Plant diseases caused by pathogenic microorganisms adversely affect the growth and yield of crops, which consequently leads to losses in food production (Peng et al., 2021). Recently, Van Dijk et al. (2021) presented a set of projections on the growth of food demand between 2010 and 2050 and the risks of food shortage and hunger. The majority of food safety research focuses on common denominators such as climate change, resistant pathogens, and the need for sustainable agriculture while addressing human health and environmental concerns (Zakirov et al., 2021). The use of technological advances, including mechanization, pesticides or fertilizers, allowed to achieve higher productivity of crop plants (Jacquet et al., 2022). It is estimated that 70%–80% of plant diseases are caused by fungal pathogens particularly by secretion of toxins that cause various physiological disorders, growth inhibition, chlorosis, wilting, etc. (Peng et al., 2021). Maize belongs to the most frequent crop plants worldwide, including Poland (Król et al., 2018), and is particularly vulnerable to pathogenic fungi, e.g., Fusarium spp. (Czembor et al., 2015). In addition, microbial diseases lead to yield losses, while mycotoxins produced by fungi contaminate and reduce the quality of maize grains (Czembor et al., 2015). A number of synthetic chemical pesticides are required to prevent crop diseases. However, some negative aspects of pesticides on the environment and human health have been unequivocally documented, therefore limiting their use is of high priority (Jacquet et al., 2022). A number of studies have been conducted to design the remedy for addressing this excessive and seemingly irreplaceable application of traditional chemicals which showed that a prospective solution may be the use of products at the nanoscale (Pestovsky and Martinez-Antonio, 2017). Metal-based nanoparticles (MNPs) show promising potential for use in agriculture field as they demonstrate broad range of antibacterial and antifungal activities (Singh et al., 2021). The use of bio-nanoparticles (bio-NPs) as antimicrobial agents might empower a significant reduction in the dose used while maintaining antimicrobial efficacy compared to conventional materials. From an agro-ecological point of view, the application of nanoparticles (NPs) obtained from biological sources may mitigate the negative consequences of agriculture procedures imposed on the environment (Hazarika et al., 2022).

To date, various studies on the potential use of MNPs as fungicides have been reported (Malandrakis et al., 2022). Silver and zinc oxide nanoparticles (AgNPS and ZnONPs, respectively) display antimicrobial activity depending, in particular, on their physicochemical properties and the targeted pathogen (Gudkov et al., 2021). Some reports showed antimicrobial activity of biosynthesized AgNPs and ZnONPs against, e.g., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Xanthomonas oryzae pv. oryzae, Alternaria alternata, Pyricularia oryzae, Sclerotinia sclerotiorum, and many more (Consolo et al., 2020; El Sayed and El-Sayed, 2020; Ogunyemi et al., 2020; Pillai et al., 2020). It is suggested that the high ability of biosynthesized NPs to control microbial pathogens is due to their large surface area, small size, and high concentration of released ions or capping agents on the surface of the metallic core (Gudkov et al., 2021). In addition, zinc plays an important role in cell function and proliferation (membrane stability, metabolic processes, enzymatic activity, photosynthesis) and consequently in plant physiology (Cakmak et al., 2010; Faizan et al., 2020). It has been reported that the foliar and soil use of ZnONPs for maize biofortification resulted in higher Zn availability and uptake and affected Zn content in edible crop organs than the commonly used zinc sulfate (ZnSO₄). The higher bioavailability of zinc in nano form is attributed to its lower solubility than that of ZnSO₄ salts (Umar et al., 2021). In another study, maize and wheat supplementation with ZnONPs in lower doses (100 mg L^-1) improved seedling length and biomass production, and enzyme activity (α-amlyase, antioxidative system enzymes), which suggests potential application of ZnONPs as stimulators of crop plant growth (Srivastav et al., 2021). However, some phytotoxic effects of MNPs to plant development has been summarized by Abedi et al. (2022). This aspect is important before the evaluation of plant growth protection and/or promotion with the use of nanoparticles in the field. The diverse properties (small sizes, variable shapes, chemical nature) of MNPs determine their high reactivity and subsequently their uptake, translocation, and interactions in plants (de la Rosa et al., 2021). Some studies reported, that MNPs affect basic cellular processes by inducing oxidative stress, disrupting cell membrane transport or altering gene expression (Lee et al., 2013; Akhtar et al., 2022). Recently, Wan and coworkers (2019) found that ZnONPs (at concentration >100 mg L^-1) caused endocytosis and led to the rearrangement of microfilament in the epidermal cells of elongation zones of Arabidopsis seedlings. However, above changes were temporary, and plants after NPs-related stress recovered faster than those treated with Zn²⁺ ions. In addition, MNPs introduced into the soil environment may cause changes in soil fertility, as well as affect microorganisms and invertebrates. Wei and coworkers (2021) reported diversified effects of ZnO, Cu, and γ-Fe₂O₃ NPs on plant (Salvia miltiorrhiza) growth and soil environment. For instance, the effect of NPs on seedling aboveground biomass depended on the type of NPs and dose applied (100 and 700 mg kg^-1). CuNPs showed no effect on plant biomass production at tested concentrations. In the case of ZnONPs, their lower concentrations (100 mg kg^-1) promoted growth, while higher concentrations (700 mg kg^-1) reduced it. In turn, γ-Fe₂O₃ NPs at both tested concentrations promoted plant growth. Moreover, the increase in the relative abundance of S. miltiorrhiza rhizosphere microorganisms, namely, the plant growth-promoting bacteria (Sphingomonas), superoxide dismutase producers (Aminobacter), and the metal-tolerant bacteria (Thiobacillus and Metarhizium) after MNPs-treatment was observed (Wei et al., 2021). The above findings indicate that various nanomaterials introduced into the environment might have undesirable effects, thus there is an urgent need to properly identify and study the effects of plant exposure to nanoparticles.

The synthesis of nanoparticles covers a variety of approaches, which include physical, chemical, biological, and their combination (Rai et al., 2021). The formation and structural parameters of nanoparticles depend on the reaction conditions and type of the stabilizing agent used in the synthesis (Lallo da Silva et al., 2019). Numerous studies have shown that the ability to form nanoparticles is demonstrated by plants (Balachandar et al., 2019; Verma and Bharadvaja, 2022), bacteria (Quinteros et al., 2019; Saeed et al., 2020), actinomycetes (Wypij et al., 2021, 2022), fungi (Feroze et al., 2020), and viruses (Le et al., 2017) by using their live cultures, biomass, extracts, and metabolites. Mycosynthesis of various nanoparticles is intensively studied by many researchers (Ingle et al., 2009; Abd El-Aziz et al., 2015; Clarance et al., 2020). The benefits of fungal-mediated synthesis of nanoparticles include the simplicity of preparation and the relatively undemanding stages of the synthesis process (Ganachari et al., 2012; Zare et al., 2017). Moreover, these microorganisms seem to possess an outstanding ability to tolerate metals, and fungal metabolites are involved in the reduction of metal salts as well as the further formation of metallic nanoparticles (Kobashigawa et al., 2019; Rai et al., 2021). Further research needs to focus on optimizing the synthesis process to obtain efficient scale production and nanoproducts with the sought-after bioactivity important for their potential application (Sasani et al., 2023). The adaptation of environmental conditions to the growth of microorganisms as well as synthesis conditions assists in the efficient production of nanoparticles with well-defined morphology and biological activity (Paul and Roychoudhury, 2021; Murillo-Rábago et al., 2022).

The aim of the study was to evaluate the ability of fungal extract from F. solani IOR 825 to synthesize AgNPs and ZnONPs and to optimize the reaction parameters for high production yields and biological activity of generated nanoproducts. It is the first report on the synthesis of silver and zinc oxide nanoparticles from Fusarium solani IOR 825 strain. The novel nanoparticles were assessed for antimicrobial activities against a wide set of phytopathogenic bacteria, fungi, and oomycetes. Moreover, the potential of these AgNPs and ZnONPs for sterilization of maize (Zea mays) seeds and the effect of seeds nanopriming on their germination and seedling growth (shoot and root elongation, fresh and dry mass production) was also evaluated. This is also the first report on the potential use of nanoparticles from F. solani species in agriculture for the protection and promotion of maize growth.

Fungi play a pivotal role in synthesis of different kinds of nanoparticles. Among these, various Fusarium species have garnered attention by the researchers (Rai et al., 2021). Several researchers successfully synthesized variety of nanoparticles including silver, gold, copper, and zinc oxide NPs synthesis from different strains of F. solani (Ingle et al., 2009; Abd El-Aziz et al., 2015; Vijayan et al., 2016; Clarance et al., 2020; El Sayed and El-Sayed, 2020; Sasani et al., 2023). Nevertheless, there is no standard synthesis protocol, as there are many factors that affect the synthesis yields of final product sizes, shapes, and other physicochemical characteristics (Sonawane et al., 2022). Therefore, the present work focuses on optimization of F. solani IOR 825 growth, high-throughput synthesis process, and fabrication of biologically active nanoparticles. The main requirements for the biosynthesis of NPs are solutions of metal ions as well as reducing and coating agents (fungal origin), the initial aim was to optimize the growth of the fungus in order to acquire a large portion of biomass, with a view to its subsequent use in preparing a cell-free extract after autolysis. The SDB medium was selected and used for further culture of F. solani IOR 825, as the highest fungal growth rate was recorded when compared with PDB or CDB, which may be due to the medium content consisting of dextrose, digested animal tissues (amino acids source) and casein, which provide carbon and nitrogen sources for growth. Similarly, Merlin et al. (2013) pointed out dextrose as the preferred carbon source, and the supplementation of amino acids to the growth medium that resulted in increased growth of F. solani strain LCPANF01.

The fungal extract obtained after cell autolysis, which contains variable biomolecules, is preferably used for the synthesis of NPs along with metal precursors. This extracellular process allows for more efficient synthesis of NPs and reduction of post-production steps, such as purification (Michael et al., 2022). Similarly, El Sayed and El-Sayed (2020) used cell-free extracts from F. solani KJ 623702 and silver nitrate (AgNO₃; final concentration of 0.5 mM) or zinc sulfate (ZnSO₄; 0.005 mM), as precursors, for green synthesis of corresponding nanoparticles. A basic visual observation of changes in the color of the mixture to dark brown (AgNPs) or white (ZnONPs) indicated that the synthesis reaction had been initiated. The reduction of metal ions and nanoparticles formation were confirmed by UV-visible spectroscopy and the detection of maximum absorbance peaks at wavelengths 422 nm and 375 nm for AgNPs and ZnONPs, respectively (El Sayed and El-Sayed, 2020), which is in line with our observations. It is well known, that the maximum absorption for AgNPs and ZnONPs ranges from 420 to 450 nm and 330–380 nm, respectively (Musa et al., 2017; Anjum et al., 2023). However, the slight shifts in the UV-vis spectra are due to differences in particle size or the presence of molecules from the biological extracts used for the synthesis (Saion et al., 2013; Ballotin et al., 2016; Musa et al., 2017; Guilger-Casagrande et al., 2019; Urnukhsaikhan et al., 2021). A number of studies on the synthesis of ZnONPs by precipitation method using fungal extracts have been reported (Ghorbani et al., 2015). In our study, two factors, namely, temperature, and pH, were considered for ZnONPs synthesis, as these factors are of great importance for the morphology and size of formed NPs (Moezzi et al., 2011). The heating of the reaction mixture (fungal extract and salt precursor) improves the kinetics of the reaction and affect NPs properties (Moezzi et al., 2011). Authors reported that an increase in the temperature of the reaction from 25°C to 90 °C between addition of ZnSO₄ and NaOH led to the lower solubility of the final product, which is important for further applications. In another study, Abdullah et al. (2020) indicated that strong alkaline (pH > 10) reaction environment favors the formation of ZnONPs. It is consistent with observation from this study, the ZnONPs were formed after a temperature rise to 40°C or at room temperature after adjusting the pH to 11.

TEM analysis showed the formation of small and spherical AgNPs from extract of F. solani IOR 835 which is in line with recently published reports on the fungal synthesis of such nanoparticles (Lofty et al., 2021; Sasani et al., 2023). These authors synthesized spherical and small (7–23 and 27.5–58.3 nm) AgNPs from Aspergillus terreus and F. solani, respectively. In contrast, ZnONPs synthesized from F. solani IOR 825 were irregularly shaped and bigger (54.44–209.69 nm and 64.84–443.02 nm) than those synthesized from Trichoderma asperellum (44–78 nm), it may be related to the use of another salt precursor (Zn (NO₃)₂ (Shobha et al., 2023). Whereas, Elrefaey et al., 2022 synthesized rectangular ZnONPs with size between 23 and 140 nm using extract of marine alga Cystoseira crinite, Zn SO₄ (0.05 M aqueous solution), as a precursor, and 1 M NaOH and heating the reaction mixture up to 45°C for 30 min. Apart from the reaction parameters (precursor, temperature, pH), the physicochemical properties of the bio-NPs are determined by the composition of the fungal extract (Lofty et al., 2021). Similar results, confirming the fungal-mediated formation of AgNPs and ZnONPs with crystalline structures were reported by other researchers (Talam et al., 2012; Nallal et al., 2021; Shobha et al., 2023). Furthermore, Singh D. et al. (2014) revealed that low concentration of precursor (1 mM AgNO₃) as well as room temperature (below 25°C) allowed for efficient mycosynthesis of small AgNPs (average size 18 nm) with good stability (Zeta potential–33.4 mV) which prevented their agglomeration. Jain et al. (2020) used zinc sulfate for microbial-mediated synthesis of ZnONPs and suggested that a larger hydrodynamic diameter of biologically synthesized ZnONPs might result from aggregation of particles. The zeta potential is an indicator of surface charge potential which is an important parameter for understanding the stability of nanoparticles in aqueous suspensions. In our studies, a low value of Zeta potential (−9.39 mV) indicated lower stability of ZnONPs (2) and the tendency to aggregate therefore larger particles diameter were observed in DLS analysis. It has been stated in the literature that nanoparticles with a Zeta potential higher than +30 mV or lower than −30 mV are considered to be very stable in the dispersion medium by pushing the same charges (Rai et al., 2018). In another study, Singh K. et al. (2014) pointed out that the DLS results encompass the layer of solvent at the interface and capping biomolecules on the nanoparticles’ surface. This was also indicated by the results of FTIR analysis, where bands identified suggest the presence of functional groups from biomolecules such as proteins (N–C-and C–C), aromatic compounds (C-H), amines (C-N), hydrocarbons (C-H, C=C) from the fungal extract which are employed in the synthesis and take part in the reduction of metal ions and subsequent stabilization of the nanoparticles (Abdullah et al., 2020; Wypij et al., 2022). The bands detected between 400 cm ^-1–600 cm ^-1 imply the formation of bonding between metal and biomolecules present in fungal extract. Our results agree with the findings reported by Salem (2022) and Shobha et al. (2023) where bands for biosynthesized nanoparticles were found at 609.3 and 414.6 cm⁻¹ for AgNPs and 534 cm⁻¹ for ZnONPs, respectively.

Mycosynthesized NPs from F. solani IOR 825 showed antimicrobial activity against a wide range of bacterial and fungal pathogens of plants in a dose-dependent manner, highly depending on the targeted strain. In a similar study by Namburi and coworkers (2021), antibacterial activity of biosynthesized AgNPs was found against Xanthomonas oryzae pv. oryzae at concentration of 2.5 μg mL^-1 while another report indicated effectiveness of AgNPs synthesized from Ulva fasciata extract against X. campestris pv. malvacearum at higher concentration of 40 μg mL^-1 (Rajesh et al., 2012). In a study conducted by Manosalva et al. (2019) AgNPs showed activity against E. coli, P. syringae, and Staphylococcus aureus. The activity of AgNPs was affected by both the bacterial strain and the size of the NPs (23, 92, and 220 nm); the highest sensitivity to AgNPs was detected for Escherichia coli (MIC 5–30 μg mL^-1 and MBC 10–50 μg mL^-1), followed by P. syringae (MIC 10–40 μg mL^-1 and MBC 30–60 μg mL^-1) and S. aureus (MIC 50–80 μg mL^-1 and MBC 60–80 μg mL^-1). In addition, the growth of bacterial strains, namely, Ralstonia solanacearum, P. syringae, X. campestris, and X. oryzae was inhibited by ZnONPs synthesized from C. tomentosa leaf extract at various concentrations of 125, 500, 250 and 250 μg mL^-1, respectively. A similar trend was found in our study, where P. syringae showed lower sensitivity to ZnONPs than X. campestris. The growth of P. syringae was inhibited by ZnONPs (1) and ZnONPs (2) at concentrations of 1024 and 512 μg mL^-1, respectively, while X. campestris at concentrations of 256 and 512 μg mL^-1, respectively. Several studies indicated that antibacterial mechanisms of metal nanoparticles include the destruction of membrane integrity, cell morphology changes, the release of metal ions, and the generation of reactive oxygen species generation (Reddy et al., 2007; Jiang et al., 2016; Gallón et al., 2019). To date, the antifungal activity of biosynthesized AgNPs and ZnONPs were tested against plant pathogens, e.g., Alternaria brassicae (Dhiman et al., 2021), F. oxysporum (González-Merino et al., 2021; Macías Sánchez et al., 2023) and F. graminearum (Ibrahim et al., 2020). For example, Talie et al. (2020) showed lower antifungal activity of AgNPs biosynthesized from Helvella leucopus which at a concentration of 20 mg mL^-1 inhibited spore germination of Penicillium chrysogenum, A. niger, and A. alternata by 83.21, 77.32% and 69.10%, respectively. The dose-dependent antifungal activity of AgNPs biosynthesized from Trichoderma viride against F. oxysporum and Alternaria brassicicola was reported by Kumari et al. (2019). These results corroborate our studies. Inhibition of mycelial growth was observed at AgNPs concentration of 5%, while complete suppression was determined at a nanoparticle concentration of 25%. In addition, they suggested that the action of AgNPs against A. brassicicola led to the generation of superoxide radicals, as well as the disruption of the mycelial structure (Kumari et al., 2019). Jain et al. (2020) evaluated antimicrobial activity of bio-ZnONPs against X. oryzae and Alternaria sp. with the maximum effect at concentration of 100 and 250 μg ml⁻¹, respectively. Similary to the results of the present work, Jamdagni et al. (2018) found that ZnONPs synthesized from Nyctanthes arbor-tristis flower extract showed MIC values of 16 μg mL^-1 against A. niger, 64 μg mL^-1 against A. alternata and F. oxysporum, and 128 μg mL^-1 against B. cinerea and Penicillium expansum. Whereas, Zhu et al. (2021) reported inhibitory effect of ZnONPs synthesized from Cinnamomum camphora (L.) leaf extract on mycelial growth and spore germination of A. alternata at concentrations of 20–160 mg mL^-1. The proposed mechanisms of antifungal activity of ZnONPs included excessive synthesis and accumulation of malondialdehyde in A. alternata cells and damage to the cell membrane, leading to leakage of proteins and nucleic acids (Zhu et al., 2021). In turn, Nandhini et al. (2019) observed the plasmolysis of spores of Sclerospora graminicola after treatment with ZnONPs at a concentration of 50 ppm. The differences in antimicrobial activity of both types of ZnONPs biosynthesized from F. solani IOR 825 may result from different protocols used for their biofabrication. There are several important factors that affect the synthesis of nanoparticles, including pH of the reaction solution, temperature, pressure and time of the reaction, the concentration of the extracts and precursors, and above all the protocols that are followed for the synthesis process (Patra and Baek, 2014; Wypij et al., 2019). Consequently, nanoparticles of different sizes, shapes, structures and properties are formed which affect their biological activity, including antimicrobial activity (Patra and Baek, 2014; Wypij et al., 2019), as discussed above.

A notable antimicrobial activity of mycosynthesized AgNPs was further confirmed in seeds sterilization tests. The minimum concentration of AgNPs that effectively sterilized maize seeds (32 μg mL^-1) was equal to or twice lower than the majority of MICs determined against the tested bacterial and fungal plant pathogens. It is noteworthy that at this concentration no negative effects of AgNPs on seed germination or seedling growth were observed, as mentioned previously. Previously, Morsy et al. (2014) used bioAgNPs form cyanobacteria for sterilization of maize, sorghum, soybean and sesame seeds. Although the authors used 2.5 higher AgNPs dose than in the present study, they noted incomplete sterilization of seeds indicated by the presence of fungi from genera Fusarium and Alternaria sp. Aspergillus spp. or Pencillium spp. The results of the present study showed that overall AgNPs at tested concentration range did not negatively affected seedling parameters, except of fresh biomass production at concentration of 256 μg mL^-1. It may suggest phytotoxicity of AgNPs at higher doses. Similarly, dose-dependent plant responses to AgNPs priming of seeds were reported by other authors. In the study by Labeeb et al. (2020), AgNPs at concentrations of 20 and 40 mg L^-1 increased germination of seeds and root length of green pea (Pisum sativum L.), while higher concentrations (80 and 160 mg L^-1) decreased seed germination and reduced seedling growth. It has been suggested that the phytotoxicity of AgNPs, especially at higher doses, may be related to their small size which facilitates their transport. Once penetrating into plant tissues and cells, they display cytotoxic and genotoxic effects (Scherer et al., 2019).

Although ZnONPs from F. solani IOR 825 were found to display non-sterilizing properties, they significantly improved seedling growth by stimulating shoot and root elongation, and fresh and dry matter production. A number of reports have proven the importance of Zn for plant growth and development as well as their resistance to biotic or abiotic stresses, that result from its involvement in physiological processes (Camp, 1945; Rudani et al., 2018). Zn content is essential for plants as a component of the cytochrome complex, for membrane integrity or as a cofactor or complexing ion for enzyme activity (Hacisalihoglu, 2020). Some other reports highlight a significant role of Zn in cell elongation and synthesis of tryptophan, a precursor of indole-3-acetic acid (Mašev and Kutáček, 1966; Sharifi et al., 2016; Sharma et al., 2021). The beneficial effects of ZnONPs on seeds germination and early seedling growth were reported by other authors who showed enhanced wheat grains germination and seedlings growth after priming the seeds with ZnONPs at a concentration of 10 mg L^-1 (Rai-Kalal and Jajoo, 2021). Recently, the mechanisms of ZnONPs action as a nanobiofeltilizer for plant growth promotion were studied by Sun et al. (2020), Khan et al. (2021) and El-Badri et al. (2021). They found that these NPs increased zinc uptake by plants, maximized plant production and improved plant resistance to biotic and abiotic stresses. In turn, Itroutwar and coworkers (2020a) identified the accumulation of ZnONPs in maize seeds endosperm leading to improved germination. It has been suggested that ZnONPs facilitate water uptake and increase α-amylase activity during germination (Itroutwar et al., 2020b; Rai-Kalal and Jajoo, 2021; Sharma et al., 2021). Moreover, study conducted by El-Badri and coworkers (2021) showed that ZnONPs activity as plant growth promoter was associated with increasing metabolic activity and modulating the expression of hormone genes (abscisic acid (ABA) and gibberellic acid (GA)) during seed germination. In another study, Rawashdeh et al. (2020) showed improved germination and enhanced biomass production under salt stress after nanopriming seeds with ZnONPs. The mechanism of action of ZnONPs was attributed to stimulation of enzyme biosynthesis, induction of carbohydrate decomposition and increased activity of the antioxidant system.

Both positive and negative effects of ZnONPs on seed germination and plant growth have been reported, but many studies have shown that bio-synthesized ZnONPs are more stable and biocompatible (due to capping and stabilizing agents of natural origin on their surface) compared to chemical ones (Singh et al., 2018). The use of bio-ZnONPs in the preparation of seeds of agronomically important crops can contribute to increased crop productivity and quality. Although there is still limited information on the interaction of nanoparticles with plants, further efforts should be made to clarify them (Tondey et al., 2021).

In this study, AgNPs and ZnONPs were effectively synthesized from F. solani IOR 825. They were comprehensively characterized using UV–vis, TEM, XRD, DLS, Zeta potential measurements, and FTIR which revealed the small size and spherical shape of AgNPs, the larger size of ZnONPs, and for both stability, crystalline nature, and that mycosynthesized nanoparticles were capped with biomolecules. The AgNPs were found to have strong antimicrobial potential against bacterial pathogens of plants. The lower sensitivity of pathogenic bacteria was demonstrated to ZnONPs. In addition, both types of NPs showed the potential to inhibit fungal spore germination, which is crucial in the fungal spread in the environment, and growth of fungal mycelia. The AgNPs revealed sterilization effect on maize seeds while ZnONPs demonstrated stimulatory effect on seedlings growth by improving fresh and dry biomass production. The present work highlights that mycosynthesized silver and zinc oxide nanoparticles in view of their unique properties, have a high potential as a promising agent to control or prevent the growth of pathogens in agriculture and enhance crop productivity. Nevertheless, continued investigations into their effects on plant development, growth and long-term toxicity are required.