350 Olson et al: Neonicotinoid Applications for Emerald Ash Borer Suppression and foliar samples to evaluate for insecticide residues, and stem sections to evaluate for EAB colonization. EAB Colonization When neonate larval emergence holes were evident, or larval galleries were present on the stem, EAB sur- vival was scored as positive. Larval galleries were measured by tracing transparent film on each stem, then using ImageJ (Rasband 2018) software to quan- tify the amount of phloem tissue consumed by each larva (Olson and Rieske 2018). Insecticide Residues Root, stem phloem, and foliar tissues were processed within 24 hours of harvest. Plant tissue was placed in liquid nitrogen and ground into a powder with a mor- tar and pestle and stored at −20 °C prior to analysis. For analysis, a 1:10 ratio of plant tissue to acetonitrile is placed on a rotary table for extraction over 24 hours, after which approximately 300 µL is filtered through a 0.2 µm PTFE syringe filter directly into an autosampler vial for analysis using liquid chromatog- raphy mass spectrometry (LC/MS/MS). Imidacloprid and its metabolites, and dinotefuran, were quantified at Villanova University Department of Chemistry using a Shimadzu Prominence HPLC (Shimadzu, Colombia, MD, USA) with a Phenomenex Gemini NX C-18 column (5 µm particle, 4.6 mm ID, 250 mm in length) fitted with a corresponding Gemini guard column using a 10 µL injection volume. The aqueous phase was 10 mM ammonium formate in water, and the organic phase was 10 mM ammonium formate in acetonitrile. Mass spectrometry was performed using a positive electrospray ionization mode; multiple reaction monitoring transitions were used for optimi- zation. Limits of detection (LOD) were 1.36 ppb for imidacloprid, 1.41 ppb for imidacloprid olefin, 6.69 ppb for dihydroxy imidacloprid, and 0.43 ppb for dinotefuran. Statistical Analysis For analysis, samples with concentrations of dinote- furan less than the limits of detection (LOD < 0.43) were considered left censored observations and thus were analyzed by the Cox proportional hazards model. However, the Cox model can only handle right censoring, so dinotefuran concentrations, y, were transformed using y* is arbitrary, but is chosen so that y* = 40 – y. The choice of 40 > 0. Therefore, a y that was left censored becomes right censored under ©2020 International Society of Arboriculture the transformation y* right censored if y* fit to y* , where now an observation is > 39.57. The Cox model was then by site, plot, treatment, tissue type, and the interaction of treatment by tissue. The goodness of fit was assessed by the Cox-Snell and Schoenfeld resid- uals. Tissue samples of imidacloprid olefin were han- dled similarly using LOD < 1.41, except that the stem tissue was removed from the analysis due to a high percentage of censored observations. Both analyses were conducted in PROC PHREG (SAS 9.3, SAS Institute Inc. 2011). Dihydroxy imidacloprid was also not analyzed due to the number of missing values. Finally, imidacloprid concentrations were log trans- formed due to right-skewness. Imidacloprid concen- trations had no censoring and were analyzed via a mixed model ANOVA using Proc GLIMMIX, where treatment, tissue type, and treatment by tissue type were designated as fixed effects, and site and plot were designated as random effects. The data for EAB larval phloem consumption have many zeroes, and the residuals of a linear regression are not normally dis- tributed, so a nonparametric Kruskal-Wallis test was used to evaluate differences in EAB larval phloem consumption among the 5 treatments using Proc NPAR1WAY. Pairwise differences were assessed using the Dwass, Steel, Critchlow-Fligner method. RESULTS Residue concentrations in trees treated with the full rate of imidacloprid were above the limit of detection (1.36 ppb), and ranged from 8.77 ppb to 567 ppb. For trees receiving the half rate, imidacloprid residues ranged from 1.83 to 598 ppb. Concentrations of the metabolite imidacloprid olefin ranged from below the limit of detection (1.41 ppb) to 142 ppb in trees receiving the full rate of imidacloprid; trees receiving the half rate had imidacloprid olefin concentrations ranging from 1.41 (the LOD) to 17.3 ppb. Of the imidacloprid-treated trees (full and half rate), 17 had imidacloprid olefin levels above the limit of detec- tion, including 9 leaf tissue samples, 6 root tissue samples, and 3 stem tissue samples. The range of the metabolite dihydroxy imidacloprid was below the limit of detection (6.69) to 18.9 ppb for trees receiv- ing a full rate, and 6.69 (the LOD) to 16.3 ppb for trees receiving the half rate. Only 2 of the full rate and 2 of the half rate imidacloprid-treated trees had dihy- droxy imidacloprid concentrations above the limit of detection, in leaf tissue and root tissue, respectively.
September 2020
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