Biochar, Halloysite, and Alginite Improve the Quality of Soil Contaminated with Petroleum Products

Abstract

Investigations into the effective, fast, and economically viable remediation of soils polluted with petroleum-derived products are still relevant. The vegetative pot experiment was conducted at the Didactic-Experimental Garden greenhouse (NE, Poland, 53.759° N, 20.452° E) on loamy sand (LS) and sandy loam (SL) soils. Its main research objective was to assess the effectiveness of biochar (B), halloysite (H) and alginite (A) in the biological regeneration of contaminated soil diesel oil (DO) and petrol (P). The assessment was conducted by determining the magnitude of the adverse impact of these xenobiotics on the growth and development of Zea mays, as well as the activity of seven soil enzymes. The impact of the tested contaminants and sorbents was assessed based on the impact factors (IF) of DO and P, as well as B, H, and A on Zea mays biomass and enzymatic activity of the soil. Soil contamination with petroleum-derived products disrupted the growth and development of Zea mays. DO had a stronger inhibitory effect on plant growth compared to P. Zea mays cultivated in LS, which was less resistant to the effects of these contaminants compared to that cultivated in SL. The impact of DO and P on enzyme activity depended on the soil texture. DO stimulated enzyme activity in LS and SL, while P only did so in LS. All remediation substances, and biochar in particular, led to an increase in plant biomass in the DO-contaminated soils. Both biochar, halloysite and alginite also improved the biochemical quality index (BA) of SL and LS. Despite the unquestionable remediation potential of the analyzed sorbents, their highest efficacy can only be achieved by their application on soils with physicochemical properties corresponding to their characteristics, which is a valuable guideline for further research.

1. Introduction

Viable techniques for the separation of petroleum-derived substances have long been a challenge in the oil industry [1,2,3,4]. Issues encountered during production, transportation, and storage have been regulated for years by the Seveso III Directive (2012/18/EU) [5], which sets provisions regarding hazard control. It obliges for entrepreneurs to obtain necessary permits, conduct audits, and monitoring. The Seveso III Directive (2012/18/EU) mandates that industrial facilities develop and implement a risk management system and take preventive actions to minimize the risk of major industrial accidents. Enterprises are also obliged to prepare emergency plans. It is important to note that obligations and regulations may vary among individual Member States of the European Union. Therefore, it is crucial for enterprises to comply with the regulations and guidelines stipulated by the relevant regulatory authorities in their respective countries [5].

Preventive actions are highly justified, due to the fact that soils contaminated with petroleum-derived products contain numerous toxic compounds such as benzene, toluene, xylene (BTEX) or polycyclic aromatic hydrocarbons (PAHs) [6]. They raise multiple concerns due to their hydrophobic nature, low biodegradability, and consequently high persistence in the soil environment [7]. This translates into disturbances in soil properties, such as mechanical properties, permeability, shear strength, texture, and porosity [8,9], fertility characteristics (N, P, and K contents) and its chemical properties (soil conductivity, pH, and organic matter content) [10]. Soil homeostasis disruption also reflects the plant’s response to the pressure of these pollutants. It involves changes in plant physiology and biochemistry, including reductions in plant biomass, chlorophyll, and root vitality [11,12]. These findings are consistent with the observed highly toxic impact of diesel oil on the growth and development of Avena sativa L. [13], Lolium perenne L. × hybridum, Poa pratensis, Festuca rubra, F. arundinacea, Phleum pratense, and Dactylis glomerata, with plants showing significantly greater sensitivity to petrol. Moreover, the plant species also influence the extent of degradation of individual PAH groups [14].

One of the techniques used to separate petroleum-derived substances from soil involves the use of minerals [15,16,17]. Iron- and calcium-rich minerals can react with pollutants in the soil binding and stabilize them, thus causing their immobilization [18,19]. Therefore, phytostabilization is a stage that can contribute to reducing the toxicity and availability of pollutants to living organisms by adsorbing pollutants on mineral surfaces [20,21,22]. This process leads to the formation of less toxic compounds. The coupling of phytostabilization and phytoextraction, which utilizes plants capable of accumulating contaminants, can yield even better results [23,24,25]. Hence, novel techniques are required for the effective protection of soil, water in the soil, and essential nutrients crucial for plants and microorganisms, ultimately leading to the enhancement of environmental quality.

Biochar, also known as plant charcoal or pyrolytic carbon, is a product obtained through the pyrolysis of biomass, involving its decomposition under limited oxygen conditions. It has been found capable of effectively mitigating the emission of toxic compounds [26]. Biochar exhibits immense potential in regulating soil properties and fertility, promoting microbial activity, and enhancing plant growth [26,27,28,29,30]. As described by Hamidzadeh et al. [26], biochar’s carboxylic acids and phenolic compounds, derived from biomass lignin, augment the specific surface area of biochar, enabling the elimination of pollutants and proper absorption and distribution of nutrients in the soil. Furthermore, by applying current environmental modifications to pyrolysis, soil pH and the efficacy of aromatic compounds in biochar can be improved [26,31]. Selvarajoo et al. [32] demonstrated that, apart from enhancing soil properties such as pH, biochar exerted positive effects on nutrient cycling, water retention, and exchangeable cations. It was also shown to enhance water-holding capacity, reduce soil density, nitrogen content, leaching of pesticide residues, and essential mineral loss [26,28,29,31]. Additionally, biochar can also act as a carrier for microorganisms, influencing various soil biological processes, such as organic matter decomposition and mineral transformation [30,33]. This sorbent increases the mobility of anionic toxic elements [34]. Due to its porous structure, biochar can adsorb and retain organic pollutants [35,36], heavy metals [37,38,39], and other harmful compounds [15].

Halloysite is a mineral that mainly consists of silicon dioxide and aluminium oxides [40]. Clay minerals, such as kaolin, bentonite, and halloysite, belong to the kaolinite-serpentine subgroup and are described by the general chemical formula Al₂Si₂O₅(OH)₄. These clay silicate minerals are utilized as fuel additives for the thermal conversion of biomass and waste [41]. They are currently used in the energy sector to mitigate the risk of ash deposition and high-temperature corrosion in low-quality fuels such as biomass and waste. Maj and Matus [41] demonstrated that the use of clay silicates minimized combustion-related issues by immobilizing alkalis in highly volatile compounds. In turn, Masoudniaragh et al. [42] noted that the application of halloysite, particularly in agricultural fields, could lead to increased production. Additionally, according to these authors, halloysite can act as a carrier for biostimulants or bioactive components, which may contribute to their improved stability. In the study conducted by Masoudniaragh et al. [42], halloysite proved effective as a carrier of pyroligneous acid in mitigating the effects of soil salinity.

The application of alginite, similar to biochar, influences an increase in water retention capacity [43,44]. Consequently, soil amended with alginite becomes more drought-resistant, and plants have access to water for a longer period. Alginite, as a natural rock, contains essential nutrients such as macro- and micronutrients [45,46]. By adding alginite to the soil, these nutrients can be supplied to plants, leading to their healthier growth, increased productivity and condition [47,48,49]. Zhao et al. [43,50] observed that alginite significantly reduces the activity of stress enzymes, catalase, and peroxidase, indicating potential damage to the plant’s defense system. This natural, ecological, and versatile polymer is commonly used as a carrier for microbial cultures, mainly Pseudomonas and Azotobacter [51,52]. Alginite has the ability to regulate soil pH. It acts as a natural buffer, helping to maintain the optimal pH for plant growth. If the soil is too acidic or alkaline, the addition of alginite can assist in balancing and maintaining the optimal pH [53].

The described properties of the sorbents have prompted authors to formulate a research hypothesis that biochar, halloysite, and alginite are effective in the bioremediation of soil contaminated with petroleum-derived products. Therefore, a study was conducted with the aim to assess the applicability of these environmentally friendly materials in mitigating the adverse effects of diesel oil and petrol on the growth and development of Zea mays, as well as the activity of soil enzymes belonging to the oxidoreductase and hydrolase classes. For the phytoremediation of soils contaminated with petroleum-derived products, it was decided to utilize a popular crop that is distributed worldwide [19], with its cultivation area steadily increasing [54,55] characterized by a short life cycle [56,57]. The implementation of this defined objective allowed evaluating of the effectiveness of biochar, halloysite, and alginite in enhancing the quality of soils contaminated with petroleum-derived products.

2. Materials and Methods

2.1. Material Characteristics

2.1.1. Soil

Two soils with different textures were utilized, loamy sand (LS) and sandy loam (SL), in the vegetative pot experiment conducted in the greenhouse. They were selected for the study as sandy soils occupy approximately 50% of the agricultural land in Poland [58]. Based on the classification by the International Union of Soil Sciences [59], they were classified as Eutric Cambisols. LS and SL were collected from the top layer (0–0.20 m) of a field located in northeastern Poland (NE, Poland, 53,713° N, 20,432° E), where winter oilseed rape was cultivated. The soils were then transported to the greenhouse and air-dried. The soil used for the vegetative experiment was sieved through a 5 mm mesh sieve, while the soil used for the basic physico-chemical, chemical, and biochemical analyses was sieved through a 2 mm mesh sieve. A detailed characterization of the soils is presented in

Table 1. Some properties of the soil used in the experiment.

Abbreviation	Properties	Unit	Soil
			Loamy Sand (LS)	Sandy Loam (SL)
Grain-size Composition
Sand	0.05–2.0 mm	%	74.30	70.38
Silt	0.02–0.05 mm		23.69	27.19
Clay	<0.002 mm		2.01	2.43
Chemical and Physicochemical Properties
Ntot	Total Nitrogen	g kg−1 dm	0.98	1.01
Corg	Organic Carbon		11.20	11.50
Pavailable	Phosphorus Available	mg kg−1 dm	164.05	172.73
Kavailable	Potassium Available		53.95	78.85
Mgavailable	Magnesium Available		46.00	38.00
pH	Soil pHKCl Reaction	1 mol KCl dm−3	6.98	7.13
EBC	Sum of Exchangeable Base Cations	mM (+) kg−1 dm	84.20	181.80
HAC	Hydrolytic Acidity		8.00	5.70
CEC	Cation Exchange Capacity	%	92.20	187.50
ACS	Alkaline Cation Saturation		91.32	96.96
Enzymatic Activity per 1 kg dm h−1
Deh	Dehydrogenases	µM TFF	5.426	8.007
Cat	Catalase	M O2	0.211	0.369
Ure	Urease	mM N-NH4	0.153	1.114
Pac	Acid Phosphatase	mM PN	2.291	0.954
Pal	Alkaline Phosphatase		2.037	1.572
Glu	β-glucosidase		0.542	0.914
Aryl	Arylsulphatase		0.386	0.721

.

2.1.2. Plant

Zea mays PR39H32 (variety registered in the European Union) easily adapts to unfavorable environmental conditions. An additional advantage for the utilization of Zea mays in the study was the fact that its biomass can be used in alternative energy production [60,61,62].

2.1.3. Petroleum-Derived Products

The latest generation fuels meeting European requirements for sulfur-free fuels were used as the contaminating substances: VERVA diesel oil (DO) and VERVA 98 petrol (P). The density of diesel oil ranges from 820–845 kg m⁻³, whereas that of petrol ranges from 720–775 kg m⁻³. Both products were purchased from a PKN Orlen fuel station (Poland), and their detailed characteristics can be found on the website http://www.orlen.pl/ (accessed on 20 May 2021).

2.1.4. Sorbents

The vegetative experiment, aiming to reduce the mobility of petroleum-derived products in soil and neutralize their impact on the growth and development of Zea mays, as well as the biochemical properties of the soil, was conducted with three substances with sorptive properties: biochar (Fluid, Sędziszów, Poland), halloysite (Halosorb minerals sorbents, Intermark, Gliwice, Poland), and alginite (Vázsony-Szövetkezeti Kft., Nagyvázsony Hungary). Biochar is produced through the pyrolysis of various raw materials under limited oxygen conditions [47,63,64]. The biochar used in the study was produced from the biomass waste of willow and miscanthus. Halloysite (Al₂Si₂O₅(OH)₄) is a clay mineral from the kaolinite group characterized by high porosity (pore diameter of 10–20 nm). The halloysite used in the experiment was extracted from the Dunino mine, which is the only deposit of this mineral in Europe and one of three worldwide [47,65]. Alginite is a naturally occurring mineral from the liptinite group, formed from fossilized algal biomass and weathered tuffs. Its pH ranges from 7.15 to 7.78. A comprehensive characterization of biochar and halloysite was presented in our previous publications [38,39,47], and alginite [17,47] was also previously described.

2.2. Research Design

The pot experiment was conducted in the greenhouse of the University of Warmia and Mazury in Olsztyn (NE, Olsztyn, Poland, 53.759° N, 20.452° E) with 5 replications. The procedure for setting up and conducting the experiment was as follows:

(1)

Preparation of 2.5 kg of LS or SL soils whose characteristics are presented in Table 1.

(2)

Soil amendment with:

• N, P, K and Mg in the following amounts (mg kg⁻¹ dm of soil): 112, 39, 112 and 15, respectively. Nitrogen was applied as N₂H₄CO, phosphorus as—KH₂PO₄, potassium as—KH₂PO₄ and KCl, and magnesium as—MgSO₄ × 7H₂O;
• petroleum-derived products: diesel oil (DO) and petrol (P) at a rate of 0 and 7 cm³ kg⁻¹ dm of soil;
• sorbents: biochar (B), halloysite (H) or alginite (A) at a rate of 0 and 10 g kg⁻¹ dm of soil.

(3)

Mixing the soils was conducted with mineral fertilizers and, in appropriate treatments, with petroleum-derived products and sorbents. The soil was packed into polyethylene pots with a capacity of 3.0 dm³ (upper base diameter: 18.5 cm; lower base diameter: 14 cm; height: 15 cm) and the soil moisture was adjusted to 60% of the maximum water-holding capacity. This moisture level was maintained throughout the entire plant growth period (60 days).

(4)

Sowing 8 Zea mays seeds and leaving 5 plants in each pot after germination.

(5)

Harvesting the aerial and root parts of Zea mays at the Biologische Bundesanstalt, Bundessortenamt and Chemical (BBCH) stage 59. On the same day as the plant harvest, soil samples were collected for biochemical analyses. These samples were sieved through a 2.0 mm mesh sieve.

2.3. Methodology for Physico-Chemical and Chemical Determinations

Prior to the experiment setup, using the methods described in our previous studies [66,67], the soil texture was determined using the aerometric method, and pH was measured in a 1 mol KCl dm⁻³ solution using a potentiometric method with an HI 2221 m (Hanna Instruments, Washington, UK) in three replicates of air-dried soils. Additionally, the sum of exchangeable base cations (EBC) and hydrolytic acidity (HAC) were determined, based on which the cation exchange capacity (CEC) and alkaline cation saturation (ACS) were calculated. The chemical properties of the soil were assessed by determining the contents of total nitrogen (N_tot), organic carbon (C_org), and available P, K, and Mg. The Buchi B-324 distillation unit (Buchi, Flawil, Switzerland), Jenway 6705 UV/VIS spectrophotometer (Jenway Ltd., Staffordshire, UK), Jenway PFP 7 flame photometer (Jenway Ltd., Staffordshire, UK), and GBC 932AA atomic absorption spectrophotometer (GBC Scientific Equipment, Braeside, Australia) were used for these analyses.

2.4. Methodology for Determining Soil Enzyme Activities

Before the initiation of the vegetation experiment and after the harvest of Zea mays, the activities of dehydrogenases (Deh), catalase (Cat), and β-glucosidase (Glu) were used as indicators of the carbon cycle, urease (Ure) as an indicator of the nitrogen cycle, acid phosphatase (Pac) and alkaline phosphatase (Pal) as indicators of the phosphorus cycle, and arylsulfatase (Aryl) as an indicator of the sulphur cycle, and were determined using specific enzyme assay procedures (buffers, incubation times, termination of reactions, and temperature) described in our previous studies [37,67], in soils with a moisture content of approximately 60% of the maximum water-holding capacity (three replicates). The activities of Deh, Ure, Pac, Pal, Glu, and Aryl were measured using a Perkin-Elmer Lambda 25 spectrophotometer (Waltham, MA, USA).

2.5. Calculations

By considering the biomass of Zea mays’ aerial and root parts, as well as the activity of seven enzymes involved in C, N, P, and S metabolism, in both uncontaminated and petroleum-contaminated soils supplemented and non-supplemented with of biochar (B), halloysite (H), and alginite (A), the Impact Index (IF) of DO, P, and Ad on these parameters was determined using Formulas (1)–(4).

$${\mathrm{IF}}_{\mathrm{DO}}=\frac{\mathrm{D}\mathrm{O}}{\mathrm{C}2}-1\mathrm{or}{\mathrm{IF}}_{\mathrm{P}}=\frac{\mathrm{P}}{\mathrm{C}2}-1$$

(1)

where:

• IF—impact index of DO or P,
• DO—biomass of Zea mays or the activity value of the tested enzyme in DO-contaminated soil or P-contaminated soil without Ad,
• C2—biomass of Zea mays or the activity value of the tested enzyme in the control soil uncontaminated by DO or P without Ad.

$${\mathrm{IF}}_{\mathrm{Ad}}=\frac{\mathrm{A}\mathrm{d}\_\mathrm{C}2}{\mathrm{C}2}-1$$

(2)

where:

• IF—impact index of sorbents: B, H or A,
• Ad_C2—biomass of Zea mays or the activity value of the tested enzyme in the control soil uncontaminated by DO or P with the addition of Ad (B, H, A),
• C2—the explanation is provided in formula number 1.

$${\mathrm{IF}}_{\mathrm{Ad}\text{\_}\mathrm{DO}}=\frac{\mathrm{A}\mathrm{d}\_\mathrm{D}\mathrm{O}}{\mathrm{D}\mathrm{O}}-1$$

(3)

where:

• IF_{Ad_DO}—impact index of sorbents: B, H lub A in soil contaminated DO,
• Ad_DO—biomass of Zea mays or the activity value of the tested enzyme in DO-contaminated soil with the addition of Ad (B, H, or A),
• DO—biomass of Zea mays or the activity value of the tested enzyme in DO-contaminated soil without Ad.

$${\mathrm{IF}}_{\mathrm{Ad}\text{\_}\mathrm{P}}=\frac{\mathrm{A}\mathrm{d}\_\mathrm{P}}{\mathrm{P}}-1$$

(4)

where:

• IF_{Ad_P}—impact index of sorbents: B, H lub A in soil contaminated P,
• Ad_P—biomass of Zea mays or the activity value of the tested enzyme in P-contaminated soil with the addition of Ad (B, H, or A),
• P—biomass of Zea mays or the activity value of the tested enzyme in P-contaminated soil without Ad.

The above indexes can range from −1 to 1. An index of 1.00 indicates a 100% increase in the value of the measured parameter, whereas −1.00 represents a 100% decrease in its value. The indexes were presented on heat maps using RStudio 2023.06.0 [68] software with R 4.2.2 [69] and the gplots library [70].

Based on the activities of the analyzed enzymes, the soil quality index (BA) was calculated using formula number 5:

BA = Deh + Cat + Ure + Pac +Pal + Aryl + Glu

(5)

The BA index of soil quality assessment has been recognized as one of the most reliable, composite indicators of soil fertility from the pool of 21 indicators determining the condition of the soil. This is justified by the fact that it takes into account the activity of as many as seven soil enzymes presented in the formula, recognized not only as very sensitive indicators of early processes indicating the disturbance of soil equilibrium under the pressure of xenobiotics, but also indicators of the intensity of biological processes related to the physicochemical properties of soils [71].

2.6. Statistical Analyses

η2 coefficients were calculated and presented in a pie chart created using the Circos 0.68 package [72]. Principal Component Analysis (PCA) was performed to highlight the interdependencies between the data. The Shapiro–Wilk test was used to check for data normality. Statistical analysis, conducted separately for each tested soil, was carried out using the Tukey test with a significance level of p < 0.05. Standard deviations (SD) were also calculated for the yield of aerial parts and roots of Zea mays and for soil enzyme activity [73].

3. Results

3.1. The Biomass Yield of Zea mays under the Influence of Diesel Oil, Petrol, Biochar, Halloysite, and Alginite

Soil contamination with both DO and P had an adverse effect on the growth and development of Zea mays. However, plant response varied depending on the type of petroleum product and the soil texture (

Table 2. The effect of soil contamination with diesel oil (DO) and petrol (P) and sorbents on Zea mays biomass.

Type of Sorbent	Objects	Loamy Sand (LS)			Sandy Loam (SL)
		Aerial Parts (Ap)	Roots (r)	Ap/r	Aerial Parts (Ap)	Roots (r)	Ap/r
		g dm of pot−1			g dm of pot−1
Control (C1)	C2	40.712 b ±1.721	8.818 bc ±0.192	4.617	46.708 a ±1.031	8.533 ab ±0.293	5.474
	DO	4.903 e ±0.690	1.656 f ±0.238	2.960	28.122 d ±2.060	4.545 e ±0.816	6.188
	P	33.378 d ±3.270	6.826 d ±0.131	4.890	42.422 b ±1.594	5.723 de ±0.468	7.412
Biochar (B)	C2	41.930 b ±0.408	11.022 a ±1.495	3.804	46.733 a ± 0.726	9.501 a ±0.206	4.919
	DO	7.214 e ±0.148	4.953 e ±0.488	1.457	31.745 c ±1.011	5.302 de ±1.462	5.987
	P	35.805 d ±1.940	8.486 cd ±0.523	4.219	43.042 b ±0.489	6.588 cd ±0.363	6.534
Halloysite (H)	C2	47.858 a ±1.433	10.186 ab ±0.623	4.698	48.907 a ±0.893	9.212 a ± 0.629	5.309
	DO	6.025 e ±0.022	3.885 e ±0.188	1.551	31.124 cd ±1.421	8.385 ab ±0.855	3.712
	P	32.799 d ± 1.070	8.492 cd ± 0.603	3.862	40.200 b ±1.270	8.181 abc ±0.819	4.914
Alginite (A)	C2	40.097 bc ± 1.850	9.234 bc ± 0.835	4.342	47.796 a ±2.553	8.404 ab ±0.418	5.687
	DO	7.304 e ± 0.768	3.876 e ± 0.012	1.884	33.092 c ±0.699	7.031 bcd ±0.922	4.707
	P	36.641 cd ± 2.069	8.058 cd ± 1.098	4.547	46.807 a ±1.064	5.682 de ±0.367	8.237

C1—soil without sorbent, C2—soil uncontaminated with DO or P; DO—diesel oil VERVA; P—VERVA 98 petrol. Homogeneous groups denoted with letters (a–f) were calculated separately for each part of the plant and each kind of soil and each kind of soil.

).

DO had a stronger inhibitory impact on the growth of the aerial parts and roots of Zea mays compared to P (Figure 1a,b). This effect was observed in both loamy sand (LS) and sandy loam (SL). Under DO pressure, the biomass of aerial parts and roots of Zea mays in LS was reduced by 88.0% and 81.2%, respectively, whereas in SL it was reduced by 39.8% and 46.7%, respectively. As a result of the different effects of DO and P in LS and SL, higher values of the aerial parts-to-root biomass ratio were determined in Zea mays grown in SL compared to LS. When P was added to both LS and SL, it had a significantly smaller disruptive effect on the growth of Zea mays, as indicated by the higher IF_P values compared to IF_DO (Figure 1a,b). Specifically, the IF_B values of the aerial parts of Zea mays in LS and SL were higher by 0.700 and 0.306, respectively, whereas the values for roots were higher by 0.586 and 0.138, respectively.

In both soils not exposed to petroleum products, two out of the three sorbents (biochar and halloysite) among biochar, halloysite, and alginite induced the growth of the aerial parts and roots of Zea mays (Table 2, Figure 1a,b).

The greatest increase in the aboveground biomass (17.6%) was observed with the application of halloysite in LS, whereas for roots, the highest increase (25.0%) was observed with biochar, also in LS. Generally, the effectiveness of these sorbents was more pronounced in LS compared to SL.

The assessment of biochar, halloysite, and alginite for effectiveness in mitigating the toxic impact of DO and P revealed their potential in alleviating the adverse impact of petroleum products on the yield of Zea mays. However, this potential varied depending on contaminant type and soil texture. In LS, the effects of all sorbents on the yield of aerial parts and roots were significantly higher in the case of DO-contaminated soil compared to the P-contaminated soil, whereas in SL, such effects were only observed for the aboveground biomass (Table 2). These findings were supported by the influence indices of individual sorbents on the aboveground and roots biomass of Zea mays, which consistently showed positive values in DO contaminated LS and SL. Positive values were observed only in LS and SL supplemented with biochar, as well as LS supplemented with alginite in P-contaminated soils (Figure 1a,b).

3.2. Changes in Soil Enzyme Activity under the Influence of Diesel Oil, Petrol, Biochar, Halloysite, and Alginite

The contamination of LS and SL soils with petroleum products resulted in a significant destabilization of the enzymatic properties of the soils, as shown in

Table 3. The impact of diesel oil (DO) and petrol (P) contamination, as well as sorbents, on enzyme activity in 1 kg dm of soil × h⁻¹ of loamy sand (LS).

Type of Sorbent	Object	Deh	Cat	Ure	Pac	Pal	Glu	Aryl
C1	C2	5.901 h ±0.090	0.227 g ±00.008	0.160 g ±0.125	2.473 g ±0.004	2.086 de ±0.001	0.571 cd ±0.004	0.401 d ±0.002
	DO	6.824 g ±0.087	0.339 c ±0.000	0.264 f ±0.027	3.551 bc ±0.012	2.606 b ±0.010	0.476 fg ±0.003	0.385 de ±0.003
	P	7.388 ef ±0.219	0.386 a ±0.011	0.266 f ±0.045	3.369 cd ±0.094	1.986 e ±0.005	0.446 h ±0.007	0.256 g ±0.007
B	C2	9.075 c ±0.001	0.246 f ±0.004	0.421 d ±0.025	3.079 e ±0.024	2.131 cd ±0.060	0.614 b ±0.006	0.443 c ±0.009
	DO	7.614 e ±0.183	0.347 bc ±0.008	0.311 e ±0.027	3.722 ab ±0.190	2.619 b ±0.017	0.515 e ±0.004	0.009
	P	7.105 fg ±0.242	0.310 d ±0.008	0.334 e ±0.025	3.829 a ±0.113	2.123 cd ±0.026	0.494 ef ±0.024	0.275 g ±0.003
H	C2	10.240 b ±0.069	0.227 g ±0.008	0.683 b ±0.025	2.752 f ±0.010	2.204 c ±0.009	0.657 a ±0.002	0.428 c ±0.009
	DO	7.707 e ±0.173	0.384 a ±0.004	0.287 e ±0.013	3.289 de ±0.020	2.738 a ±0.006	0.493 ef ±0.001	0.476 b ±0.007
	P	6.686 g ±0.090	0.287 e ±.0.000	0.421 d ±0.025	3.180 de ±0.002	2.110 cd ±0.010	0.501 ef ±0.004	0.298 f ±0.003
A	C2	8.173 d ±0.007	0.234 fg ±0.005	0.595 c ±0.025	2.715 f ±0.008	2.148 cd ±0.009	0.589 bc ±0.005	0.551 a ±0.005
	DO	11.594 a ±0.256	0.364 b ±0.008	0.893 a ±0.013	3.591 b ±0.055	2.754 a ±0.026	0.558 d ±0.017	0.009
	P	5.732 h ±0.135	0.227 g ±0.000	0.465 d ±0.025	3.314 d ±0.015	2.046 de ±0.006	0.453 gh ±0.003	0.375 e ±0.003

C1—soil without sorbent, C2—soil uncontaminated with DO or P; B—biochar, H—halloysite, A—alginite, DO—VERVA diesel oil; P—VERVA 98 petrol; Deh—dehydrogenases; Cat—catalase; Ure—urease; Pac—acid phosphatase; Pal—alkaline phosphatase; Glu—β-glucosidase; Aryl—arylsulfatase. Homogeneous groups denoted with letters (a–h) were calculated separately for each enzyme.

and

Table 4. The impact of diesel oil (DO) and petrol (P) contamination, as well as sorbents, on enzyme activity in 1 dm of soil × h⁻¹ of sandy loam (SL).

Type of Sorbent	Object	Deh	Cat	Ure	Pac	Pal	Glu	Aryl
C1	C2	8.550 d ±0.327	0.393 b ±0.004	1.238 de ±0.066	1.011 h ±0.014	1.619 de ±0.013	0.986 e ±0.004	0.765 de ±0.014
	DO	11.395 c ±0.116	0.437 a ±0.012	1.664 b ±0.069	1.640 d ±0.011	2.318 c ±0.018	1.092 cd ±0.020	0.934 a ±0.021
	P	7.588 e ±0.125	0.303 ef ±0.004	0.979 g ±0.020	1.454 f ±0.031	1.553 de ±0.044	1.044 cde ±0.041	0.677 f ±0.034
B	C2	8.746 d ±0.490	0.244 g ±0.004	0.785 h ±0.012	1.539 e ±0.025	1.679 d ±0.011	0.998 e ±0.009	0.757 e ±0.004
	DO	11.168 c ±0.162	0.338 d ±0.008	1.074 fg ±0.069	1.927 b ±0.008	2.499 b ±0.128	1.100 c ±0.020	0.858 bc ±0.018
	P	6.206 f ±0.276	0.310 e ±0.008	1.118 ef ±0.025	1.400 g ±0.006	1.515 ef ±0.008	1.126 c ±0.004	0.681 f ±0.017
H	C2	8.769 d ±0.207	0.225 h ±0.007	1.282 d ±0.025	1.376 g ± 0.006	1.628 de ±0.011	1.004 de ±0.009	0.815 cde ±0.035
	DO	12.351 b ±0.300	0.374 c ±0.004	2.163 a ±0.026	1.644 d ±0.003	2.531 b ±0.019	1.127 c ±0.012	0.914 ab ±0.017
	P	7.651 e ±0.139	0.287 f ±0.004	1.118 ef ±0.025	1.956 b ±0.012	1.408 f ±0.064	1.418 a ±0.086	0.680 f ±0.021
A	C2	9.210 d ±0.149	0.232 gh ±0.004	1.519 c ±0.045	1.412 fg ±0.012	1.621 de ±0.011	1.004 de ±0.005	0.817 cd ±0.008
	DO	15.779 a ±0.466	0.354 d ±0.008	2.072 a ±0.026	1.859 c ±0.036	2.733 a ±0.021	1.109 c ±0.026	0.908 ab ±0.008
	P	6.884 ef ±0.344	0.227 gh ±0.004	1.162 def ±0.025	2.299 a ±0.015	1.568 de ±0.009	1.233 b ±0.020	0.654 f ±0.013

Abbreviations are explained under the Table 3. Homogeneous groups denoted with letters (a–h) were calculated separately for each enzyme.

and Figure 2 and Figure 3. The response of the enzymes to these pollutants varied depending on soil type. In LS, both DO and P stimulated the activity of Deh, Cat, Ure, Pac, and inhibited Glu and Aryl (Table 3), whereas in SL, they stimulated Pac and Glu activity (Table 4). The response of the other enzymes differed depending on contaminant type. In LS, DO significantly stimulated the activity of Pal, whereas in SL, it enhanced the activity of Deh, Cat, Ure, Pal, and Aryl. On the other hand, P decreased the activity of these enzymes. The response of soil enzymes to the application of sorbents to the soil not exposed to petroleum products was determined by multiple independent variables (Table 3 and Table 4, Figure 2 and Figure 3). Biochar, halloysite, and alginite significantly increased Deh activity (IF values were 0.538, 0.735, 0.385, respectively), Ure activity (IF—1.631, 3.269, 2.719), Pac activity (IF—0.245, 0.113, 0.098), and Aryl activity (IF—0.105, 0.067, 0.374) in LS. In SL, they significantly enhanced Pac activity (IF—0.522, 0.361, 0.397) and inhibited Cat activity (IF −0.379, −0.427, −0.410). Additionally, in LS, biochar induced Cat activity (IF 0.084) and Glu activity (IF 0.075), and halloysite induced Glu activity (IF 0.151), while in SL, Ure activity was stimulated by alginite (IF 0.227) and inhibited by biochar (IF −0.366).

Sorbents exhibited different effects on enzyme activity in the heavier soil contaminated with DO and P (Figure 3). In the DO series, Cat activity was significantly suppressed by all sorbents, while Ure and Aryl activities were reduced only by biochar. In the soil amended with P, the inhibitory effect of biochar was noted for Deh (IF −0.182) and Pac (IF −0.037), that of halloysite on Pal (IF −0.093), and that of alginite on Cat (IF −0.251). Conversely, in the soil contaminated with DO, biochar exhibited a stimulating effect on Pac (IF 0.175) and Pal (IF 0.078) activity, while P stimulated Ure (IF 0.142) and Glu (IF 0.079) activity. Halloysite increased the activity of Deh (IF 0.084) and Ure (IF 0.300) in the soil samples with DO and that of Ure (IF 0.142), Pac (IF 0.345), and Glu (IF 0.358) in the objects with P. Alginite enhanced Ure (IF 0.245–0.187) and Pac (IF 0.134–0.581) activity in the soil with DO and P, as well as Deh (IF 0.385) and Pal (IF 0.179) activity in the SL with DO, and Glu (IF 0.181) activity in P-treated soil.

The changes in soil enzyme activity observed under the influence of petroleum products and sorbents were reflected in the biochemical soil quality index (BA), which was 11.819 in LS and 14.563 in SL (Figure 4).

In the LS, both DO and P increased the BA value, while in SL, only DO exerted this effect. In the LS, all sorbents applied to the uncontaminated soil significantly increased the BA value, whereas only alginite exerted this effect in the SL. The greatest changes in the BA index in soil contaminated with DO were observed under the influence of halloysite and alginite, which increased the BA value by 6.4% to 39.5% in LS and by 8.3% to 27.4% in the SL. The sorbents tested for their effect on P caused significantly smaller fluctuations in the BA index in the experimental series.

3.3. Correlations between Zea mays Biomass and Soil Enzyme Activity—Independent Variable Analysis and PCA

The analysis of independent variables (η2) indicates that, in both soils, enzyme activity was mainly determined by soil contamination with petroleum products (Figure 5). An exception was the activity of Ure in LS, which was more dependent on soil amendment with sorbents than its contamination with DO and P. In LS, DO and P predominantly determined the activity of Pal (94.98% and 95.25% respectively), while sorbents influenced Ure activity (59.27% in LS) and Cat activity (35.00% in SL) to a greater extent. The biomass of the aerial parts of Zea mays in LS was primarily influenced by DO and P to a significant extent (97.51%), followed by root biomass (84.73%), whereas in SL, the influence was 91.28% and 47.08% respectively. Sorbents, on the other hand, influenced the above-ground biomass by 0.37% and 9.57% in LS and 3.01% and 24.25% in SL.

PCA analysis, considering all sorbents (B, H, and A) as well as petroleum products (DO and P), was performed separately for LS (Figure 6a) and SL (Figure 6b). In LS, positive correlations were observed between the activities of Pac and Pal with Cat, as well as Deh with Ure and Glu. In SL, Pal was correlated with Deh; Cat, and Ure; Aryl with Deh; Ure, and Pal, Deh with Ure, and Glu with Pac (Figure 7). Significant negative correlations were found between Cat, Pac, and Pal activities with the biomass of the aerial parts and roots of Zea mays in LS, as well as between Deh, Cat, Ure, Pal, and Aryl activities with the biomass of the aerial parts in SL. In both soils, DO had a greater destabilizing effect on Zea mays growth compared to P, resulting in a lower biomass yield of this plant (Figure 6a,b). The independent variables formed three groups: one in the soil not exposed to the pressure of petroleum products, the second in the soil with DO, and the third in the soil with P.

4. Discussion

4.1. Response of Zea mays and Soil Enzymes to Soil Contamination with Petroleum-Derived Products

The toxicity of petroleum-derived substances determines the quality of soils as well as the health of plants, animals, and even humans [20,22,74,75]. Therefore, the remediation of soil contaminated with organic compounds poses a challenge for scientists and practitioners [76,77,78], considering that there are approximately 2.8 million sites contaminated with diesel fuel, petrol, mineral oils, and heating oil in Europe alone [79]. Bica [80] estimates that the number of these sites will continue to increase. Therefore, the classification of contaminated areas into risk classes [80,81] and the selection of appropriate remediation measures are crucial for the restoration of these soils [82,83,84]. When choosing a remediation method, the type and extent of contamination, soil texture, size of the contaminated area, and its economic purpose use should be taken into account [85,86]. Therefore, the plants can play an invaluable role in mitigating potential hazards associated with the impact of petroleum-derived products [14,86,87]. They have the ability to eliminate organic pollutants through the acceleration of their degradation by microorganisms inhabiting the rhizosphere [14,88,89]. For successful phytoremediation, plants, especially those from the Poaceae family, should adapt well to the contaminated environment, be easy to cultivate, and produce a high biomass [89,90]. Worldwide research on phytoremediation potential [14,86,91] demonstrates that leguminous plants belonging to the Fabaceae family [85] and plants belonging to the Poaceae family [14,92,93,94,95] are most effective in degrading petroleum hydrocarbons in contaminated soils. All these plants have well-developed root systems capable of reaching deep soil layers where petroleum pollutants can accumulate [96,97,98]. The success of these plants in phytoremediation relies on the synergistic cooperation between plant roots and microorganisms inhabiting the rhizosphere [99], which participate in the breakdown and degradation of petroleum compounds [14,23]. Bacteria, fungi, and protozoa are the main producers of enzymes in the soil [100]. They secrete enzymes involved in the breakdown and degradation of organic compounds into simpler forms, releasing nutrients.

In the present research, we utilized Zea mays, belonging to the Poaceae family, whose biomass obtained from areas contaminated with organic compounds can be used for bioenergy production [62,101]. In our studies, both diesel oil and petrol disrupted the growth and development of Zea mays, although to varying degrees. The use of plants, particularly Poaceae, for the remediation of soils contaminated with petroleum substances offer a viable remediation strategy [85,102,103]. Diesel fuel and petrol containing aromatic hydrocarbons, benzene, toluene, and other toxic compounds, can exhibit strong phytotoxic effects [74]. The adverse impact of petroleum-derived products on plants, as observed in the present study, could be attributed to the reduction in nutrient levels and available water for plants, as well as disturbances in nutrient uptake [85,104], leading to oxidative stress in plants resulting from the overproduction of reactive oxygen species (ROS) impairing the synthesis of amino acids, enzymes, and proteins [105,106]. Dian et al. [104] and Meudec et al. [107] highlight the negative influence of hydrocarbons and heavy metals contained in petroleum-derived products on the reduction of chlorophyll levels and inhibition of photosynthesis. The extent of the impact of the tested petroleum-derived products in our studies, which are mixtures of organic compounds primarily composed of hydrogen and carbon atoms, as well as smaller amounts of nitrogen, oxygen, sulfur, and trace amounts of metallic components [108,109] on the growth and development of Zea mays, was determined by contaminant type and soil texture. Diesel oil exhibited more toxic effects on plants compared to petrol.

According to Al-Rubaye et al. [75], the toxicological effects of hydrocarbons depend on their molecular weight. Toxic substances present in diesel fuel can also increase the concentration of heavy metal salts [102,110,111], negatively affecting populations and activity of soil microorganisms, which play a significant role in the nutrient cycling and biological processes in the soil [4,67,74,103,112,113]. These changes can have an adverse impact on root development and the absorption of nutrients by plants. Compounds present in diesel fuel can therefore cause metabolic dysfunctions in plants, leading to their growth inhibition [85]. Since hydrocarbons persist in ecosystems for a long time, they can accumulate in animal and plant tissues, transferring from one trophic level to another in the food chain, causing death or genetic mutations. The interaction between petroleum-derived products and plants, as well as the soil enzyme activity, depends on the soil particle size distribution [67,87,112]. The present study results demonstrated that diesel fuel and petrol had significantly less disruptive effects on the growth of Zea mays cultivated on sandy loam compared to loamy sand. In loamy sand, both products increased the value of the biochemical soil quality index, which reflects the activity of seven enzymes, whereas in sandy loam, only diesel fuel did so. The differences in the effects of petroleum-derived products on soils with different agronomic categories arise from changes in the bioavailability of these products in individual soils [114,115].

4.2. The Role of Sorbents in Improving the Quality of Soil Contaminated with Petroleum-Derived Products

In recent years, there has been an intensive development of methods supporting the phytoremediation of soils contaminated with petroleum-derived compounds [90]. A modern approach to soil restoration under the pressure of organic compounds not only focuses on the use of biostimulation, bioaugmentation, and the application of biosurfactants [116], but also necessitates the search for newer remediation compounds. In our research, biochar, halloysite, and alginite were applied to enhance the phytoremediation potential. The higher impact indices noted for biochar, halloysite, and alginite in the soil exposed to DO pressure than P are a due to the stronger negative influence of DO on Zea mays biomass in both soils compared to P, as well as the unique properties of sorbents such as large surface area, porosity, carbon content, and increased water-retention capacity [43,44,90].

These properties of biochar, halloysite, and alginite induced an increase in the activity of dehydrogenases, catalase, urease, alkaline phosphatase, β-glucosidase, and arylsulfatase in loamy sand, as well as acidic and alkaline phosphatases in sandy loam. As a result of our research, a negative correlation was observed between Zea mays yield and soil enzymatic activity, most likely due to the negative, toxic interaction of petroleum-derived pollutants with plants. This negative correlation explains the positive response of Zea mays cultivated on soils contaminated with petroleum products to the applied sorbents (biochar, halloysite, and alginite). The sorbents used may have contributed to the improvement of nutrient availability for plants. Disturbances in the development of Zea mays and soil enzymatic activity were mitigated by the applied sorbents, although their effects varied depending on the tested soil. All remediation substances resulted in increased plant biomass in soils contaminated with DO. This is likely due to the adsorption of nutrients on the surface or within the sorbents, thereby protecting them from leaching or oxidation in the soil. The successive release of nutrients in a controlled and plant-adapted manner contributed to improved nutrient availability for plants, thus supporting the degradation of pollutants. This concept is supported by studies conducted by Fahimizadeh et al. [117], Guopeng et al. [118], and Wang et al. [119]. The use of sorbents for the remediation of soil contaminated with petroleum-derived products in combination with plants can be highly valuable. Based on our own research, it was found that biochar, halloysite, and alginite can be recommended for remediating soils contaminated with petroleum products, especially in coarser-textured soils. Differences in plant and enzyme response to soil contamination with petroleum products are attributable to the variability in colloidal clay content, which plays a similar role in the soil as the tested sorbents. This explains the underlying mechanism of the positive influence of sorbents on the development of Zea mays and enzyme activity in loamy sand. According to Zhang et al. [120], phosphorus-enriched biochar has great potential for use as a soil fertilizer. Additionally, Khosravi et al. [121] states that the use of biochar-based fertilizers can prevent nutrient leaching. The safe application of this sorbent is also supported by its certification based on two main credentials: the certification program of the Swiss-based European Biochar Certificate (EBC) [122] and the US-based International Biochar Initiative (IBI) [123].

5. Conclusions

The study demonstrates a negative correlation between the yield of Zea mays and soil enzymatic activity, which was mainly caused by the toxic effect of diesel oil and petrol on this crop. The magnitude of disturbances in the growth and development of Zea mays was determined by the type of contaminant and the texture of soils. Diesel oil exerted a more toxic effect on plants than petrol. Both pollutants caused greater disturbances in the development of aerial parts and roots of plants grown in loamy sand soils compared to sandy loam soils. This finding explains the positive response of Zea mays, cultivated in soils contaminated with petroleum-derived products, to the applied sorbents (biochar, halloysite, and alginite). All of these sorbents can be recommended for the remediation of soils contaminated with petroleum-derived products, especially in coarser-textured soils, as in the case of loamy sand, where they can improve enzymatic activity, as indicated by the magnitude of the biochemical soil quality index. The present study results demonstrate that combining the determination of biochemical indicators with the evaluation of plant responses to diesel oil and petrol pressure is a viable approach in soil quality assessment. The conducted research indicates that effective soil remediation depends on a thoughtful selection of a sorbent, based on the knowledge of its characteristics combined with well-defined physicochemical properties of the soil and the type of petroleum-derived substances. In the case of selecting the appropriate sorbent, one should consider its sorption capacity, ability to immobilize pollutants, biodegradability, and environmental impact. Simultaneously, we suggest that further research is necessary to better understand the successful phytoremediation processes, particularly regarding changes in soil microbiome quality, plants cultivated in contaminated areas, and the interactions between soil, plants, pollutants, and soil enzyme activity.