The miticidal activity of silver nanoparticles towards date palm mite (Oligonychus afrasiaticus (McGregor)): efficacy, selectivity, and risk assessment

Promising bioactivities of silver nanoparticles SNP urged researchers of different specialties to evaluate their field- respective activities. Bioactivity towards agricultural pests were the subject of limited publications. In the current study, SNP were synthesized and miticidal activity was evaluated towards old world date mite Oligonychus afrasiaticus (McGregor) (Acari: Tetranychidae) and an associated predatory mite Neoseiulus barkeri Hughes (Phytoseiidae). Under laboratory conditions, SNP displayed significantly higher activity towards O . afrasiaticus (LC 50 was 39.7 µg/mL) than N . barkeri (LC 50 was 1587.9 µg/mL) which accounts for about 40 folds of selectivity against the pest. SNP exhibited ovicidal activity against laid eggs of O. afrasiaticus (LC 50 was 67.8 µg/mL). In field, SNP (at 216 µg/mL) achieved slightly higher efficiency than in laboratory study, 86.5% of population reduction of O. afrasiaticus was achieved and only 18.5% of N. barkeri population was affected. SNP suppressed hatching of 57.1% of laid eggs of O. afrasiaticus . Residues of silver were determined using ICP-OES spectrometry. Initial residues reached 1.83 µg/mL after application then declined with time passing. Estimated daily intake (EDI) reached 1.28 µg/kg/day, calculated for the highest residues obtained and the highest consumption rate of date in the world. Hazard index (H i) was 0.17 in average. The obtained level of residues appeared to be safe in terms of acute and chronic toxicity references.


Introduction
Date palm (Phoenix dactylifera L.) is cultivated in forty countries that distributed in all continents. World production of date exceeded nine million tonnes in the year 2019, of which 16% was produced in Saudi Arabia with a value of about 3.5 billion US Dollars (FAO, 2019). World production is increasing every year by cultivating more Abstract Promising bioactivities of silver nanoparticles SNP urged researchers of different specialties to evaluate their fieldrespective activities. Bioactivity towards agricultural pests were the subject of limited publications. In the current study, SNP were synthesized and miticidal activity was evaluated towards old world date mite Oligonychus afrasiaticus (McGregor) (Acari: Tetranychidae) and an associated predatory mite Neoseiulus barkeri Hughes (Phytoseiidae). Under laboratory conditions, SNP displayed significantly higher activity towards O. afrasiaticus (LC 50 was 39.7 µg/mL) than N. barkeri (LC 50 was 1587.9 µg/mL) which accounts for about 40 folds of selectivity against the pest. SNP exhibited ovicidal activity against laid eggs of O. afrasiaticus (LC 50 was 67.8 µg/mL). In field, SNP (at 216 µg/mL) achieved slightly higher efficiency than in laboratory study, 86.5% of population reduction of O. afrasiaticus was achieved and only 18.5% of N. barkeri population was affected. SNP suppressed hatching of 57.1% of laid eggs of O. afrasiaticus. Residues of silver were determined using ICP-OES spectrometry. Initial residues reached 1.83 µg/mL after application then declined with time passing. Estimated daily intake (EDI) reached 1.28 µg/kg/day, calculated for the highest residues obtained and the highest consumption rate of date in the world. Hazard index (Hi) was 0.17 in average. The obtained level of residues appeared to be safe in terms of acute and chronic toxicity references.
Quite few investigations were conducted to test the activity of SNP towards phytophagous mites (Jalalizand et al., 2013;Pavela et al., 2017;Al-Azzazy et al., 2019). The current study was dedicated to assessing the activity of SNP against O. afrasiaticus mite, one of the key pests of date palm, and give special focus to one of its natural enemies, the predatory mite N. barkeri in laboratory and in field. In addition, shed light on the concerns of residues of the SNP in the produce and presumed risks to the consumer.

Synthesis and characterization of silver nanoparticles
Silver nano particles (SNP) were synthesized using the method described by Lee and Meisel (1982). Spectroscopic characterization of the synthesized SNP was performed to confirm the achievement of the required particles in terms of size and shape. A particle size of 40 nm (average) was obtained, as shown from the laser particle size analyzer (S3500 Microtrac Bluewave, USA). EDX analysis confirmed the existence of the silver metal. Scanning electron microscopy (JEOL SEM, USA) was also run to show, basically, the particle shape and its dimension as well. Particles were mostly spherical ( Figure 1). palm trees. Pests play an infamous role in agricultural production as a limiting factor. Old world date mite O. afrasiaticus or (dust mite of palm) is a key pest of date palm in The Middle East and North Africa (Talhouk, 1991;Blumberg, 2008;El-Shafie, 2012). It is responsible for great losses of date produce in terms of quantity and quality (Jeppson et al., 1975;Alatawi, 2020). It develops netting of thin filament produced by nymphs and adults where dust and remains of mites accumulate (Yahia and Kader, 2011). It causes fruit drop while still immature (Yahia and Kader, 2011). Palm dust mite affects date fruits mainly at the early stages of development and continues at later stages (Alatawi, 2020). According to Codex Alimentarius, date fruits should be practically free of any visible extraneous matter such as, dust, living or dead pests of different life-stages and remnants of pests per se or their life's products (FAO, 2017). Pesticides are applied to control date pests and to enhance the quality of the produce. Several pesticides are recommended, by authorities, to control date palm mite (i.e. ethion, bifenthrin, abamectin and sulfur). Residues of pesticides in crops are proportional to rates and times of application that increase with incidence of pesticide resistance. The exceeding acceptable limits residues of pesticides in palm dates present a risk to consumers (Abdel Ghani et al., 2018). New selective and effective pesticides are always looked-for to accomplish control of normal and resistant pests. Silver nanoparticles SNP showed activity towards various arthropods (Govindarajan et al., 2016;Pavela et al., 2017;Avinash et al., 2017). As a metal, SNP may bind to several enzymes and impair vital biological systems (Benelli, 2018). It was reported that SNP can affect superoxide dismutase (Nair et al., 2013), glutathione s transferase , acetylcholine esterase, phosphatase, carboxylesterase  as well as genes' expression Nair and Choi, 2012). This multi-target biosite mechanism of action may qualify SNP to be implemented in agricultural pest control programs. SNP is already registered in USA as an algicide and bactericide in water (EPA, 1992). SNP are produced on an industrial level to supply the increasing world demand that has exceeded 70 tonnes in the year 2017 and it is expected to be 160 tonnes in the year 2020

Evaluation of SNP activity on infested dates in laboratory
Bunches of infested date fruits were brought from the Experimental Farm of Qassim University, Qassim, Saudi Arabia. Dates were naturally infested with the phytophagous mite O. afrasiaticus. In addition, the predacious mite N. barkeri was also found. Both mites were identified according to Meyer (1987); Chant and McMurtry (2003, 2005. Dates were separated from the strands and placed in plastic plates. Pre-treatment counts of moving individuals of both mites and laid eggs (for only O. afrasiaticus) were recorded before treatment. Serial concentrations of SNP i.e. 13.5, 27, 54, 108 and 216 µg/mL were freshly prepared from the stock solution and sprayed using a one-liter volume hand atomizer. Untreated control was sprayed with tap water. Ten replicates were employed for each concentration of SNP and untreated control. Numbers of moving stages were recorded after three days of treatment. Hatched larvae were monitored for 7 days or until untreated control was fully hatched and newly hatched larvae were removed daily. Corrected mortality percent in each treatment was computed using Equation 1 (Henderson and Tilton, 1955).
where: Cb is the number of mites or laid eggs in untreated control before spray, Ca is the number of mites or hatched larvae in untreated control after spray, Tb is the number of mites or laid eggs in treatment before spray and Ta is the number of mites or hatched larvae in treatment after spray.

Evaluation of contact activity of SNP in petri dishes
In order to assess the contact activity of SNP on both studied mites. Moving individuals of the phytophagous mite O. afrasiaticus and the predacious mite N. barkeri (100 individuals) were placed in petri dishes separately in triplicates. Mites were sprayed with SNP solutions viz 13.5, 27, 54, 108 and 216 µg/mL using a one-liter hand atomizer. Mortality was checked after 24 h. Corrected mortality was calculated using Abbot Formula Equation 2 (Abbott, 1925).
nT is the number of mites in treatment and nC is the number of mites in untreated control.

Evaluation of the miticidal activity of SNP in the field
The experiment was conducted using short heighted medium-laden date palm trees of Barhi variety in the Experimental Farm of Qassim University, from August to September 2020. The selected trees were not sprayed with any pesticide during the season and were already infested with the phytophagous mite O. afrasiaticus. The predacious mite N. barkeri was also identified on the selected palm trees. Palm trees were sprayed once with SNP solution (216 µg/mL). Spraying solution were applied using a ten-liter fixed pressure hand sprayer (obtained from the local market). Spray solution was directed mainly to date bunches until run-off. Untreated control trees were sprayed with the available irrigation well water. Each SNP or control treatments were conducted in triplicate using randomly selected palm trees. Date fruits, representing all bunches, were picked randomly from each tree before treatment to determine the initial population of both mites. Post treatment population was counted after three days of application. Date samples were transferred to the laboratory for counting of moving individuals of both mites. Hatching of laid eggs of O. afrasiaticus was also recorded. Corrected mortality percent in each treatment was calculated using Equation 1.

Determination of silver residues in dates and risk assessment
In order to determine residues of silver in date fruits, bulk representative samples of about 1 Kg of date fruits from the treated date palm trees were collected at zero time (after 3 h), 7, 15 days of application. Untreated date samples were also collected once. Samples were transferred to an (ISO 17025) accredited laboratory for analysis. Samples were digested and then subjected to determination using Varian 720ES ICP-OES Spectrometer.
To assess the risk caused by silver residues in date fruits, Hazard index (Hi) was calculated using Equations 3 and 4 (FAO, 2016).
where EDI is Estimated Daily Intake (µg/Kg body weight/day) and RfD is the Reference Dose (µg/Kg body weight/day) of silver.

EDI AgRC FC
where AgRC is Silver Residues Concentration (µg/Kg) in dates generated from field trial and FC is Food Consumption (g/day).

Statistical analysis
Statistical tests were performed using IBM SPSS Statistics version 23. All data were initially investigated through the Shapiro-Wilk's test for normality (Shapiro and Wilk, 1965). One way ANOVA was performed for statistical analysis within the same treatment using transformed corrected mortality percentage data. Differences in the means were considered statistically significant when P ≤ 0.05. Probit regression analysis was performed, according to Finney (1971), using average mortality percent versus the normal logarithm of applied concentration.

Evaluation of efficiency of SNP towards moving individuals on date fruits in laboratory
Probit analysis of the acquired mortality versus used concentrations was performed for SNP against both tested phytophagous and predatory mites. Toxicity parameters were calculated and reported in  (Table 1). In addition, the ANOVA test showed a significant difference between O. afrasiaticus and N. barkeri (P < 0.001), indicating lower toxicity of SNP towards non-target predacious mites. SNP may work as digestive and /or contact poison. As well, body openings or interskeletal membranes might be an easy route to enter inside a mite's body. This can explain the lower activity towards predatory mites that possess thicker body wall with shields on dorsal and ventral sides (Zannou et al., 2007) preventing the penetration of the nanoparticles. In addition, predatory mites feed only on alive phytophagous mites and it is unlikely to feed on intoxicated individuals. Thus decreasing the dose of SNP received by predatory mites. This is not the case for phytophagous mites that have thin body wall and feed on plant sap. Tripathi and coworkers reported that SNP translocated through vascular tissues in plant in Ag 0 /Ag + form (Tripathi et al., 2017), which might increase the exposure of plant sap-feeding mites to SNP by increasing the received dose.
Quite few studies have tested the miticidal activity of SNP towards phytophagous mites. Silver ion impregnated chitosan textile increased mortality of different dust mites by about 65% more than the only chitosan textile without Ag+ (Rahel et al., 2013). Moving stages of Tetranychus urticae were moderately affected by commercial SNP of 18-34 nm diameter size (Jalalizand et al. 2013). Plant extract assisted synthesis of SNP was achieved and the obtained silver particles showed miticidal activity towards eggs, nymphs and adults of T. urticae mite (Pavela et al., 2017). Synthesized SNP showed miticidal activity towards tomato russet mite Aculops lycopersici and two-spotted mite T. urticae (Al-Azzazy et al., 2019).
Acaricidal activity of SNP in ticks was previously tested against the cattle tick Rhipicephalus (Boophilus) microplus. Marimuthu  Several studies investigated the effect of SNP on several insects (Benelli, 2018). On a genetic level, SNP produced up and/or down-regulation of genes responsible for ribosomal protein gene , glutathione S-transferase (GST) , ecdysone receptor gene (Nair and Choi, 2012) and Mn superoxide dismutase (Nair et al., 2013) in Chironomus riparius midge. In relation to oxidative stress, SNP caused accumulation of reactive oxygen species (ROS) and related apoptosis in Drosophila melanogaster (Mao et al., 2018). Regarding the activity of essential enzymes, SNP decreased the activity of esterase enzymes (carboxylesterase and acetylcholinesterase), phosphatase and a noticeable decrease in total protein levels, in general, in mosquitos Ga'al et al., 2018).
Mechanisms of toxicity of SNP were not explored in either phytophagous mites or animal parasitic ticks . Such studies are instrumental in comprehending how SNP, as a control agent, work and unveiling real active site(s).

Evaluation of the ovicidal activity of SNP in laboratory
The ovicidal activity of SNP was investigated and dose-response regression lines were composed and data were summarized in Table 1. Chi-square significance and  (Madhiyazhagan et al., 2015). Ovicidal activity of plant extract synthesized SNP towards eggs of T. urticae was demonstrated (Pavela et al., 2017). SNP (216 µg/mL) caused 70% reduction of hatching of laid eggs in T. urtica (Al-Azzazy et al., 2019). SNP affected moving individuals more significantly than egg hatching as no overlap is noted between the obtained probit lines of O. afrasiaticus for moving individuals and egg hatching (Table 1). Nonfeeding and stationary behaviors of eggs may decrease the received dose of SNP. Upon contact, SNP can penetrate inside the developing ova. As mentioned above, SNP can affect several active sites in living organisms or cells.

Evaluation of contact activity of SNP in petri dishes in laboratory
In order to assess only the contact activity of SNP, moving individuals of both O. afrasiaticus and N. barkeri mites were placed in petri dishes separately without diet and were exposed to a spray of SNP at the same concentrations used above. Toxicity regression lines were plotted. Chi-square and R 2 values showed the quality of regression assumption fitness (Table 2). LC 50 of SNP for O. afrasiaticus (155.9 µg/mL) was significantly different than in case of N. barkeri (848.1 µg/mL) as appeared from the non-overlapping confidence limits and ANOVA (P = 0.001). In case of LC 90 s, a slight overlap in CL 95 was noted; however, LC 90 values were estimates and not practically tested. A significant difference is noted between toxicity regression lines of SNP for O. afrasiaticus in this petri dishes experiment (Table 2) and the above-mentioned (Table 1) one as both LC 50 and LC 90 confidence limits values were not overlapped. This might suggest that ingestion of SNP through the digestive system has a significant effect on toxicity of SNP towards phytophagous mites. However, there was an overlap of CL 95 values, of both LC 50 and LC 90 values, in the case of N. barkeri implying that contact with SNP droplets might be the main rout of SNP's entry in predatory mites, and supporting the opinion that predatory mites feed only on non-intoxicated and alive phytophagous mite individuals.

Evaluation of efficacy of SNP in the field
The efficacy of the highest concentration solution (216 µg/mL) used in the laboratory experiment was evaluated in the field. The obtained results are illustrated in Figure 2. In the case of O. afrasiaticus, SNP achieved a mortality percent of 86.5. For N. barkeri, SNP caused about 18.5% of mortality. Selectivity was about 4.7 times for O. afrasiaticus, over N. barkeri. In addition, SNP showed ovicidal activity of 57.1% against the laid eggs of O. afrasiaticus. These results of the field showed that SNP was more effective towards the target phytophagous mite and safer towards the non-target predacious mite. Ovicidal activity of SNP is an added benefit to the activity of SNP in control of the phytophagous mite O. afrasiaticus, making it a multi-life stage poison for mite pests. SNP could be used in mite control programs to control mite populations in place of the traditional non-selective pesticides or interchangeably with them to suppress the development of mite resistance for the sake of sustainable mite control. Studies on the evaluation of the biological activity of SNP in the field are scarce and more investigation is required.

Residues of silver generated under field conditions and risk assessment
Silver is allowed to be topically applied to burned or damaged skin or mucous tissues for therapeutic purposes. Silver is also used as a coloring agent for food, beverages and medical food products under the code E174 (European Commission, 2000). Excessive exposure to silver via ingestion, inhalation and dermal applications may cause a medical condition called argyrosis or argyria (greyishblue coloration of the exposed organs) (Chan et al., 2014). American Environmental Protection Agency EPA established the Reference Dose RfD for silver to be 5 µg/Kg/day, which is the estimate of the amount of silver to be consumed  in life time course without presenting a risk to human health (EPA, 1992), this dose accounts for 300 µg/day for the average person of 60 Kg bodyweight (EPA, 1992). In order to assess the anticipated risk that might be caused by remained residues of silver in date fruits, silver's residues were determined. Silver residues were not detected in the untreated date sample. The initial deposit of silver ( (WHO, 2003), which happens to be the highest consumption rate worldwide. Average body weight is 60 Kg in the Middle East. The highest found residues of silver (initial deposit) produced an EDI value of 1.28 (µg/Kg body weight/day) which is only 26% of the set RfD of silver intake implying that neither chronic nor acute toxicity is predicted under such exposure. Risk assessment as Hazard index (Hi) was estimated using the set RfD of 5 µg/Kg/day and consumer body weight of 60 Kg. Hi was calculated to be 0.26 at the initial deposit level (1830 µg/Kg), which is the highest expected exposure and the Hi values declined using residues data of 7 and 14 days after application. In average Hi was 0.17, meaning consumers, by consuming such silver-containing dates, are exposed to only 17% of the RfD. However, residues were determined as total silver regardless of its chemical and physical real form in date fruits. Further studies on the risk assessment of silver in nanoparticle form are crucial. Table 3 summarizes residues concentrations of silver in palm dates in successive time intervals and respective EDI and Hi values.

Conclusion
Nanoparticles of silver metal were synthesized using a chemical method. Evaluation of the miticidal activity of the synthesized particles against date palm phytophagous mite O. afrasiaticus and an associated predacious mite N. barkeri was the chief focus of this study. SNP showed miticidal activity against moving stages and laid eggs of the phytophagous mite. SNP showed miticidal activity towards O. afrasiaticus in both laboratory and field trials. SNP showed remarkable selectivity of about five folds towards the pest over the non-target predatory mite at the field application rate. SNP showed ovicidal activity for O. afrasiaticus that may increase its overall control efficacy. Field results confirmed the practical usefulness of SNP as an acaricide. Phytotoxic effects were not observed in both laboratory and field experiments. Risk assessment using hazard index showed that residues of metal silver remained in date fruits pose no risk to the consumer.  Estimated Daily Intake (EDI) of silver (µg/Kg body weight/day) at 41.8 g dates/person/day according to GEMS/ Food Regional Consumption (WHO, 2003) and at average body weight of 60 Kg; b Hazard index (Hi) = EDI/RfD. RfD is set at 5 µg/Kg/day according to EPA RED Facts of Silver (EPA, 1992).