate the remediation process and for expanding knowledge related to biochemical transformations that lead to the neutralization of these pollutants. The goal of this study was to determine and compare the sensitivity of soil enzymes to petroleum-derived products in soil sown with Zea mays, remediated using four sorbents. The study was conducted in a pot experiment, with loamy sand (LS) and sandy loam (SL) polluted with VERVA diesel oil (DO) and VERVA 98 petrol (P). Soil samples were collected from arable lands, and the effects of the tested pollutants were compared with those used as control uncontaminated soil samples in terms of Zea mays biomass and the activity of seven enzymes in the soil. The following sorbents were applied to mitigate DO and P effects on the test plants and enzymatic activity: molecular sieve (M), expanded clay (E), sepiolite (S), and Ikasorb (I). Both DO and P exerted a toxic effect on Zea mays, with DO more strongly disturbing its growth and development and the activities of soil enzymes than P. In sandy clay (SL), P was found to be a significant inhibitor of dehydrogenases (Deh), catalase (Cat), urease (Ure), alkaline phosphatase (Pal), and arylsulfatase (Aryl) activities, while DO stimulated the activity of all enzymes in this soil. The study results suggest that the sorbents tested, mainlya molecular sieve, may be useful in remediating DO-polluted soils, especially when alleviating the effects of these pollutants in soils of lower agronomic value.


Introduction
Environmental pollution poses the most severe threat to the health and welfare of the global population [1,2]. This threat stems from progressing industrialization and urbanization, both being sources of pollutant emissions, which in turn exert an adverse impact on soil properties, various ecosystems as well as health of soils and human populations [3,4]. Within the last two decades, the worldwide death rate due to environmental pollution has increased by 66%. According to the Lancet Commission on Pollution and Health, pollution is the cause of approximately nine million deaths annually, which accounts for one sixth of the total deaths recorded worldwide [4]. One of the most frequent environmental challenges stems from the pollution with petroleum-derived products [5][6][7]. Annually, 1.7-8.8 million metric tons (MMT) of petroleum compounds are discharged into the environment. A total of 90% of this is related to human activity [8]. In addition, according to the IEA Oil Market Report [9], global demand for crude oil will increase to a record level of 101.9 mb d −1 . Based on estimates published by the Organization of the Petroleum Exporting Countries [10], global oil supplies in 2021 will increase to 95.06 mb d −1 , and production

Study Design
The study was conducted in a greenhouse of the Warmia and Mazury University in Olsztyn (NE Poland, 53.72 • N, 20.42 • E) in the form of a pot experiment. The subject of the study included brown eutrophic soils (Eutric Cambisols) with the particle composition of loamy sand (LS) and sandy loam (SL) [85]. Their characteristics are given in Table 1. The soils derived from the area of the Olsztyn Lake District belonging to the East European Plain, which is devoid of the pressure of anthropogenic factors, and their samples were collected from the accumulation stratum (Ap) from winter rape crops. The full characteristics of the molecular sieve, expanded clay and sepiolite have been presented in our earlier publications [56,57], as were those of the sorbent Ikasorb [86]. Table 1. Characteristics of materials and design of the greenhouse experiment with Zea mays.

Parameter Description
Loamy Hydrated aluminosilicate with a micropore size of 0.3 nm. Silosiv A3 manufactured by Grace, Columbia, SC, USA, was used in the study. It is a zeolite with a three-dimensional pore system with pH KCl = 8.5.

Expanded clay (E)
Lightweight ceramic aggregate produced by firing loamy clay at a temperature of about 1200 • C. It is characterized by high porosity. The aggregate used in the study was obtained from Garden Guru (Piła, Poland). It consists of 85% of particles with a size of 75 to 710 µm, with a pH KCl  Fuel for the latest generation compression ignition engines. This product meets the stringent European requirements for the so-called "sulfur-free" fuels. Diesel oil density is 820-845 kg m −3 . It contains a mixture of C 9 -C 25 petroleum-derived hydrocarbons. Diesel oil was purchased at a PKN Orlen (Olsztyn, Poland) gas station. The detailed characteristics of VERVA diesel oil is provided on the following website: http://www.orlen.pl/ (accessed on 5 March 2023) petrol VERVA 98 (P) Unleaded motor petrol with octane number 98. Its density is 720-775 kg m −3 . It was purchased at a PKN Orlen (Poland) gas station. Its detailed characteristics are available on the following website: http://www.orlen.pl/ (accessed on 5 March 2023)

The Design of the Greenhouse Experiment with Zea mays
Experiment Steps/Factors Treatments 1 factor soil type The experiment was carried out in 3.0 dm 3 polyethylene pots with 2.5 kg portions of loamy sand (LS) or sandy loam (SL). Before the physicochemical analyses, the soil was air-dried and sieved through a screen with a 5 mm mesh diameter.

Parameter Description
Maximum water capacity Subsequently, 7 cm 3 of a suitable petroleum substance, measured with a cylinder, was added to the individual pots and mixed with the soil material in order to homogenize the samples. Once the soil had been packed in pots, its moisture content was brought to 60% of the maximum water capacity.
An experimental plant After 7 days, the soil was sown with eight seeds of Zea mays PR39H32 (variety registered in the European Union). Five plants were left in each pot after emergence. Vegetation growth was continued for 60 days, while maintaining the stable moisture content of the soil using distilled water. The pots were located in random complete blocks on tables.
Zea mays was used as the test plant. Its choice was driven by its high adaptability to stressful environmental conditions, short life cycle, ease of cultivation, broad distribution across the globe and high biomass productivity [87,88]. Morales-Máximo et al. [89] and Liao et al. [90] emphasized that maize biomass produced on polluted areas can be used in alternative energy production. Analyses were carried out in 5 replications. The aerial parts and roots of Zea mays were harvested at the BBCH 59 stage. Immediately after plant harvest, soil samples were collected for biochemical analyses. The samples were sifted through a screen with mesh diameter of 2 mm.

Methodology of Physicochemical and Chemical Determinations
In the collected soil samples, the following were determined: fraction size by aerometric method; pH in 1 mol KCl dm -3 ; hydrolytic acidity (HAC); and the sum of base exchangeable cations (EBCs).The HAC and EBC values obtained were used to compute the cation exchange capacity (CEC) and alkaline cation saturation (ACS). In addition, analyses were conducted to determine contents of total nitrogen (N tot ), organic carbon (C org ) and available P, K and Mg. The above determinations were conducted in 3 replications following the methods described in our previous work [79].

Methodology of Determination of the Activity of Soil Enzymes
The soil samples with natural moisture content were determined for the activities of the following enzymes: dehydrogenases (Deh), catalase (Cat), urease (Ure), acidic phosphatase (Pac), alkaline phosphatase (Pal), β-glucosidase (Glu) and arylsulfatase (Aryl). The activity of each of the individual enzymes was determined in a weighed soil sample, incubated for a designated period of time and at a constant temperature. The activity of each of the individual enzymes was determined in a weighed soil sample, incubated for a designated period of time and at a constant temperature. The parameters of each sample were as follows: for Deh-6 g of soil, 24 h of incubation (37 • C); for Cat-2 g of soil, 20 min of incubation (21 • C); for Pac, Pal, Glu and Aryl-1 g of soil, 1 h of incubation (37 • C); and for Ure-10 g of soil, 24 h of incubation (37 • C).
All determinations were made in triplicate. The following chemical compounds were used as substrates: 2,3,5-triphenyltetrazolium chloride for dehydrogenases; hydrogen peroxide for catalase; urea for urease; 4-nitrophenyl phosphate disodium salt 6-hydrate for phosphatases; potassium 4-nitrophenyl sulfate for arylsulfatase; and 4-nitrophenyl-β-Dglucopyranoside for β-glucosidase. Enzyme activity units were expressed in moles or in the subunits of their reaction products produced within 1 h in 1 kg of soil, i.e., formazane in the case of dehydrogenases, O 2 in the case of catalase, N-NH 4 in the case of urease, as well as 4-nitrophenol in the case of acidic and alkaline phosphatase, arylsulfatase and b-glucosidase. The extinction of the reaction products was measured using a Perkin-Elmer Lambda 25 spectrophotometer (Peabody, MA, USA) at the following wavelengths: 485 nm (dehydrogenases), 410 nm (urease, acidic and alkaline phosphatase), 420 nm (arylsulfatase) and 400 nm (β-glucosidase). Detailed procedures of enzyme determinations are provided in our previous works [56,91].

Calculations and Statistical Analysis
In order to determine the impact of soil pollution with petroleum-derived products and sorbents (Ad) on the biomass of aerial parts and roots of Zea mays and on the activities of individual soil enzymes, the index of influence (IF) of DO, P and Ad on these dependent variables was determined in this study. The IF was computed from Formula (1).
where y-respectively: DO, P or sorbent (Ad); Po-weight of Zea mays biomass or activity of the analyzed enzyme in the soil polluted with DO or P; Co-weight of Zea mays biomass or activity of the analyzed enzyme in the control soil (uncontaminated).
The index value of 1 denotes a 100% increase in the value of the analyzed trait, whereas the index value of −1 indicates a 100% decrease in the value of the analyzed trait.
Values of the computed indices were presented on heat maps, developed using R v1.2.5033 software (Boston, MA, USA) [92] with R v3.6.2 addition [93] and a gplots library [94]. The "color key and histogram" was used to signalize the frequency of the results obtained on the heat map matrix.
The determined activities of dehydrogenases (Deh), catalase (Cat), urease (Ure), acidic phosphatase (Pac), alkaline phosphatase (Pal), arylsulfatase (Aryl) and β-glucosidase (Glu) enabled the computation of the soil quality index (BA), according to Formula (2) proposed by Wyszkowska et al. [74]: The results of the determinations of Zea mays biomass and enzymatic activity were analyzed in terms of the normality of their distribution and the homogeneity of their variance. The analysis of variance (ANOVA) with a post hoc test (Tukey) at P = 0.05 was conducted separately for each soil type tested. It aimed to determine the percentage contribution (coefficient η 2 ) of soil pollution with petroleum-derived products, sorbents and their interactions in changes observed in the Zea mays biomass and activities of individual soil enzymes. In addition, the principal component analysis (PCA) was performed in order to highlight the correlations between the results obtained. Computations were carried out using Statistica 13.0 software (Palo Alto, CA, USA) [95]. The values of the η 2 coefficient, describing the contribution of an independent variable to the modification of the values of dependent variables, were presented on a circular diagram plotted using the Circos 0.68 package [96].

Effect of Diesel Oil, Petrol and Sorbents on Zea mays Biomass
The analyzed petroleum-derived products significantly inhibited the growth and development of Zea mays. The weight of aerial part biomass produced on loamy sand (LS) polluted with DO was 88.0% lower compared to that obtained in the control, non-polluted soil, whereas the biomass produced by roots in this soil was 81.2% lower. In the case of sandy loam (SL), the respective biomass weights were 39.8% and 47.1% lower compared to those produced on the control soil (Table 2, Figure 1). Petrol was found to exert a less toxic effect on the test plants than DO. The biomass of the aerial parts produced in LS soil polluted with P was lower by 18.0% and that of roots was decreased by 22.6% compared to the non-polluted LS, whereas the respective weights of biomass produced on SL were 9.2% and 32.9% lower compared to those produced on the control soil. The ratio of biomass of the aerial parts to the biomass of roots of Zea mays grown on LS averaged 4.566, and the respective ratio determined for the test plants grown on SL averaged 6.247, with the biomass of the aerial parts being higher, along with an increasing root biomass. toxic effect on the test plants than DO. The biomass of the aerial parts produced in LS soil polluted with P was lower by 18.0% and that of roots was decreased by 22.6% compared to the non-polluted LS, whereas the respective weights of biomass produced on SL were 9.2% and 32.9% lower compared to those produced on the control soil. The ratio of biomass of the aerial parts to the biomass of roots of Zea mays grown on LS averaged 4.566, and the respective ratio determined for the test plants grown on SL averaged 6.247, with the biomass of the aerial parts being higher, along with an increasing root biomass.   However, this dependency was distorted once the non-polluted soils were amended with sorbents ( Table 3). The molecular sieve (M) increased the biomass of the aerial parts of Zea mays grown on LS by 18.8% and that of maize grown on SL by 12.7% (Table 3, Figure 1). Expanded clay (E) also exerted a positive effect on the biomass of the aerial parts on LS and that of roots on SL. The growth and development of roots were stimulated by expanded clay (E) and Ikasorb (I) but only on LS.
The analyzed sorbents were observed to mitigate the toxic effects of DO on Zea mays (Table 4, Figure 1). Under their influence, the biomass of aerial parts increased by 42.2% in LS and by 9.2% in SL, on average. In LS, M had the most favorable effect on the biomass of aerial parts, while in SL E and S had the most positive effect on this parameter.All sorbents tested turned out to promote root growth and development; however, their stronger effects on root biomass were noted in LS as opposed to SL.  The effects of sorbents on the biomass of Zea mays grown on the soils polluted with P were significantly weaker compared to the DO-polluted soils (Table 5, Figure 1), which was due to a less toxic effect P than DO on this crop.

Effect of Diesel Oil, Petrol and Sorbents on the Activity of Soil Enzymes
Soil pollution with DO and P significantly suppressed the activities of soil enzymes (Table 6, Figure 2). Changes observed in the enzymatic activity in both soil types were mainly due to the effect of P, which suppressed the activities of Deh, Cat, Ure, Pal and Aryl in sandy loam (SL) as well as stimulating the activities of the first three mentioned enzymes and Pac and inhibiting those of Pal, Glu and Aryl in loamy sand (LS). Soil pollution with DO significantly stimulated the activities of all analyzed enzymes in SL and almost all in LS, where it inhibited the activities of Glu and Aryl. The effects of the tested sorbents on enzymatic activity varied depending on soil type, sorbent and enzyme (Table 7, Figure 2). The molecular sieve turned out to be the greatest promoter of Ure activity, regardless of soil type. It suppressed Cat activity by 42.9% in SL but did not affect it in LS. In turn, it exerted a weak effect on Pal, Glu and Aryl in both soil types. Deh also proved minimally susceptible to its effects in both types of soil, as their activities increased by merely 8.3% in LS and 7.8% in SL. The other sorbents tested stimulated Deh activities but only in the lighter soil, i.e., in LS, by 27.2% (expanded clay), 37.8% (sepiolite) and 37.2% (Ikasorb). All sorbents contributed to Ure activity enhancement in LS, while causing no significant changes in its activity in SL. In turn, they increased Pac activity in the range of 10.1% to 17.0% in LS and of 14.8% to 37.8% in SL. In contrast, Pal activity was stimulated only by sepiolite and Ikasorb and only in LS.
Enzymatic activity in the DO-polluted soils was modified by the tested sorbents to a various extent (Table 8, Figure 2). In loamy sand (SL), the activities of dehydrogenases and urease were enhanced by all sorbents tested, with the strongest stimulating effect exerted by the molecular sieve which increased Deh activity by 61.7% and Ure activity by 229.4%. This extent of activity boosting was indicated by the values of the index of influence presented in Figure 2. In LS, the molecular sieve additionally had a positive effect on catalase activity. The activity of this enzyme was also enhanced by Ikasorb. The activity of the other enzymes analyzed in LS was not significantly changed. Slightly different observations were made regarding the effects of the tested sorbents on enzymatic activity in sandy loam (SL) polluted with DO. The stimulation of Deh and Ure activities was substantially weaker. The molecular sieve stimulated Deh activities by only 19.8% and Ure activity by 62.7%. All sorbents inhibited catalase activity, whereas their usability in modifying the activities of the other enzymes was negligible.  The effects of the tested sorbents on enzymatic activity varied depending on soil type, sorbent and enzyme (Table 7, Figure 2). The molecular sieve turned out to be the greatest promoter of Ure activity, regardless of soil type. It suppressed Cat activity by 42.9% in SL but did not affect it in LS. In turn, it exerted a weak effect on Pal, Glu and Aryl in both soil types. Deh also proved minimally susceptible to its effects in both types of soil, as their activities increased by merely 8.3% in LS and 7.8% in SL. The other sorbents tested stimulated Deh activities but only in the lighter soil, i.e., in LS, by 27.2% (expanded clay), 37.8% (sepiolite) and 37.2% (Ikasorb). All sorbents contributed to Ure activity enhancement in LS, while causing no significant changes in its activity in SL. In turn, they increased Pac activity in the range of 10.1% to 17.0% in LS and of 14.8% to 37.8% in SL. In contrast, Pal activity was stimulated only by sepiolite and Ikasorb and only in LS.   The effects of sorbents in the soil polluted with P varied depending on the analyzed enzyme and soil type (Table 9, Figure 2).
All sorbents significantly suppressed the activities of Deh and Cat, and stimulated those of Ure and Glu in both LS and SL. In LS, all sorbents additionally stimulated activities of Pal and Aryl, whereas in SL, the added sorbents induced an increase in Pac activity. The above observations are confirmed by the values of the indices of influence presented in Figure 2. In LS, Ikasorb caused the greatest changes in Deh activities (IF Ad = −0.240); expanded clay caused the greatest changes in the activities of Cat (IF Ad = −0.383), Pac (IF Ad = 0.141), Pal (IF Ad = 0.123) and Aryl (IF Ad = 0.398); and the molecular sieve caused the greatest changes in Ure activity (IF Ad = 1.726). In turn, in SL, the molecular sieve had a particularly negative effect on the activity of Deh (IF Ad = −0.168) and positively on the activity of Ure (IF Ad = 1.099); expanded clay caused the greatest changes in the activities of Cat (IF Ad = −0.213) and Aryl (IF Ad = 0.143); sepiolite caused the greatest changes in Pac activity (IF Ad = 0.657); and finally, the molecular sieve and Ikasorb contributed to a significant increase in Glu activity (IF Ad = 0.224-0.225). Regardless of enzyme type, the molecular sieve was proven to be the most effective remediating agent in the soils polluted with DO and P. The mean value of the soil quality index (BA) reached 14.752 for loamy sand (LM) and 16.541 for sandy loam (SL), regardless of the independent variables examined ( Figure 3). Soil pollution with DO caused a 22.2% increase in its value in LS and a 33.8% increase in SL, whereas soil pollution with P led to BA value increase by 19.3% in LS and its decrease by 6.6% in SL. Soil amendment with sorbents increased the values of BA index in the nonpolluted soils by 9.4% (expanded clay) to 15.7% (Ikasorb) on average. In the DO-polluted soils, the observed increase ranged from 8.1% (expanded clay) to 22.1% (molecular sieve), whereas in those polluted with P, the BA values remained unaffected by the sorbents.
The sorbents tested exerted a stronger effect on the values of dependent variables in SL than in LS (Figure 4). In SL, they affected 67.8% of Ure activity, 32.3% of Glu activity, 31.1% of Cat activity and 19.5% of Pac activity, as well as 7.9% to 11.1% of Zea mays biomass. In LS, the sorbents contributed to barely 1.2-2.8% of changes in Zea mays biomass and modified 89.0% of Ure activity and 23.6% of Aryl activity, as well as over 10.0% of Deh, Pal and Pac activities.
In both soil types, pollution with DO and P had a significantly stronger effect on changes in the values of dependent variables compared to sorbents. Their contribution in the changes observed in SL ranged from 26.2% (Ure) to 94.3% (Pal) and in LS from 49.1% (Deh) to 95.7% (biomass of aerial parts of Zea mays). Urease activity in LS was an exception, as merely 3.2% of its value was affected by soil pollution with DO and P. In both soil types, pollution with DO and P had a significantly stronger effect on changes in the values of dependent variables compared to sorbents. Their contribution in the changes observed in SL ranged from 26.2% (Ure) to 94.3% (Pal) and in LS from 49.1% (Deh) to 95.7% (biomass of aerial parts of Zea mays). Urease activity in LS was an exception, as merely 3.2% of its value was affected by soil pollution with DO and P. Results of the principal component analysis ( Figure 5) indicate that the weight of Zea mays biomass was negatively correlated with activities of most of the enzymes tested in both LS and SL. This negative correlation was probably due to the adverse impact of DO and P on maize growth and development and their stimulating effect on activity of enzymes such as Deh, Pal, Cat, Aryl and sometimes Ure. The adverse effect of DO on the  In both soil types, pollution with DO and P had a significantly stronger effect on changes in the values of dependent variables compared to sorbents. Their contribution in the changes observed in SL ranged from 26.2% (Ure) to 94.3% (Pal) and in LS from 49.1% (Deh) to 95.7% (biomass of aerial parts of Zea mays). Urease activity in LS was an exception, as merely 3.2% of its value was affected by soil pollution with DO and P. Results of the principal component analysis ( Figure 5) indicate that the weight of Zea mays biomass was negatively correlated with activities of most of the enzymes tested in both LS and SL. This negative correlation was probably due to the adverse impact of DO and P on maize growth and development and their stimulating effect on activity of enzymes such as Deh, Pal, Cat, Aryl and sometimes Ure. The adverse effect of DO on the Results of the principal component analysis ( Figure 5) indicate that the weight of Zea mays biomass was negatively correlated with activities of most of the enzymes tested in both LS and SL. This negative correlation was probably due to the adverse impact of DO and P on maize growth and development and their stimulating effect on activity of enzymes such as Deh, Pal, Cat, Aryl and sometimes Ure. The adverse effect of DO on the test plant was stronger than that of P. In both soil types, in the experimental series without DO and P, the sorbents not only did not impair the growth and development of Zea mays but even stimulated them. Figure 5 shows three distinct groups of effects of independent variables on changes in the values of dependent variables in both soil types. The first group includes the sorbents applied on the soils non-polluted with DO and P, the second includes those used on P-polluted soils and the third one includes those used on soils polluted with DO. DO and P, the sorbents not only did not impair the growth and development of Zea mays but even stimulated them. Figure 5 shows three distinct groups of effects of independent variables on changes in the values of dependent variables in both soil types. The first group includes the sorbents applied on the soils non-polluted with DO and P, the second includes those used on P-polluted soils and the third one includes those used on soils polluted with DO.

Response of Zea mays to Soil Contamination with Petroleum-Derived Products
Soil pollution with petroleum-derived products poses a common problem, which requires deploying an environmentally friendly and economically effective solution [97,98]. A viable option for the decontamination of areas polluted with petroleum-derived hydrocarbons is offered by phytoremediation, especially as the biomass of plants applicable for in situ remediation can later be used to produce bioenergy [90,97]. Hence, the success of the effective remediation of soils polluted with these compounds depends on the appropriate choice of plants, considering their lasting and stable effects in the natural environment [99][100][101][102]. The choice of the plants should also be driven by the concentration and type of petroleum-derived hydrocarbons polluting the soil [98,103] and-consequently-by an appropriate phytoremediation strategy [99,104]. Native plants are preferred in this case, as they easily adapt to the local climate conditions, thereby increasing the chances for successive phytoremediation [105]. The plants intended for phytoremediation should have a well-developed root system [18,27], e.g., those from the Poaceae family [21,27,30,[106][107][108] used for the phytoremediation of soils polluted with petroleum-derived hydrocarbons. This plant family also includes Zea mays, used in the present study.
Hydrocarbons of petroleum products modify texture, structure and pore size, as well as the water-air and physicochemical properties of soil [8,28], thereby exerting a phytotoxic effect on seed germination and plant growth and development [27,28,109]. The negative impact of soil pollution from diesel oil and petrol on the growth and development of Zea mays, observed in the present study, could have been due to the inhibition of water and mineral salts uptake, contributing to the arrestment of metabolic processes in plants [8]. Their immediate effect on the test plants cannot be excluded either. Petroleum-derived hydrocarbons contribute to, most of all, the deformation and reduced length of roots, decreased leaf surface area, the deformation of leaf pigments, and the accumulation of H2O2, often leading to their chlorosis and necrosis [28,110,111]. The leaf chlorosis and

Response of Zea mays to Soil Contamination with Petroleum-Derived Products
Soil pollution with petroleum-derived products poses a common problem, which requires deploying an environmentally friendly and economically effective solution [97,98]. A viable option for the decontamination of areas polluted with petroleum-derived hydrocarbons is offered by phytoremediation, especially as the biomass of plants applicable for in situ remediation can later be used to produce bioenergy [90,97]. Hence, the success of the effective remediation of soils polluted with these compounds depends on the appropriate choice of plants, considering their lasting and stable effects in the natural environment [99][100][101][102]. The choice of the plants should also be driven by the concentration and type of petroleum-derived hydrocarbons polluting the soil [98,103] and-consequently-by an appropriate phytoremediation strategy [99,104]. Native plants are preferred in this case, as they easily adapt to the local climate conditions, thereby increasing the chances for successive phytoremediation [105]. The plants intended for phytoremediation should have a well-developed root system [18,27], e.g., those from the Poaceae family [21,27,30,[106][107][108] used for the phytoremediation of soils polluted with petroleum-derived hydrocarbons. This plant family also includes Zea mays, used in the present study.
Hydrocarbons of petroleum products modify texture, structure and pore size, as well as the water-air and physicochemical properties of soil [8,28], thereby exerting a phytotoxic effect on seed germination and plant growth and development [27,28,109]. The negative impact of soil pollution from diesel oil and petrol on the growth and development of Zea mays, observed in the present study, could have been due to the inhibition of water and mineral salts uptake, contributing to the arrestment of metabolic processes in plants [8]. Their immediate effect on the test plants cannot be excluded either. Petroleum-derived hydrocarbons contribute to, most of all, the deformation and reduced length of roots, decreased leaf surface area, the deformation of leaf pigments, and the accumulation of H 2 O 2 , often leading to their chlorosis and necrosis [28,110,111]. The leaf chlorosis and deformation of aerial parts were also observed in the case of Zea mays in the present study, with diesel oil inducing a stronger effect in this respect than petrol. Our previous research also demonstrated a stronger effect of DO on Elymus elongatus [23] and Lolium [21] compared to P. According to Wyszkowska et al. [21], Lolium hybridum and Phleum proved effective in degrading hydrocarbons in the soil contaminated with diesel oil and petrol, respectively.The phytotoxicity of petroleum-derived hydrocarbons depends on petroleum product type [21,98], plant species [8,28,112,113] and the plant's capability of the immediate absorption and metabolization of these hydrocarbons [109]. Plants absorb and then metabolize (phytotransformation, phytodegradation) organic pollutants, which are degraded by enzymes such as dehalogenase and oxygenase, secreted by plants. Plants are deemed to be the "green liver" of the biosphere [99]. Meištininkas et al. [98] have demonstrated the usability of the following legumes: Medicago sativa, Lotus corniculatus and Melilotus albus, in the remediation of soils polluted with heavy fuel oils, whereas Panchenko et al. [104] have recommended a mixture of Medicago sativa L. and Lolium perenne L. for the remediation of soils polluted with oils.
According to Malik et al. [114], only few plant species, e.g., Cucurbita, are capable of absorbing permanent pollutants (POPs) from soil. Therefore, breeding transgenic, hyperaccumulative plants encoding for POP-degrading enzymes may foster certain potential in the phytoremediation of these pollutants. The adverse impact of DO and P on the biomass of aerial parts of Zea mays in the present study was weaker in sandy loam (SL) than in loamy sand (LS), probably due to the stronger binding of petroleum-derived hydrocarbons with soil particles in SL than in LS and, consequently, due to their lower bioavailability [107]. The analyzed sorbents were more effective in mitigating the adverse effects of DO than P on Zea mays, which was due to the less toxic effects of P than DO on this crop. The positive impact of sorbents on Zea mays yield in soils polluted with petroleum-derived products stems from the improvement of the physical properties of soil, resulting in upgraded water-air conditions that could promote the degradation of hydrocarbons. High-porosity materials can improve the effectiveness of their bioremediation as well [115].

Response of Soil Enzymes to Soil Pollution with Petroleum-Derived Products
Soil pollution with petroleum-derived products causes disorders in soil metabolism [116], which need to be considered from the perspective of sustainable development [100], health of soil and plants [29,33,34,96] and the feasibility of homeostasis restoration in the ecosystem [117,118]. For these reasons, it is essential to determine the activity of soil enzymes in real time when establishing soil quality [19,75,76,102,119,120]. In the present study, enzymatic activity was significantly modified by petroleum-derived products; however, DO stimulated it significantly in both soil types, whereas P only in LS. DO served as an energy substrate to microorganisms, owing to which we were able to observe enhanced activities of Deh, Cat, Ure, Pac and Pal in LS and SL as well as Glu and Aryl in SL. The high enzymatic activity determined in the soil polluted with this petroleum-derived product could be due to the sorption of exoenzymes in mineral and organic colloids [120,121]. In turn, the inhibiting effect of P on most analyzed enzymes could stem from modifications induced in the molecular structure of enzymes by inhibitors [122]. Petroleum-derived hydrocarbons are considered to be potent enough to disrupt the structure of protein molecules [121].
The modified activities of all soil enzymes were reflected in the values of the index of DO and P influence on these enzymes. The presented IF values provide an unbiased assessment of soil ecosystem's stability [123]. The present study results demonstrate that the extent of enzymatic activity changes under the influence of DO and P depended on the fraction composition of the soil. Considering the extent of changes triggered by DO in LS, the analyzed enzymes can be ordered as follows (from the most to the least sensitive), Ure > Cat > Pac > Pal > Glu > Deh > Aryl, whereas in the case of soil pollution with P, the order is Cat > Ure > Pac = Aryl > Deh > Glu > Pal. In SL, terms of their sensitivity to soil pollution with petroleum products, the enzymes can be ordered as Pac > Pal > Ure > Deh > Aryl > Cat > Glu in the case of DO and Pac > Cat > Ure > Aryl > Deh > Glu > Pal in the case of P. The above changes in the biochemical activity of soil reflected its response to the biotic stress induced by DO and P, both being a mixture of highly hydrophobic compounds, which causes their binding with soil particles and consequently leads to their diminished bioavailability in the soil [75,124].
Other authors also demonstrated the destructive effect of petroleum-derived products on the biochemical properties of soil [75,125,126]. Taking into account the resistance (RS) of enzymes to diesel oil, Wyszkowska et al. [23] ordered them as follows (in descending order from the most to the least resistant): Glu > Pac > Aryl > Pal > Ure > Cat > Deh, whereas considering their resistance to unleaded petrol, their order was: Pac > Glu > Cat > Aryl > Deh > Pal > Ure. Among the analyzed enzymes, worthy of notice is β-glucosidase, which turned out to be one of the most resistant to the tested pollutants [91,126]. A similar observation was made in the present study, in both LS and SL.
The exchange of matter and energy between soil, rhizospheric microorganisms and plants represents a strong and specific relationship, which allows the biochemical activity of soil to provide information highly valuable to soil health [19,75,119]. According to Qu et al. [72], dehydrogenases and polyphenolic oxidase, which provide information on the bioactivity and population of micro-organisms in the soil and play an important role in degradation of resistant organic compounds. Dehydrogenases are involved in the oxidation of organic matter [69,76], whereas polyphenolic oxidase is involved in the conversion of aromatic organic compounds into humus in the soil [83,116]. The determination of the biochemical activity may provide information about the function and structure of microbial communities in soils polluted with petroleum [83,120,123].
To achieve the present study goal, soil quality was assessed using the biochemical soil quality index (BA), which is based on activities of Deh, Cat, Ure, Pac, Pal, Glu and Aryl. The importance of these enzymes as sensitive indicators of changes in soil condition has been proven in the cases of soils polluted with heavy metals [46,74]. Computed values demonstrated that soil pollution through DO caused greater disorders in the metabolic profile of LS and SL than soil pollution through P. Presumably, DO represents a better source of nutrients to microorganisms, which in the present study could affect enzyme biosynthesis [91]. Soil polluted with petroleum may modify the structure of a bacterial community [127], whereas petroleum itself can act as a substrate for certain bacteria, facilitating soil transformation and reclamation [111,128].
A holistic approach should be implemented while discussing the disorders of soil metabolism. Special attention should be paid to the feasibility of restoring homeostasis in the ecosystem [128]. The present study results indicate that the biochemical activity of soil should be taken into account when planning the remediation measures undertaken to restore its productivity. Although phytoremediation is a broadly deployed and costeffective method for the remediation of soils polluted with petroleum-derived products, it is not effective enough to significantly stimulate the biochemical activity of soils under the pressure of these xenobiotics. [129]. From an environmental perspective, it is essential to search for innovative remediation technologies useful for environments polluted with petroleum products [8,42,43] that are non-invasive and ecologically friendly [44,47]. In the present study, the phytoremediation of soils polluted with petroleum-derived products using Zea mays was aided by four sorbents. The molecular sieve applied to the non-polluted soil enhanced biochemical activity in both LS and SL, whereas expanded clay, sepiolite and Ikasorb enhanced it only in LS. These effects of sorbents on the activities of soil enzymes are due to their microporous structure, large specific surface areas and high ion adsorption capability [59,60,130]. It is worth emphasizing that the sorption capacity of adsorbents in relation to petroleum derivatives is usually in the range of 0.20-0.50 g·g −1 and the bulk density is 0.45-0.90 kg·dm −3 [66]. Low values of bulk density correspond to the formation of capillaries between the grains of the material, which leads to an increase in the surface available for petroleum substances. Sorption on the tested sorbents takes place by filling the available pores and capillaries between the sorbent grains. This is a capillary action that depends on the effective diameter of the capillary, the viscosity of the absorbed petroleum substance and the surface energy of the inner wall of the capillary, with the micropores present in the structure of minerals being available only for substances of low molecular weight [131]. The second mechanism of sorption concerns the outer surface of the sorbent, on which a uniform layer of petroleum substance or its irregular clusters is formed. Importantly, substances with a higher molecular weight are usually adsorbed preferentially over substances with a lower molecular weight, and the sorption capacity of sorbents is higher than petroleum substances with a higher density and viscosity [132,133].
In addition, the positive impact of sorbents on the enzymatic activity of soil reflects their effect on its physicochemical and chemical properties, including their potential to reduce hydrolytic acidity and increase the exchange capacity of the soil [134]. In the present study, all sorbents stimulated the enzymatic activity in LS polluted with DO, whereas in SL, this effect was triggered by the molecular sieve, Ikasorb and expanded clay. Sorbents had no significant effect on the enzymatic activity of the soils polluted with P, which could be due to the chemical composition of both the petroleum-derived products and sorbents themselves [128,135].

Conclusions
DO and P turned out to be toxic to Zea mays, with the DO found to more strongly impair its growth and development than P, and especially when maize was grown in LS rather than in SL. The sorbents proved useful in alleviating the effects of these petroleumderived products on the growth and development of this crop, especially in LS. They were more effective in neutralizing the toxic effect of DO than P, with the molecular sieve the most effective in LS, whereas the expanded clay and sepiolite were the most effective in SL. Both soil types polluted with DO exhibited higher enzymatic activity compared to the non-polluted soils. However, similar effects of P and DO were observed only in LS. In sandy clay (SL), P was found to be a significant inhibitor of Deh, Cat, Ure, Pal and Aryl activities, while DO stimulated the activity of all enzymes in this soil. Moreover, in loamy sand (SL) contaminated with DO, all the tested sorbents increased the activity of urease to the greatest extent, with the most stimulating effect attributed to the molecular sieve. The molecular sieve stimulated the biochemical activity of both non-polluted soils. The effects of the sorbents in the soils polluted with P were negligible. Regardless of enzyme type, the molecular sieve was proven to be the most effective remediating agent in the soils polluted with DO and P, although all sorbents tested can be recommended for the remediation of soils polluted with DO, especially soils of a lower agronomic value.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, interpretation of data; in the writing of the manuscript; or in the decision to publish the results.