Zinc Oxide Nanoparticles in the “Soil–Bacterial Community–Plant” System: Impact on the Stability of Soil Ecosystems

: The use of man-made nanoparticles (NPs) has increased exponentially in recent years, many of which accumulate in significant quantities in soil, including through use in agriculture as nanofertilizers and nanopesticides. ZnO NPs are more environmentally friendly but have specific antimicrobial activity, which can affect soil microbiota, thereby influencing key microbial processes such as mineralization, nitrogen fixation and plant growth-promoting activities. Their behavior and persistence in soil depend on their chemical nature and soil characteristics. This review summarizes the applications of ZnO NPs in soil systems and their effects on various plants and soil microorganisms, particularly rhizobacteria that promote plant growth. A stimulating effect of ZnO NPs on the morphometric and biochemical characteristics of plants, as well as on soil microbiota and its activity at relatively low concentrations of up to 500 mg/mL and 250 mg/kg, respectively, is observed. As the concentration of ZnO NPs increases above these limits, toxic effects appear. The different effects of ZnO NPs are related to their size, dose, duration of exposure, solubility in water, as well as soil type, acidity and organic matter content. The review substantiates the need to study the behavior of ZnO NPs in the “soil-plant-microbiota” system for the possibility of using nanotechnologies in the agricultural industry and ensuring the safety of agricultural products.


Introduction
In the last few decades, industrial nanomaterials (NMs) have attracted much attention due to their increasing applications in almost all aspects of human activities [1].Nanotechnology offers enormous potential applications in the agricultural sector, including nanofertilizers, nanopesticides, nanoherbicides, nanosensors and intelligent delivery systems for the controlled release of agrochemicals [2].
Among the various types of nanoparticles (NPs) that are gaining increasing popularity, zinc oxide NPs (ZnO NPs) are considered one of the most used [3].However, for the effective use of ZnO NPs it is necessary to understand the mechanisms of their influence Agronomy 2024, 14, 1588 2 of 28 and key interactions with both the physical and biological environment of the soil, but information in this area is extremely limited.This review aims to fill this knowledge gap.Zinc (Zn) itself is one of the vital and essential microelements for plants and a component of more than 300 plant enzymes and vital proteins that are needed for proper plant growth and development.This trace element is essential for a wide range of physiological and biochemical processes, such as photosynthesis, protein synthesis, antioxidant function, maintaining the structural and functional integrity of biological membranes, detoxification of highly toxic oxygen free radicals, participation in protein synthesis and gene expression under normal and stress conditions, regulation of pollination and plant growth [4,5].Its normal requirement is met naturally; however, under certain environmental and physical conditions, toxicity due to excess Zn or problems associated with its deficiency can occur, which can lead to a number of diseases of crops and other plants and lead to their death or damage.Zn deficiency is one of the main problems limiting agricultural productivity in alkaline soil conditions [6].However, Zn deficiency in crops can lead to its deficiency in humans [7].A successful breeding program to biofortify food crops with Zn depends largely on the amount of Zn reserves available to plants in the soil.The use of nanofertilizers enriched with Zn offers a quick solution to the problem.To increase the accumulation of Zn in crops necessary for measurable biological effects, sufficient amounts of Zn must be maintained in the soil and available to plants [8].Despite a number of advantages, NMs are also known to have certain ecotoxicological consequences [9].They can become potential soil pollutants [10].Because soil is the main sink of NMs, which enter it both accidentally and intentionally (as a result of the use of NPs for the immobilization of toxic metals during land reclamation and as nanofertilizers and nanopesticides), increased concentrations of NMs can potentially increase adverse effects on living organisms in the soil.Soil microbiota and plants are among the main targets of NPs.Soil microorganisms are sensitive bioindicators that instantly respond to any changes in the soil environment.They are essential for most soil ecosystem processes and functions.They play a key role in soil fertility, plant nutrition and global biogeochemical cycles and are responsible for primary productivity, plant and animal diversity and environmental changes [9,11,12].Thus, nanofertilizers may unintentionally have adverse effects on microbial communities beneficial to soil and plants, which in turn may affect consumers such as animals and humans.However, studies of the toxicity of NPs in comparison with their chemical composition and properties are clearly insufficient [9].The belowground soil microbial community has received much less attention regarding improving soil quality and fertility compared to aboveground diversity and function.
Thus, it is important to understand the impact of ZnO NPs (nano-ZnO) on soil, microbial community, plants and microbial-plant interactions, as this will help researchers and agronomists determine the actual fate of NPs in the agricultural ecosystem with the aim of future mitigation of potential negative impacts caused by excess NPs in the soil ecosystem [13].
Therefore, the main objective of this review was to provide a comprehensive assessment of the effect of ZnO NPs on the most important components of the agroecosystem (such as soil, plants and soil microbiota) that might help assess environmental risks and control soil pollution.

Main Effects of ZnO Nanoparticles on the Physical and Chemical Properties of Soil
Soil is a key ecosystem that supports plants and animals and hosts many activities that can help organisms mitigate or adapt to changing conditions [14][15][16].Most soils (50%) are either deficient in Zn or contain it in a fixed form inaccessible to plants [17].Zn deficiency is common in calcareous and neutral soils, paddy soils, intensively used and poorly drained soils, saline and brackish soils, peat soils, soils high in phosphorus and silicon, sandy soils, highly weathered acidic and coarse soils [18].Soil is a non-renewable, endangered resource, extremely vulnerable to climate change and intensive agroecosystem management [19].Thus, conservation and improvement of soil are the most important requirements of the Agronomy 2024, 14, 1588 3 of 28 modern era in order to reduce the negative effects associated with population growth and the reduction of arable land as a result of anthropogenic activities.
Soil is considered the main sink for artificial NPs due to their inevitable release into the subsurface during production, transportation, use and disposal [20].Figure 1 shows the main sources and possible migration routes of ZnO NPs into the soil.Understanding the behavior of NPs in soil and assessing their adverse effects on arable and other soil ecosystems is very relevant today [9].NMs have the ability to interact with the environment, abiotic and biotic, soon after their introduction into the environment.The mentioned nanobioecosystem interface usually includes the process of interaction and transformation of NMs, which is influenced by their internal characteristics and environmental dynamics [21].
Agronomy 2024, 14, x FOR PEER REVIEW 3 of 29 management [19].Thus, conservation and improvement of soil are the most important requirements of the modern era in order to reduce the negative effects associated with population growth and the reduction of arable land as a result of anthropogenic activities.
Soil is considered the main sink for artificial NPs due to their inevitable release into the subsurface during production, transportation, use and disposal [20].Figure 1 shows the main sources and possible migration routes of ZnO NPs into the soil.Understanding the behavior of NPs in soil and assessing their adverse effects on arable and other soil ecosystems is very relevant today [9].NMs have the ability to interact with the environment, abiotic and biotic, soon after their introduction into the environment.The mentioned nano-bioecosystem interface usually includes the process of interaction and transformation of NMs, which is influenced by their internal characteristics and environmental dynamics [21].The soil consists of liquid, solid (organic and mineral substances) and gaseous components.It is considered a complex and extremely dynamic system containing mineral, organic and organomineral soil colloids that contribute to pronounced biotic diversity.The interaction between the soil matrix and NM can occur in both aqueous and solid phases.Pores present in soils (micro-and macro-pores), which control the diffusion of air and water, in addition to the biotransformation of NMs, can act as sorption sites.In addition, the transport and release of NPs are influenced by biopasses formed by soil organisms and plant roots, which are an ideal habitat for microorganisms [22].
NMs may undergo transformation and degradation steps such as dissolution in soil water, oxidation, photodegradation and passivation by coexisting substances or be absorbed by organisms, leading to their bioaccumulation.The latter depends on the bioavailability of artificial NMs, which are able to reach lower water bodies and sediments.The fate of NMs in soil and other environments varies depending on their inherent properties as well as on the properties of the environment.Size, shape, chemical nature and surface properties are key characteristics that determine the behavior and fate of NMs in soil.All NMs are subject to aging (weathering); as particles age, they may undergo chemical The soil consists of liquid, solid (organic and mineral substances) and gaseous components.It is considered a complex and extremely dynamic system containing mineral, organic and organomineral soil colloids that contribute to pronounced biotic diversity.The interaction between the soil matrix and NM can occur in both aqueous and solid phases.Pores present in soils (micro-and macro-pores), which control the diffusion of air and water, in addition to the biotransformation of NMs, can act as sorption sites.In addition, the transport and release of NPs are influenced by biopasses formed by soil organisms and plant roots, which are an ideal habitat for microorganisms [22].
NMs may undergo transformation and degradation steps such as dissolution in soil water, oxidation, photodegradation and passivation by coexisting substances or be absorbed by organisms, leading to their bioaccumulation.The latter depends on the bioavailability of artificial NMs, which are able to reach lower water bodies and sediments.The fate of NMs in soil and other environments varies depending on their inherent properties as well as on the properties of the environment.Size, shape, chemical nature and surface properties are key characteristics that determine the behavior and fate of NMs in soil.All NMs are subject to aging (weathering); as particles age, they may undergo chemical transformation, aggregation and disaggregation.Chemical transformations of NMs in soil include dissolution, sulfidation, adsorption and desorption [23] (see also Figure 2).
Agronomy 2024, 14, x FOR PEER REVIEW 4 of 29 transformation, aggregation and disaggregation.Chemical transformations of NMs in soil include dissolution, sulfidation, adsorption and desorption [23] (see also Figure 2).Aggregation and agglomeration of ZnO NPs in the soil environment occur when the attractive energy exceeds the repulsive energy under proper contact [24,25] and under the influence of Brownian motion, gravity and fluid movement [26].The aggregation and dissolution of ZnO NPs are usually influenced by a number of soil factors, such as pH, organic matter, ionic compounds and colloids [27,28].The aggregation process largely affects their colloidal stability, which is one of the key factors controlling the fate of NPs and their toxic effect [28].The size distribution of aggregates depends on the type of ZnO NPs and the chemical conditions promoting sorption in the soil.Aggregation of ZnO NPs leads to the formation of particle aggregations that settle out of solution under the influence of gravity, and increasing the size of ZnO NPs leads to a decrease in their movement in the soil.Thus, smaller particles penetrate the depth of the groundwater, while larger aggregates tend to linger in the upper layers of the soil, potentially causing soil clogging.Consequently, the properties of the soil matrix may change once the voids are filled with a significant amount of ZnO NPs [29][30][31].Therefore, the physical properties of soil (bulk density, texture, permeability, plasticity, total porosity, solid density, etc.) can definitely be changed by ZnO NPs over long-term exposure [32,33].In general, ZnO NPs strongly influence the physical and engineering properties of soil, even in small quantities, due to their large specific surface area and nanoporosity.ZnO NPs successfully increase soil moisture content and water-holding capacity in sandy soils.Moreover, adding ZnO NPs to soil improves soil porosity and reduces bulk density, which provides better conditions for root growth [33].In addition, the physical properties of soil can be influenced by the concentrations and sizes of ZnO NPs used.Komendová et al. [34] observed an increase in the strength of water molecule bridges and the structural stiffness of soil after using platinum NPs (size 3 nm) at concentrations of 0.1, 1 and 10 µg per 300 mg of soil.When the concentration was increased to 1000 µg per 300 mg of soil, a decrease in water content in the soil was observed with the preservation of soil organic matter [35].Aggregation and agglomeration of ZnO NPs in the soil environment occur when the attractive energy exceeds the repulsive energy under proper contact [24,25] and under the influence of Brownian motion, gravity and fluid movement [26].The aggregation and dissolution of ZnO NPs are usually influenced by a number of soil factors, such as pH, organic matter, ionic compounds and colloids [27,28].The aggregation process largely affects their colloidal stability, which is one of the key factors controlling the fate of NPs and their toxic effect [28].The size distribution of aggregates depends on the type of ZnO NPs and the chemical conditions promoting sorption in the soil.Aggregation of ZnO NPs leads to the formation of particle aggregations that settle out of solution under the influence of gravity, and increasing the size of ZnO NPs leads to a decrease in their movement in the soil.Thus, smaller particles penetrate the depth of the groundwater, while larger aggregates tend to linger in the upper layers of the soil, potentially causing soil clogging.Consequently, the properties of the soil matrix may change once the voids are filled with a significant amount of ZnO NPs [29][30][31].Therefore, the physical properties of soil (bulk density, texture, permeability, plasticity, total porosity, solid density, etc.) can definitely be changed by ZnO NPs over long-term exposure [32,33].In general, ZnO NPs strongly influence the physical and engineering properties of soil, even in small quantities, due to their large specific surface area and nanoporosity.ZnO NPs successfully increase soil moisture content and water-holding capacity in sandy soils.Moreover, adding ZnO NPs to soil improves soil porosity and reduces bulk density, which provides better conditions for root growth [33].In addition, the physical properties of soil can be influenced by the concentrations and sizes of ZnO NPs used.Komendová et al. [34] observed an increase in the strength of water molecule bridges and the structural stiffness of soil after using platinum NPs (size 3 nm) at concentrations of 0.1, 1 and 10 µg per 300 mg of soil.When the concentration was increased to 1000 µg per 300 mg of soil, a decrease in water content in the soil was observed with the preservation of soil organic matter [35].
However, the stability, mobility and toxicity of ZnO NPs in soil depend on water chemistry, ionic strength, aggregation and sedimentation.Natural soil organic matter influences the bioavailability of NPs in the soil through multiple mechanisms, such as electrostatic interactions, ligand exchange, hydrophobic effect, hydrogen bonding and complexation.An additional important factor that occurs at the nano-bio-ecosystem boundary is the dissolution of the NM, which depends on its surface area, charge, surface chemistry, state of aggregation, morphology, size, composition and concentration.Dissolution affects its movement in the environment [28].Dissolved ZnO 2+ ions released from ZnO NPs increase soil pH [24,36].Dissolution of ZnO NPs occurs when the ion detaches from the particle and migrates through the electrical double layer into solution [37,38].ZnO NPs have a surface charge formed by hydroxyl (-OH) groups, which can capture and release protons and absorb dissolved chemical species.The surface charge leads to the formation of an electrical double layer, including charged areas of the surface and a diffuse layer containing ions attracted from the solution to the surface of the particles in response to the charge [31].
Bulk ZnO particles (equivalent spherical diameter > 100 nm) are poorly soluble in water.On the contrary, ZnO NPs dissolve faster and to a greater extent in soils based on thermodynamic and kinetic principles.In addition, the presence of organic acids in the soil, such as citric and oxalic acids, enhances the dissolution of NPs, which, in turn, increases their mobility and bioavailability for plants and soil organisms.ZnO NPs also have good solubility at low pH values, while their solubility decreases with increasing pH.In acidic/neutral environments, ZnO NPs exhibit the ability to release ions, which makes them more toxic [39].NPs always agglomerate to some extent.The use of high concentrations of NPs can lead to increased agglomeration [40], which in turn will reduce the surface area of the particles and also slow down or even stop the dissolution of the particles [41].Nano-ZnO can be firmly attached to soil colloids.It exhibits low mobility at different ionic strengths [42] and exhibits higher sorption compared with ionic Zn 2+ .The sorption of these two forms of metal increases with increasing soil pH.

Effects of ZnO NPs on Crops
Zn is one of the essential elements for plant growth and development.Zn deficiency can cause leaf chlorosis, stunted shoot growth and reduced plant yield, leading to human health problems [43].According to the literature, ZnO NPs have both positive and negative effects on modulating the growth of agricultural crops.
Various methods have been adopted for the application of Zn, such as soil fertilization, root treatment, foliar feeding, etc. Soil application is generally considered the most promising approach to the application of macronutrients.In contrast, foliar spraying of Zn on crops appears to be effective in achieving this effect, especially where there is a low level of Zn intake from the soil.Seed priming is also used to increase grain yields [44].In agriculture, seeds are an important factor in determining the yield and productivity of crops.For these reasons, the application of seed treatment technologies to improve seed quality and physiological parameters is a promising area [45].An agronomic approach to solving the problem of nutrient deficiency in plants, which consists of treating seeds with missing nutrients, is an economically feasible and simple way to prevent nutrient deficiency at subsequent stages of plant growth [46].

Seed Priming
Nanomaterials in crop production are of interest as potential agents for stimulating plant growth processes and increasing resistance to phytopathogens.Therefore, most nanocompounds are tested for the presence of growth-stimulating ability during the germination of seeds of cultivated plants-the nanopriming effect.Currently, most studies conducted on the effects of ZnO NPs on seed germination have shown that the effects of NPs range from positive to negative effects, and this is largely determined by their concentration, size, shape, coagulation, reactivity in solution and stability.The most common physiological processes responsible for the positive and negative regulation of seed germination, growth and development of plants affected by the use of ZnO NPs are shown in Table 1.
Seed priming with ZnO NPs has been shown to increase the Zn content in primed seeds, thereby promoting better seedling growth and yield [47]. ZnO NPs in the form of 35-40 nm aggregates with rod-shaped morphology improved germination and seedling vigor of old chili pepper (Capsicum annum L.) seeds at a dose of 1000 mg per kg of treated seeds and also caused maximum shoot and root length and seedling viability index compared to control [48].Afrayeem and Chaurasia [49] reported that seed germination and root and shoot elongation of chili peppers C. annum reached maximum values at high levels (750 mg/L) of ZnO NPs, while decreases were observed at lower concentrations (250 and 500 mg/L).
Priming wheat (Triticum aestivum L.) seeds with ZnO NPs in the form of 20-30 nm spherical aggregates contributed to a significant increase in root length at a low concentration (10 mg/L), which also improved water absorption and led to an increase in α-amylase activity and the content of photosynthetic pigments such as chlorophyll a and b, as well as the total chlorophyll content [50].The chlorophyll content in the crop is an important vegetative characteristic.Micronutrient deficiency can inhibit chlorophyll formation due to decreased protein synthesis [51].When using NPs of an amorphous form with a scaly structure (31.5 nm), a decrease in the percentage of germination of soft varieties of wheat and rye is observed, while the germination of durum varieties of wheat and barley increases, as well as the elongation of roots and the viability of seedlings [52].Awasthi et al. [53] and Solanki et al. [54] reported a positive effect of ZnO NPs on wheat T. aestivum with an average NP size of about 13 nm (50 mg/L) and 40-50 nm (250 and 500 mg/L), respectively.On the other hand, Chanda et al. [55] noted that ZnO NPs (average size 3-5 nm) had a negative effect on wheat at a concentration of 1000-2000 mg/L.Seed germination increases in Petri dishes containing ZnO NPs (crystalline form, 9 nm) at concentrations of 10, 25, 50 and 100 mg/L, compared to a control dish containing only distilled water.Moreover, an increase in the length of roots, shoots and leaves and the corresponding biomass was observed with increasing concentration of NPs, with a concentration of 50 mg/L having the greatest positive effect [53].
Peanut (Arachis hypogea L.) seeds treated with ZnO NPs (25 nm) at a concentration of 1000 mg/L showed increased germination, seedling viability and active growth, which in turn manifested itself in early flowering and higher chlorophyll content in leaves and overall high productivity [56].While at a higher concentration of NPs (2000 mg/L), an inhibitory effect was observed [56], which indicates the need for judicious use of NPs.The above results were also confirmed at 500 mg/L ZnO NPs in another study of A. hypogea [57], where the studied concentration significantly improved seed quality indicators, such as germination, root and shoot length, and α-amylase activity.
When corn (Zea mays L.) grains were treated with ZnO NPs (25 nm) at a concentration of 1500 mg/L, the highest percentage of germination (80%) and the length of roots and shoots were observed, which differed significantly from those in the control and when treated with 2000 mg/L ZnSO 4 [58].In another study, when corn seeds were treated with biogenic ZnO NPs (rod and spherical particle structures with an average size of ~37 nm), synthesized using the seaweed Turbinaria ornata, at a concentration of 100 mg/L, a high percentage of germination (87%) was observed already at the 8th day after treatment, as well as the greatest length of the shoot, root, root and shoot width, significantly different from the control [45].Under cobalt stress and soil salinity, seed treatment with ZnO NPs (with a size of 20 nm and <100 nm at concentrations of 500 and 100 mg/L, respectively) significantly increased the cumulative percentage of germination, grain weight, potassium content and α-amylase activity, which further improved plant growth, biomass growth and photosynthesis mechanisms of corn.In addition, seed treatment with ZnO NPs significantly reduced the negative effects of these abiotic stresses [59,60].
Agronomy 2024, 14, 1588 7 of 28 The concentration of ZnO NPs and the aging period of rice (Oryza sativa L.) seeds largely influence the toxicity.Thus, higher concentrations caused a decrease in the length and number of roots starting from 100 mg/L, with an increase in the inhibitory effect with increasing concentrations (500 and 1000 mg/L) and a longer soaking time for seeds [61].Low concentrations (25 mg/L) of ZnO NPs (35 nm) promoted rice growth, pigment content and grain quality parameters (number of grains per ear, grain dry weight), and also stimulated the accumulation of Zn (2%) and Fe (5%) [62].
Thus, seed priming with ZnO NPs helps to reduce abiotic and biotic stress in plants, acts as a biostimulant, causing an increase in the rate of seed germination, seedling and plant growth, and the total mass of the fresh harvest, and also improves biomass and photosynthetic mechanisms [72].

Foliar Treatment
When applied foliarly, NPs pass through the cuticle layer in two different ways: (1) Hydrophilic path-penetration into the cuticle through water pores, the size of which varies from 0.6 to 5 nm in diameter depending on the type of plant; (2) lipophilic pathway-through the diffusion process.The cuticle serves as the main barrier to prevent the penetration of NPs larger than 5 nm [73][74][75].
Foliar application (leaf spray) of rice (O.sativa) with ZnO NPs (50 nm) at three different concentrations (500, 1000 and 5000 mg/L) at an interval of 15 days significantly improved growth and yield parameters compared with control (0 mg/L) [76].Burman et al. [77] observed a significant acceleration of root growth of chickpea (Cicer arietinum) seedlings when using leaf spraying with ZnO NPs at a concentration of 1.5 mg/L, while a slowdown in root growth was observed at a dose of 10 mg/L.Enhanced root growth may be due to the uptake of NPs through stomatal openings followed by their mobilization through the apoplast/symplast to reach the roots.However, at the optimal concentration below and above 1000 mg/L, spraying roots with ZnO NPs had a negative effect.Similar results were also obtained on ryegrass [78], rice [61], mung bean (Vigna radiate) and chickpea seedlings [79] and clusterbean (Cyamopsis tetragonoloba L.) [80], where the application of nano-ZnO inhibited root growth at higher concentrations.The slowdown in root growth was likely due to structural changes in the root surface.Zn content in shoots, roots and grains was also improved by foliar spraying with nano-ZnO, and these results are consistent with [56,81].Foliar treatment with nano-ZnO also affected the content of other microelements in crop plants and showed an increase in iron (Fe) content in rice shoots treated with NPs [81].It is likely that the interaction of Zn with macronutrients affected the nitrogen content of treated plants.However, the application of Zn to rice (Oryza sativa L.) resulted in a decrease in grain phosphorus (P) content, likely due to an antagonistic relationship between Zn and P [82].ZnO NPs or their aggregates, having a diameter smaller than the size of stomatal pores, have the ability to penetrate and move inside plant tissues.Moreover, enlargement of pores or induction of new cell wall pores upon the interaction of plant leaf tissue with NPs have been reported [83].This may be due to increased transport and deposition of Zn in leaves.Thus, these results highlight the role of ZnO NPs in relieving Zn deficiency and enhancing the yield and Zn content of rice cultivars.

Application to the Soil
When the soil was treated with a suspension of ZnO NPs (<50 nm) at a concentration of 200 and 300 mg/L, a decrease in Arabidopsis thaliana L. growth by ~20 and 80% was observed, respectively, compared with the control.The content of chlorophylls a and b, the intensity of photosynthesis, leaf stomatal conductance, intercellular CO 2 concentration and transpiration rate decreased by more than 50% in Arabidopsis plants treated with ZnO NPs at a concentration of 300 mg/L [84].
In another study, when the soil was treated with ZnO NPs (spherical, 51 nm), the maximum increase in wheat (T.aestivum) growth, total chlorophyll content, grain yield and improvement in Zn content in shoots and grain was noted relative to the corresponding control [85].The authors obtained similar data for rice.The stimulating effect of ZnO NPs on the developmental morphometric traits in wheat is also confirmed by other studies [86,87].
The addition of ZnO NPs (20 nm) to the soil (pH 7.12) at doses of 100 and 700 mg/L increased the Zn content in sage (Salvia miltiorrhiza Bge.) roots compared to the control [88].Similar to this study, exposure to ZnO NPs solution resulted in the rapid uptake of Zn by rice roots and its increase in the roots [89].A concentration of 100 mg/L ZnO NPs caused an increase in above-ground biomass, but 700 mg/L resulted in a significant decrease.The use of ZnO NPs at doses of 100 and 700 mg/L also led to an increase in underground biomass and root diameter but did not have a significant effect on either plant height or the number of roots, regardless of the dose.
ZnO NPs (16-31 nm) significantly improved tomato (Solanum lycopersicum) growth (length, fresh weight and dry weight), increased the number of cellular antioxidant enzymes and reduced the production of reactive oxygen species (ROS) when infected with a soil-borne bacterial pathogen (Ralstonia solanacearum), which causes bacterial wilting of tomatoes (bacteriosis), mitigating the consequences of the disease compared with the control treatment [90].In addition, this study has shown that in acidic soils, direct addition of Zn to the soil causes severe iron deficiency in dicotyledonous plants, indicating a preference in such cases for foliar application of Zn nanofertilizers [90].Nanofertilizers represent a good option for Zn enrichment of cereals and other crops, but when applied to soil, it is necessary to better understand the interaction of NPs with various soil properties and components, mainly with soil and rhizosphere microbiota.
The effects of ZnO NPs on the growth and development of various plant species are also summarized in Table 1.

Effects of ZnO NPs on the Microbiota of Soil and Rhizosphere
Key phyla of microorganisms, such as Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria and Firmicutes, widely represented in the soil, participate in the regulation of biogeochemical cycles (carbon, nitrogen, sulfur and phosphorus), facilitate the absorption of nutrients by plants, stimulate the growth and suppression of plant pathogens, acting as biocontrol agents [91][92][93][94][95][96][97][98][99].The rhizosphere microbiota community and root microbiota play a critical role in the growth and health of terrestrial plants.The rhizosphere microbiota participates in the regulation of plant growth, nutrition, physiology and resistance to adverse effects following the feedback principle, providing bioavailable nutrients and synthesizing phytohormones.Root exudates secreted by plant roots also affect the microbiota of the rhizosphere, and enrichment with beneficial microorganisms promotes plant growth and development [100,101].Recently, the manipulation of soil microorganisms has received much attention from the scientific community to overcome adverse environmental stresses and factors affecting plant growth and soil maintenance.Soil microorganisms play a crucial role in plant growth even under stress conditions, which have been reported in a large number of scientific publications [102,103].
Zn is an essential element needed by plants, humans and microorganisms.Biofortification is a modern approach aimed at increasing the bioavailability of trace elements such as Zn and Fe in staple crops.In this regard, beneficial free-living soil bacteria that have been shown to improve plant health or increase crop yields can also mobilize these micronutrients [18].An ideal Zn source should release only small amounts of Zn into the soil over a longer period of time, a property associated with the nanoform of ZnO [104].Nanoagrochemicals are active ingredients designed to improve the properties and qualities of active molecules used in agriculture, such as biocides, herbicides and nutrients.The ability to use nanotechnology to improve the efficiency of pesticides, nutrients and their delivery will likely lead to a reduction in the number of resources used in agriculture.In addition to directly affecting plant growth, NMs also influence the soil microbial community and plant microbiomes, which in turn influence plant health.However, the effect of these nanoagrochemicals on soil microbiota has been underestimated until recently [105].Because ZnO NPs are often added directly to soil as fertilizers, regulators or pesticides, this inevitably affects the rhizosphere environment, a microecological system in which plants, soil elements, and microorganisms interact [10,106].
An earlier study reported that ZnO NPs (spheroidal shape, 20-30 nm) reduced both microbial biomass and diversity.A stronger negative effect was noted at the maximum tested concentration of 500 mg/kg.At the same time, the soil was characterized by a slightly acidic reaction (pH 6.0) [107].After treating soil samples (pH 7.5) with ZnO NPs (15 nm) at a dose of 1000 mg/kg, both a decrease in the number of Azotobacter, phosphate-and potassium-solubilizing bacteria and a significant decrease in the enzymatic activity of the soil (urease, catalase, hydrolysis of fluorescein diacetate) were noted, which were proposed as an indicator of its biological activity.A decrease in catalase (CAT) activity with increasing soil pollution is a convenient marker of the unstable ecological situation of the observation site.A change in urease activity can serve as an indicator of a violation of the soil's ability to effectively carry out nitrogen exchange and, consequently, maintain ecological balance in the ecosystem as a whole [108].
Minor changes in the microbiota were noted after soil treatment (pH 6.9) with 500 mg/kg ZnO NPs.The number of representatives of the phyla Proteobacteria and Actinobacteria decreased to 40% from 43.7% and 20.1% from 22.6%, respectively.On the contrary, the number of members of the Firmicutes phylum increased slightly from 20.99% to 22.6%, as did the Bacteroidetes phylum from 2.9% to 3.4%.An increase was observed in representatives of the orders Bacillales, Burkholderiales, Lactobacillales and Pseudomonadales and the genera Pseudomonas, Streptococcus and Dialister.The relative abundance of members of the orders Actinomycetales, Desulfivibrionales, Alteromonadales, Oceanospirillales and the genus Halomonas decreased [98].Previously, an increase in representatives of Burkholderiales and Bacillales in the presence of ZnO NPs was also reported in [109].The tolerance of bacilli to ZnO NPs can be explained by their ability to form endospores and also produce extracellular polymeric substances (EPS) [110,111].It is also known that representatives of this class persist in soils contaminated with metals [112,113].The increase in the abundance of Pseudomonadales and Burkholderiales may not be due to the enhancing effect of specific NPs per se but most likely as a result of reduced competition from bacteria that were suppressed.An increase in the number of representatives of Pseudomonadales and Burkholderiales can have both positive and negative implications.Both orders include plant pathogens that cause blight, necrosis and galls, but strains of the genus Burkholderia (β-proteobacteria) are also important to the environment because they are good biodegraders of polychlorinated biphenyls.It is known that Pseudomonas spp.promotes plant growth by synthesizing plant hormones and suppressing phytopathogens [114].It is worth noting that in the study by Chavan and Nadanathangam [98] there is a decrease in the number of representatives of the order Rhizobiales.These representatives of bacteria are of agricultural importance because they oxidize methane to CO 2 and are also able to bind inorganic atmospheric nitrogen, producing organic nitrogen-containing substances.However, the relative abundance of the order Sphingomonadales, which plays a role in stimulating plant growth and decomposing polycyclic aromatic hydrocarbons (PAHs), did not change [115].Collins et al. [116] reported that representatives of Rhizobiales showed lower sensitivity, and Sphingomonadales showed high sensitivity to ZnO NPs (elongated 15-50 nm in width and 50-20 nm in length, without clearly defined crystal edges) at a dose of 550 mg/kg in weakly alkaline soil (pH 7.5).While Ge et al. [117] observed a decrease in Rhizobiales and an increase in Sphingomonadaceae in response to ZnO NPs (spheroidal form, 20-30 nm) in slightly acidic soil (pH 6.0).
In a study by Chen et al. [12], silty loamy soils (pH of soil samples varied between 7.90-8.20)were exposed to low (100 mg/kg) and high (500 mg/kg) concentrations of ZnO NPs (spherical shape, 25 nm).These treatments significantly affected soil bacterial communities after 30 and 90 days of exposure.The number of bacteria of the Proteobacteria and Nitrospirae types increased slightly when the soil was treated with the studied NP compared with the control.In contrast, the abundance of bacteria of the phyla Bacteroidetes, Acidobacteria, Actinobacteria and Firmicutes decreased.Compared with bacterial taxa, Ascomycota, Zygomycota and Basidiomycota were the dominant fungal phyla in the treated soils.Another study also demonstrated the negative impact of ZnO NPs (40 nm), which was reflected in a significant decrease in the number of soil bacterial species and changes in the organization of the entire community [118].
When the soil was treated (pH 8.58) with 0.5 mg/kg, 1.25 mg/kg and 2.5 mg/kg ZnO NPs (65.8 nm), there was a significant decrease in the number of soil bacteria, actinomycetes and fungi compared with control [119].The higher application rate (2.5 mg/kg) resulted in a greater reduction in enzymatic activity compared with the lower application rate (0.5 mg/kg), which may be due to the higher specific surface area leading to higher adsorption.There is a significant decrease in the amount of soil dehydrogenases and the proportion of carbon in microbial biomass, which indicates a negative effect on the microbial activity of the studied soil [119].Among all soil enzymes, dehydrogenases are among the most important and are used as an indicator of overall soil microbial activity because they occur intracellularly in all living microbial cells.Dehydrogenases play a significant role in the biological oxidation of soil organic matter by transferring hydrogen from organic substrates to inorganic acceptors.As a result, they serve as an indicator of microbiological redox systems and are considered a good and adequate indicator of the oxidative activity of microorganisms in the soil [120].The proportion of microbial biomass carbon in soil organic carbon is an important indicator of the quality of organic matter.It characterizes the state and diversity of the microbial community, as well as the degree of its maturity [121].In contrast, in another study, ZnO NPs (hemispherical shape, 50 ± 10 nm) increased the richness of the soil microbial community (pH 7.24) [100].Thus, among the treated soil samples, the dominant types of bacteria and fungi with the highest relative abundance were Proteobacteria (38.7-44.6%)and Ascomycota (59.9-80.9%).The relative abundance of members of the order Sphingomonadales, associated with the degradation of persistent organic pollutants, was significantly enriched by increasing concentrations of ZnO NPs.The relative abundance of the orders Solirubrobacterales and Catenulisporales, as well as the phylum Armatimonadetes, decreased.At the same time, it is difficult to determine a specific group that is responsible for certain soil functions (for example, the N-cycle) because all representatives of the listed groups of microorganisms contain representatives that oxidize ammonia/nitrite and anammox bacteria, and denitrifying bacteria involved in nitrogen fixation, removal of nitrogen or during the process of (de)nitrification.At higher doses of ZnO NPs (>250 mg/kg of soil), the stimulating effect became weaker; moreover, at increased concentrations (500 and 1000 mg/kg), ZnO NPs become toxic to microorganisms [100].
In [122] 2 h after exposure to ZnO NPs (spherical shape, particle size 50 nm) at 40.9%, Proteobacteria (α, δ, γ) were the most abundant phylum in the studied soil (pH 7.56) followed by Actinobacteria (19%).On the 30th day of NP exposure, the relative abundance of different types changed.Some species with specific functions responded differently to different concentrations of ZnO NPs 30 days after exposure.In particular, the abundance of the genera Altererythrobacter, Massilia and Ohtaekwangia, which are associated with the carbon cycle and energy metabolism [123], increased at high NP levels but decreased at low NP levels at 30 days.Representatives of Bacteroidetes, which are associated with the degradation of polycyclic aromatic hydrocarbons (Terrimonas) and degradation of organic matter (Flavitalea and Ohtaekwangia), were significantly suppressed by ZnO NPs treatment.Reducing the number of these microflora representatives that destroy PAHs will negatively affect the restoration of hydrocarbons in the soil.Similarly, the relative abundance of Pseudomonas spp., one of the phosphate-mobilizing microorganisms of the Gammaproteobacteria class, decreased, which could lead to a lower content of phosphorus available in the soil.In contrast, the abundance of the methyl tert-butyl ether (MTBE) degrading genus Piscinibacter, Actinobacteria (including the family Micrococcaceae and the genus Streptomyces) and Burkholderiales associated with the suppression of phytopathogens in soil increased with increasing amounts of NPs.
The effect of soil treatment (pH 6.4) at a dose of 30 mg/kg with nanosized ZnO (50 nm) on the rhizosphere microbiota was expressed in an increase in the relative abundance of bacterial types involved in the nitrogen cycle and having the ability to regulate the concentration of nitrates in the soil-Bacteroidetes and Actinobacteria [124].The authors considered elevated levels of these types as a biomarker of exposure to ZnO NPs.At the family level, declines in the relative abundance of Gemmatimonadaceae (polyphosphate-storing), Sphingomonadaceae and Haliangiaceae were observed.On the other hand, an increase in the abundance of Streptomycetaceae, Rhizobiaceae, Oxalobacteraceae (nitrogen-fixing members), Chitinophagaceae and Solibacteraceae was observed [125].Streptomycetaceae plays a vital role in the degradation of complex organic molecules such as lignocellulose, cellulose, xylan and lignin, which are essential for the catabolism of soil organic matter [126].According to the study, soil treatment with ZnO NPs improved the levels of functional categories of rhizosphere soil bacteria.In particular, it led to a significant improvement in membrane transport, which is necessary for cell survival and can help overcome environmental stresses [127].In addition, treatment with ZnO NPs improved carbohydrate metabolism, promoting the rate of breakdown of available carbon by microorganisms [128].
At low concentrations of ZnO NPs (35 nM, 25 mg/kg), soil microorganisms had greater diversity and richness than in the control soil group (pH 7.4) [62].The strongest effects were observed when changing the abundance of three types: proteobacteria, actinobacteria and planctomycetes.The rhizosphere environment was noticeably enriched with potential producers of streptomycin, carbon and nitrogen fixers, and lignin destructors according to functional groups of microorganisms.However, a decrease in the number of microbial communities mainly responsible for chitin degradation, ammonia oxidation and nitrite reduction was found [62].
Changes in the microbial community after 120 days of application of ZnO NPs (polygonal shape with a smooth surface, ~20 nm) to slightly alkaline soil (pH 7.7-7.8)at the concentration of 5 kg/ha was characterized by a significant increase in the population of the genera Bacillus, Acinetobacter, Pedobacter, Massilia, Lysobacter and Pseudomonas in [129].Findings suggested roles for most of these genera in promoting plant growth, suppressing plant pathogens (through the production of antimicrobial enzymes), and alleviating stress [130,131].Representative of gammaproteobacteria Acinetobacter spp. is known to dissolve phosphate and produce phytohormones to enhance plant growth [132].The results presented in this study indicate that soil treatment with ZnO NPs significantly promotes plant growth and improves grain Zn content without significantly altering microbial community structure.When ZnO NPs (20 nm) were added to the soil (pH 7.12) at a concentration of 700 mg/kg, an increase in the relative abundance of proteobacteria was noted.Doses of 100 and 700 mg/kg led to a significant increase in the relative abundance of some superoxide dismutase (SOD) producers (Methylobacillus, Humicola and Aminobacter-700 mg/kg NPs), acidophilus bacteria (Arenimonas-700 mg/kg) and metal-tolerant microorganisms (Thiobacillus-700 mg /kg and Metarhizium-100 mg/kg) [88].In another case, the bacterial community structure of the lettuce rhizosphere was altered by ZnO NPs (90 nm) by enriching the relative abundance of the phylum Cyanobacteria, genera Nostoc, Scenedesmus, Xanthomonas, Galbibacter and Burkholderia [133].This phenomenon can be explained by the important role of Zn in promoting the spread of cyanobacteria.
After soil treatment with ZnO NPs (16-31 nm, 500 mg/kg), the presence of Proteobacteria and Patescibacteria was significantly reduced compared with the control group, but the relative abundance of the phylum Chloroflexi significantly increased [90].In addition, compared with the control group, the relative abundance of actinobacteria was reduced by the fourth week of exposure.Interestingly, the relative abundance of Ralstonia spp.(bacterial plant pathogen) in the NP exposure group decreased significantly in the second week with complete destruction by the fourth week compared to the corresponding control.In addition, the soil after nanotreatment was significantly enriched with families Sphingomonadaceae, Rhizobiaceae, Rhodanobacteraceae, Xanthomonadaceae and other bacteria in the second week.Representatives of the family Nitrosomonadaceae, Methylophilaceae, Microscillaceae and Gemmatimonadaceae soil were significantly enriched in the fourth week of NP treatment [90].All of the above families belong to the Proteobacteria, which are important companion bacteria to plants that help resist abiotic stress [134].
Soil treatment (pH 5.59) with ZnO NPs (30 nm) significantly altered the soil microbial community structure in [135].Both an increase in the relative abundance of bacteria (phyla Proteobacteria, Chloroflexi, Gemmatimonadota, Bacteroidota, Myxococcota, Cyanobacteria) and a decrease (Firmicutes, Acidobacteriota, Nitrospirota, Verrucomicrobiota) were observed.The relative abundance of fungi of the genera Tausonia, Chaetomium and Mrakia increased.In contrast, the relative abundance of the genera Neocosmospora, Gibberella and Fusarium decreased significantly compared with the control.Surprisingly, ZnO NPs at a concentration of 250 mg/L had a better antifungal effect than those at a concentration of 1000 mg/L [135].This may be due to the fact that NMs in high concentrations can aggregate in solution, impairing the antifungal effect [136].
Published data on the effects of ZnO NPs on soil and rhizosphere microbiota are also summarized in Table 2.

Effects of ZnO NPs on Soil Invertebrates
Soil invertebrates play an important role in the functioning of the soil ecosystem.During their life activity, invertebrates enrich the soil with biologically active substances that have a stimulating effect on seed germination and plant development [24].ZnO NPs can be toxic to soil invertebrates.The level of toxicity depends on factors such as the concentration of nanoparticles, the size of the particles, the exposure duration and the specific species of invertebrate.Soil invertebrates can accumulate ZnO NPs, leading to potential adverse effects on their physiology and behavior [137].This accumulation can vary among different species and environmental conditions.Therefore, eliminating the exposure of these organisms to ZnO NPs is critical to understanding the potential impacts of NPs in the soil environment [24].
Californian red worms or earthworms Eisenia andrei are very often used as model invertebrate organisms in research.Earthworms are keystone species of soil ecosystems and are constantly exposed to the solid phase of soil through oral ingestion and the porewater phase of soil, both through oral ingestion and through the skin.Therefore, these taxa represent an ideal group to study the influence of soil properties on metal NPs and, consequently, their bioavailability and toxicity in different soil types [138].ZnO NPs can cause toxic damage to soil invertebrates after direct contact through the skin or entry into the body through the digestive tract [139,140].In a study by Hu et al. [140], ZnO NPs accumulated in earthworm tissues.
It has been shown that the growth and reproduction of earthworms, such as Eisenia fetida, E. veneta and others, can be negatively affected by ZnO NPs due to their enzymatic alterations and cellular dysfunction (see [141] for review).ZnO nanoparticles suppressed the reproduction of E. andrei at a concentration of ≥400 mg/kg [142].These data are consistent with the results obtained by Samarasinghe et al. [143]: ZnO NPs caused reproductive toxicity at high dosages (500 and 1000 mg/kg).However, no significant chronic toxic effects were observed on earthworms in terms of their survival, growth and bioaccumulation after 28 days of exposure to soil with ZnO NPs at concentrations of 0.1, 1, 10 and 100 mg/kg.Moreover, the calculated half-maximal effective concentration (EC50) of ZnO NPs for earthworm reproduction based on tissue concentrations ranged from 160 to 360 mg/kg [138].In contrast, in another study, ZnO NPs at a dose of 300 mg/kg improved the growth and survival of juvenile E. fetida earthworms [144].
ZnO NPs can reduce the survival and reproduction of nematodes such as Caenorhabditis elegans [139,145].It has been shown that the negatively charged nematode cuticle attracts ZnO NPs to a greater extent than bulk ZnO particles [139].Changes in gene expression related to stress responses and metabolism have also been observed.However, ZnO/graphene oxide nanocomposites decreased cadmium-induced ecotoxicity in soil nematodes C. elegans [146].
Soil-dwelling arthropods, including collembolans (springtails) and mites, can experience decreased survival and reproduction rates when exposed to ZnO NPs (see [147] for review).The adverse effect of ZnO NPs added to soil with sewage sludge on mortality and reproduction of springtails Folsomia candida was observed at ZnO concentrations in sewage sludge ≥250 mg/kg.In most cases, toxicity decreased after 45 days of exposure [148].ZnO NPs had a significant effect on the reproduction rate of F. candida at a dose of 4000 mg/kg [142].
In conclusion, while there is evidence that ZnO nanoparticles can be toxic to soil invertebrates, the extent and nature of these effects can vary widely depending on several factors.Continued research is needed to better understand these interactions and inform regulatory decisions to protect soil health and ecosystems.

Mechanisms of Toxicity of ZnO NPs to Plants, Microorganisms and Invertebrates of Soil and Rhizosphere
Zn, including its nanoforms, is needed for plants throughout the growing season, but at high doses, it has a toxic effect on plants.In this regard, it is necessary to take into account both the vital and toxic roles of ZnO NPs with an understanding of the mechanisms underlying them.Tables 1 and 2 demonstrate the main reported effects of ZnO NPs on various plants and microorganisms in the soil and rhizosphere.However, the mechanisms of toxicity of ZnO NPs to soil biocommunities are poorly understood.The main factors determining the toxicity of ZnO NPs to plants and microorganisms are the characteristics of the nanomaterials themselves.They can be related to the solubility of NPs, chemical composition and structure, size, surface area, concentration, stability and their type [149][150][151].In addition, soil as an ecosystem habitat, as well as growth and plant species, play a significant role in the interaction with ZnO NPs [150].

Effect of Soil on Toxicity of ZnO NPs to Plants and Bacteria
The characteristics of NPs vary greatly depending on environmental conditions [152].Soil properties such as organic matter content, ionic strength, redox potential, pH, texture and edaphic factors largely determine the behavior of ZnO NPs, Zn speciation in soil, as well as its bioavailability [153].

Soil Acidity
Soil acidity (pH) is a major factor determining the concentration and distribution of soluble metals between the solid and water phases of the soil [154][155][156], including solubility and toxicity of ZnO NPs [156,157].In acidic soils, compared with neutral ones, there is a high concentration of Zn in the form of Zn 2+ and Zn(OH) + due to increased solubility of ZnO NPs [158].With an increase in the concentration of ZnO NPs in acidic soil, there was an increase in pH by approximately 1 unit compared with the control.Dissolution of Zn 2+ ions released from NPs leads to a decrease in protons and an increase in soil pH.The respiratory potential of soil microorganisms decreased when ZnO NPs were added to calcareous soil at a dose of 10, 100 and 1000 mg/kg and in acidic soil at 100 and 1000 mg/kg [157,159,160].In another study, soil treatment by ZnO NPs and Zn 2+ ions affected the activity of soil enzymes, with a particularly strong decrease in the activity of dehydrogenase, which is involved in the respiration process [161].Soil acidity appears to directly or indirectly influence the behavior of NPs in the respiratory potential of soil organisms [162].Inhibition of soil heterotrophic respiration caused by soil substrate at high doses of ZnO NPs has also been shown in other studies [107,163,164].Acidity influences nutrient availability, alters soil solids balance, and controls soil microbial community association and diversity [157,165].However, a pH close to neutral is considered optimal for the functioning of soil organisms, and ecotoxicity tests conducted over a wide acidity range demonstrated that the soil reaction plays a dominant role in determining toxic effects [156].
When growing wheat Triticum aestivum L. in alkaline soil with the addition of ZnO NPs (125-500 mg/kg), no changes in morphometric traits were recorded, with the exception of an increase in the formation of lateral roots [166].In alkaline soil, the solubility of ZnO NPs decreases, as does the sorption of Zn by organic soil components (iron oxides and carbonates) and clay minerals, thereby reducing the availability of micronutrients for the plant [156].However, Watson et al. [166] noted an increased content of Zn in wheat shoots, while the dose had a minor effect.A twofold increase in Zn content was observed in tissues, reaching 100 mg/kg compared with control plants grown without adding Zn to the soil.The addition of NPs to acidic soil led to a sharp decrease in root growth, probably due to the high solubility and availability of Zn in high concentrations [166].Wu et al. [167] showed that ZnO NPs rapidly dissolve when exposed to acidic and near-neutral soil; solubilized Zn 2+ ions released from ZnO NPs were completely converted into stable forms.However, a number of studies have shown the toxicity of ZnO NPs upon dissolution due to the active release of Zn 2+ ions [168,169].Shen et al. [170] showed that the toxicity of ZnO NPs was highest in acidic soil, followed by neutral soil.In alkaline soil, the toxicity of NPs was relatively low.Moreover, the toxicity of NPs was not due to the release of Zn 2+ ions from ZnO NPs; one of the possible reasons for this was the direct interaction of ZnO NPs with biological targets [170].

Soil Organic Matter
Soil organic matter (SOM) is an important renewable natural resource that is formed as a result of the breakdown of plant and animal tissues in the environment.Its main components are humic and fulvic acids and the hydrophilic fraction [171,172].The interactions of ZnO NPs with SOM can affect the stability, aggregation and dissolution of NPs [173].SOM reduces the aggregation of NPs and increases their colloidal stability [159].SOM has been shown to be able to coat NPs and thus alter their aggregation and their interaction with soil and plants [174][175][176].As soil organic matter concentration increases, the coating of NPs thickens [177], which ensures charge neutralization and colloidal stability of NPs because the surface of metal-based NPs is mostly positively charged at near-neutral pH, and the SOM molecules are partially deprotonated [173,177,178] (see also Figure 2).SOM adsorption reduced the short-term bactericidal effects of NPs by reducing the release of toxic Zn 2+ ions but thereby increased bioaccumulation [176,179,180].Due to its bioavailability, SOM is biodegradable and removed by bacteria from NPs over time [181].[180] study on the availability of applied ZnO NPs in SOM-treated and untreated soil showed that at low concentrations of NPs in soil (<100 mg/kg), they had a positive effect, but with increasing concentrations to 1000 mg/kg, they showed a toxic effect on plants.There was a decrease in shoot dry biomass by 52% in soil not treated with SOM supplemented with ZnO NPs and an increase in shoot dry biomass by 35% and Zn concentration in cucumber Cucumis sativus tissues in soil treated with SOM and ZnO NPs.In soil not treated with SOM at a high concentration of NPs (1000 mg/kg), deformation of the plant root apex was also observed, which suppressed plant growth and nutrient uptake by roots.Transmission electron microscopy showed aggregation of ZnO NPs within the plant cytoplasm and their accumulation near the cell membrane.

Moghaddasi et al.'s
Soil amended with leaf litter and ZnO NPs exhibited significant reductions in heterotrophic bacteria and fungi, as well as reduced carbon and nitrogen contents of soil microbial biomass compared with soil amended with leaf litter alone.Thus, the applied ZnO NPs, in addition to the toxicological effects on soil microbiota, influenced their associated functions [182].

Toxic Effects of ZnO NPs on Plants
The effect of NPs is directly related to the mechanism of their internalization.ZnO NPs penetrate into the plant through the roots after adhesion to the root surface [183].The roots anatomically establish direct contact with the soil and rhizosphere microorganisms, and after absorption of NPs, they spread apoplastically throughout the plant.The distribution of NPs is increased due to their ability to move throughout the plant using the vascular system and is directly dependent on the size of the NPs [183][184][185].Penetration into cells occurs due to the interaction between the cell wall and transport proteins, the formation of pores, the process of endocytosis, the interaction between the net charge of NPs and roots, as well as the participation of the apoplast [184,186].Cell wall pores are typically 3.5-3.8nm in root hairs and 4.5-5.2nm in parenchyma cells.NPs less than 5 nm in diameter are able to penetrate the cell walls of intact plant cells [187].Ghosh et al. [188] assessed the cytotoxicity, genotoxicity and biochemical effects of ZnO NPs (≈85 nm) on Allium cepa, Nicotiana tabacum and Vicia faba plants.Loss of membrane integrity increased chromosomal aberrations, formation of micronuclei, DNA strand breaks and cell cycle arrest at the G2/M checkpoint were observed in root meristem cells of Allium cepa.Increased intracellular ROS production, lipid peroxidation (LPO) and changes in the activity of some antioxidant enzymes were observed in Vicia faba and Nicotiana tabacum.Transmission electron microscopy revealed gross morphological changes and internalization of NPs.ZnO NPs were localized along the plasma membrane [188].The observed ultrastructural changes may be associated with apoptotic/necrotic or necrotic/vacuolar cell death [189].In this case, the amount of dissolved Zn 2+ ions was recorded in the range of 3 mg/L, and it did not depend on the concentration of ZnO NPs.However, plant growth inhibition may not be directly related to the phytotoxicity of NPs.Rather, physical interactions between NP transport pathways in plants may result in inhibition of symplastic communication between cells through plasmodesmal occlusion by ZnO NPs or by occlusion of intercellular spaces in the cell wall or cell wall pores.The solubility of ZnO NPs themselves is another factor that needs to be considered in phytotoxicity studies, but the synergistic effects of NPs stabilizers should also be taken into account [190].In addition, the protective response of plants to the influence of ZnO NPs is associated with an increase in the activity of antioxidant enzymes.ZnO NPs (2 mg/mL) inhibited the growth of maize (Zea mays L.) and stimulated the production of superoxide radicals, LPO and apoptosis [191].Increased production of ROS and malondialdehyde (MDA) has been reported in okra seedlings (Abelmoschus esculentus L. Moench) exposed to ZnO NPs at concentrations greater than 250 mg/L [192].A study of the antioxidant defense system in barley seedlings (Hordeum vulgare L.) after 7 days of exposure to ZnO NPs (300 and 2000 mg/L) revealed an increase in the level of reduced glutathione (GSH) and the activity of SOD, CAT, glutathione reductase (GR) and glutathione S-transferase (GST) [193].A three-fold increase in GST activity in roots was found when exposed to 2000 mg/L ZnO NPs.The antioxidant defense system was activated in H. vulgare in response to ZnO NPs, which effectively prevented the progression of oxidative stress in the early stages of plant ontogenesis.However, with constant exposure to ZnO NPs in high concentrations, such activation leads to depletion of the plant's energy resources, which negatively affects its growth and development [193].
Thus, a review of the available published data allows us to conclude that in most cases, ZnO NPs do not penetrate into the plant cell, accumulating on its surface.They cause oxidative stress inside the plant cell by increasing the synthesis of ROS, influencing the level of activity of antioxidant enzymes, and increasing the intensity of LPO processes, which leads to damage to the membrane and DNA.Such events can lead to cell destruction.

Mechanism of ZnO NPs Toxicity on Bacteria
NPs use a number of mechanisms to act as an antibacterial agent, which are the same for all bacteria, regardless of their biotope.There are the following main antibacterial mechanisms of ZnO NPs: (1) direct contact of ZnO NPs with the cell walls of bacteria, leading to the destruction of the integrity of bacterial cells, (2) release of Zn 2+ ions and (3) formation of ROS, which destroy the cell [194].
ROS production by metal oxide NPs is one of the mechanisms responsible for the antimicrobial activity most commonly reported in the literature.ROS include superoxide anions (O 2− ), hydroxyl radicals (HO −2 ) and hydrogen peroxide (H 2 O 2 ), which can cause the destruction of cellular components such as DNA, proteins and lipids [195][196][197].The bacterial cell wall is negatively charged, as are hydroxyl radicals and superoxides, so they cannot penetrate the membrane, but direct contact can cause damage.Conversely, hydrogen peroxide is able to pass through the cell wall and can be absorbed into the bacterial cell, causing its death [198,199].Using scanning and transmission microscopy of bacterial cells, it was confirmed that ZnO NPs disrupt the cell membrane and accumulate in the cytoplasm, where they interact with biomolecules, causing cell apoptosis and leading to the death of bacterial cells [200].ZnO NPs typically induce elevated concentrations of Zn 2+ ions, leading to excessive intracellular ROS production, mitochondrial dysfunction and cell death [201].
Thus, the uptake of ZnO NPs by bacterial cells, their intracellular dissolution and subsequent release of Zn 2+ ions are the intrinsic reasons for the high toxicity of ZnO NPs.This toxicity is explained by oxidative stress, which can destroy the macromolecules of the bacterial cell due to the high production of ROS.

Mechanism of ZnO NPs Toxicity on Invertebrates
One of the primary mechanisms by which ZnO NPs exert toxicity on invertebrates is through the generation of ROS [202].This can lead to oxidative stress, damaging cellular components such as lipids, proteins and DNA [203].ZnO NPs can dissolve in soil, releasing Zn2+ ions, which can be toxic to soil invertebrates, and distinguishing between the effects of NPs and ions is essential in toxicity assessments.The NPs can physically interact with cell membranes and tissues of invertebrates, leading to mechanical damage and disruptions in physiological processes [202,204].

Conclusions
Analysis of the literature allows us to draw the following conclusions: 1.
Nanopriming of seeds with ZnO NPs at a concentration not exceeding 500 mg/L showed good results on the germination and viability of plant seeds, and, therefore, NPs can be used as a potential fertilizer to increase crop yields.These results indicate that relatively low concentrations of ZnO NPs can have a stimulating effect on the germination and growth parameters of various crop plants.

2.
Soil acidity affected the solubility of NPs and their toxicity.Acidic soil promotes the dissolution of ZnO NPs with the release of free ions and a decrease in the aggregation of NPs, which manifests itself in an increased toxic effect on soil microorganisms.

3.
Smaller NPs (up to ~35-40 nm) tended to have a stronger inhibitory effect; with increasing size, the inhibitory effect decreased (>50 nm).Smaller NPs are characterized by a larger specific surface area, which determines their surface charge density and is critical for attachment to the cell membrane and subsequent penetration into the cell, high free surface energy, promoting the formation of free radicals and ROS.All this contributes to causing significant damage to microbial cells.4.
ZnO NPs had a significant negative impact on the diversity, biomass, activity and functions of the soil microbiome.It is noteworthy that the decrease in microbial biomass and soil enzyme activity was more pronounced than the decrease in microbial diversity.

5.
The toxicity of NPs towards soil microbiota had a dose-dependent nature, also known as the hormesis effect: low doses (up to 250 mg/kg) promoted stimulation, high doses (>500 mg/kg)-inhibition.
Thus, future research should focus on analyzing the comprehensive characteristics of NMs and soil, as well as their short-term and long-term effects on plants and microbial communities, to determine the threshold of NM exposure and develop sustainable management of soil resources and the effective application of NMs in agricultural and soil environments.

Figure 1 .
Figure 1.Schematic illustration of the main sources and routes of entry of artificial ZnO NPs into the soil ecosystem.

Figure 1 .
Figure 1.Schematic illustration of the main sources and routes of entry of artificial ZnO NPs into the soil ecosystem.

Figure 2 .
Figure 2. Key processes determining the fate of ZnO NPs in soil under the influence of soil factors and microorganisms.SOM-soil organic matter.

Figure 2 .
Figure 2. Key processes determining the fate of ZnO NPs in soil under the influence of soil factors and microorganisms.SOM-soil organic matter.

Table 1 .
Effect of ZnO NPs on the growth and development of various plant species.

Table 2 .
Effect of ZnO NPs on soil microbiota, rhizobacteria and enzymes.