**Current Progress and Open Challenges for Combined Toxic Effects of Manufactured Nano-Sized Objects (MNO's) on Soil Biota and Microbial Community**

**Bismillah Mubeen 1,2,† , Ammarah Hasnain 1,2, Jie Wang 1,\* ,†, Hanxian Zheng <sup>1</sup> , Syed Atif Hasan Naqvi 3,\* , Ram Prasad <sup>4</sup> , Ateeq ur Rehman <sup>3</sup> , Muhammad Amir Sohail <sup>5</sup> , Muhammad Zeeshan Hassan <sup>3</sup> , Muhammad Farhan <sup>3</sup> , Muhammad Altaf Khan <sup>6</sup> and Mahmoud Moustafa 7,8**

	- <sup>4</sup> Department of Botany, Mahatma Gandhi Central University, Motihari 845401, India
	- <sup>5</sup> Hubei Key Laboratory of Plant Pathology, Huazhong Agricultural University, Wuhan 430070, China <sup>6</sup> Legume Research Group, Plant Production Department, College of Food and Agricultural Sciences,
	- King Saud University, Riyadh 11451, Saudi Arabia <sup>7</sup> Department of Biology, Faculty of Science, King Khalid University, Abha 62529, Saudi Arabia
	- <sup>8</sup> Department of Botany and Microbiology, Faculty of Science, South Valley University, Qena 83523, Egypt
	- **\*** Correspondence: wangjie@caas.cn (J.W.); atifnaqvi@bzu.edu.pk (S.A.H.N.)
	- † These authors contributed equally to this work.

**Abstract:** Soil is a porous matrix containing organic matter and minerals as well as living organisms that vary physically, geographically, and temporally. Plants choose a particular microbiome from a pool of soil microorganisms which helps them grow and stay healthy. Many ecosystem functions in agrosystems are provided by soil microbes just like the ecosystem of soil, the completion of cyclic activity of vital nutrients like C, N, S, and P is carried out by soil microorganisms. Soil microorganisms affect carbon nanotubes (CNTs), nanoparticles (NPs), and a nanopesticide; these are called manufactured nano-objects (MNOs), that are added to the environment intentionally or reach the soil in the form of contaminants of nanomaterials. It is critical to assess the influence of MNOs on important plant-microbe symbiosis including mycorrhiza, which are critical for the health, function, and sustainability of both natural and agricultural ecosystems. Toxic compounds are released into rural and urban ecosystems as a result of anthropogenic contamination from industrial processes, agricultural practices, and consumer products. Once discharged, these pollutants travel through the atmosphere and water, settling in matrices like sediments and groundwater, potentially rendering broad areas uninhabitable. With the rapid growth of nanotechnology, the application of manufactured nano-objects in the form of nano-agrochemicals has expanded for their greater potential or their appearance in products of users, raising worries about possible eco-toxicological impacts. MNOs are added throughout the life cycle and are accumulated not only in the soils but also in other components of the environment causing mostly negative impacts on soil biota and processes. MNOs interfere with soil physicochemical qualities as well as microbial metabolic activity in rhizospheric soils. This review examines the harmful effect of MNOs on soil, as well as the pathways used by microbes to deal with MNOs and the fate and behavior of NPs inside the soils.

**Keywords:** manufactured nano-objects (MNOs); nano-agrochemicals (NAgs); nanoplastics (NPs); nano-scale materials (NSMs)

### **1. Introduction**

According to some scientists, nanoparticles and nanostructured materials may have been formed during the Big Bang and brought to Earth via meteorites. The term "nanotech-

**Citation:** Mubeen, B.; Hasnain, A.; Wang, J.; Zheng, H.; Naqvi, S.A.H.; Prasad, R.; Rehman, A.u.; Sohail, M.A.; Hassan, M.Z.; Farhan, M.; et al. Current Progress and Open Challenges for Combined Toxic Effects of Manufactured Nano-Sized Objects (MNO's) on Soil Biota and Microbial Community. *Coatings* **2023**, *13*, 212. https://doi.org/10.3390/ coatings13010212

Academic Editors: Yu Shen and Manuel Miguel Jordan-Vidal

Received: 5 December 2022 Revised: 3 January 2023 Accepted: 6 January 2023 Published: 16 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

nology" gained widespread attention in the 1990s due to advances in imaging technologies that enabled practical applications in various industries. Seashells, skeletons, and other Nanostructures developed later in nature. Early people used fire to create nano-scaled smoke particles. The scientific story of nanomaterials, on the other hand, started considerably later. The colloidal gold particles created by Michael Faraday in 1857 are one of the first scientific reports. Nanostructured catalysts have also been researched for almost 70 years. By the early 1940s, precipitated and fumed silica nanoparticles were produced and supplied in Germany and the United States as alternatives to ultrafine carbon black for rubber reinforcements [1].

Nano sized objects have piqued people's curiosity for more than 30 years. These items are recognized in environmental sciences as having a major role in the biogeochemical cycles of chemical elements and organisms in their colloidal condition [2]. This is a result of their small size, commonplaceness, enormous specific surface area, and capacity to cling to the surface of aqueous solutions for extended periods of time. Analytical methods for characterizing naturally occurring colloidal objects have been developed as a result of the need to comprehend the behavior and fate of trace components [3]. The ability to synthesize nano-scale items has vastly increased since the early 2000s. The development of nanotechnologies has been aided by this increased knowledge. The qualities of the end goods generated are intimately tied to the properties of their nano-components. As a result, approaches for nano-scale analytical control has to be developed as well [4].

Nanotechnology advancements have driven the production of MNOs with fascinating and valuable material features, like nanoparticles (NPs) and nano-fibers (NFs) are defined as particles with diameters ranging from 1–100nm and many industrial products contain harmful substances like heavy metals. MNOs are widely used in medicine [5] and have a number of well-known benefits, like improving H2O management [6], preservation of food and enhancement in agricultural production [7], preserving storage of energy [8], and a variety of other applications in working for the environment and improving it [9]. Unintentional releases of MNOs into the environment grow as production quantities increase, and this can happen throughout the life of goods containing manufactured nanoobjects [10,11]. Furthermore, MNOs employed in agriculture as pesticides or fertilizers (nano-agrochemicals) are viewed as a viable approach for ensuring future food supply, although they may penetrate the environment in enormous numbers [12]. Several research initiatives have evaluated the environmental health and human safety consequences during the previous two decades [13]. Due to their tiny size, needle-like morphologies reduce the stability and enhance the area of surface-to-volume ratio, MNOs have nanoscale-specific detrimental effects when non-NSMs come in comparison with them [14]. MNOs, on the other hand, offer beneficial nano-specific physiochemical features that might be advantageous in applications like agriculture. Nano-agrochemicals, which include nanopesticides and nanofertilizers, are new nanoformulations that mix various surfactants, polymers, and inorganic NPs to enhance the dissolving power of substances that cannot dissolve in water to provide a managed and slower delivery [15]. Among nano-bio confluence, manufactured nano-objects are very helpful for plant defense because they can be employed to segregate phyto viruses, serve as a release system of organic nutrition, or enhance the role of enzymes as antioxidants [16]. If the application of manufactured nano-objects is excessive and straight in agriculture, current research focuses on enhancing MNO efficacy as well as risk evaluation. Along with physical and chemical changes, biological transformation occurs when MNOs and nano-agrochemicals are added to the soil. The pH, pore water, electrolyte, and organic matter in the soil all have an impact on these processes. The characteristics of the soil, such as pH, the make-up of the pore water and electrolyte, the quantity of natural organic matter (NOM), and other elements have a significant impact on these activities. During nano-bio interaction heteroaggregation, dissolution and oxidation-reduction takes place. For instance, the adsorption of protein NOM onto the surface of MNO might result in the creation of a corona, which increases particle mobility in the environment, as opposed to the adsorption of electrolytes (such as Ca2+), which decreases particle mobility [17].

It is important to investigate the transformation mechanisms of MNOs, including through bioassays, in order to understand exposure routes and potential absorption by biota in risk assessments. Invertebrates, which play a crucial role in maintaining and improving soil fertility and structure through their participation in various biochemical and biological processes in agri-soils, are particularly important to consider. Invertebrates in the soil are significant indicators of soil characteristics and are crucial in assessing the risk of potentially contaminating substances. According to literature, among the negative impacts of manufactured nano-objects that are composed of basically carbon and metal, include effects on the community and morphology of soil, manufactured nano-objects completely change both of these and can also have negative effects on the environment and living things present there [18,19]. Scientists and industry are becoming increasingly aware of nanomaterial impacts while also realizing the enormous potential of nanomaterials. As a result, they're attempting to strike a balance between the two, such as using the "safe by design method." The physico-chemical properties of nanoparticles are investigated in this method. Then, by experimenting with various physicochemical factors, they strive to discover a strategy to minimize nanomaterial toxicity to the lowest possible level [20]. The objective of this review is to give a succinct overview of the existing knowledge on MNO behavior and its impact on soil biota (Figure 1). opposed to the adsorption of electrolytes (such as Ca2+), which decreases particle mobility [17]. It is important to investigate the transformation mechanisms of MNOs, including through bioassays, in order to understand exposure routes and potential absorption by biota in risk assessments. Invertebrates, which play a crucial role in maintaining and improving soil fertility and structure through their participation in various biochemical and biological processes in agri‐soils, are particularly important to consider. Inverte‐ brates in the soil are significant indicators of soil characteristics and are crucial in as‐ sessing the risk of potentially contaminating substances. According to literature, among the negative impacts of manufactured nano‐objects that are composed of basically carbon and metal,include effects on the community and morphology of soil, manufactured nano‐objects completely change both of these and can also have negative effects on the environment and living things present there [18,19]. Scientists and industry are becoming increasingly aware of nanomaterial impacts while also realizing the enormous potential of nanomaterials. As a result, they're attempting to strike a balance between the two, such as using the "safe by design method." The physico‐chemical properties of nanopar‐ ticles are investigated in this method. Then, by experimenting with various physico‐ chemical factors, they strive to discover a strategy to minimize nanomaterial toxicity to the lowest possible level [20].The objective of this review is to give a succinct overview of the existing knowledge on MNO behavior and its impact on soil biota (Figure 1).

dants [16]. If the application of manufactured nano‐objects is excessive and straight in agriculture, current research focuses on enhancing MNO efficacy as well as risk evalua‐ tion. Along with physical and chemical changes, biological transformation occurs when MNOs and nano‐agrochemicals are added to the soil. The pH, pore water, electrolyte, and organic matter in the soil all have an impact on these processes. The characteristics of the soil, such as pH, the make‐up of the pore water and electrolyte, the quantity of natu‐ ral organic matter (NOM), and other elements have a significant impact on these activi‐ ties. During nano‐bio interaction heteroaggregation, dissolution and oxidation‐reduction takes place. For instance, the adsorption of protein NOM onto the surface of MNO might result in the creation of a corona, which increases particle mobility in the environment, as

*Coatings* **2023**, *13*, x FOR PEER REVIEW 3 of 25

**Figure 1.** Possible role of nanomaterials in sustainable agriculture.

#### **2. Occurrence of Manufactured Nano-Objects in Soil**

Soil is a porous matrix containing organic and mineral substances as well as living creatures that are well organized and dynamic in all aspects, physically, geographically, and temporally. Soil microbiota, for example, serve as a reservoir in which plants choose a certain microbiome, which helps them develop and stay healthy. Microorganisms in the soil also take part in several ecosystemic functions in agrosystems, such as nutrient recycling in the soil ecosystem. Nano-agrochemicals are active ingredients developed using nanotechnologies and nanoformulations to enhance the features and qualities of active molecules used in pesticides in agriculture, such as biocides, herbicides, and nutrients. The promise of using nanotechnologies to improve pesticide, nutrient, and delivery efficiency has caused an explosion in the field of agronomy and will likely result in a reduction in

the amount of input used in farming. However, the effect of these nanopesticides as a non-target organism on the soil microbiota has been underappreciated until recently [21,22].

#### **3. Existence of MNOs in Environment**

Nanotechnology enables the manipulation of matter, atoms, and molecules to change the properties of materials. This vitally important emerging technology has made it possible to create nano crystalline semiconductors, nano-agrochemicals and nanotherapeutics for cancer cures and a number of other purposes. Between 267,000 and 318,300 tonnes of MNOs, including Ag, Al2O3, CNTs, CeO2, Cu, Fe, SiO2, ZnO, TiO2, and nanoclays are produced globally [22,23]. According to one market study, the United States generated 50% of MNOs, with the EU producing 19%, China producing 12%, Korea 6%, Japan 4%, Canada 3%, Taiwan 2%, and other nations producing 4%. According to a recent market assessment, worldwide MNO production volume in 2020 will range between 400,000 and 3,150,000 tonnes for nano-SiO<sup>2</sup> and 2 to 4 tonnes for nano-Ags [24].

MNO production volumes, according to the authors of the reference [24], represent a small portion of the mining industry's total ore production, such as 1% of the entire production of Silver or 0.000002% of the entire production of Fe, and as a result, has a negligible impact on the whole life of harmful elements in the extracting and synthesizing or production areas [24]. More research is needed to evaluate the mass concentrations of manufactured nano-objects in the universal cycle of handled or unprocessed materials because the quantities that have already been declared vary widely and occasionally are not accurate [18]. Using data and dynamic material flow models, it has been possible to predict the mass fluxes related to releases throughout the whole life in H2O, soil, or the atmosphere [25,26]. According to the authors of the reference [25], by 2020 the use of nano-enabled products (such as building materials, packaging, medical products, etc.) was responsible for about 51% i.e., 12,200 tones of the worldwide release of Fe2O3, SiO2, and TiO<sup>2</sup> while end-of-life releases accounted for 43% (9890 tones). The remaining leaks occurred during manufacture [25].

Release models were established by the authors of the reference [27,28], to predict MNO concentrations in urban, natural, and sludge-treated soils in Europe. Results show that most MNOs are converted or kept in operation through processes like wastewater treatment or rubbish incineration. Released nano-Ags, for instance, bind to biosolids which dissolve slightly in wastewater of acidic nature or convert into soluble Ag2S or silver chloride types which flows with water or adheres to the wastewater and waste materials of sewage that is heated to remove contaminations and is applied to the agricultural lands to enhance the fertility of soil [27,28]. Al2O3, Fe2O3, SiO2, or TiO2, which are chemically more stable MNOs, bind to biosolids in the treatment time of wastewater and can build up in measureable concentrations in the soil of sludge treatment [27,28]. Literature estimates that the lowest MNO concentrations are of quantum dots. MNOs are approximately 8.4109 mg·kg−<sup>1</sup> in the urban and natural soils, while the highest concentrations of nano-SiO<sup>2</sup> is anticipated to be around 4.9102 mg·kg−<sup>1</sup> in landfills are predicted to be around that same level. The largest concentrations of MNOs are anticipated for the agricultural soils which are treated with sewage water. According to the authors of the reference [29,30], simulation of ten years of release of CeO<sup>2</sup> NPs, CuO NPs, SiO<sup>2</sup> NPs, and ZnO NPs in the San Francisco Bay region of California (The United States).

MNO release routes are influenced by the type of application, the product's lifespan of use, and the product's treatment for disposal. Soils treated with sludge in rural or urban areas are the most important MNOs sinks. Regarding risk evaluation and projected increase in MNO manufacturing volumes, adverse effects onland species cannot be ruled out anytime soon. Release models should be regularly updated with pertinent data because they serve as crucial guidelines for testing toxicity under realistic conditions and concentration ranges. Incorporating different MNO transformation processes into the environment is also essential for quantitative risk assessment [18,30]. Methods of expected toxicity tests or analyses of the relationship of structural activity suggest that dissolution of the particle is not the main cause of nano-toxicity. Other toxicological factors include membrane lysis, formation of ROS, redox activity, cationic stress, oxidative stress, and interference in embryo hatching and photoactivation [31]. To identify commonalities in toxicological effects among various MNO types, grouping approaches for regulatory testing have been developed in the last ten years. The connection of physiological and chemical characteristics with toxic effects is also being further investigated [32–34]. dissolution of the particle is not the main cause of nano‐toxicity. Other toxicological fac‐ tors include membrane lysis, formation of ROS, redox activity, cationic stress, oxidative stress, and interference in embryo hatching and photoactivation [31]. To identify com‐ monalities in toxicological effects among various MNO types, grouping approaches for regulatory testing have been developed in the last ten years. The connection of physio‐ logical and chemical characteristics with toxic effects is also being further investigated

trations of nano‐SiO2is anticipated to be around 4.9102 mg∙kg−<sup>1</sup> in landfills are predicted to be around that same level. The largest concentrations of MNOs are anticipated for the agricultural soils which are treated with sewage water. According to the authors of the reference [29,30], simulation of ten years of release of CeO2 NPs, CuO NPs, SiO2 NPs, and

MNO release routes are influenced by the type of application, the product's lifespan of use, and the product's treatment for disposal. Soils treated with sludge in rural or ur‐ ban areas are the most important MNOs sinks. Regarding risk evaluation and projected increase in MNO manufacturing volumes, adverse effects onland species cannot be ruled out anytime soon. Release models should be regularly updated with pertinent data be‐ cause they serve as crucial guidelines for testing toxicity under realistic conditions and concentration ranges. Incorporating different MNO transformation processes into the environment is also essential for quantitative risk assessment [18,30]. Methods of ex‐ pected toxicity tests or analyses of the relationship of structural activity suggest that

ZnO NPs in the San Francisco Bay region of California (The United States).

*Coatings* **2023**, *13*, x FOR PEER REVIEW 5 of 25

MNOs can be discharged accidentally into the soil or used purposely as biocides in agricultural applications, such as the usage of nanopesticides or nanofertilizers [35]. As a result of particle ingestion and transmission via trophic levels from bioaccumulation or biomagnifications, extensive ecosystem exposure may occur [36]. As a result of particle ingestion and transmission via trophic levels from bioaccumulation or biomagnification, extensive ecosystem exposure may occur. For instance, laboratory studies showed that the soil fungus *Penicillium solium* attracted the amino acid conjugated quantum dots; however, no MNO absorption was observed in the absence of this coating. According to the authors of the reference [37], invertebrates like *Eisenia fetida* (Earthworm) could be used to quantify the accumulation of MNOs by determining the ratios of MNOs present in tissues to the amounts present in water and in bio-contaminants, in which the ratio of MNOs is determined in prey and predator (Figure 2). [32–34]. MNOs can be discharged accidentally into the soil or used purposely as biocides in agricultural applications, such as the usage of nanopesticides or nanofertilizers [35]. As a result of particle ingestion and transmission via trophic levels from bioaccumulation or biomagnifications, extensive ecosystem exposure may occur [36]. As a result of particle ingestion and transmission via trophic levels from bioaccumulation or biomagnification, extensive ecosystem exposure may occur. For instance, laboratory studies showed that the soil fungus *Penicillium solium* attracted the amino acid conjugated quantum dots; however, no MNO absorption was observed in the absence of this coating. According to the authors of the reference [37], invertebrates like *Eisenia fetida* (Earthworm) could be used to quantify the accumulation of MNOs by determining the ratios of MNOs present in tissues to the amounts present in water and in bio‐contaminants, in which the ratio of MNOs is determined in prey and predator (Figure 2).

**Figure 2.** Soil matrix, aggregation and organic allocation induced by plant and microbial activity in the rhizosphere.

#### **4. Nanoparticles and Mycorrhizas/Rhizobia Interactions**

Certain nanoparticles like AgNPs, ZnO NPs, and TiO<sup>2</sup> have long been known to have antimicrobial effects against bacteria and fungi. Fungal hyphae and bacterial cells can be damaged by nanoparticles [38–40]. Limited or positive impacts of NPs on microbial populations of soil and function studies imply that NPs and microbe interactions depend on the environment [41–45]. Root and microbe symbioses occur in the rhizosphere, where factors like interactions between the soil biota, the complexity and availability of resources, and biophysical heterogeneities may affect how much NPs impact both free-living and root colonization microorganisms [46,47]. Overall, it appears that NP effects on the emergence of mycorrhizal (Arbuscular mycorrhiza (AM) is a common type of symbiotic relationship between plants and microbes. These fungi are found in many natural habitats and are known to offer a variety of ecological benefits, including improved plant nutrition and stress resistance, better soil structure and fertility, and increased tolerance to environmental

challenges) and rhizobial symbioses are extremely context-specific. NPs have been shown in numerous studies to negatively affect mycorrhizal and rhizobial interactions [48–51]. Symbioses of mycorrhiza and rhizobia are the most significant relationship on the earth and have importance in the earth's ecosystem. It plays role in nutrient cycling in the soil, mineralization of the organic matter, modeling of microbial communities and plants, and eventually in the functioning of an ecosystem [52–54]. The characteristics of structure and plant fungus species involved have led to the reports of various advantageous root-fungal symbioses so far [55]. Although few of the studies have partly addressed other associations, the majority of research in the field of nanotechnology has focused on the common mycorrhiza, notably arbuscularmycorrhiza. The gymnosperms, angiosperms, bryophytes, and pteridophytes [56], associate with fungus from the Mucoromycota subphylum Glomeromycotina to establish arbuscularmycorrhizal (AM) symbioses. Rotating legume and non-legume crops, rhizobial symbioses, and resilience to environmental stresses like acidity, heavy metals, and organic pollutants are all essential aspects of the Fabaceae, the thirdlargest plant family [57]. Roots produce nodules to make room for N2-fixing rhizobia at the symbiotic contact [58]. The overall impact of NPs on mycorrhizal colonization of nodule growth is influenced by the NP characteristics, concentration of fungal or bacterial species, and characteristics of the interaction matrix, where mycorrhizas and rhizobia reside and interact with plant roots. There is little study on how NPs affect these and how these symbiotic interactions function. However, available evidence indicates that studies on the toxicity of NPs against these advantageous root and microbe symbioses should concentrate on both structural and functional aspects [36].

Physical characteristics of NPs have a significant influence on the colonization or nodulation of root mycorrhizal fungi (e.g., kind, speciation, and size). AgNPs appear to be more toxic to mycorrhizas than ZnO NPs because of their negative effects on root colonization at approximately 5600 times lower soil concentrations [48,50,51,59,60]. In a pea of rhizobium leguminosarum symbiosis, exposure to Fe2O<sup>3</sup> nanoparticles at 6 g·L <sup>−</sup><sup>1</sup> had no effect on nodulation. ZnO NPs and TiO<sup>2</sup> NPs both had unfavorable impacts on nodule growth 35 days after treatment, despite equal concentrations and exposure times [61]. In a soybean-*Bradyrhizobium japonicum* symbiosis, CeO NPs at 50 g·kg−<sup>1</sup> had no effect on nodulation, but ZnO NPs at the same concentration increased nodulation [62]. Depending on the NP types and coating, the bioavailability and effects of NPs on mycorrhizas and rhizobia may differ. For instance, functionalized silver nanoparticles (PVP-AgNPs) were sown to reduce arbuscular mycorrhiza (AM) growth in tomato roots when applied at the same treatment rate of 100 mg·kg−<sup>1</sup> in tomato roots when applied at the same treatment rate of 100 mg·kg−<sup>1</sup> at the same treatment rate of 100 mg·kg−<sup>1</sup> soil, however, silver sulphide NPs had no discernible effect [63]. The authors of the reference [64] used functionalized Fe3O<sup>4</sup> NPs with positive and negative surface charges (carrying an amine and a carboxylic acid, respectively) to study NPs and rhizobia interactions and discovered that positively charged Fe2O<sup>4</sup> NPs improved nodulation in soybean more than negatively charged Fe2O<sup>4</sup> NPs. This demonstrates that NP surface coating and modification may affect their toxicity towards mycorrhizas and rhizobia before exposure, and as a result, NP physicochemical characteristics can be changed to achieve acceptable results or avoid unwanted results in NP interactions with rhizobia and mycorrhiza. It was also found that NP size affects interactions between NP and mycorrhiza. Tomato root colonization was reduced when exposed to 2 nm-AgNPs at the same dosage of 12 mg·kg−<sup>1</sup> soil but was unaffected when exposed to larger AgNPs of 15 nm [64]. Moreover, soil spiked with TiO<sup>2</sup> NPs exhibited considerably greater Ti concentrations in the microcosm leachates than soil spiked with P25- TiO<sup>2</sup> NPs [65], indicating that NP shape and size may have an impact on the bioavailability. More research is necessary because our knowledge of how NP size influences NP–rhizobia interactions are currently relatively limited. Because partially or completely converted NPs may have a different toxicity potential than their pure counterparts, chemical alteration of NPs may have an effect on rhizobia [66,67]. The amount of NPs in the soil influences the interactions between mycorrhizas and rhizobia. No negative effects on Arbuscular

mycorrhiza (AM) colonization in tomatoes were seen at low ZnO NP concentrations i.e., 25 and 400 mg·kg−<sup>1</sup> soil, respectively [68] and maize plants [69], whereas, greater amounts (500 to 3200 mg·kg−<sup>1</sup> soil) prevented colonization in maize [70]. In contrast to a low concentration of AgNPs higher concentrations i.e., 0.1 and 1 mg·kg−<sup>1</sup> soil significantly enhanced root colonization in white clover (*Trifolium repens*) [53].

White clover grew AM fungi when exposed to a low concentration of FeO NPs i.e., 0.032 mg·kg−<sup>1</sup> soil to a low concentration of FeO NPs (0.032 mg·kg−<sup>1</sup> soil), but not when exposed to a concentration 100 times higher (3.2 mg·kg−<sup>1</sup> soil) [62]. The concentration of NPs appears to play a role in its interaction with rhizobia, comparable to the responses observed for mycorrhizal colonization, despite the paucity of experimental evidence. The effects of ZnO NPs on nodulation in *Bradyrhizobium japonicum*-infected soybean plants changed from neutral to positive when the concentration was increased from 5 to 50 g·kg−<sup>1</sup> . When exposed to Kocide, a fungicide made of copper that contains a substantial quantity of copper NPs [71], nodulation and N<sup>2</sup> fixation were unaffected at the authorized rate (1.7 mg·kg−<sup>1</sup> ), whereas nodulation was prevented at higher doses of 3.4 and 6.8 mg·kg−<sup>1</sup> of treatment [72]. Negative NP and rhizobia interaction do not show a negative association between the concentration of NO and nodule growth [61,73]. The used concentration range may have exceeded the NPs' toxicity threshold in the relevant experimental circumstances (soil-less media), causing all concentrations to have a detrimental effect on the results [36]. According to the study, several mycorrhizal fungal species may respond to NPs differently. *Glomus caledonium*, an AM fungus, was shown to be more resistant to the toxicity of ZnO NPs than *G. versiforme* based on the extent of the unfavorable impact of ZnO NPs on root colonization [74]. This was attributed to the stronger resistance of *G. caledonium* to heavy metals like copper, zinc, cadmium, etc. [69] (Figure 3). *Coatings* **2023**, *13*, x FOR PEER REVIEW 8 of 25

**Figure 3.** Toxic effects of manufactured nano‐objects (MNOs) on earthworms. **Figure 3.** Toxic effects of manufactured nano-objects (MNOs) on earthworms.

The knowledge gained about the toxicity mechanism of NPs will aid in the antici‐ pated remodeling of nanoparticles to reduce their environmental impact. Non toxicity's main mechanisms include directly attaching to the external membrane surface of cells,

tion techniques for NP toxicity in the environment after elaborating on the nano‐toxicity mechanism, providing an understanding of lowering toxicity and promoting long‐term

Direct NP surface binding is a primary mechanism of inducing toxicity among sev‐ eral others and surprisingly, when NPs are found in near proximity to cells or animals, their interface is governed by electrostatic attraction. A bacterial cell's surface is usually found to have a negative charge [77]. According to the aforementioned finding, posi‐ tively charged NPs are more closely linked to bacteria than negatively charged ones. A subset of cells in multicellular model organisms may take up NP as a result of such in‐ teractions [78]. When applied to bacterial culture, NPs persist on the surface of the cell, thus destruct membrane lipid, causing membrane loss or disruption [79]. The stimulation of an internal signaling cascade as a result of such a charge in membrane physical prop‐ erties disturbs cells [80]. NPs then get dissolved in and release harmful ions on the sur‐ face of the cell which are cell permeable [81]. The authors of the reference [82] asserted that the relationship between NPs and gram‐positive bacteria was likely caused by the attachment of negatively charged entities to lipopolysaccharides on the cell surface, which led to cell death. In another study, the presence of colloidal semiconductor nano‐ crystals made of poly diallydimethylammonium chloride covered with cadmium sele‐ nide (CdSe) quantum dots embedded in bilayer of lipid was demonstrated using tools

**5. NPs and Soil Biota: Mechanisms of Action**

use of NPs [76].

#### **5. NPs and Soil Biota: Mechanisms of Action**

The knowledge gained about the toxicity mechanism of NPs will aid in the anticipated remodeling of nanoparticles to reduce their environmental impact. Non toxicity's main mechanisms include directly attaching to the external membrane surface of cells, dissolution of hazardous ions, and stimulation of oxidative stress [75]. Researchers clarified the influence of NPs on nanoparticles association with microorganisms and mitigation techniques for NP toxicity in the environment after elaborating on the nano-toxicity mechanism, providing an understanding of lowering toxicity and promoting long-term use of NPs [76].

Direct NP surface binding is a primary mechanism of inducing toxicity among several others and surprisingly, when NPs are found in near proximity to cells or animals, their interface is governed by electrostatic attraction. A bacterial cell's surface is usually found to have a negative charge [77]. According to the aforementioned finding, positively charged NPs are more closely linked to bacteria than negatively charged ones. A subset of cells in multicellular model organisms may take up NP as a result of such interactions [78]. When applied to bacterial culture, NPs persist on the surface of the cell, thus destruct membrane lipid, causing membrane loss or disruption [79]. The stimulation of an internal signaling cascade as a result of such a charge in membrane physical properties disturbs cells [80]. NPs then get dissolved in and release harmful ions on the surface of the cell which are cell permeable [81]. The authors of the reference [82] asserted that the relationship between NPs and gram-positive bacteria was likely caused by the attachment of negatively charged entities to lipopolysaccharides on the cell surface, which led to cell death. In another study, the presence of colloidal semiconductor nanocrystals made of poly diallydimethylammonium chloride covered with cadmium selenide (CdSe) quantum dots embedded in bilayer of lipid was demonstrated using tools like quartz crystal microbalance and atomic force microscopy [83]. When NPs bind to the cells; hydrophilic regions of the lipid bilayer collapse, disrupting cell membranes. Both eukaryotic and prokaryotic cells require these liquid domains for signaling and membrane transport [84–86].

The breakdown of harmful components from NPs, which causes an oxidative burst in affected organisms, is the most important mechanism of toxicity induced by NPs. Toxic ions can be released from NPs in a variety of ways, all of which are dependent on the identity of the released ions. A small number of ions attach to important proteins and enzymes, thus changing their metabolism and eventually causing major biological activities to be suppressed [87]. The hazardous ions are slowly released from metal oxides and get absorbed by membranes resulting in direct interactions with amino, mercapto, and carboxyl groups in nucleic acids and proteins. These interactions have a significant effect on the structure of cells and enzymatic activity, ultimately disrupting the exposed organisms overall physiology. Another method is to directly associate harmful ions with the damaged organism's phospholipid bilayer or even its genetic material [88]. As a result, metal ions cause an oxidative burst in organisms by increasing reactive oxygen species (ROS) levels [89]. The major form of hazardous ion dissolution in AgNPs is one of the primary sources of toxicity in organisms [90]. Due to the release of Ag and Pd ions into the solution, Pd nanolayers and nanowires with diameters ranging from 0.4 to 22.4 nm medium of polyethylene naphthalate were discovered to show antibacterial activity [91]. The authors of the reference [92] found that metaloxides had only minimal antibacterial activity when added to the culture, indicating that metal ions dissolution may not be the NPs antibacterial action. The toxicity of a few complex oxides including lithium (Li), nickel (Ni), manganese (Mn), and cobalt oxides to *Shewanella oneidensis* MR-1 bacterial cells was confirmed to be largely caused by the dissolution of ions [93]. To assess bacterial respiration, the researchers used a respirometer and optical density analysis to evaluate bacterial multiplication. According to this study, the complex oxides of Li, Ni, Mn, and Co were broken down to produce the ionic Li, Ni, Mn, and Co respectively. The continual dose and release of these nanomaterials, however, was straight linked to toxicity that was observed. An equi-stoichiometric NMC with different morphologies and bacterial toxicity was produced in order to highlight the importance of dissolution to Ni, Mn

and Co oxide toxicity [94]. These were chosen because they show different crystal faces, demonstrating how variations in crystal faces affect the transition of metal coordination and how dissolution depends on them. Dissolution is connected to a material's exposed surface area, as was previously stated. Additionally, the surface area of the NMC was used to determine its toxicity rather than its mass. When using the surface area as the basis of dose, morphologies with different crystal faces demonstrated equal toxicity to the bacterial strain. As a result of the results made above, it was determined that neither crystal faces nor surface area significantly affects NMC toxicity to *Shewanella oneidensis* [76].

There are numerous studies that have been published recently that support the importance of ROS production and oxidative burst in relation to NP toxicity [95,96]. The four main kinds of ROS are singlet oxygen, hydrogen peroxide, hydroxyl ions, and superoxide anion, which are produced by short-term stress induced processes [97]. It has been determined that physiological harm is caused by singlet oxygen. Under typical environmental cues, exposed organisms maintain a balance between ROS generation and scavenging. On the other hand, when too much ROS is produced, the intercellular redox equilibrium is upset, which sets the stage for oxidation [98]. The main effects of ROS include lipid peroxidation and disruption of important enzymes such as mononuclear iron proteins [99]. Additionally, ROS production leads to the oxidation of DNA bases and deoxyribose, which causes mutations and DNA damage [95]. It is being determined whether NPs' electrical structures, ROS, and toxicity are correlated. Similar behaviors were also demonstrated in *E. coli* by the authors of the reference [100] using 7-metal oxide NPs with band edges close to the redox potential of reactive redox couples. It was discovered that the level of abiotic ROs produced in response to NPs exposure was directly correlated with the organism's level of toxicity. Another experimental study also showed the toxicity of 24 distinct metal oxide nanoparticles in *E. coli*. Only seven of the 24 metal oxides found in NPs were hazardous and increased the levels of intercellular ROS. It was established through the use of nanostructure activity association analysis that NPs' toxicity was directly related to (1) the conduction band energy of the nanomaterials and (2) the hydration enthalpy dictating their capacity to dissolve. Additionally, it was hypothesized that nanomaterials and biomolecules conduction bands might overlap if the materials were hazardous and disintegrated quickly. The NPs have also been shown to activate a chain of biological signals that results in an oxidative burst [66]. Another finding obtained by the authors of the reference [101] in the guts of *Daphia magna* was that when the guts were exposed to positively or negativity charged nano diamond particles of 5 nm to 15 nm size, the larger particle was observed to stimulate ROS production in a dose-dependent way in comparison to lesser sized NPs. The expression of genes related to oxidative burst was also confirmed to be decreased; indicating that cells were preventing ROS clarification as the basic mechanism of NPs-toxicity [101].

According to several pieces of research, NPs antifungal properties, which include the release of metal ions, have a detrimental effect on mycorrhizal colonization like Zn+2 and Ag [102]. The detrimental effects of NPs on mycorrhizal colonization may be directly related to their antifungal properties, which include adhering to cell surfaces and physically harming cell walls and membranes, increasing membrane permeability, obstructing water channels, and killing cells through NP penetration and deposition. Considering that NPs have sharp edges, they might also be able to cut through fungal structures and cell walls [101,103]. Inhibition of germination of spore due to formation of aggregations of NPs by Van der Waals forces [101] buildup of ROS by disruption of ROS scavenging defence mechanisms such as glutathione cycle and regulation [104]; ion discharge from metal-based NPs [105]; and capability of certain NPs, like TiO2, of being photocatalytic are also related to negative effects of NPs on the colonization of mycorrhiza [106].

The mechanisms behind the beneficial NP-mycorrhiza interactions need to be studied more thoroughly. AgNPs have been discovered to promote AM fungal colonization [53]. Additionally, plants under heavy metal stress showed an increase in AM fungus colonization [107]. Plants exposed to NPs may see a decline in the growth of rhizobial nodules as a

direct result of the NPs' antibacterial properties. The antibacterial effects of NPs may generally result from simultaneous oxidative stress induction and metal ion release processes such as cell membrane disruption [108–110]. Additionally, it has been verified that NP concentrations that are environmentally relevant have a significant impact on greenhouse gas emissions, crucial ecosystem services like nitrogen cycling, and soil microbial populations [111]. Applying any material to soil that is both persistent and immobile should generally be done with utmost caution. Due to their elemental nature, nanoparticles do not degrade in the environment. For instance, hazardous stages may be achieved in soil after ten years of continuous applications. Several NPs seem to be determined and mostly fixed in the soil, depending on the NPs and the soil properties. They also incorporate into plant tissues and the soil biota [111,112]. Therefore, it is necessary to undertake spatial and temporal trials for nano-agrochemicals and NP-containing amendments (such as AgNPs provided through sludge application) to assess the impact of repeated NP treatments on these significant root microbial symbioses. Cu concentrations in soil are up to an order of magnitude higher than in natural soils as a result of decades of historical use of Cu-based fungicides, which can have detrimental environmental consequences on soil fertility, water resources, and species [113] (Figure 4). *Coatings* **2023**, *13*, x FOR PEER REVIEW 11 of 25

**Figure 4.** An outlook of MNOs possible pathways for earthworms. **Figure 4.** An outlook of MNOs possible pathways for earthworms.

#### *5.1. Silver 5.1. Silver*

to lessen stress [115,116].

Silver has long been used as a biocide. The antimicrobial/biocidal effects of sil‐ ver‐based nanopesticides are demonstrated against a variety of kinds of microorganisms, including bacteria, fungi, and viruses [114]. Ag‐based nanopesticides, which have been commercialized and patented for use in plant protection, seed processing, and plant de‐ velopment improvement, have already been introduced in the plant protection measures. Even though these nanopesticides are advertised as having the ability to efficiently pre‐ vent phytopathogen illnesses in a variety of plants to boost the plant immune system and Silver has long been used as a biocide. The antimicrobial/biocidal effects of silverbased nanopesticides are demonstrated against a variety of kinds of microorganisms, including bacteria, fungi, and viruses [114]. Ag-based nanopesticides, which have been commercialized and patented for use in plant protection, seed processing, and plant development improvement, have already been introduced in the plant protection measures. Even though these nanopesticides are advertised as having the ability to efficiently prevent

Due to their widespread use in commercial and industrial products, AgNPs may

The majority of the information that has been published in this field comes from studies about the effects of nanopesticides that are formed of silver on bacteria and mi‐ croorganisms that are not intended targets. The authors of the reference [119] used bio‐ solids and AgNPs to modify a clayey SiO2 with low pH (SiO2 73, Silt 22 and Clay 5%, pH 5.6, and small amounts of organic components) to a target concentration of 0.19 to 15 mg∙kg**−**<sup>1</sup> soil (particle size 15 nm). This mixture was kept at 22°C in the dark for 30 days. Exoenzyme activity, soil respiration and potential ammonium oxidation (PAO) along

nano‐agrochemicals. Predicted environmental concentration (PEC) values for Ag‐NPs in soil vary by location around the world; in Denmark, native soil has a PEC value of 13–61 mg∙kg**−**1, whereas agricultural soil has a PEC value of 6–12 mg∙kg**−**<sup>1</sup> [117,118], while the expected values of Ag‐NPs in American soils range from 6.6 to 29.8 ng∙kg**−**1, they are

predicted to be between 17.4 and 58.7 ng∙kg**−**<sup>1</sup> in European soils [117].

phytopathogen illnesses in a variety of plants to boost the plant immune system and to lessen stress [115,116].

Due to their widespread use in commercial and industrial products, AgNPs may have accumulated in the soil accidently or on purpose, for example as fungicides and nano-agrochemicals. Predicted environmental concentration (PEC) values for Ag-NPs in soil vary by location around the world; in Denmark, native soil has a PEC value of 13–61 mg·kg−<sup>1</sup> , whereas agricultural soil has a PEC value of 6–12 mg·kg−<sup>1</sup> [117,118], while the expected values of Ag-NPs in American soils range from 6.6 to 29.8 ng·kg−<sup>1</sup> , they are predicted to be between 17.4 and 58.7 ng·kg−<sup>1</sup> in European soils [117].

The majority of the information that has been published in this field comes from studies about the effects of nanopesticides that are formed of silver on bacteria and microorganisms that are not intended targets. The authors of the reference [119] used biosolids and AgNPs to modify a clayey SiO<sup>2</sup> with low pH (SiO<sup>2</sup> 73, Silt 22 and Clay 5%, pH 5.6, and small amounts of organic components) to a target concentration of 0.19 to 15 mg·kg−<sup>1</sup> soil (particle size 15 nm). This mixture was kept at 22 ◦C in the dark for 30 days. Exoenzyme activity, soil respiration and potential ammonium oxidation (PAO) along with patterns of coming generations of bacteria were then carried out to assess the influence of AgNPs and examine bacterial diversity. Similar AgNP sensitivity was shown in these assays, with effects starting at levels of at least 1.67 mg·kg−<sup>1</sup> . With an enrichment of proteobacteria, cytophagales, and spirobacteria, next-generation sequencing demonstrated a shift in the microbial population and variable sensitivity of bacterial groups. Some nitrifiers (nitrosomonadales) were adversely impacted, and this correlated with the reduction of PAO activity [119].

The authors of the reference [120] incubated AgNPs and added AgNO<sup>3</sup> to SiO<sup>2</sup> loamy soil with a hydrogen ion concentration of 5.61 and 0.93 percent organic content in order to study the effects of Silver nanoparticles on NH4+ oxidizing bacteria (AOB) for 140 days. Amounts of 0.56, 1.67, and 5 milligrams per killograms of silver nanoparticles kg−<sup>1</sup> of AgNO<sup>3</sup> were applied. They showed relative hindrance of AOB with 1.67 and 5 milligrams per Kg's of silver nanoparticles starts at day fourteen and enhanced to 140 days, in contrast to silver ionic form, where inhibition begins on the first day and shows growth even during deficiency [121,122]. AgNPs impacts on a black poplar tree's associated phyllosphere and rhizosphere bacteria were examined by the authors of the reference [123]. Three years old poplar trees were chronically supplied with nano powder, amorphous carbon coated AgNPs of 25 nm size, and the dose applied was 1 mg·L −1 for a 10-week period, single supply was applied for 4 weeks and double supply was applied for 6 weeks. AgNPs foliar exposure was done with the soil covered, and no fertilizer was used during the experiment.

Using next-generation sequencing, the ITS 1 region and the V3-V4 section of the 16S rRNA, respectively, were used to study the bacterial and fungal microbiome. Root AgNPs treatment reduced the biodiversity of microorganisms like bacteria and fungi; application of AgNPs to leaves enhances the evenness of bacteria and fungi and discovered a significant decrease in both microbial groups. According to a bioinformatics functional analysis, the use of AgNPs enhanced the bacteria that do not require too much oxygen and can bear oxygen stress while lowering aerobic bacteria. However, the AgNPs treatments in this study mimicked the application of Ag nanopesticides on agricultural soil rather than a contaminated environment. For example, Zerebra**®**Agro, a commercial nanopesticides based on silver, has an Ag content of 0.5 g·L −1 . Additionally, 0.1 L·t −1 is the suggested amount for plant therapy, 0.1 L−<sup>1</sup> and seed hectare-1 for use on cultivated crops from 1–3 times during the vegetative period as opposed to twenty grams per hectare [122].

The authors of the reference [38] investigated how citrate-coated AgNPs of 50 nm and their ions disturb the functioning of enzymes present in soil and the make-up of the microbial community in a bio-solid amended agricultural soil. During the treatment of wastewater solid part that was organic in nature was added to surface soil at the Macdonald campus of McGill University (soil/biosolid weight ratio: 50/1; depth: 35 cm; pH: 6.7). The amount of total AgNPs supplied to the soil was 1, 10, and 100 mg. The material disintegrated within the first two hours and maintained its stability for up to

30 days. AgNPs at one and ten mg per kg of enzymes that are present outside involved in phosphorous, carbon, and nitrogen cycling did not have any effect at short-term (2 h) concentrations. Because only 37% of the AgNPs were dissolved at 2 h, AgNPs had a milder effect on these enzymatic activities at 100 mg·kg−<sup>1</sup> compared to Ag<sup>+</sup> . After two hours and thirty days of exposure, the microbial population of the soil was tested using 16S rRNA gene amplicon sequencing. In comparison to all other treatments, the relative abundance of the Gamma proteobacteria category was remarkably greater for Ag<sup>+</sup> ions and AgNPs at 100 mg·kg−<sup>1</sup> of soil response to dissolve Ag and AgNPs [38]. According to the authors of the reference [123], decline in the abundance of AOB has been reported due to the application of 0.01 mg nano particles of silver per kilogram per year that showed bad effects on nitrogen fixation and soil microorganisms' environment, leucine amino peptidase activity and nitrogen-fixing microorganisms.

The authors of the reference [63] focused on distinct Ag speciation and NPs coating while examining the effects of AgNPs. They introduced 1, 10, or 100 mg of Ag2S NPs, AgNPs coated in polyvinylpyrrolidone (PVP), and Ag<sup>+</sup> to a sandy loam soil that had been modified with biosolids (pH 6.8). Before planting tomato seeds, the soil mixture was infected with an arbuscular mycorrhizal fungus (AMF) or a commercial inoculum (*Solanum lycopersicum*). The authors measured neutral lipid fatty acid (and phospholipids fatty acid) analyses, ammonium nitrate extractable Ag concentrations, and the overall microbial community structure in soil modified with biosolids. All silver applications at the rate of 1 mg·kg−<sup>1</sup> and 10 mg·kg−<sup>1</sup> did not substantially differ from the control, with the exception of three treatments i.e., 100 mg·kg−<sup>1</sup> for Silver-PVP and Ag+ and 10 mg·kg−<sup>1</sup> for Ag2S NPs [63]. Ag-PVP, Ag+, and Ag2S NPs had an effect on fungus and bacteria, including Actinomycetes and microbial community even at concentrations of 1 mg·kg−<sup>1</sup> [124,125].

#### *5.2. Copper*

There has been a rise in the release of copper oxide NPs (CuO NPs) into terrestrial and aquatic environments as a result of their use in different sectors like goods against microorganisms and nano-agrochemicals [126]. About 79,000 tons of CuO-NPs are consumed annually in North America, which contributes about fifty percent of the world marketplace [127]. There has been extensive research on the effects of metal and metal oxide NPs on the soil and rhizosphere microbiome, mostly with an eye toward the effects of environmental pollution [128,129]. NPs of Silver, Zinc oxide, copper, and iron are the most extensively studied in toxicity studies. There are now two types of nanomaterials that have produced commercial agrochemicals that are nano-enabled and on the market: colloidal silver and copper nanoparticles for treating fungal infections on seed, vegetative parts, and tubers respectively [130]. Copper is a well-known biocide that has been used for centuries, as well as an important nutrient for living things like plants and bacteria. As chemicals to kill bacteria and fungi on veins, plants, and reproductive parts, some copper-based insecticides are currently permitted in organic farming. Originally employed as lime, copper sulfate was neutralized in the Bordeaux mixture to treat downy mildewinfected grapes [131]. Products to kill pests that are formed of copper include those with the chemical compositions copper hydroxide, cuprous oxide copper ammonium carbonate, copper oxychloride, and copper octanoate. Since copper sulfate's solubility encourages phytotoxicity and reduces the persistence on tree leaves and fruits as well as the fungicide action, fixed copper-less soluble variations have really been developed. These particles are known as fixed-coppers, and their size affects how well they cover and cling to plant leaves as well as how much copper ions they discharge. Copper nanoparticles have been developed and marketed for their ability to improve the growth of plants by preventing the release of copper ions. These nanoparticles were originally sold as micronized particles. There are two brands of nanosized copper formulations available: NANOCU and Kocide**®**3000, both produced by the same company [131].

The authors of the reference [132] examined the effect of nano-sized CuO vs copper ions (CuSO4) in five various agricultural soils with pH ranging from 6.4 to 8.21 in order to understand the complexes in biology and physio-chemical diversity of the soil. After the soil water content had been maintained to a holding capacity of H2O particular to every soil, microbes present in soil were incubated for 90 days without light at a temperature of twenty-eight centigrade. When maximum dosage was applied, 100 mg·kg−<sup>1</sup> , CuONPs dramatically inhibit microbial activity involved in C and N cycles, respiration, denitrification, and nitrification in the five soils under study. These results worsen overtime. The lowest doses usually have little to no impact, with respiration in sandy loam soils reducing at 1 mg·kg−<sup>1</sup> and denitrification in loamy soils decreasing at 1 mg·kg−<sup>1</sup> after 90 days. Copper oxide nanoparticles have a different impact on soil microbial activity in relation to the carbon and nitrogen cycles compared to ionic copper. They are most effective at promoting denitrification in maximum soils, while nitrification and soil respiration are more influenced in coarse soils. However, when used at agriculturally suitable levels, copper oxide nanoparticles have little impact on soil microbes. Soils with a coarse texture and low levels of organic matter or clay may be more susceptible to the effects of copper oxide nanoparticles. In a study, plants cultivated for 50 days in climatic chambers showed that copper oxide nanoparticles had a negative impact on microbial respiration and denitrification when applied at a dose of 1 mg·kg−<sup>1</sup> . Copper-based nanoparticles have also been found to be harmful to nitrifiers, with copper oxide nanoparticles and copper ions exhibiting distinct behaviors such as the release and uptake of copper ions and their effects on microbial activity [133].

In a study, copper nanoparticles between 40 and 60 nm in size were incubated for 30 days in soil with high levels of organic matter at concentrations of 0.05% and 0.15% by weight and 3 mg·kg−<sup>1</sup> ATZ. The bacterial, fungal, and nitrifying bacterial population profiles, as identified by PCR denaturing Gradient Gel Electrophoresis, remained relatively stable throughout the experiment. However, the 0.15% weight concentration of copper nanoparticles significantly reduced the dissipation of ATZ, indicating increased persistence of ATZ in soil. Most of the interactions between this persistence and soil particles were physical-chemical in nature. Paddy soils, which are commonly used for agriculture in China and are exposed to cycles of flood and dry conditions and frequent changes in soil oxidation-reduction environments, are the most common type of agricultural soils in the country [116,133].

Cu and AgNPs' behavior and fate in soil are influenced by factors that are intrinsic to the NPs, such as their size, charge on the surface, and pH, as well as factors that are extrinsically linked to the characteristics of the intricate soil matrix. Nanoparticle dissolution and interactions with cells are significantly influenced by their form. Exposure time also influences the characteristics of common nanoparticles and of how silver can change the makeup of communities of microorganisms [134]. Nanoparticles may dissolve, undergo changes due to redox reactions, form aggregations with particles of soil and adsorb, particularly to clays, on a global scale [135,136]. Acidity tends to source solubilization of Silver nanoparticles, while high pH of soil facilitates Ag-sorption. According to the authors of the reference [120,133], across a range of soils, AgNP toxicity towards microbial processes including substrates persuade breakdown of glusose and to bacteria that oxidize NH<sup>3</sup> decreased as clay content and pH increased [120,133]. The similar line of reasoning is reached in the conclusion by the authors of the reference [132] regarding the sporadic effects of copper oxide nanoparticles at agriculturally related concentrations on coarse soil texture having lower content of organic matter or clay [133–135]. Almost 4.5% of the world's soils with low pH are used for cultivation purposes. This is despite the fact that acidic soils make up roughly 30% of the ice-free terrain of the world [137]. Short-term effects of the nanoparticles can be increased by using acidic soil, which encourages the breakdown of copper- and Silver nanoparticles releasing free ions. An intriguing finding in the literature is that the pesticide's ionic or nanoform can have distinct effects, perhaps due to the ion release percentage. Ionic and nanoforms of metals may exhibit parallels and variations in the mode of antibacterial action or the influence on a microbial population when exposed in vitro to AgNPs, according to certain scientists [134,138]. The toxicity of nanoparticles

is kinetic in long-term investigations and appears to be connected to soil breakdown or transformation processes that result in momentary adjustments and adaptations of the microbial population. As demonstrated by the authors of the reference [139], adjusting the surface features of NPs could aid in controlling the dissolution and phase shifts and probably lessen the toxicity towards microbial cells.

#### *5.3. Zinc Oxide*

The third most common metal-based NP is zinc oxide nanoparticles (ZnO NPs) [140]. Wastewater from factories and waste material from homes that are used in agricultural systems to enhance fertility is the basic source of entry of Zinc oxide nanoparticles into the environment [141]. Zinc oxide nanoparticles create a network of soil particles that preserve NPs' emulsion characteristics, but they can also form bigger aggregates or become soluble and excrete zinc ions [142]. ZnO-nanoparticles have a regionally variable PEC, similar to Ag-NPs. In Europe, PECs of 0.085–0.661 g·kg−<sup>1</sup> were projected, while in the United States, PECs of 0.041–0.271g·kg−<sup>1</sup> were predicted [39]. The values viz., 0.018–0.9 and 0.008–0.35 g·kg−<sup>1</sup> were anticipated by a Danish study comparing uncultivated and agricultural soil [39]. Among the various varieties of NPs, ZnO NPs are becoming more common and are now the third most widely used nanomaterial. As a result, the direct influence of ZnO NPs discharged into the environment on soil microbial communities and processes must be carefully assessed. Despite many potential benefits, various experimental research studies have found that ZnO NPs can affect soil productivity by modulating microbial community features [143]. ZnO-NPs have an ecotoxicological impact on soil microorganisms wherein according to the authors of the reference [144], the microbial respiration, dehydrogenase, ammonification, and fluorescent diacetate hydrolase activity decrease in the soil.

Another study demonstrated that ZnO NPs inhibit enzymatic activities such as diacetatehydrolysis, urease, and catalase. They also reduced thermogenic metabolism, lowered Azotobacter, Phosphate-solubilizing, and potassium solubilizing bacterial colonies [145]. According to reports ammonification was suppressed by up to 37.8% in soil that had received three months' worth of treatment with 1 mg of ZnO NPs per gram of soil. Respiration inhibition was 14.2% during first the month. In soil treated with 1–10 mg ZnO NPs a similar pattern was observed for dehydrogenase and fluorescent diacetate hydrolase activities. A study by the authors of the reference [146] was conducted on Staphylococcus aureus and Escherichia coli which indicate that ZnO NPs are hazardous for both of these gram-positive and gram-negative bacteria. ZnO-NPs inhibited completely the growth of *S. aureus* at doses of 1 mmol/L and *E. coli* at doses of 3.4 mmol/L [146]. These findings validated ZnO-NPs' toxicity to many bacterial systems, paving the way for biomedical and antimicrobial applications. It has been demonstrated that ZnO-NPs are antibacterial against a variety of bacteria, Including *B. subtilis*, *E. coli*, *P. flourescens*, *S. aureus*, and *S. typhimurium* as well as fungus including *A. flavus* and *A. fumigates* [147]. According to the authors of the reference [148], exposure to ZnO-NPs results in bacterial morphological abnormalities and ultimately mortality for food-borne and water-borne diseases such *E. coli*, *C. jejuni*, and *V. cholera*. Although there is little research using environmental strains, there is a lot of data concerning the environmental influence of ZnO-NPs [149].

It also has been proven that NPs have an antimicrobial effect against the helpful soil bacterium *P. putida*. ZnO-NPs were bacteriostatic, but CuO and AgO-NPs were largely bactericidal [150]. An intriguing set of findings were produced by *P. chlororaphis*, a member of the same genus. These plant-growth-promoting rhizobacteria produced the phytohormones more quickly thanks to CuO-NPs than ZnO-NPs [151]. It appears that ZnO-NPs have a species-specific impact. For instance, *P. putida* is bacteriostatically affected by these NPs [150]. These intricate mechanisms must surely be further investigated because both species are gram-negative rods with the same general cell layout. ZnO NPs have been widely used in environmental cleanup and as an antibacterial agent [152].

In an alternative study, the effects of ZnO NPs on the marine alga *Chlorella vulgaris* were studied by the authors of the reference [153], who discovered that cell viability was inversely correlated with nano-ZnO concentration and exposure time. The authors of the reference [154] investigated the capability of ZnO-NPs to eradicate clinical isolates of *B. subtilis*, *E. coli*, *K. pneumoniae*, *P. aeuginosa*, *S. typhi*, and S. *aureus* clinical isolates of *B. subtilis*, *E. coli*, *K. pneumoniae*, *P. aeruginosa*, *S. typhi*, and *S. aureus* [139]. ZnO NPs were found to have a greater impact than TiO<sup>2</sup> NPs at the same exposure dose, as shown by decreased DNA content and more pronounced changes in the genetic makeup of the bacterial population (Figure 5) [155,156]. *Coatings* **2023**, *13*, x FOR PEER REVIEW 16 of 25

**Figure 5.** How manufactured nano‐objects are deteriorating the soil microbiome. **Figure 5.** How manufactured nano-objects are deteriorating the soil microbiome.

#### *5.4. Titanium Oxide 5.4. Titanium Oxide*

There are many applications for TiO2‐NPs, the bulk of which are commercial [157]. TiO2 can potentially be employed in agriculture [158]. In soil PEC value of TiO2 NPs has been determined to be 1.01 to 4.45 g per kilogram, in soil that is treated from wastes PEC value of these NPs was determined as 70.6–310 g∙kg−<sup>1</sup> while in sewage sludge its PEC value is 100–433 mg∙kg−<sup>1</sup> [39,117] and the most hazardous factor of titanium dioxide na‐ noparticles for soil microorganisms is sewage sludge. Importantly, even the 1000mg∙kg**−**1concentrations claimed in several papers are not found in soils exposed toTiO2 NPs polluted sewage sludge [157]. There are many applications for TiO2-NPs, the bulk of which are commercial [157]. TiO<sup>2</sup> can potentially be employed in agriculture [158]. In soil PEC value of TiO<sup>2</sup> NPs has been determined to be 1.01 to 4.45 g per kilogram, in soil that is treated from wastes PEC value of these NPs was determined as 70.6–310 g·kg−<sup>1</sup> while in sewage sludge its PEC value is 100–433 mg·kg−<sup>1</sup> [39,117] and the most hazardous factor of titanium dioxide nanoparticles for soil microorganisms is sewage sludge. Importantly, even the 1000 mg·kg−<sup>1</sup> concentrations claimed in several papers are not found in soils exposed to TiO<sup>2</sup> NPs polluted sewage sludge [157].

#### *5.5. Cerium Oxide 5.5. Cerium Oxide*

*5.6. Silica*

ng∙kg−1and 10 to 530 ng∙kg−1[137].

Cerium (Ce) is frequently utilized as a catalyst and a fuel additive as CeO2‐NPs in optics [159] which has increased nanoscale form manufacturing levels in the worldwide market level up to 1000 NT∙yr**−**1. In terms of weight cerium makes up about 0.0046% of the planet earth [160], Ce can share its electrons in three ways (1) gaining two electrons (2) gaining three electrons (3) gaining four electrons. The PEC of CeO2‐NPs is unknown; the Cerium (Ce) is frequently utilized as a catalyst and a fuel additive as CeO2-NPs in optics [159] which has increased nanoscale form manufacturing levels in the worldwide market level up to 1000 NT·yr−<sup>1</sup> . In terms of weight cerium makes up about 0.0046% of the planet earth [160], Ce can share its electrons in three ways (1) gaining two electrons

PECs of uncultivated and agricultural soils in Denmark is estimated to be 24 to 1500

Silica nanoparticles (SiNPs) are crystalline solids [143] where farming and trading items are the most common applications [15]. For instance, hollow porous sand nano‐ particles are responsible as agents for the managed release of neurotoxins or medicines while also shielding them from UV light [161]. The annual output of Si‐NPs was around (2) gaining three electrons (3) gaining four electrons. The PEC of CeO2-NPs is unknown; the PECs of uncultivated and agricultural soils in Denmark is estimated to be 24 to 1500 ng·kg−<sup>1</sup> and 10 to 530 ng·kg−<sup>1</sup> [137].

#### *5.6. Silica*

Silica nanoparticles (SiNPs) are crystalline solids [143] where farming and trading items are the most common applications [15]. For instance, hollow porous sand nanoparticles are responsible as agents for the managed release of neurotoxins or medicines while also shielding them from UV light [161]. The annual output of Si-NPs was around 93,300 tons in 2016, which was the third highest production after nTiO<sup>2</sup> and nFeOx and rose yearly [162]. The PEC value of sand nanoparticles in different types of soils may range from 86 to 150,000 g·kg−<sup>1</sup> [28]. Negative impacts on soil organic communities have not been fully recognized.

### *5.7. Quantum Dots*

The prospective applications of semiconductor quantum dots (QDs) in electronics, solar cells, and healthcare have generated a lot of attention. These luminescent nanocrystals range in size depending on the materials employed and the synthesis process, but most are between 1 and 10 nanometers in size [70]. Global QD production peaked at around 135 tones in 2012 and has steadily climbed since then [14]. In nanosafety research, QDs are utilized as fluorescent indicators at the laboratory scale [140]. They are not yet used as nano-agrochemicals. Less is known about their possible negative effect on human health and the environment. The PEC value for QDs in soil in Europe was determined lower and that was about 9 pg·kg−<sup>1</sup> [28], nevertheless, due to a lack of data on the QD production ratio, published exposure model findings are currently quite erroneous.

#### **6. Carbon Nanotubs (CNTs)**

CNTs are a cylindrical nanostructure and allotrope of carbon. Single-wall carbon nanotubes (SWCNTs) and multi-wall carbon nanotubes (MWCNTs) are the two most popular forms of CNTs [163]. CNTs used in the electronic industry and have other applications too [158]. The worldwide requirement for carbon nanotubes is estimated to be over US\$ 4.5 billion in 2026, with a rise of more than US\$ 10–15 billion [14]. CNTs could be used as a nano-agrochemical to stop viral multiplication and spread, for example [14]. However, nanosafety issues continue to limit their use in agriculture. The soil PEC value in Europe is 35 ng·kg−<sup>1</sup> in urban and natural soil and 12 g per kg in soils treated from wastes projected for CNTs [26]. It is also critical to understand their behavior and potential environmental implications. CNTs may behave differently in natural environments depending on their length, diameter, functionalization, and environmental conditions [164].

CNTs' impact on the microbiological activity of soil is debatable and has gotten little study. However, the majority of these studies seem to indicate that CNTs lessen soil microbial activity [165–167]. Both MWCNTs and SWCNTs prevented soil bacteria from producing enzymes at 500 mg·kg−<sup>1</sup> , and MWCNTs decreased the enzyme production of two natural soils [165]. Similarly, the authors of the reference [166] discovered that SWCNTs at concentrations of 30 to 300 mg·kg−<sup>1</sup> dramatically reduced enzyme activity in a natural sandy loam soil. Another study found that after 3 days, SWCNTs had a substantial effect on the bacterial soil community, but after 14 days, the bacteria had entirely recovered [168,169]. The authors of the reference [162] discovered that MWCNTs at low concentrations (0.2 mg·kg−<sup>1</sup> ) promoted microorganisms to mineralize an agricultural soil. The authors of the reference [170] used long-term dry soil research to examine the effects of MWCNTs in contrast to natural or manufactured carbonaceous materials on soil microbial populations. After one year of exposure, they discovered that MWCNTs reduced soil DNA diversity and altered bacterial populations. These results are equivalent to those of carbonaceous materials, both man-made and natural. As of now, there aren't enough studies to determine if functionalized and unfunctionalized CNTs affect soil microbial

activity differently [170]. There have been few investigations on the effects of CNTs on soil microorganisms to date. They were all interested in earthworms that were uncovered in the soil [170–172].

#### **7. Nanoplastics**

Nanoplastics, which are closely connected to microplastics, are an increasing pollutant of concern. Microplastics are most commonly found in macroplastic objects that are mistakenly discharged into the environment and breakdown into secondary microplastics [86,173]. Due to the analytical equipment's resolution limits in identifying and quantifying these materials, the majority of research has focused on microplastics with particles larger than 10 micrometrers [147]. As a result, there aren't many articles that examine the relationship between earthworm species and nanoplastics. In particular, the inclusion of traceable materials like fluorescently labeled polystyrene (PS) beads enables higher-resolution identification using fluorescence microscopy [28]. The mobility and bioavailability of metallic MNOs in the soil is influenced by the material's physico-chemical characteristics and a number of soil variables, the most important of which are pH and organic matter. Other soil parameters are pore size, texture and other surface properties of soil are also important [28].

#### **8. Nano-Agrochemicals/Nanopesticides**

These are active compounds that have been improved by nanotechnology and nanoformulation. Nanopesticides, nanofertilizers, and nanosensors are all examples of nanoenabled agrochemical [127]. Nanoparticles can interface with the soil community when they are applied to soils, plants or used for coating seeds potentially influencing soil microbiome and soil fertility. The microorganisms that dwell in the soil make up the soil microbiota. Cycles of C, N, P, S, and other elements are significantly influenced by microbiota, as well as soil formation, pollution, degradation, and water and microbial modification of rock is also affected by microorganisms. Soil organisms are vital to the ecosystem services provided by the agricultural landscape, including pest control, biodiversity, soil structure, and nutrient cycling. Significantly, for the cultivation of land, soil offers a collection of microbes out of which plants select a community of microbes to support its production and wellbeing. As a result, the plant responds more quickly to stress, whether it be biotic (pathogens) or abiotic (drought, floods, chemical toxins) (plant pathogens). The plant microbiome and the gut microbiome in humans are frequently compared. The second genome of the plant and its potential for agriculture are believed to reside in the soil microbiome. Some soil microbiome can naturally help to prevent plant illnesses by acting as pathogen suppressors. The creation of smart nano-agrochemicals that combine efficacy and eco-compatibility while protecting soil microbial diversity depends on an understanding of how nanopesticides interact with soil and plant microbiome [21].

#### **9. Conclusions**

It is undeniable that scientific breakthroughs in nanotechnology have become extremely important. However, it has had some negative consequences for the ecosystem. The abundant production of nano-goods, as well as their discharge and permanence within the soil ecosystem, have damaged beneficial microorganisms and soil composites. Positive interactions between soil, plant, and bacteria are prevented by surface charges, area, size, and responsiveness. Some of them, for instance, cling to or enter microbial cells and cause significant harm. Our comprehensive review of the literature reveals that MNOs may have adverse, beneficial, or even neutral impacts on soil microbiota. Extensive literature assessment indicates that MNOs may have negative, neutral, or even positive effects on soil microbiota. To maintain ecosystem functioning and resilience, the study of interactions between MNOs and these vital root microbial symbioses must be increased. Procedures for safe disposal in soil agro ecosystems are widespread and safe, and they should be devised to avoid contact with soil microflora given the structural and functional toxicity of NPs to the environment. To put it another way, the items should be tailored to their intended

use. Many illusive outcomes could be established to open new roads in this discipline by undertaking microcosm study based on this. These studies are critical for a thorough understanding of this topic and the protection of the ecosystem.

**Author Contributions:** Conceptualization, B.M., A.H., S.A.H.N., and A.u.R.; methodology, B.M. and A.H.; validation, S.A.H.N. and M.M.; formal analysis, M.A.S.; investigation, B.M. and A.H.; resources, A.H. and S.A.H.N.; data curation, B.M.; writing—original draft preparation, B.M., A.H., S.A.H.N., M.A.S., and M.M.; writing—review and editing, J.W., H.Z., M.A.K., R.P., M.F., and M.Z.H.; visualization, R.P.; supervision, S.A.H.N.; project administration, M.A.S.; funding acquisition, M.A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** Central Public-Interest Scientific Institution Basal Research Fund (No. 1610232022002) and Science and Technology Cooperation Project of Shandong and Gansu (YDZX2021098, YDZX2022162).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Authors extend their sincere thanks to Central Public-Interest Scientific Institution Basal Research Fund (No. 1610232022002) and Science and Technology Cooperation Project of Shandong and Gansu (YDZX2021098, YDZX2022162).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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