Nanoclays and mineral derivates applied to pesticide water remediation.

Although pesticides are vital in agroecosystems to control pests, their indiscriminate use generates innumerable environmental problems daily. Groundwater and surface water networks are the most affected environmental matrices. Since these water basins are mainly used to obtain water for human consumption, it is a challenge to find solutions to pesticide contamination. For these reasons, development of efficient and sustainable remedial technologies is key. Based on their unique properties including high surface area, recyclability, environmental friendliness, tunable surface chemistry and low cost, nanoclays and derived minerals emerged as effective adsorbents towards environmental remediation of pesticides. This study provides a comprehensive review of the use of nanoclays and mineral derivatives as adsorbents for pesticides in water. For this purpose, the characteristics of existing pesticides and general aspects of the relevant clays and minerals are discussed. Furthermore, the study provides insightful discussion on the potential application of nanoclays and their derivatives toward the mitigation of pesticide pollution in the environment. Finally, the outlook and future prospects on nanoclay implications and their environmental implementation are elucidated.

Titanium biogenic nanoparticles to help the growth of Trichoderma harzianum to be used in biological control.

BACKGROUND: The biogenic synthesis of metallic nanoparticles is a green alternative that reduces the toxicity of this nanomaterials and may enable a synergy between the metallic core and the biomolecules employed in the process enhancing biological activity. The aim of this study was to synthesize biogenic titanium nanoparticles using the filtrate of the fungus Trichoderma harzianum as a stabilizing agent, to obtain a potential biological activity against phytopathogens and mainly stimulate the growth of T. harzianum, enhancing its efficacy for biological control. RESULTS: The synthesis was successful and reproductive structures remained in the suspension, showing faster and larger mycelial growth compared to commercial T. harzianum and filtrate. The nanoparticles with residual T. harzianum growth showed inhibitory potential against Sclerotinia sclerotiorum mycelial growth and the formation of new resistant structures. A great chitinolytic activity of the nanoparticles was observed in comparison with T. harzianum. In regard to toxicity evaluation, an absence of cytotoxicity and a protective effect of the nanoparticles was observed through MTT and Trypan blue assay. No genotoxicity was observed on V79-4 and 3T3 cell lines while HaCat showed higher sensitivity. Microorganisms of agricultural importance were not affected by the exposure to the nanoparticles, however a decrease in the number of nitrogen cycling bacteria was observed. In regard to phytotoxicity, the nanoparticles did not cause morphological and biochemical changes on soybean plants. CONCLUSION: The production of biogenic nanoparticles was an essential factor in stimulating or maintaining structures that are important for biological control, showing that this may be an essential strategy to stimulate the growth of biocontrol organisms to promote more sustainable agriculture.

Encapsulated plant growth regulators and associative microorganisms: Nature-based solutions to mitigate the effects of climate change on plants.

Over the past decades, the atmospheric CO2 concentration and global average temperature have been increasing, and this trend is projected to soon become more severe. This scenario of climate change intensifies abiotic stress factors (such as drought, flooding, salinity, and ultraviolet radiation) that threaten forest and associated ecosystems as well as crop production. These factors can negatively affect plant growth and development with a consequent reduction in plant biomass accumulation and yield, in addition to increasing plant susceptibility to biotic stresses. Recently, biostimulants have become a hotspot as an effective and sustainable alternative to alleviate the negative effects of stresses on plants. However, the majority of biostimulants have poor stability under environmental conditions, which leads to premature degradation, shortening their biological activity. To solve these bottlenecks, micro- and nano-based formulations containing biostimulant molecules and/or microorganisms are gaining attention, as they demonstrate several advantages over their conventional formulations. In this review, we focus on the encapsulation of plant growth regulators and plant associative microorganisms as a strategy to boost their application for plant protection against abiotic stresses. We also address the potential limitations and challenges faced for the implementation of this technology, as well as possibilities regarding future research.

Development and biological evaluation of nanoencapsulated-based pyrethroids with synergists for resistance management of two soybean pests: insights for new insecticide formulations.

BACKGROUND: Chemical control is commonly used against Euschistus heros (F.) and Chrysodeixis includens (Walker) in soybean fields in South America. However, previous studies reported that these pests have reduced susceptibility to pyrethroids in Brazil. On this basis, we developed and evaluated nanoencapsulated-based bifenthrin (BFT) and λ-cyhalothrin (LAM) with the synergists piperonyl butoxide (PBO) and diethyl maleate (DEM) for insect resistance management (IRM). RESULTS: Nanoformulations of BFT and LAM with PBO and DEM presented good physical-chemical characteristics and were stable. The spherical morphology of all systems and the encapsulation efficiency in nanostructured lipid carriers did not change when synergists were added. Nanoencapsulated BFT with DEM applied topically increased the susceptibility of E. heros to BFT by 3.50-fold. Similarly, nanoencapsulated BFT and LAM with PBO in diet-overlay bioassays increased the susceptibility of C. includens to both chemicals by up to 2.16-fold. Nanoencapsulated BFT and LAM with synergists also improve control efficacy of both species, causing higher mortality than commercial products containing these chemistries. CONCLUSIONS: It is possible to develop nanoencapsulated-based formulations of BFT and LAM with PBO or DEM, and these nanoformulations have the potential to improve control of E. heros and C. includens with recognized low susceptibility to pyrethroids. This study provides updates for designing new insecticide formulations for IRM. © 2022 Society of Chemical Industry.

Combined in-silico and in-vitro experiments support acid-base equilibrium as a tool to estimate the localization depth of 4-nitrophenol within a phospholipid bilayer.

The interaction and location of 4-nitrophenol (PNP) in biomembranes are relevant in the bioaccumulation and potentiation of the intensive toxic effects of this persistent organic pollutant. In this work, in-silico analyses predicted that, in a fluid phospholipid bilayer, the minimum energy of the protonated (PNPH) and deprotonated (PNP-) species is located within the glycerol and choline region, respectively. This was experimentally confirmed by acid-base equilibrium experiments and theory, allowing the estimation of the mean location of PNP within a bilayer region with a dielectric constant D = 50.6 compatible with the phosphate/choline moiety of egg-yolk phosphatidylcholine unilamellar (EPC) vesicles. The comparison with the D = 43.2 value obtained in Triton X-100 micelles allow predicting a mean surface potential of ψ = 25.37 mV for the EPC-water interface. Changes in the chemical shifts and longitudinal relaxation times of EPC hydrogens by 1H NMR confirm the deeper location of the PNPH within the glycerol region and at the choline region (PNP-) at higher pH. Intermolecular PNP-EPC dipolar interactions within the choline region was also demonstrated at pH 10.2 using ROESY experiments. Additional information was obtained trough 31P NMR, that detected an increase in the anisotropy at the membrane interface after insertion of PNP which probably act as a bridge between choline moieties rigidizing the crystalline structure at that spot. Concluding, here we provide experimental support to the "pH-piston hypothesis" proposed some decades ago in the pharmaceutical field, and that reinforce the importance of the environmental conditions (e.g. pH) to modulate the bioavailability of this highly toxic pollutant.

Fluorescent labeling as a strategy to evaluate uptake and transport of polymeric nanoparticles in plants.

The use of biodegradable nanopolymers in agriculture offers an excellent alternative for the efficient delivery of agrochemicals that promote plant protection and development. However, tracking of these systems inside plants requires complex probe tagging strategies. In addition to providing a basis for better understanding such nanostructures to optimize delivery system design, these probes allow monitoring the migration of nanoparticles through plant tissues, and determine accumulation sites. Thus, these probes are powerful tools that can be used to quantify and visualize nanoparticle accumulation in plant cells and tissues. This review is an overview of the methods involved in labeling nanocarriers, mainly based on polymeric matrices, for the delivery of nanoagrochemicals and the recent advances in this field.

Using Chitosan-Coated Polymeric Nanoparticles-Thermosensitive Hydrogels in association with Limonene as Skin Drug Delivery Strategy.

Topical delivery of local anesthetics (LAs) is commonly used to decrease painful sensations, block pain throughout procedures, and alleviate pain after surgery. Dermal and/or transdermal delivery of LAs has other advantages, such as sustained drug delivery and decreased systemic adverse effects. This study reports the development of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles coated with chitosan for the sustained release and topicality of benzocaine (BZC) and topical delivery. BZC PLGA nanoparticles or nonencapsulated drugs were further incorporated into Poloxamer hydrogels (Pluronic™ F-127). The nanoparticles showed a mean diameter of 380 ± 4 nm, positive zeta potential after coating with chitosan (23.3 ± 1.7 mV), and high encapsulation efficiency (96.7 ± 0.02%). Cellular viability greater than 70% for both fibroblasts and keratinocytes was observed after treatment with nanoparticles, which is in accordance with the preconized guidelines for biomedical devices and delivery systems. Both the nanoparticles and hydrogels were able to modulate BZC delivery and increase drug permeation when compared to the nonencapsulated drug. Furthermore, the incorporation of limonene into hydrogels containing BZC-loaded nanoparticles increased the BZC permeation rates. Non-Newtonian and pseudoplastic behaviors were observed for all hydrogel nanoformulations with or without nanoparticles. These results demonstrate that the hydrogel-nanoparticle hybrids could be a promising delivery system for prolonged local anesthetic therapy.

Ecotoxicity evaluation of polymeric nanoparticles loaded with ascorbic acid for fish nutrition in aquaculture.

BACKGROUND: Ascorbic acid (AA) is a micronutrient essential for the mechanisms of reproduction, growth, and defense in fish. However, the biosynthesis of this micronutrient does not occur in fish, so it must be supplied with food. A difficulty is that plain AA is unstable, due to the effects of light, high temperature, and oxygen, among others. The use of nanoencapsulation may provide protection and preserve the physicochemical characteristics of AA for extended periods of time, decreasing losses due to environmental factors. METHOD: This study evaluated the protective effect of nanoencapsulation in polymeric nanoparticles (chitosan and polycaprolactone) against AA degradation. Evaluation was made of the physicochemical stability of the nanoformulations over time, as well as the toxicological effects in zebrafish (Danio rerio), considering behavior, development, and enzymatic activity. For the statistical tests, ANOVA (two-way, significance of p < 0.05) was used. RESULTS: Both nanoparticle formulations showed high encapsulation efficiency and good physicochemical stability during 90 days. Chitosan (CS) and polycaprolactone (PCL) nanoparticles loaded with AA had mean diameters of 314 and 303 nm and polydispersity indexes of 0.36 and 0.28, respectively. Both nanosystems provided protection against degradation of AA exposed to an oxidizing agent, compared to plain AA. Total degradation of AA was observed after 7, 20, and 480 min for plain AA, the CS nanoparticle formulation, and the PCL nanoparticle formulation, respectively. For zebrafish larvae, the LC50 values were 330.7, 57.4, and 179.6 mg/L for plain AA, the CS nanoparticle formulation, and the PCL nanoparticle formulation, respectively. In toxicity assays using AA at a concentration of 50 mg/L, both types of nanoparticles loaded with AA showed lower toxicity towards the development of the zebrafish, compared to plain AA at the same concentration. Although decreased activity of the enzyme acetylcholinesterase (AChE) did not affect the swimming behavior of zebrafish larvae in the groups evaluated, it may have been associated with the observed morphometric changes, such as curvature of the tail. CONCLUSIONS: This study showed that the use of nanosystems is promising for fish nutritional supplementation in aquaculture. In particular, PCL nanoparticles loaded with AA seemed to be most promising, due to higher protection against AA degradation, as well as lower toxicity to zebrafish, compared to the chitosan nanoparticles. The use of nanotechnology opens new perspectives for aquaculture, enabling the reduction of feed nutrient losses, leading to faster fish growth and improved sustainability of this activity.

Influence of the capping of biogenic silver nanoparticles on their toxicity and mechanism of action towards Sclerotinia sclerotiorum.

BACKGROUND: Biogenic nanoparticles possess a capping of biomolecules derived from the organism employed in the synthesis, which contributes to their stability and biological activity. These nanoparticles have been highlighted for the control of phytopathogens, so there is a need to understand their composition, mechanisms of action, and toxicity. This study aimed to investigate the importance of the capping and compare the effects of capped and uncapped biogenic silver nanoparticles synthesized using the filtrate of Trichoderma harzianum against the phytopathogenic fungus Sclerotinia sclerotiorum. Capping removal, investigation of the composition of the capping and physico-chemical characterization of the capped and uncapped nanoparticles were performed. The effects of the nanoparticles on S. sclerotiorum were evaluated in vitro. Cytotoxicity and genotoxicity of the nanoparticles on different cell lines and its effects on nontarget microorganisms were also investigated. RESULTS: The capped and uncapped nanoparticles showed spherical morphology, with greater diameter of the uncapped ones. Functional groups of biomolecules, protein bands and the hydrolytic enzymes NAGase, ß-1,3-glucanase, chitinase and acid protease from T. harzianum were detected in the capping. The capped nanoparticles showed great inhibitory potential against S. sclerotiorum, while the uncapped nanoparticles were ineffective. There was no difference in cytotoxicity comparing capped and uncapped nanoparticles, however higher genotoxicity of the uncapped nanoparticles was observed towards the cell lines. Regarding the effects on nontarget microorganisms, in the minimal inhibitory concentration assay only the capped nanoparticles inhibited microorganisms of agricultural importance, while in the molecular analysis of the soil microbiota there were major changes in the soils exposed to the uncapped nanoparticles. CONCLUSIONS: The results suggest that the capping played an important role in controlling nanoparticle size and contributed to the biological activity of the nanoparticles against S. sclerotiorum. This study opens perspectives for investigations concerning the application of these nanoparticles for the control of phytopathogens.

Ecotoxicological and regulatory aspects of environmental sustainability of nanopesticides.

Recent years have seen the development of various colloidal formulations of pesticides and other agrochemicals aimed at use in sustainable agriculture. These formulations include inorganic, organic or hybrid particulates, or nanocarriers composed of biodegradable polymers, that can provide a better control of the release of active ingredients. The very small particle sizes and high surface areas of nanopesticides may however also lead to some unintended (eco)toxicological effects due to the way in which they interact with the target and non-target species and the environment. The current level of knowledge on ecotoxicological effects of nanopesticides is scarce, especially in regard to the fate and behaviour of such formulations in the environment. Nanopesticides will however have to cross a stringent regulatory scrutiny before marketing in most countries for health and environmental risks under a range of regulatory frameworks that require pre-market notification, risk assessment and approval, followed by labelling, post-market monitoring and surveillance. This review provides an overview of the key regulatory and ecotoxicological aspects relating to nanopesticides that will need to be considered for environmentally-sustainable use in agriculture.

Fabrication and Characterization of a Novel Herbicide Delivery System with Magnetic Collectability and Its Phytotoxic Effect on Photosystem II of Aquatic Macrophyte.

The use of nano- and microparticles as a release system for agrochemicals has been increasing in agricultural sector. However, the production of eco-friendly and smart carriers that can be easily handled in the environment is still a challenge for this technology. In this context, we have developed a biodegradable release system for the herbicide atrazine with magnetic properties. Herein, we investigated the (a) physicochemical properties of the atrazine-loaded magnetic poly(ε-caprolactone) microparticles (MPs:ATZ), (b) in vitro release kinetic profile of the herbicide, and (c) phytotoxicity toward photosynthesis in the aquatic fern Azolla caroliniana. The encapsulation efficiency of the herbicide in the MPs:ATZ was ca. 69%, yielding spherical microparticles with a diameter of ca. 100 µm, a sustained-release profile, and easily manipulated with an external magnetic field. Also, phytotoxicity issues showed that the MPs:ATZ maintained their herbicidal activity via inhibition of PSII, showing lower toxicity compared with the nonencapsulated ATZ at 0.01 and 0.02 µmol·L-1. Therefore, this technology may conveniently promote a novel magnetic controlled release of the herbicide ATZ (with the potential to be collected from a watercourse) and act as a nutrient boost to the nontarget plant, with good herbicidal activity and reduced risk to the environment.

Zein Nanoparticles Impregnated with Eugenol and Garlic Essential Oils for Treating Fish Pathogens.

The supply of food derived from aquaculture has increased significantly in recent years. The aim of this industrial sector is to produce sustainable products to meet the needs of consumers, providing food security and nutritional benefits. The development of aquaculture has faced challenges including disease outbreaks that can cause substantial economic losses. These diseases can be controlled using chemicals such as antibiotics. However, the indiscriminate use of these substances can have major negative impacts on human health and the environment with the additional risk of the emergence of resistant organisms. The present manuscript describes the use of phytotherapy in association with nanotechnology in order to obtain a more effective and less harmful system for the control of bacterial diseases in fish. Zein nanoparticles associated with eugenol and garlic essential oil were prepared through antisolvent precipitation and characterized. Zein nanoparticles are promising carrier systems as zein proteins are biodegradable and biocompatible and, in this way, good candidates for encapsulation of active ingredients. The system presented good physicochemical properties with an average particle diameter of approximately 150 nm, a polydispersity index lower than 0.2, and a zeta potential of approximately 30 mV. High encapsulation efficiency was obtained for the active compounds with values higher than 90%, and the compounds were protected against degradation during storage (90 days). The nanoparticle formulations containing the botanical compounds also showed less toxicity in the tests performed with a biomarker (Artemia salina). In addition, the systems showed bactericidal activity against the important fish pathogenic bacteria Aeromonas hydrophila, Edwardsiella tarda, and Streptococcus iniae in vitro. The present study opens new perspectives for the use of botanical compounds in combination with nanotechnology to treat fish diseases caused by bacteria, contributing to a more sustainable fish chain production.

Poloxamer micellar system for intra-articular injection of 15-deoxy-Δ12,14-prostaglandin J2 with improved bioavailability and anti-inflammatory properties in the temporomandibular joint of rats.

Painful conditions of the temporomandibular joint (TMJ) are challenging to manage and most attempts often result in unsatisfactory outcomes. In such context, nanocarrier systems, such as polymeric micelles, have been showing encouraging results in solving therapeutic limitations. Poloxamers are widely used, especially PL 407, because of their high biocompatibility and approval by the Food and Drug Administration (FDA) for clinical use. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has shown important antinociceptive and anti-inflammatory activity. The present study evaluated the efficacy and viability of the micellar system of PL-15dPGJ2 in a formalin-induced acute pain model in the temporomandibular joint of rats. The PL-15dPGJ2 was prepared and characterized. The animals were pretreated with an intra-articular injection of PL-15dPGJ2 followed by the formalin challenge. The nociceptive response was evaluated at different time-periods and the periarticular tissue and articular wash were collected for analysis. We found that intra-articular injection of PL-15d-PGJ2 produced pain relief at lower concentrations and in a sustained manner compared with free 15d-PGJ2. Moreover, a strong anti-inflammatory effect was observed with decreased levels of key pro-inflammatory cytokines and modulation of the leukocyte migration process. Our findings suggest that 15d-PGJ2 combined with a poloxamer micellar system provided clinical relevance in terms of bioavailability, long-lasting effect, and safe dosage. The formulation investigated herein is a promising micellar carrier system for managing pain conditions of the TMJ.

Localization of Coated Iron Oxide (Fe3O4) Nanoparticles on Tomato Seeds and Their Effects on Growth.

Food demand due to the growing global population has been stretching the agriculture sector to the limit. This demands the cultivation of plants in shrinking land areas which makes the search for highly effective systems for plant nutrition and pest control important. In this context, the application of nanoparticles (NPs) in agriculture can have a transformative effect on food production techniques as it can enable the delivery of bioactive agents (including growth factors, pesticides, and fungicides) directly to plants. Herein, we report the application of unfunctionalized as well as amine-functionalized and polycaprolactone-coated Fe3O4 NPs to seed treatment in tomato (Solanum lycopersicum). The study reveals that the treatment has no side effects on plant germination and development. Furthermore, the translocation of NPs in seeds and seedlings posttreatment depends on the surface functionalization of the NPs. X-ray fluorescence spectroscopy analysis of seedlings suggested that around 66% of unfunctionalized Fe3O4 NPs were translocated in the cotyledons, while only 50% of functionalized NPs (both amine and polycaprolactone) were translocated. Our results demonstrate that all particles were taken up by the seeds, thus suggesting that the functionalized NPs can act as a versatile platform for delivering of active compounds, such as fungicides and growth factor agents.

Atrazine nanoencapsulation improves pre-emergence herbicidal activity against Bidens pilosa without enhancing long-term residual effect on Glycine max.

BACKGROUND: Poly(ϵ-caprolactone) nanocapsules (NC + ATZ) are an efficient carrier system for atrazine and were developed as an alternative to reduce the harmful environmental effects of this herbicide. Here, we analyzed the pre-emergence herbicidal activity of NC + ATZ against Bidens pilosa and evaluated its residual effect on soybean plants after different periods of soil treatment with the formulations. RESULTS: In contrast to non-nanoatrazine, NC + ATZ treatment led to very high mortality rates of B. pilosa seedlings even after a tenfold dilution, which suggests that atrazine nanoencapsulation improved its pre-emergence herbicidal activity. In a short-term assay (17 days), soil treatment with all atrazine-containing formulations resulted in intense toxicity to soybean plants. NC + ATZ at 200 g ha-1 had the same inhibitory effects on the physiological and growth parameters of soybean plants compared with non-nanoatrazine at 2000 g ha-1 , which suggests that atrazine nanoencapsulation increased the short-term residual effect of the herbicide. In a long-term assay (60 days), a gradual recovery of soybean plants from atrazine phytotoxicity was observed. When comparing the effects of nano- and non-nanoatrazine at the same concentrations, the growth and physiological parameters of soybean plants were mainly affected to the same extent. This indicates that encapsulation of atrazine into poly(ϵ-caprolactone) nanocapsules did not enhance the long-term residual effect of the herbicide on soybean. CONCLUSION: NC + ATZ could be applied for efficient weed control without additional phytotoxicity to susceptible crops compared with non-nanoatrazine, provided that a safe interval is respected from atrazine application to sowing. © 2019 Society of Chemical Industry.

Interference of goethite in the effects of glyphosate and Roundup® on ZFL cell line.

Goethite (α-FeOOH) brings important perspectives in environmental remediation, as, due to its physicochemical properties, this iron oxide can adsorb a wide variety of compounds, including glyphosate. This study aimed to evaluate the effects of goethite nanoparticles (NPs), glyphosate (Gly), Roundup® (Rd), and co-exposures (Gly + NPs and Rd + NPs) on zebrafish liver cell line (ZFL). ZFL cells were exposed to NPs (1, 10, and 100 mg L-1), Gly (3.6 mg L-1), Rd (10 mg L-1), and co-exposures (Gly + NPs and Rd + NPs), or only to saline for 1, 6, and 12 h. Cell viability was assessed by Trypan blue, MTT, and neutral red assays. The generation of reactive oxygen species and total antioxidant capacity were also determined, while genotoxicity was quantified by the comet assay. Both NPs and Rd in isolation produced cytotoxic effects at 6 h and genotoxic effects at 1 and 6 h. Rd + NPs resulted in synergistic effects, intensifying the toxicity. Cells exposed to Gly did not present toxic effects and Gly + NPs resulted in the suppression of toxic effects observed for NPs. The presence of other components in Roundup® seems to favor its toxicity compared to the active ingredient. In conclusion, according to the in vitro model, the concentrations used were not safe for the ZFL lineage.

Nanopesticide based on botanical insecticide pyrethrum and its potential effects on honeybees.

Nanotechnology has the potential to overcome the challenges of sustainable agriculture, and nanopesticides can control agricultural pests and increase farm productivity with little environmental impact. However, it is important to evaluate their toxicity on non-target organisms, such as honeybees (Apis mellifera) that forage on crops. The aims of this study were to develop a nanopesticide that was based on solid lipid nanoparticles (SLNs) loaded with pyrethrum extract (PYR) and evaluate its physicochemical properties and short-term toxicity on a non-target organism (honeybee). SLN + PYR was physicochemically stable after 120 days. SLN + PYR had a final diameter of 260.8 ± 3.7 nm and a polydispersion index of 0.15 ± 0.02 nm, in comparison with SLN alone that had a diameter of 406.7 ± 6.7 nm and a polydispersion index of 0.39 ± 0.12 nm. SLN + PYR had an encapsulation efficiency of 99%. The survival analysis of honeybees indicated that PYR10ng presented shorter longevity than those in the control group (P ≤ 0.01). Empty nanoparticles and PYR10ng caused morphological alterations in the bees' midguts, whereas pyrethrum-loaded nanoparticles had no significant effect on digestive cells, so are considered safer, at least in the short term, for honeybees. These results are important in understanding the effects of nanopesticides on beneficial insects and may decrease the environmental impacts of pesticides.

Re-addressing the biosafety issues of plant growth promoting rhizobacteria.

To promote agronomic sustainability, extensive research is being carried out globally, investigating biofertilizer development. Recently, it has been realized that some microorganisms used as biofertilizers behave as opportunistic pathogens and belong to the biosafety level 2 (BSL-2) classification. This poses serious risk to the environmental and human health. Evidence presented in various scientific forums is increasingly favoring the merits of using BSL-2 microorganisms as biofertilizers. In this review, we emphasize that partial characterization based on traditional microbiological approaches and small subunit rRNA gene sequences/conserved regions are insufficient for the characterization of biofertilizer strains. It is advised herein, that research and industrial laboratories developing biofertilizers for commercialization or environmental release must characterize microorganisms of interest using a multilateral polyphasic approach of microbial systematics. This will determine their risk group and biosafety characteristics before proceeding with formulation development and environmental application. It has also been suggested that microorganisms belonging to risk-group-1 and BSL-1 category should be used for formulation development and for field scale applications. While, BSL-2 microorganisms should be restricted for research using containment practices compliant with strict regulations.