Effect of Platinum Nanoparticles (PtNPs) Pollution on the Biological Properties of Haplic Cambisols Eutric of the Caucasus Forests

Abstract

Pollution by platinum (Pt) is an emerging threat to forest soil health. The widespread use of Pt nanoparticles (NPs) in gas neutralizers for automobile exhaust has sharply increased the amount of PtNP pollution in the environment, including forest ecosystems. Recently, territories with Pt concentrations greater than 0.3 mg/kg in soil have been discovered. This concentration is 750 times greater than the background content in the earth’s crust. Cambisols, the most prevalent forest soil type in boreal forests that determines the functioning of the entire forest ecosystem, occupy a significant share of the Earth’s soil cover, which is about 1.5 billion hectares worldwide, or 12% of the entire continental land area. This shows the importance of studying the effect of pollution on this type of soil. In this study, laboratory simulations of PtNP contamination of the Haplic Cambisols Eutric at concentrations of 0.01, 0.1, 1, 10, and 100 mg/kg were carried out. The effect of PtNPs on soil properties was assessed using the most sensitive and informative biological indicators. The total number of bacteria was studied by the methods of luminescent microscopy, catalase activity (gasometrically), dehydrogenases activity (spectrophotometrically), germination, and length of roots by the method of seedlings. It was found that at the concentrations of 0.01, 0.1, and 1 mg/kg of PtNPs, there was either no effect or a slight, statistically insignificant decrease in the biological state of Haplic Cambisols Eutric. Concentrations of 10 and 100 mg/kg of PtNPs had a toxic effect on all the studied parameters. No statistically significant stimulating effect (hormesis) of PtNPs on the biological properties of Haplic Cambisols Eutric was observed, which indicates the high toxicity of PtNPs and the importance of studying the consequences of soil and ecosystem contamination with PtNPs. However, when the content of Pt in the soil was 1 mg/kg, there was a tendency to stimulate germination, the length of radish roots, and the total number of bacteria. The toxicity of PtNPs measured by biochemical indicators (activity of catalase and dehydrogenases) starts at a concentration of 100 mg/kg for phytotoxic effects (germination and root length of radish) and 10 mg/kg for microbiological effects (total number of bacteria).

1. Introduction

Pollution by platinum (Pt) is an emerging threat to forest soil health. The widespread use of Pt nanoparticles (PtNPs) in gas neutralizers for automobile exhaust has sharply increased the amount of PtNP pollution in the environment, including forest ecosystems. Recently, territories with Pt concentrations greater than 0.3 mg/kg in soil have been discovered.

Haplic Cambisols Eutric are found in boreal forests, account for a significant portion of the Earth’s soil cover (approximately 1.5 billion hectares worldwide, or 12% of the total continental land area), and play an important role in forest ecosystems [1,2]. The functioning of the entire forest ecosystem depends on the ecological state of forest soils. Forest soils are often polluted with heavy metals [3,4], which leads to a decrease in biological activity and soil fertility. In addition, heavy metals have a direct toxic effect on forest vegetation, reduce forest productivity, and degrade the wood’s quality [5,6]. In addition to their ecological and economic importance, the cambisols of the boreal forests play a vital role in ensuring the food security of the growing population.

Since the 1970s, platinum (Pt) has been actively used in motor vehicles as a catalyst, which has led to a sharp increase in Pt contamination in the soils along roads. It was only since the late 1980s that the Pt content in the soils of roadside landscapes began to be studied [7]. Automotive catalysts have become the main source of anthropogenic pollution of the environment with Pt, mostly in the form of nanoparticles (NPs) [8,9,10]. During the operation of vehicles, Pt is released as both nano- and micrometer-sized particles on the surface of road dust, roadside soils, and on plants [11,12]. The global release of catalysts is 0.8–6.0 tons of Pt per year. If one assumes that 500 million cars are equipped with catalysts and that the average annual mileage is 15,000 km per car, then the average level of Pt emissions per year is 0.0001–0.0008 mg/km [13]. Natural Pt content in the Earth’s crust is 0.0004 mg/kg [14]. Volcanism, weathering of rocks, and deposition of extraterrestrial matter are natural sources of platinoids on the Earth’s surface [15]. Considering that the concentrations of both substances in the continental crust are low and that natural sources provide a limited amount of platinoids on the Earth’s surface, the fate of these elements in the environment should not be of particular concern. However, anthropogenic emissions of Pt group elements (PGE) began early in the industrial period, around the 1750s. This is evidenced by the fact that, since that time, there has been an accumulation of PGE in some soils.

The increased release of Pt into the environment has led to the fact that one can already observe territories with a concentration of Pt in the roadside soil of more than 0.3 mg/kg [16] and in the road dust of more than 2.2 mg/kg [17], which exceed the content of Pt in the earth’s crust by 750 and 5500 times, respectively. In addition to the automotive industry, other industries in which Pt is widely used can be sources of Pt release into the environment: medicine [18,19,20], chemicals [21,22], electrical engineering, and the glass industry [7,23]. The widespread use of Pt in various industries can lead to the achievement of toxic levels of this metal in the environment and, consequently, an imbalance in the ecosystem, a decrease in the microbial population, the decomposition of soil organic matter, and a decrease in soil fertility. Due to the increase in Pt emissions into the environment, the need to determine its background levels and behavior in the environment increases.

Information on the content of Pt in forest soils is scarce. There are data on the content of Pt in the forest soils of Berlin (0.00583 mg/kg) [24] and in the nature reserve of Italy (0.00251 mg/kg) [25]. Previous studies have determined an increased Pt content in soils subject to anthropogenic influence in comparison with untouched soils [24,26,27]. An increased Pt content in soils, especially in the form of NPs, will unavoidably negatively affect the soil biota. Because nanoscale HMs have different or improved properties compared to their conventional “bulk” (micro) counterparts due to their increased relative surface area, this results in higher reactivity and, therefore, a stronger impact on performance and soil conditions [28]. It was found that low concentrations of platinum can have both stimulating and toxic effects on the enzymatic activity of soils, depending on the enzyme under study [29]. Toxicity is noted, and PtNPs are known to reduce the biological activity of microorganisms [29,30,31]. A 2.5 mg/kg dose of PtNPs reduced the total number of bacteria in all soil horizons and inhibited nitrogen fixation and nitrification in loamy horizons [32]. In addition, there are data on the effect of PtNPs on plant growth [33,34] and how they accumulate in the plant tissues [35,36,37]. The most studied plant is grass, which usually grows on roadside soil. The maximum Pt concentrations in grass were 0.03 and 0.256 mg/kg, respectively, near highways and smelters [38,39]. Kołton and Czaja [33] studied the effect of different forms and concentrations of Pt on the germination and growth of the roots of different plants (oats (Avena sativa), watercress (Lepidium sativum), and tomato (Lycopersicon esculentum). The toxicity of Pt-containing compounds for root growth in young seedlings varied depending on the dose and plant type tested. A dose of Pt at 1 mg/kg had a stimulating effect on the growth of the length of the roots of watercress (Lepidium sativum) [33]. It is critical to assess the condition of Haplic Cambisols Eutric under chemical contamination with PtNPs in this regard.

The hypothesis of the study was that PtNPs would show a toxic effect with a certain dose of content in the soil. In addition, it was interesting to see if the “low dose effect” (hormesis) that had been noted earlier for other heavy metals would manifest itself.

The purpose of this research is to evaluate the ecotoxicity of PtNPs using biological indicators of the state of Haplic Cambisols Eutric.

2. Materials and Methods

2.1. Characteristics of Platinum Nanoparticles (PtNPs)

Metal-based NPs have high mobility and the ability to show high toxicity to soil biota and plants [40,41]. Spherical PtNPs with a size of 20–30 nm and a purity of 99.99% were obtained from Guangzhou Hongwu Material Technology Co., Ltd. (Guangzhou, China).

2.2. Study Object

Haplic Cambisols Eutric is widely distributed throughout the world, particularly in Europe, the East Coast of America, and East Asia [42,43]. The Haplic Cambisols Eutric samples were taken from the territory of the Republic of Adygea (Maykop district, Nikel village, 44°10.649′ N, 40°9.469′ E). This territory is characterized by a beech-hornbeam wood type ecosystem. The soil for the model experiments was selected from the topsoil (0–10 cm). The pollutants mainly accumulate in this very topmost layer of the soil [44]. The results of the analysis showed that the soil selected for this study is characterized by a low content of organic matter in the upper horizon (2.7%), an acidic reaction (pH = 5.6), a medium-loamy texture, and low biological activity. The soil for the model experiments was selected far from sources of platinum pollution, primarily mining sites, highways, etc. An analysis of the content of 30 heavy metals (HM) in the soil was carried out, none of which were found to be contaminated. The content of Pt in Haplic Cambisols Eutric was determined by inductively coupled plasma mass spectrometry (ICP-MS), complete opening from a sample of 10 g, on ELAN-DRC-e or Agilent 7700× instruments at the Federal State Unitary Enterprise All-Russian Scientific and Research Geological Institute (FSUI RSRGI), directed by A.P. Karpinsky, and amounted to 0.0025 mg/kg. At the same time, in the area of the tailing dump of the Urup Mining and Processing Plant (Karachay-Cherkess Republic, Russia), the content of Pt in Haplic Cambisols was up to 0.28 mg/kg, which is 112 times higher than the content of Pt in uncontaminated brown soils of the Caucasus.

2.3. Model Experiment

After sampling, the soil was dried, cleaned of plant residues, ground, and sieved through a sieve with 3 mm holes. Then, 300 g were weighed into 500 mL plastic containers, and the contaminant was added. PtNPs do not dissolve in water; therefore, initially, PtNPs were mixed with a small amount of soil and then added to the total mass, which ensured an even distribution of the contaminant. Vegetative vessels with soil were maintained in the laboratory under controlled conditions: room temperature (20–22 °C) and optimal hydration (25%). Soil moisture was controlled by the gravimetric method. The PtNPs were introduced into the soil of Haplic Cambisols Eutric in the form of a dry, fine powder with concentrations of 0.01, 0.1, 1, 10, and 100 mg/kg of dry soil. The investigated doses of Pt were chosen to cover a wide range of possible pollutant concentrations in the soil. A 10-fold interval between doses was chosen. The dose of 0.01 mg/kg exceeds the natural background content of Pt in the soil by 25 times. Even though the highest concentrations of PtNPs (10, 100 mg/kg) are not found in soils today, they will become relevant very soon. Our goal was to establish the lowest environmentally safe concentration of Pt in the soil, which does not cause toxic effects on living organisms. We assumed that the smallest of the studied doses would not have a toxic effect, which was confirmed by the study’s results.

The soil was incubated for 10 days. We chose a period of 10 days since changes in the biological properties of the soil are already noted in this period, and a longer incubation period increases the difference in the state of the soil incubated in the laboratory from its state in natural conditions [45].

After the end of incubation, the soil was dried and crushed through sieves with a diameter of 3 mm to determine the activity of enzymes. Soil samples were used to determine the following biological indicators: the total number of soil bacteria, catalase and dehydrogenase activities, seed germination, and length of radish (Raphanus sativus L.) roots. Biological indicators are the first to react to anthropogenic impact and demonstrate soil deviation from its normal state and functioning [41].

2.4. Biological Indicators

2.4.1. Total Number of Soil Bacteria

Soil microorganisms are highly sensitive to heavy metal contamination [46]. Bacterial growth and reproduction rates are so high that it is possible to track the effectiveness of any environmental factor in a short period of time. The sensitivity of soil bacteria in Haplic Cambisols Eutric to PtNPs was determined based on the total number of bacteria using the luminescent microscopy technique reported by Zvyagintsev et al. [47]. The luminescent microscopy method is based on the optical study of bacteria stained with acridine orange, which emits a glow when exposed to UV radiation, with further counting of microbial cells (n = 720: 3 incubation vessels with soil × 3 soil samples × 4 square centimeters on slides × 20 fields of view).

2.4.2. Enzymatic Activity

Soil enzymes have great importance among numerous indicators of soil biological activity. Their diversity and richness make it possible to carry out sequential biochemical transformations of organic residues that enter the soil [48]. The use of enzymatic activity as a diagnostic indicator is facilitated by lower error rates during the experimentation (not more than 5%–8%) and high enzyme stability during sample storage [49]. This study concentrated on the enzymatic activities of the oxidoreductase class, specifically catalase and dehydrogenases, because these enzymes are involved in the redox process of humus component synthesis. The catalase enzyme catalyzes the decomposition of hydrogen peroxide into water and molecular oxygen. Enzymatic activity was determined by the gasometrically method, which determines the decomposition rate of 3% hydrogen peroxide (H₂O₂) after contact with soil (temperatures of 20–22 °C). Catalase activity is expressed in ml of O₂ per 1 g of soil released in 1 min. Dehydrogenases catalyze redox reactions by dehydrogenating organic substances. The optical density of colored solutions was spectrophotometrically determined on a PE 5800VI spectrophotometer at a wavelength of 540 nm. Dehydrogenases’ activity is expressed in mg of triphenyl formazan (TPF) per 10 g of soil in 24 h.

2.4.3. Phytotoxicity Analysis

Plant seeds or seedlings appear to be appropriate test organisms for determining the soil toxicity. Radish (R. sativus L.) seeds of variety “18 days” were used for germination. Radishes are a traditional test object for determining soil phytotoxicity, in particular when polluted with toxic substances [50]. Their advantage is a small supply of nutrients in the seed, which makes it very sensitive to adverse environmental factors, in particular pollution. Our studies have shown the high sensitivity and informativeness of radish germination and root length indicators in assessing the phytotoxicity of various soils with different pH, humus content, granulometric composition, etc., including forest soils.

The method is based on the high responsiveness of radish seeds to toxic substances by considering the reduction in germination and root length of seedlings compared to their respective controls, expressed as a percentage. Soils contaminated with PtNP pollutant were placed in a Petri dish after incubation and growth. Radish seeds were germinated in a Petri dish of soil mass—25 seeds in a Petri dish at a humidity of 60% and a temperature 24–25 °C. Seven days after the beginning of the experiment, the radish was pulled out of the soil, and seed germination (in pieces) and length of radish roots (in mm) were determined.

2.5. Data Analysis

The total number of soil bacteria was expressed as 10⁹ bacteria per 1 g of dry soil weight (Equation (1)):

$$\mathit{M}=\frac{\mathit{b}\times \mathit{A}\times \mathit{H}\times \mathit{T}}{\mathit{P}}$$

(1)

where: M—number of cells per 1 g of fresh soil; b—magnification coefficient (b = 4); A—average number of cells within one field of view; H—Dilution index; T—Conversion rate in billions of bacteria per 1 g of soil (T = 10¹⁰); P—Area of the field of view, µm².

The integral indicator of the biological state (IIBS) of soil was calculated according to Kazeev and Kolesnikov [50]. To do this, in the sample, the maximum value of each indicator is taken as 100%, and in relation to it, the indicator value in the remaining samples is expressed as a percentage (Equation (2)).

$${\mathit{B}}_{\mathit{1}}=\frac{{\mathit{B}}_{\mathit{x}}}{{\mathit{B}}_{\mathit{m}\mathit{a}\mathit{x}}}\times \mathit{100}\%$$

(2)

where: B₁—the relative score indicator; B_x—the actual value indicator; B_max—the maximum value indicator.

Thereafter, the average evaluation score of the studied indicators for the sample (option) was calculated, the absolute values of which cannot be summed up since they have different units of measurement (mg, mm, %). The integral indicator of the biological state IIBS of soil was calculated similarly to Equation (3):

$$\mathit{I}\mathit{I}\mathit{B}\mathit{S}=\frac{\mathit{B}}{{\mathit{B}}_{\mathit{m}\mathit{a}\mathit{x}}}\times \mathit{100}\%$$

(3)

where B represents the average evaluation score of all indicators and B_max represents maximum evaluation score of all indicators.

The methodology used makes it possible to integrate the relative values of various indicators, which have different units of measurement.

When calculating the integral indicator, the most informative and sensitive indicators of soil biological activity should be used. The total number of bacteria is one of the best indicators of the state of decomposers in an ecosystem. The activity of catalase and dehydrogenases reflects the intensity of organic substance mineralization processes in the soil and is used to assess soil biological activity. Enzymes of the oxidoreductase class (activity of catalase and dehydrogenases) are much more sensitive to chemical soil pollution than other classes of enzymes. The germination rate and length of radish roots are the most informative of the numerous indicators of phytotoxicity recovery [46].

Thanks to this IIBS methodology, it is possible to compare the biological characteristics of soils in terms of relative values (relative to the control, 100%). The soil condition was assessed based on the change in IIBS on the scale of the change in the indicator relative to the control (%). When IIBS changes <5%, no significant changes in soil condition are recorded (no changes). If a change is found in the range of 5%–10% of the control, then a change in information functions is detected; with a decrease of 10%–25% from the control, a violation of the chemical, physical, and biochemical functions of the soil is detected. When IIBS changes by more than 25%, the physical functions of the soil are disturbed. In this case, the soil needs a long recovery period [46].

The degree of sensitivity of the indicator was judged by the degree of decrease in its values in the options with contamination that were compared with the control. The correlation between the biological indicator and substance concentration in the soil was used to assess the indicators’ informative value. The closer the correlation coefficient R = −1 is, the higher the informational content of this biological indicator.

2.6. Statistical Analyses

The rate of change (standard deviation) was analyzed at p ≤ 0.05 to determine the significance of the results. The results of threefold repetitions were used to collect data. Statistical data processing was carried out using the software package Statistica 12.0 and Python 3.6.5 Matpolib. The nonparametric Spearman correlation coefficient was calculated between the concentration of NPs and the average value of biological indicators. The results are presented using one-way ANOVA on the mean and standard error (S.E.) of three separate replications. Fisher’s LSD test was used to compare means with the control treatment (0.0025 mg/kg), and differences were deemed significant at a probability level of less than 0.05.

3. Results

3.1. Effect of PtNPs on the Enzymatic Activities of Haplic Cambisols Eutric

Regarding the degree of influence of different PtNP concentrations on catalase and dehydrogenase activity, it was found that PtNPs at concentrations of 0.01, 0.1, 1, and 10 mg/kg had no significant effect on either catalase or dehydrogenase activities in Haplic Cambisols Eutric. However, at the maximum concentration of 100 mg/kg, catalase activity was significantly decreased (p = 2.8021 × 10⁻⁴) as compared to the control (0.0025 mg/kg). As shown in Figure 1a, this reduction accounted for 20% (5.6) relative to the control (7.0) and dehydrogenases activity by 28% (15.4) relative to their control (19.1). In the case of dehydrogenases activity, the only significant changes were observed in the case of 100 mg/kg dose treatment (p = 9.18643 × 10⁻⁴). All other doses had no significant effect (Figure 1). The stimulation of enzyme activity (hormesis) was also not recorded.

Early research results show both a negative effect of NPs on soil enzymatic activity [34,35,51] and a stimulating effect [36]. There are studies that have found the “effect of small doses” or hormesis of Pt on enzyme activity [29].

The mechanism of the inhibitory effect of metal NPs on enzymes seems to be due to their interaction with sulfhydryl groups [52,53]. The NPs’ surface could induce conformational changes in adsorbed protein molecules, which may affect the overall bioreactivity of the NPs. Functionalized NPs would selectively bind to cytochrome c or cytochrome c peroxidase and inhibit enzyme turnover.

3.2. Effect of PtNPs on the Phytotoxicity of Haplic Cambisols Eutric

The phytotoxicity of Haplic Cambisols Eutric was determined by the inhibition of seed germination and measuring the length of radish roots. It was found that after 10 days of contamination, at lower concentrations (0.01 and 0.1 mg/kg) of PtNPs, they had not shown any statistically significant effect on phytotoxicity indicators. The concentration of 1 mg/kg had no effect on radish seed germination but increased root length by 7% (45) compared to the control (41.7). However, this stimulation was statistically significant, and higher doses (10 and 100 mg/kg) inhibited both seed germination and root length of radish, as shown in Figure 2a,b. The maximum decrease was observed at a concentration of 100 mg/kg; the seed germination was decreased by 40% (60) to the control (100), and the root length was decreased by 45% (22.8) to the control (41.7), as shown in Figure 2.

Delay in germination and inhibition of plant root growth caused by exposure to PtNPs have also been noted by other researchers [34,54]. Mechanisms of toxicity of NPs for plants are associated with the inhibition of the main physiological processes, including photosynthesis, mineral nutrition, and connection with water [55,56]. As noted above, NPs cause the inactivation of enzymes because of their interaction with sulfhydryl groups of proteins and impair the permeability of cell membranes, which leads to metabolic disorders and causes chlorosis, necrosis, and stunting of shoots and roots [53,57].

Available literature suggests that PGE is received from the soil through the roots, and then PGE binds to biologically active substances rich in sulfur [58,59]. The PGE accumulation process takes place mainly in the vegetative parts of plants and decreases in the following order: root > stem > leaves [60].

3.3. Effect of PtNPs on the Total Number of Bacteria in Haplic Cambisols Eutric

The total number of bacteria was selected as a microbiological indicator of the state of Haplic Cambisols Eutric after PtNPs contamination. The PtNPs at concentrations of 0.01 and 0.1 mg/kg did not show any effect on the total number of bacteria. The concentration of 1 mg/kg was found to not significantly stimulate this indicator by 10% (2.6) compared to the control (2.4). At the higher doses of 10 and 100 mg/kg, the bacterial growth was suppressed by 17% (2.0) and 42% (1.4), respectively (Figure 3). As shown in Figure 3, at 10 and 100 mg/kg, the total number of bacteria was found to change significantly as compared to 0.0025 mg/kg treatment (control), respectively (p = 0.00531 for 10 mg/kg and p = 2.03866 × 10⁻⁶ for 100 mg/kg).

The high antibacterial activity of PtNPs was also noted by other researchers [31,32]. The reason for observing the toxicity induced by PtNPs for bacteria is increased levels of reactive oxygen species (ROS) in the cell [61]. Through physicochemical interaction at the cell membrane, NPs cause oxidative stress by generating ions that cause toxicity in the cell membrane surface [62]. The penetration of NPs occurs through intrusion at the diffusion site, endocytosis, and membrane proteins such as the phospholipid layer. Mitochondrial ROS can accumulate at higher levels, causing oxidative stress and disrupting the protein folding process, which causes ER stress and induces DNA damage, ultimately leading to the activation of cell death pathways [63].

To determine the ecological state of polluted soil, the IIBS was calculated. To do this, the maximum value of each indicator in the sample was taken as 100%, and in relation to it, the value of the same indicator in other samples was expressed as a percentage, i.e., a relative indicator. The IIBS allows for the establishment of general patterns of PtNPs’ influence on the state of Haplic Cambisols Eutric, depending on pollution parameters. Figure 4 depicts the results of the influence of PtNPs on the IIBS of Haplic Cambisols Eutric. It was found that at concentrations of 0.01, 0.1, and 1 mg/kg, no significant effect was observed on IIBS or the ecological state of Haplic Cambisols Eutric, while at higher concentrations of PtNPs, the biological properties of the soil deteriorated significantly. When applying 10 mg/kg of PtNPs, there is a decrease in IIBS by 12% compared to the control. The maximum decrease was noted in IIBS at the highest dose used (100 mg/kg), where a 35% decrease was observed relative to the control. No statistically significant stimulating effect (hormesis) of PtNPs was recorded on the biological properties of Haplic Cambisols Eutric.

4. Discussion

Contamination of Haplic Cambisols Eutric with PtNPs leads to a decrease in enzymatic activity only at the maximum studied concentration—100 mg/kg, where the reduction was found to be 20% compared to the control. Although this dose is quite a bit higher than the background Pt level in soil, with the increasing rate of Pt intake into the environment, this concentration may become actual in the coming decades. When Haplic Cambisols Eutric was contaminated with PtNPs at concentrations of 0.01, 0.1, 1, and 10 mg/kg, the activity of dehydrogenases decreased to a lesser extent than catalase activity. However, the dehydrogenases’ activity was strongly inhibited at the highest dose of 100 mg/kg, showing a reduction of 28% compared to the control. A similar pattern was reported earlier by Kolesnikov et al. [41] when Haplic chernozem was observed upon contamination with silver (Ag) NPs. NPs of other metals, such as Zn, Ti, Ce, Cu, Ni, and Fe, also have a negative effect on soil enzymatic activity, as has been demonstrated in other studies [64,65,66].

The results of the study revealed a significant negative impact of PtNPs at doses of 10 and 100 mg/kg on the phytotoxicity indicators (seed germination and root length of radish). When applying doses up to 1 mg/kg, PtNPs did not affect these indicators; however, higher doses (10–100 mg/kg) caused a decrease in seed germination by 16%–30% and root length by 21%–45%. Similar results for the sensitivity of phytotoxic parameters in Haplic Cambisols Eutric were recorded for the NPs of other elements such as Cu, Zn, and Ni [66]. Many authors reported the negative impact of heavy metal NPs such as Ti, Fe, Au, Ag, Zn, Cu, and Ce on the germination, seedling growth, physiological responses, and photosynthetic apparatus of plants [67,68,69,70,71].

The higher toxicity of PtNPs at concentrations of 10 and 100 mg/kg was also noted for the microbiological indicator of the state of Cambisols, i.e., the total number of bacteria. This indicator decreased significantly by 17% at 10 mg/kg and 42% at 100 mg/kg compared to the control. This indicates a high sensitivity of bacteria to PtNP contamination at the above-mentioned doses. Previously, the negative influence of other NPs such as Cu, Zn, Ni, and Ag on the microbiological parameters of soils was reported elsewhere [41,45,66,72]. It has been found that NPs pose a serious threat to the environment because of their accumulation potential. In the literature, the data on the increased content of PtNPs in various environmental samples, e.g., air [13], road dust [10], sewage [73], plants [74,75], and roadside soils [8,9,37], was documented.

The IIBS at the highest dose of 100 mg/kg PtNPs showed a significant decrease of 35% compared to the control. Based on the obtained IIBS results, an assessment of their informativeness and sensitivity was given to determine the effectiveness of their use in monitoring, diagnosing, and normalizing soil contamination with PtNPs, as given in

Table 1. The sensitivity and information value of biological indicators of Haplic Cambisols Eutric after PtNPs contamination.

Biological Indicator	Sensitivity 1	Informativeness 2
Catalase activity	92	−0.93 *
Dehydrogenases activity	93	−0.87 *
Germination of radish (R. sativus L.) seeds	88	−0.95 *
Length of radish (R. sativus L.) roots	89	−0.91 *
Total number of bacteria	90	−0.92 *

Note: ¹ Sensitivity indicator is the degree of decrease in the biological indicator when the soil is contaminated with PtNPs, % of the control (values are averaged overdoses). ² Informativeness indicator is the correlation coefficient (r) between the content of PtNPs in the soil and the biological indicator (p = 0.05). (* p ≤ 0.05)

. In this context, the decrease in the values of the biological indicator compared to the control was used to calculate the sensitivity.

After 10 days of contamination, several sensitivity indicators of Haplic Cambisols Eutric were discovered in the following order, as shown in Table 1: radish seed germination (88) ≥ root length of radish (89) ≥ total number of bacteria (90) ≥ catalase activity (92) dehydrogenases activity (93). Thus, seed germination and the length of radish roots (phytotoxicity indicators) were found to be the most sensitive to PtNPs contamination. On the other hand, catalase and dehydrogenases activities were the least sensitive. It was earlier reported that when Haplic Cambisols Eutric was contaminated with Ni and Zn NPs, phytotoxicity indicators were the most sensitive [48], in agreement with our findings. Perhaps this is due to the fact that exoenzymes adsorbed on soil particles exhibit enzymatic activity, which continues to work even after the death of living organisms.

To assess IIBS, the closeness of the correlation between the indicator and the concentration of PtNPs in soil was evaluated (Figure 5). The order of informative indicators was noted as follows: radish seed germination (−0.95) > catalase activity (−0.93) > total number of bacteria (−0.92) ≥ radish root length (−0.91) > dehydrogenases activity (−0.87). According to the results obtained during the statistical analysis, the most informative indicators were seed germination and catalase activity, and the least was dehydrogenases activity. Thus, catalase activity is one of the less-sensitive indicators. Moreover, seed germination was both the most sensitive and the most informative indicator.

5. Conclusions

The objective of this study was to understand the impact of PtNPs on the biological properties of Haplic Cambisols Eutric. The content of PtNPs at 1 mg/kg was found to positively stimulate germination, root length, and the total number of bacteria compared to the control. However, the toxicity of PtNPs increased with concentration. 100 mg/kg PtNPs were found toxic for catalase and dehydrogenases, decreasing the activity by 20 and 28%, respectively, compared to the control. The toxic effect was observed in concentrations of 10–100 mg/kg for germination, root length, and the total number of bacteria. A negative correlation was found between the values of biological parameters and the concentration of PtNP in the soil. Moreover, it was established for the study of PtNP contamination that the most sensitive and informative indicator was seed germination, while dehydrogenase activity provided the least. The obtained results make it possible to assess the ecological state of soils polluted with PtNPs, develop monitoring methods, detect environmental risks, and establish maximum permissible concentrations.