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Determination of Stability from Multi-Component Pesticide Mixes Kelly J. Dorweiler, Jagdish N. Gurav, James S. Walbridge, Vishwas S. Ghatge, and Rahul H. Savant J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05681 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 4, 2016

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Journal of Agricultural and Food Chemistry

REVISED MANUSCRIPT

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Determination of Stability from Multi-Component Pesticide Mixes

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Kelly J. Dorweiler1*, Jagdish N. Gurav2, James S. Walbridge3, Vishwas S. Ghatge2, Rahul

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H. Savant2

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1. Medallion Laboratories/General Mills Inc., James Ford Bell Technical Center; 9000 Plymouth Ave N. Minneapolis, MN 55422, USA. 2. Medallion Laboratories/General Mills Inc., Spectra Building; Hiranandani Business Park, Powai, Mumbai 400076, India. 3. Supelco Division of Sigma-Aldrich, 595 North Harrison Road Bellefonte, Pennsylvania 16823, USA.

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*Corresponding author contact information: 763-764-5647;

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[email protected]

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1 ACS Paragon Plus Environment


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A study was conducted to evaluate the stability of 528 pesticides, metabolites, and

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contaminants prepared in large multi-component mixes, to enhance laboratory efficiency

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by allowing maximum use of the useful shelf life of the mixtures. Accelerated aging at

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50ºC simulated six month, one year, and two year storage periods at -20°C. Initial mixture

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composition was based upon the instrument of analysis. After obtaining preliminary

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stability data, mixtures were reformulated and re-evaluated. In all, 344 compounds showed

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satisfactory stability across all treatment groups, 100 compounds showed statistically

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significant changes between the control and the six month simulated storage period (27

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with losses >20%), and the remainder showed borderline stability or were tested in one

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protocol. Stability behavior for organophosphates agreed with the proposed reaction

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mechanism responsible for acetylcholinesterase inhibition. A small number of compounds

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increased in response over time, suggesting the occurrence of degradation of precursor

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pesticides into these respective compounds.

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KEYWORDS: Pesticides; stability; degradation; mass spectrometry, accelerated aging


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INTRODUCTION A critical factor for successful quantitation of any analytical procedure relies on the

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stability of the analytical standards. Improper treatment and storage of such standards, or

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use beyond a validated shelf life for quantitation may result in compromised data. For

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pesticide residue analysis, this is particularly important since analytical results are the basis

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for taking regulatory or other action. Studies have been conducted to determine ideal

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solvent compositions versus duration of stable storage of analytical standards. Besamusca

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et.al. presented data supporting the use of 90:10 iso-octane/toluene as a suitable solvent

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composition for many single analyte standards with shelf lives exceeding fifteen years

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when stored at -18ºC.1 Stefanelli et.al. performed a stability evaluation for 82 individual

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pesticide compounds relevant to the European member states participating in the European

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Food Safety Authority pesticide monitoring program that demonstrated the suitability of

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extending shelf lives of prepared standards up to one year at 4ºC.2

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The International Organization for Standardization (ISO) has recommended

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suppliers become Guide 34 compliant for the sale of certified reference materials (CRMs)

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for use in ISO/IEC 17025:2005 accredited laboratories,3 allowing laboratories to have

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confidence that, when unopened, CRMs will remain stable up to their documented expiry

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date. However, after laboratories receive standards, they are opened, combined, and mixed

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at various concentrations in solvents (often based on analytical method suitability criteria)

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and used without necessarily knowing the stability the resulting solutions. Therefore, some

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laboratories look to chemical suppliers to prepare custom mixes containing analytes based

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upon method(s) of analysis. Unfortunately, custom mixes containing hundreds of

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components prepared as CRMs are very costly. Alternatively, conducting stability



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evaluations on prepared analytical standard mixtures within the laboratory offers a higher

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degree of confidence that the integrity of standard solutions has not been compromised.

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The use of accelerated aging studies is widely accepted in the pharmaceutical

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industry for drug development, for food and packaging stability, and is regularly employed

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by suppliers when preparing CRMs.4-9 These studies are based upon the Arrhenius model,

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where k = the rate constant, A = pre-exponential factor, Ea = activation energy, R = gas

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constant, and T = temperature. ݇ = ‫݁ܣ‬

ିாೌൗ ோ்

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In practical applications as described by Anderson et. al., when the Ea of a chemical is

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known, the analyte reaction rate will decrease by a constant factor (Q10) for every ten

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degree temperature decrease, otherwise known as the Q Rule.9 In accelerated aging

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studies, this rule assumes the Ea of all target analytes will exist within specified limits.

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Stability experiments may also be conducted at four different temperatures to derive the

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Ea.4 In both techniques, stability results have served as conservative indicators of apparent

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stability. Once the sufficient time has elapsed, a classical aging study under real-time

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temperature conditions may be performed if desired.9 Accelerated aging studies offer the

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benefit of direct comparisons between controls and treatment groups using analytical

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systems under consistent operating conditions, such as the inlet, column, and detector

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performance without contributing bias to the analysis, provided that the experiment is fully

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randomized.

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In the study presented here, large multi-component pesticide mixtures dissolved in

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solvents were temperature stressed to simulate six, twelve, and twenty-four month storage

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time periods at -20°C. Dilutions of the mixtures were randomly prepared, then evaluated at


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the requisite time periods using liquid chromatography mass spectrometry (LC/MS) and

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gas chromatography mass spectrometry (GC/MS). The data were statistically evaluated for

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significant response differences due to the apparent lack of stability of individual

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components of each mix.

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MATERIALS AND METHODS Chemicals. Two custom reference standard mixtures composed of 528 pesticides,

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metabolites, and contaminants of >95% purity in a solution of 0.1% acetic acid (HAc,

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Glacial, Macron, Center Valley, PA) in acetonitrile (MeCN, Optima LC/MS grade, Fisher,

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Pittsburgh, PA) were prepared by Supelco (Bellefonte, PA, see Table 1). Mixes 1A (204

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components) and 1B (324 components) were prepared November 2013 and stability study

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protocol #1 (SSP#1) conducted. Based on the results of SSP#1, new mixtures were

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formulated in March 2014; mixes 2A (373 components) and 2B (154 components) for a

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stability study protocol (SSP#2). Concentrations for analytes in SSP#1 were based upon

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Medallion Laboratories AOAC 2007.01 modified method which has defined matrix

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matched standard concentrations at the limit of quantitation (LOQ). Component

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concentrations in SSP#2 were double the SSP#1 concentrations. The internal standard

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used was d5-atrazine (Cambridge Isotope Laboratories, Andover, MA). Liquid

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chromatographic mobile phases were prepared using formic acid (EMD Millipore,

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Billerica, MA), ammonium formate (Sigma-Aldrich, St. Louis, MO), and Optima LC/MS

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grade solvents water and MeCN.

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Heat Treatments of Standard Solutions. For SSP#1, ampoules (Mix 1A, n=12;

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Mix 1B, n=12) were grouped into four treatment groups to simulate time treatments: zero



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(0m), six months (6m), twelve months (12m) and twenty-four months (24m). Post filling,

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and prior to the start of the study, all ampoules were maintained at -20°C, then nine of the

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twelve ampoules from each mix were placed into a VWR 1530 oven (Radnor, PA) set to

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50ºC to simulate accelerated rate, time-specific degradation, assuming Ea for all analytes

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were between 10 and 20 kcal/mol (Table 2)15. At the end of each heat treatment period, the

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designated ampoules were returned to -20ºC. Following the conclusion of the final

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treatment period, 24 ampoules (6 controls 1A:0m, 1B:0m, and 18 from treatment groups

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1A:6m, 12m ,24m and 1B:6m, 12m, 24m) were shipped overnight on ice to Medallion

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Laboratories (Golden Valley, MN) in December 2013. Following the conclusion of data

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collection and evaluation for SSP#1, ampoules for SSP#2 (Mix 2A, n=12; Mix 2B, n=12)

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were prepared, grouped and simulated aged in the same manner as in the SSP#1 evaluation.

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Upon completion, all twenty-four ampoules from SSP#2 were shipped overnight on ice to

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the same Medallion Laboratories location in May 2014.

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Sample preparation. On the day received, the ampoules for SSP#1 were placed in

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-20ºC storage until the day of analysis, when the ampoules were allowed to thaw to room

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temperature. Subsequent preparation and analysis of the contents of each ampoule was

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fully randomized to minimize bias. The same protocol was repeated for SSP#2. A single

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internal standard solution (IS, 5 µg/mL in acetone), and one acidified MeCN solution

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(0.1% HAc) were prepared prior to each instrumental quantitation sequence and used for

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subsample preparation. For both evaluations the same analytical procedures were

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performed, with the exception that only half the volume was used from each ampoule for

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SSP#2.


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The content of each ampoule was prepared for instrumental analysis in triplicate

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resulting in a total of nine subsamples prepared for each treatment group. The final

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concentrations of the analytes in the injection solutions were targeted at five times the LOQ

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of the instrumentation, concentration levels selected to ensure a sufficient response across

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all acquiring instrumentation for all variations studied. From each ampoule, 100 µL (50µL

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for SSP#2) of solution was delivered into three individual Class A 2 mL volumetric tubes.

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To each tube, 50 µL of the IS was added. The solution was brought to volume with

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acidified MeCN and thoroughly mixed fifteen times by inversion. Four aliquots of the

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solution thus prepared (300 µL, volume utilized to minimize headspace between the

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solution and the vial cap) were delivered into individual amber auto sampler vials

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containing deactivated glass low recovery inserts. The content of each individual ampoule

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was prepared through this entire process prior to processing the next ampoule to minimize

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bias from inconsistent atmospheric exposure and cross contamination. Once completed,

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four sets of thirty-six vials per set containing the prepared ampoule contents under assay

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were placed in randomized order on four chromatography-mass spectrometer systems

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programmed to quantitate all compounds of interest. To assure against carryover, solvent

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blanks were injected following every sixth assay injection. Water was avoided in solutions

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prior to LC-MS to minimize the chance of hydrolysis reactions with the pesticides. Thus,

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this project evaluated the analytes in solvent only; standards were not prepared as matrix

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matched standards, as the objective was to determine the stability of standards in ampoule

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containers under frozen conditions.

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Large Volume Sample Introduction for GC-MS Analysis. Injections of

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preparations from Mixes 1A, 2A, and 2B were made into an Automated Tube Exchanger



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(ATEX) sample introduction system (Gerstel, Baltimore, MA). The inlet program was

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controlled using Maestro software. Vials were placed in a cooled auto sampler tray (8°C)

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regulated by Peltier cooling. Each 10 µL injection was made into an individual thermal

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desorption tube containing a disposable microvial. The tube was transported to the

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Thermal Desorption Unit (TDU) held at 20°C using a Universal Peltier Cooling unit. The

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TDU was set to Solvent Venting Desorption Mode to concentrate the injected volume to ~1

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µL. The temperature was ramped at 200°C/min to 65°C, held for one minute, and then

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ramped at 720°C/min to 350°C, held for three minutes, thermally transferring analytes onto

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the Cooled Injection System (CIS) inlet. The CIS containing a liner packed with

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deactivated quartz wool was maintained at 0°C during the desorption step using liquid

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nitrogen as the coolant. From there, the CIS temperature ramped at 720°C/min to 300°C

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and held for three minutes to transfer most analytes from the inlet to the analytical column.

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A final ramp rate of 720°C/min increased the temperature to 350°C which was held for six

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minutes to transfer the remaining analytes to the analytical column.

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GC/TOF-MS Analysis. Analyte quantitation was performed on an Agilent 6890

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gas chromatograph coupled to a Leco Pegasus 4D GC×GC time of flight mass

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spectrometer (TOF-MS) operating in the one-dimensional GC mode, controlled by

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ChromaTOF acquiring software (Saint Joseph, MI). Inlet conditions controlled by the GC

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systems operated in the Programmable Thermal Vaporization (PTV) solvent vent mode;

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septum purge flow of 30 mL/min for three minutes; solvent vent flow of 50 mL/min (4

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psi); synchronized with the thermal desorption program. Analytes were chromatographed

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on a 20 m × 0.25 mm i.d. × 0.25 µm film thickness Restek Rxi-5ms analytical column

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(Bellefonte, PA). GC temperature program, TOF-MS conditions, and some analyte8 ACS Paragon Plus Environment

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specific quantitation ions were acquired per Mastovska et. al.10

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previously published were identified using peak find and forward library searching (Table

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S1-Supplemental Information).

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Analyte-specific ions not

GC/MS/MS Analysis. GC-amenable pesticides data was also acquired using an

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Agilent 7890 GC system coupled to an Agilent 7000 triple-quadrupole mass spectrometer

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(MS/MS) controlled by MassHunter software (Agilent Technologies, Palo Alto, CA). GC

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controlled inlet, carrier gas, and chromatographic separation conditions matched the TOF-

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MS system. The MS/MS was operated using EI ionization (source temperature 250°C, N2

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collision gas), with data collected in the multiple reaction monitoring (MRM) mode.

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Analyte specific transitions were optimized using full scan acquisitions followed by

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product ion scans at four collision energies for each analyte (Table S2-Supplemental

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Information). Data analysis was performed using the MassHunter Quantitation software.

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UPLC/MS/MS Analysis. Samples under study prepared from Mixes 1B, 2A, and

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2B were injected (volume 2 µL) onto a Waters Acquity I-Class Ultra Performance Liquid

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Chromatograph with an Agilent Zorbax RRHD Eclipse Plus C18 2.1 × 150 mm, 1.8 µm

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analytical column at 30°C coupled to a TQD MS/MS (Milford, MA) using a mobile phase

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composition of (A) 5 mM ammonium formate in water with 0.01% formic acid and (B) 5

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mM ammonium formate in 95:5 acetonitrile:water with 0.01% formic acid at a flow of 300

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µL/min. The gradient program was modified to accommodate the 150 mm column

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length.11 Starting at 6% B (0-0.75 min) the program linearly increasing to 95% B over

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20.25 minutes where it was held for 4.5 minutes, returned to 6% B over 0.15 minutes and

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equilibrated for 4.5 minutes before the next injection. The MS/MS utilized electrospray

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ionization (ESI) with positive polarity. The source temperature (130°C), capillary voltage 9 ACS Paragon Plus Environment


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(1.7 kV), desolvation temperature (350°C), and desolvation and cone flows (N2 at 600 and

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100 L/hr, respectively), were maintained throughout the analysis. Argon was used as the

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collision gas. Target analytes acquired in the MRM mode produced two transitions per

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compound in a single injection. Retention times for previously published analyte specific

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transitions are listed in Table S3 (Supplemental Information).10 Additional analyte specific

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transitions optimized using the MassLynx QuanOptimizer software are listed in Table S4

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(Supplemental Information). Analysis of data was performed using TargetLynx.

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UHPLC-MS/MS Analysis. LC-MS target analytes not acquired using the TQD

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MS/MS were acquired using an Agilent 1290 Infinity ultra-high performance LC coupled

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to a 6460 MS/MS. For the UHPLC separation, mobile phase configuration, column

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selection and gradient program matched the UPLC-MS/MS system. The 6460 MS/MS

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operated under electrospray ionization (ESI) with positive polarity. Nitrogen served as

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both the nebulizing and collision cell gas. The source temperature (300°C), nebulizer gas

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flow (11 L/min), sheath gas temperature and flow (350°C and 11 L/hr, respectively),

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capillary voltage (4500 V) and nozzle voltage (500 V) were all maintained throughout the

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analysis. Acquisition and analysis was performed using MassHunter packages. Analyte

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specific MS/MS transitions not obtained from the Agilent Dynamic MRM Pesticide

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Database were optimized using the MassHunter Optimizer package (Table S5-

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Supplemental Information).

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Statistical Analysis. The treatment groups were examined for trending and

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statistically evaluated to determine differences between 0m, 6m, 12m, and 24m samples.

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Response areas from analytes were exported to Excel 2010, compiled into sequential order,

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and categorized into the four treatments. Nine data points were obtained for each treatment


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for a total of thirty-six data points. Response factors (RF) were calculated by dividing the

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chromatographic area of the target analyte with the IS area. Analysis of Variance

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(ANOVA) was carried out on both the RF and integrated area count data for analytes at the

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95% confidence interval. ANOVAs were also conducted on the IS areas on all systems to

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check for bias. Due to a mis-injection on one sample on the GC-TOF/MS, ANOVA

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calculations involved eight data points for each treatment in SSP#2. All ANOVA

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calculations were performed using the Excel 2010 Data Analysis Tools.

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RESULTS AND DISCUSSION Solvent Selection. The solvent selection for the mixtures to be evaluated and for

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dilution of test samples prior to data acquisition was based upon compatibility with the

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Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) technique.10 Although

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MeCN is the preferred QuEChERS extraction solvent, Mastovska and Lehotay reported

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variable stability of base-sensitive N-trihalomethylthio pesticides captan, folpet, and

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tolylfluanid from several solvent lots of one manufacturer and from two different

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manufacturers. With the addition of HAc, these pesticides were stable throughout the

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QuEChERS extraction.12 Therefore, the addition of 0.1% HAc to MeCN was utilized for

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the mixture preparations by Supelco and Medallion Laboratories. Additionally, the choice

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of MeCN with HAc purposefully to minimize the analyst’s potential exposure to pesticides,

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since the procedural steps of the method allowed the analyst to open an ampoule and

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deliver appropriate amounts for spiking, without the need of combining solutions or

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diluting into an alternative method-compatible solvent. The authors acknowledge that

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MeCN may not be the ideal storage solvent for each pesticide evaluated since other



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solvents such as isooctane, toluene, methanol, ethyl acetate, or acetone have been shown to

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improve the shelf stability of some pesticides in solution.1,10,12 In these cases however, not

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all listed solvents are well suited for QuEChERS methodology given their polarity indices

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and miscibility (or lack thereof) with MeCN, and thus require dilution into a more polar

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solvent, such as MeCN or methanol prior to use in a QuEChERS extract.13 Importantly

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this study addresses the suitability of acidified MeCN for long term storage thus easing the

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efforts of the analyst.

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Stability evaluation. The objective of this study was to monitor the stability of

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pesticide analytes in large component mixes using accelerated aging to simulate prescribed

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storage conditions for the routine laboratory. We defined a stable analyte as one resistant

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to degradation within a multi-component mixture solution. Thus the effect of analyte-

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solvent interactions, analyte-analyte interactions, temperature, or all of the above were

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studied over the simulated time period. The evaluation of the control and treatment groups

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data using RF- and area-based ANOVAs and trending showed satisfactory stability for 161

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(Mix 1A) and 223 (Mix 1B) pesticides, metabolites, and contaminants across all treatments

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in a two year simulated storage condition for SSP#1. Likewise, for SSP#2, the same held

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true for 342 (Mix 2A) and 44 (Mix 2B) compounds. Combining the data for both SSP#1

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and SSP#2, a total of 345 (65%) analytes showed satisfactory stability. In contrast, 100

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analytes (19%) produced statistically significant changes (28 degraded rapidly exceeding

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20% loss between the control and 6m treatment group). Such an occurrence, if not

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detected, particularly when only a single calibration standard is used near the LOQ, poses a

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risk of false negative detection during analysis. As validation of the first study, SSP#2

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offered confidence that the change in mix composition was reasonable. Though 84% of


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compounds demonstrated similar stability between both protocols, 68 (13%) had borderline

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stability, and 15 (3%) were tested in only one of the two protocols. Table 3 presents results

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from all analytes demonstrating statistically significant changes in concentration in SSP#1

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and/or SSP#2. The remaining analytes which were considered stable with 95% confidence

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and no observed trending are reported in Table S6 (Supplemental Information). Compound

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classes exhibiting predominantly stable analytes were the organohalogens (OH) and

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pyrethroid pesticides. Classes with unstable pesticides included acidic herbicides,

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carbamates, miscellaneous pesticides (not included in any class), organonitrogens (ON) and

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organophosphates (OP). We focus on the OP and ON classes in further detail, but this by

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no means lessens the importance of the remaining classes.

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Organophosphate stability. For most OP insecticides and metabolites included in

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the study, inferences can be made based upon the structure. In general, the effectiveness of

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a given OP as an insecticide relies on the presence of a suitable electron-withdrawing

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leaving group and a phosphate or phosphorothioate substrate as an acetylcholinesterase

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inhibitor following a second order nucleophilic substitution (SN2) reaction mechanism.14

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Stability data support this as a possible degradation pathway for reactive OP

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compounds in which the source of the nucleophile is the acetic acid (present at 0.1%),

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which attacks the OP at the phosphorus. The lack of hydrogen-bonding with the aprotic

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acetonitrile makes the acetate ion available for nucleophilic substitution. Peak intensities

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for the dimethyl phosphorothioates bromophos, chlorpyrifos-methyl, and parathion-methyl

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across treatments 0m, 6m, 12m, and 24m demonstrate significant loss in response over

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time, as compared to the diethyl phosphorothioates bromophos-ethyl, chlorpyrifos, and

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parathion (Figure 1). For analytes with a susceptible substrate (i.e. dimethyl



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phosphorothioate) and an electron-withdrawing leaving group, such as a halogen

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substituted phenoxy or a nitro substituted phenoxy group, rapid degradation occurred. In

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contrast, the diethyl phosphorothioate substrate limited degradation, likely due to stearic

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hindrance of the larger substituent. OP structures in Figure 2 further support the SN2

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reaction mechanism for degradation or lack thereof. The presence of propyl, butyl, and

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substituted phenyl groups on the substrate produced a similar effect in other OPs; for

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example, (RS)-(O-ethyl O-(4-nitrophenyl) phenylphosphonthioate (EPN), comprised of an

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ethyl and nitro substituted phenyl phosphonthioate substrate, showed no evidence of

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degradation. Leptophos, also with a substituted phenyl group on the phosphorothioate,

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contained a methyl group, exposing the phosphorus to slight degradation. Interestingly, the

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diethyl phosphate chlorpyrifos oxon, degraded across the treatment groups, suggesting the

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presence of the double-bonded oxygen (covalent radius of 66 pm), enabled a nucleophilic

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attack. The double-bonded sulfur (covalent radius of 105 pm) on chlorpyrifos most likely

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prevented degradation by stearic hindrance.15

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In contrast, numerous OPs were reported with satisfactory stability across both

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protocols. Azinphos-ethyl and methyl moieties (Figure 3), both of which contained a weak

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methyl substituted leaving group and ethyl or methyl phosphorothioate substrates, showed

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no evidence of trending and no statistical significant changes in response (p-values 0.74

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and 0.33, respectively), in spite of the alkyl groups on the phosphorothioate. Other OPs

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with electron-releasing groups such as acephate, methamidophos, dicrotophos,

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monocrotophos, and tetrachlorvinphos, all with dimethyl phosphorothioates remained

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relatively stable across the treatment groups. The proposed nucleophilic attack of the

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acetate ion suggests a possible future study; an evaluation without the addition of acetic


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acid, or with the use of a protic solvent such as methanol which may improve the stability

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across the entire OP class. Consequently, such a change may affect N-trihalomethylthio

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pesticides, a class requiring acidic conditions for stability.

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Organonitrogen stability. In general, ON compounds were more stable than the

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OPs, however, few generalities could be drawn from this class. Noteworthy ON subgroups

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which contained predominantly stable compounds included azole compounds (i.e. conazole

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fungicides, pyrazole insecticides), triazine herbicides, and dinitroamine herbicides (Figure

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4). In contrast, ureas and phenylureas degraded rapidly in all mix formulations studied.

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This finding was not surprising given the reactivity of urea and phenylurea herbicides.16-20

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Certain pesticides were classified based on their mode of action, though other functional

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group(s) affected their apparent stability (Figure 5). The highly reactive site at the oxygen

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on the [1,4,5]oxadiazepane ring has been reported to be the source of instability of the

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phenylpyrazole herbicide pinoxaden, while the mode of action and subsequent

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classification was based on the pyrazole functional group.21 Likewise, chlorantranilipole, a

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diamide insecticide, sharing characteristics of the pyrazole subgroup subsequently

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degraded in both protocols.22, 23 Thiophanate-methyl, a highly reactive carbamate

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insecticide, was dually classified as a benzimidazole.23 These observations, identified

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within the 39 individual subgroups of the ON class, many of which contained as little as

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one compound within a given subgroup made generalizations regarding stability difficult.

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Comparison between SSP#1 and SSP#2. As might be expected in such a large

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study, some analytes did not have clear cut stability/lack of stability results. There were 68

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(13%) such analytes, 37 (4%) showed more stability in SSP#2, while 31 (4%) were less

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stable in SSP#2 compared to SSP#1. Propazine and procyazine showed modest though



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statistically significant changes, while methacrifos, prochloraz, fenfuram, and resmethrin

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produced more visible downward trending when included in Mix 2A compared to inclusion

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in Mix 1B. The statistics also show 15 analytes (3%) wherein the stability does not agree

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between SSP#1 and SSP#2 because they were not evaluated in both protocols. Although

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the compositions of the mixes changed between SSP#1 and SSP#2, it is unclear if these

335

compositional changes contributed to the observed changes in stability. Further studies are

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required to identify whether the conflicting results are attributable to differences in

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composition between SSP#1 and SSP#2, or to other possible factors such as different lots

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or ages of neat materials, solvents, or acetic acid used in preparation of the standards, or

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purely to statistical chance when dealing with such a large number of compounds.

340

This study showed degradation of some analytes resulted in enhancement of others.

341

Precursor analytes that degraded into target pesticides and metabolites are listed in Table 4.

342

While preparing these analytes in separate mixes provides a means of reducing

343

instrumental response changes, stability of the product analyte cannot necessarily be

344

shown, as the product analyte may also be subject to degradation. We observed this with

345

3,4-dichloroaniline (Figures 5 and 6). In Mix 1A, 3,4-dichloroaniline significantly

346

degraded, which prompted its placement into Mix 2B for SSP#2. However, Mix 2B also

347

contained the phenylurea herbicides diuron, linuron, and neburon, all of which were in Mix

348

1B and not in mix 1A with the 3,4-dichloroaniline for SSP#1. The ureas degraded rapidly

349

as expected, and consequently the response of 3,4-dichloroaniline increased (Figure 6).

350

Phenylurea herbicides have been reported to degrade into aniline-based products, which

351

has prompted inclusion of such products for residue screening.20 We observed the reverse

352

effect between fenitrothion and its primary degradation product 3-methyl-4-nitrophenol. In


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Page 17 of 51


353

SSP#1, both analytes were in Mix 1A, and fenitrothion rapidly deteriorated, resulting in

354

enhancement of the 3-methyl-4-nitrophenol signal. The two were separated in SSP#2;

355

fenitrothion again rapidly degraded, however in Mix 2A, 3-methyl-4-nitrophenol produced

356

no statistically significant change.

357

Some pesticides with known degradation products resulting from plant or microbial

358

metabolism, such as OP pesticides, did not undergo degradation into their respective

359

oxygen analogs or sulfoxides within these studies. However, carbamate pesticides prone to

360

microbial degradation into sulfoxide and sulfone products, such as aldicarb and thiofanox,

361

produced significant sulfoxide enhancement between the treatments (Table 3). The source

362

of the oxygen responsible for this reaction was not identified.

363

Upon completion of the studies, there were effects observed not only related to the

364

controlled heat treatments, but also between evaluations for certain analytes, including the

365

IS. During the statistical analysis, response changes were observed across different

366

instruments for the d5-Atrazine. Within SSP#1, low p-values were observed for the IS

367

during analyses by GC-TOF/MS (0.06, slightly declining trend observed) and UHPLC-

368

MS/MS (0.01, with no trends observed).

369

systems (0.41 – 0.92), indicate that sample preparation is random, the apparent bias is

370

attributed to the acquisitions from the two specified instruments. Following the statistical

371

review of the RF data, ANOVAs were conducted across raw analyte area counts.

372

Statistically relevant degradation based upon the RF data from the GC-TOF/MS for 36

373

analytes demonstrated no trending from the respective analyte counts (0.14 ≤ p ≤ 0.87).

374

Likewise on the UHPLC-MS/MS, 50 analytes showed no statistical significance or

375

trending (0.08 ≤ p ≤ 0.97) after repeating the statistical analysis with response areas.

Since all other IS p-values from the other MS



376

Conversely, eleven analytes had area-based ANOVAs which produced statistically

377

significant low trends, which previously produced no response change with RF-based

378

ANOVA results.

379

For consistency, SSP#2 was handled statistically the same as SSP#1 to ensure valid

380

conclusions. ANOVAs and trend analyses were conducted across all RF and area response

381

data. The IS ANOVA produced a wide range of p-values (0.02 ≤ p ≤ 0.96), resulting in

382

inconsistencies for 181 analytes (34%) when using the RF in the ANOVAs.

383

Chromatographic effects across all acquiring technologies and ESI suppression for LC-MS

384

instruments were suspected of contributing to the bias. The use of more internal standards

385

may have offered more options for affected compounds, or helped diagnose the issue early

386

on in the study.

387

Statistically significant non-trending behavior suggested a similar effect occurred

388

for some analytes in addition to the IS. Cyromazine, fluoroxypur-meptyl, and nitrofen

389

produced this effect in one of the two evaluations (Table 3). There are no structural

390

similarities, nor are there target precursors known to degrade into these analytes.

391

A possible explanation is to consider what can happen to analytes when exposed to

392

various matrices. The inclusion of a matrix with injections into an instrument can induce

393

either a suppressive or enhanced effect on the analyte’s signal; matrix-analyte effects often

394

manifest as ion suppression in ESI source technology, while enhancement occurs as the

395

matrix binds to active sites within the GC inlet and column prior to detection.10

396

Additionally, GC-MS and LC-MS instruments with nominal mass detection can suffer

397

from lack of mass resolution, a phenomenon not observed in high resolution time-of-flight

398

or orbitrap mass spectrometers. Consequently, matrix components co-eluting with analytes


Page 18 of 51

Page 19 of 51


399

producing very similar masses can present as an additive peak on nominal mass

400

spectrometers. For pesticide residue analysis, this behavior requires the use of matrix

401

matched standards to normalize this effect for all target analytes, particularly when

402

isotopically labeled standard compounds are not available. However, in this study, no

403

matrix was introduced as part of the injection; therefore, a suggested explanation for the

404

observed behavior may be that the collective target analyte mixture is producing a similar

405

effect, suggested here as a concentration effect. Given that the composition of the mixtures

406

contained hundreds of compounds with known and presumably unknown by products, and

407

the studies were double randomized to reduce other sources of bias, the total mass injected

408

onto the column may have been high enough to produce this effect.

409

For compounds with previously reported chemical lability, such as urea and

410

phenylurea herbicides, it is reasonable to expect the response loss was due primarily to

411

chemical degradation within the mix based upon percent analyte losses exceeding 20%

412

with 95% confidence. For analytes with statistically significant but smaller overall losses

413

(0-10%), degradation cannot be verified without support from an orthogonal analytical

414

technique demonstrating the same effect with a different mode of separation and preferably

415

a different ionization technique, or clear support from structural assessments and reaction

416

mechanisms such as SN2, hydrolysis (if water is present), or amide reduction. Regardless

417

of the reason, the end result of the study requires removal of affected analytes from the

418

large mix into a more appropriate mix with a suitable storage solvent whereby neither

419

effect occurs, or, if that cannot be achieved, then more frequent preparation of the unstable

420

analytes solution.



421

For analyte reaction behavior that arguably falls outside of the Arrhenius

422

generalization, further evaluations using natural aging techniques offer more confidence

423

into specific behaviors under frozen conditions. Analyte stability reported within this study

424

cannot necessarily be directly translated across different compositions containing these and

425

other analytes; furthermore, different instruments of analysis and variations of

426

chromatographic methods may also influence how these analytes respond. Therefore it is

427

advised to conduct SSP evaluations anytime changes to a standards mixture and/or the

428

acquiring technology have occurred.

429 430 431

ACKNOWLEDGEMENTS The authors thank Daniel Badger (Supelco) for managing study related standards.

432

Jon Wong and Kai Zhang (FDA), Steven Zbylut, Mark Sewald, and Steven Murray

433

(Medallion Laboratories), and Jonathan DeVries (formerly of Medallion Laboratories) are

434

gratefully acknowledged for helpful dialog and discussions.

435 436

CONFLICT OF INTEREST DISCLOSURE

437

The authors declare no competing financial interest.

438 439

Supporting Information Available: Table S1: Analyte-specific masses used in the GC-

440

TOF/MS method; Tables S2-S4: Analyte-specific MS/MS parameters used in the (S2) GC-

441

MS/MS method, (S3) Waters UPLC-MS/MS method, and (S4) Agilent UHPLC-MS/MS

442

method; Table S5: Retention Times for analytes acquired by UHPLC-MS/MS and UPLC-

443

MS/MS; Table S6: Analytes with satisfactory stability. This material is available free of

444

charge via the Internet at http://pubs.acs.org.


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445 446


REFERENCES 1. Besamusca, E. W.; Vreeker, C. P.; Toonen, A. A.; deKok, A. Validation Stability

447

of Pesticide Standards. Presented at the European Pesticide Residue Workshop,

448

June 10-12, 1996.

449

2. Stefanelli, P.; Barbini, D. A,; Amendola, G.; Generali, T.; Girolimetti, S.; Pelosi, P.;

450

Santilio, A. A Survey on Long-Term Stability of Stock Standard Solutions

451

in Pesticide Residue Analysis. Food Anal. Methods. 2014, 8, 1164-1172.

452

3. AOAC International. Guidelines for laboratories Performing Microbiological and

453

Chemical Analyses of Food and Pharmaceuticals. AOAC International: Maryland,

454

2010; p 29.

455 456 457 458 459

4. Bajaj, S., Singla, D. Sakhuja, N. Stability Testing of Pharmaceutical Products. J. App. Pharm. Sci. 2015, 2, 129-138. 5. Carr, R. W. Elements of Chemical Kinetics. In Modeling of Chemical Reactions. Carr. R. W. Ed.; Elsevier: The Netherlands, 2007; pp 43-100. 6. Rokugawa, H.; Fujikawa, H. Evaluation of a New Maillard Reaction Type Time-

460

Temperature Integrator at Various Temperatures. Food Control. 2015, 57, 355-

461

361.

462

7. Palmers, S.; Grauwet, T.; Celus, M.; Wibowo, S.; Kebede, B. T.; Hendrickx, M. E.

463

Loey, A. V. A Kinetic Study of Furan Formation During Storage of Shelf-Stable

464

Fruit Juices. J. Food Engineer. 2015, 165, 74-81.

465

8. Taoukis, P.S.; Labuza, T. P. Time Temperature Indicators (TTIs). In Novel Food

466

Packaging Techniques. Ahvenainen, R. Ed.; CRC Press: Florida, 2000; pp 103-

467

126.



468 469

9. Anderson, G., Scott, M. Determination of Product Shelf Life and Activation Energy for Five Drugs of Abuse. Clin. Chem. 1991, 37, 398-402.

470

10. Mastovska, K.; Dorweiler, K. J.; Lehotay, S. J.; Wegscheid, J. S.; Spzylka, K. A.

471

Pesticide Multiresidue Analysis in Cereal Grains Using Modified QuEChERS

472

Method Combined with Automated Direct Sample Introduction GC-TOFMS and

473

UPLC-MS/MS Techniques. J. Agric. Food Chem. 2009, 58, 5959–5972.

474

11. Zweigenbach, J.; Flanagan, M.; Stone, P.; Glauner, T.; Zhao, L. Multi-Residue

475

Pesticide Analysis with Dynamic Multiple Reaction Monitoring and Triple

476

Quadrupole LC/MS/MS. Agilent Technologies, Inc. 2009 5990-4253EN.

477

12. Mastovska, K.; Lehotay, S. J. Evaluation of common organic solvents for gas

478

chromatographic analysis and stability of multiclass pesticide residues. J.

479

Chromatogr. A 2004, 1040, 259-272.

480 481 482 483 484

13. Snyder, L. R. Classification off the Solvent Properties of Common Liquids. J. Chromatogr. Sci. 1978, 16, 223-234. 14. Fukuto, T. R. Mechanism of Action of Organophosphorus and Carbamate Insecticides. Environ. Health Perspect.1990, 87, 245-254. 15. Cordero, B., Gómez, V., Platero-Prats, A. E., Revés, M., Echeverría, J., Cremades,

485

E., Barragán, F., Alvarez, S. Covalent radii revisited. J. Chem. Soc., Dalton Trans.

486

2008, 21, 2832-2838.

487

16. Gatidou, G.; Iatrou, E. Investigation of photodegradation and hydrolysis of selected

488

substituted urea and organophosphate pesticides in water. Environ. Sci. and Pollut.

489

Res. 2011, 18, 949-957.


Page 22 of 51

Page 23 of 51


490

17. Amine-Khodja, A.; Boulkamh, A.; Boule, P. Photochemical behaviour of

491

phenylurea herbicides. Photochem. And Photobio. Sci. 2004, 3, 145-156.

492

18. Cullington, J. E.; Walter, A. Rapid Biodegradation of Diuron and other Phenylurea

493

Herbicides by a Soil Bacterium. Soil Biology and Biochem.1999, 31, 677-686.

494

19. ElGouzi, S.; Draoui, K.; Chtoun, E. H.; Mingorance, M. D.; Pena, A. Changes in

495

the persistence of two phenylurea herbicides in two Mediterranean soils under

496

irrigation with low- and high-quality water: A laboratory approach. Sci. Total

497

Environ.2015, 538, 16-22.

498

20. De Kok, A.; Vos, Y. J.; Van Garderen, C.; De Jong, T.; Van Optstal, M.; Frei, R.

499

W.; Geerdink, R. B.; Brinkman, U. A. Th. Chromatographic determination of

500

phenylurea herbicides and their corresponding aniline degradation products in

501

environmental samples. I. J. Chromatogr. A 1984, 288, 71-89.

502

21. Muehlebach, M.; Cederbaum, F.; Cornes, D.; Friedmann, A. A.; Glock, J.; Hall, G.;

503

Indolese, A. F.; Kloer, D. P.; Goupil, G. L.; Maetzke, T.; Meier, H.; Schneider, R.;

504

Stoller, A.; Szczepanski, H.; Wendeborn, S.; Widmer, H. Aryldiones incorporating

505

a [1,4,5]oxadiazepane ring. Part 2: Chemistry and biology of the cereal herbicide

506

pinoxaden. Pest Manage. Sci. 2011, 67, 1499-1521.

507

22. Kar, A.; Mandal, K.; Singh, B. Environmental Fate of Chlorantraniliprole Residues

508

on Cauliflower Using QuEChERS Technique. Environ. Monit. Assess. 2013, 185,

509

1255-1263.

510 511

23. Wood, A. Compendium of Common Names. http://www.alanwood.net/pesticides/index.html.



512

24. Guo, B.; Huang, Z.; Wang, M.; Wang, X.; Zhang, Y.; Chen, B.; Li, Y.; Yan, H.;

513

Yao, S. Simultaneous direct analysis of benzimidazole fungicides and relevant

514

metabolites in agricultural products based on multifunction dispersive solid-phase

515

extraction and liquid chromatography–mass spectrometry. J. Chromatogr. A 2010,

516

1217, 4796-4807.

517

25. Iesce, M. R.; Della Greca, M.; Cermola, F.; Rubino, M.; Isidori, M.; Pascarella, L.

518

Transformation and ecotoxicity of carbamic pesticides in water. Environ. Sci.

519

Pollut. Res. 2006, 13, 105-109.

520

26. Jones, R. L.; Hunt, T. W.; Norris, F. A.; Harden, C. Field research studies on the

521

movement and degradation of thiodicarb and its metabolite methomyl. J. Contam.

522

Hydrol. 1989, 4, 359-371.

523

27. Durand, G.; Bertrand, N.; Barcelo, D. Applications of thermospray liquid

524

chromatography-mass spectrometry in photochemical studies of pesticides in water.

525

J. Chromatogr. A 1991, 554, 233-250.

526

28. Abbott, P.J.; Barabas, K.; Black, A.L.; Borzelleca, J.F.; Campbell, P.J.; Costa,

527

L.G.; Dobson, S.; Dewhurst, I.; Drevenkar, V.; Erickson, W.; Finizio, A.;

528

Garvey, K.; Kocialski, A.B.; Moretto, A.; Pelkonen, O.; Ray, D.; Temmink, J.H.M.;

529

Adcock, J.W.; Arnold, D.; Bellet, E.; Chart, J.; Egli, H.; Harvey, P.; Krinke, G.;

530

McReath, A.; Scheffler, H.; Smith, A.E.; Harrison, L.; Herrman, J.L.; Jenkins, P.G.;

531

McGregor, D.; Plestina, R.; Smith, E.; Toft, P. Demeton-S-methyl. Environ.

532

Health Crit. 1997, 197, 1-76.


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FIGURE CAPTIONS

Figure 1. Structures and extracted ion chromatograms of diethyl and dimethyl phosphorothioates (a) parathion and parathion-methyl; (b) chlorpyrifos and chlorpyrifos-methyl; (c) bromophos-ethyl and bromophos obtained from stability study protocol #2 by GC-TOF/MS analysis.

Figure 2. Structures of organophosphates evaluated in stability study protocols.

Figure 3. Structures and extracted ion chromatograms of azinphos-ethyl and azinphos-methyl demonstrating satisfactory stability from stability study protocol #2, obtained by UPLC-MS/MS analysis.

Figure 4. Apparent stability of organonitrogen analytes categorized by functional group classification.23

Figure 5. Select organonitrogen analytes with multiple functional groups evaluated in stability study protocols.

Figure 6. Structures of phenylurea herbicides and 3,4-dichloroaniline degradation product.



Figure 7. Response changes for 3,4-dichloroaniline between simulated 0m, 6m, 12m, and 24m treatment groups at 50°°C.


Page 26 of 51

Page 27 of 51


Table 1. Composition of pesticides, metabolites, and contaminants in stability study protocols. Analyte Name 1,2,3,4 -Tetrachlorobenzene 1,2,4,5 -Tetrachlorobenzene 2,3,5,6-Tetrachloroaniline 2,3,5-Trimethacarb 2,6-Diethylaniline 2-Ethylhexyldiphenyl phosphate 3,4-Dichloroaniline 3-Ketocarbofuran 4,4′-Dicofold Acetamiprid Acibenzolar-S-methyl Akton Alanycarb Aldicarb sulfone Aldrin Aminocarb Aramite Atrazine-desisopropyl Azamethiphos Azinphos-methyl Beflubutamid Bendiocarb Benfuracarb Benoxacor Benzoximate BHC alpha BHC delta Bifenox Bioallethrinc Bitertanol Bromacil Bromophos-ethylb Bromuconazole Buprofezin Butafenacil Butocarboxim-sulfoxide Buturon Cadusafos Captan Carbendazim Carbofuran Carbosulfan Carfentrazone-ethyl

SSP#1 Mix 1A 1A 1A 1B 1B 1B 1A 1B 1A 1B 1B 1A 1B 1B 1A 1B 1B 1B 1B 1B 1B 1B 1B 1A 1B 1A 1A 1B 1B 1B 1A 1A 1B 1B 1B 1B 1B 1B 1A 1B 1B 1B 1A

SSP#2 Mix 2A 2A 2A 2A 2B 2A 2B 2A 2B 2A 2A 2A 2B 2A 2A 2B 2A 2A 2B 2A 2A 2A 2B 2A 2B 2A 2A 2A 2B 2A 2A 2A 2A 2A 2A 2B 2B 2A 2B 2B 2B 2B 2A

Analyte Name 1,2,3,5 -Tetrachlorobenzene 1-Naphthol 2,3,5,6-Tetrachloroanisole 2,6-Dichlorobenzamide 2,6-Diisopropylnaphthaleneb 3,4,5-Trimethacarb 3-Hydroxycarbofuran 3-Methyl-4-nitrophenol Acephate Acetochlor Aclonifen Alachlor Aldicarb Aldicarb sulfoxide Ametryn Amitraz Atrazine Azaconazole Azinphos-ethyl Azoxystrobin Benalaxyl Benfluralin Benodanil Bensulide Benzoylprop-ethyl BHC beta Bifenazate Bifenthrin Bioresmethrin Boscalid Bromophos Bromopropylate Bupirimate Butachlor Butocarboxim Butralin Butylate Captafol Carbaryl Carbetamide Carbophenothion Carboxin Chinomethionate


SSP#1 Mix 1A 1A 1A 1B 1A 1B 1B 1A 1B 1A 1B 1A 1B 1B 1B 1B 1B 1B 1B 1B 1B 1A 1A 1B 1A 1A 1B 1A 1A 1A 1A 1A 1B 1A 1B 1B 1B 1A 1B 1B 1A 1B 1A

SSP#2 Mix 2A 2B 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2B 2B 2A 2B 2A 2B 2A 2A 2B 2A 2A 2A 2A 2A 2B 2A 2B 2A 2B 2A 2B 2A 2A 2A 2A 2A 2A 2B 2B 2B 2A


Chlorantraniliprole Chlorbromuron Chlordane, cis Chlordene, transChlorethoxyfos Chlorfenson Chlorflurazon Chloridazon Chlorobenzilate Chloropropylate Chlorotoluron Chlorpropham Chlorpyrifos oxon Chlorthal-dimethyl Chlorthiophos Clethodim Clomazone Coumaphos Crufomate Cyanophenphos Cyazofamid Cycloxydim Cyfluthrin Cyhalothrin Cypermethrin Cyprazine Cyprodinil DDD, o, p'DDE, o, p'DDT, o,p'DEF Demeton-O Demeton-S-Methyl Sulfone Desmetryn Diallate Diazinon oxon Dichlobenil Dichlofluanidd Diclobutrazol Dicloran Dieldrin Diethofencarb Difenoxurone Diflufenicanc Dimethachlor Dimethametryn Dimethoate Dimetilan

1B 1B 1A 1A 1A 1A 1B 1B 1A 1A 1B 1A 1B 1A 1A 1B 1B 1B 1B 1A 1B 1B 1A 1A 1A 1B 1B 1A 1A 1A 1B 1A 1B 1B 1A 1B 1A 1A 1B 1A 1A 1B 1B 1B 1B 1B 1B 1B

2B 2B 2A 2A 2A 2A 2A 2A 2A 2A 2B 2A 2B 2A 2A 2B 2A 2A 2A 2A 2B 2B 2A 2A 2A 2B 2A 2A 2A 2A 2A 2B 2B 2A 2A 2B 2A 2B 2A 2A 2A 2A 2B 2B 2A 2A 2A 2A

Chlorbenside Chlorbufamc Chlordane, trans Chlordimeform Chlorfenapyr Chlorfenvinphos Chlorflurenol methyl ester Chlormephos Chloroneb Chlorothalonil Chloroxuron Chlorpyrifosb Chlorpyrifos-methyl Chlorthiamid Cledinafop-propargyl Clofentezine Clothianidin Crotoxyphos Cyanazine Cyanophosb Cycloate Cycluron Cyhalofop-butyl Cymoxanil Cyphenothrin Cyproconazole Cyromazine DDD, p, p'DDE, p, p'DDT, p, p'Deltamethrin Demeton-S Desmedipham Dialifos Diazinon Dicapthon Dichlofenthion Dichlorvos Diclofop-methyl Dicrotophos Diethatyl-ethyl Difenoconazole Diflubenzuron Dimefuron Dimethametryn Dimethenamide Dimethomorph Dimoxystrobin


Page 28 of 51

1A 1B 1A 1A 1A 1A 1A 1A 1A 1A 1B 1B 1A 1B 1B 1B 1B 1B 1B 1A 1A 1B 1A 1B 1A 1B 1B 1A 1A 1A 1B 1A 1B 1A 1B 1A 1A 1B 1A 1B 1A 1B 1B 1B 1B 1A 1B 1B

2A 2B 2A 2B 2A 2A 2A 2A 2A 2B 2B 2A 2B 2B 2A 2A 2A 2B 2A 2B 2A 2B 2A 2B 2A 2A 2B 2A 2A 2A 2A 2A 2B 2A 2B 2B 2A 2B 2A 2A 2A 2A 2A 2B 2A 2A 2A 2A

Page 29 of 51


Diniconazole Dinotefuran Dioxathion Diphenylamine Disulfoton Disulfoton sulfoxide Diuron Endosulfan beta Endosulfan sulfate Endrin Endrin ketone Epoxiconazole EPTCc Ethalfluralin Ethiofencarb Ethiofencarb sulfoxide Ethiprole Ethoprophos Etofenprox Etridiazole Famphur Fenamiphos Fenamiphos sulfoxide Fenazaquin Fenchlorphos Fenfuram Fenitrothion Fenoxaprop ethyl ester Fenpiclonil Fenpropimorph Fenson Fenthiocarbc Fenuron Fipronil Fipronil Sulfone Flonicamid Fluchloraline Fludioxonil b Flufenoxuron Fluoroglycofen-ethyl Fluquinconazole Flurochloridone Flurtamone Flutolanil Fluvalinate Fomesafen Forchlorfenuron Fuberidazole

1B 1B 1A 1A 1A 1B 1B 1A 1A 1A 1A 1B 1A 1A 1B 1B 1B 1A 1A 1A 1B 1A 1B 1B 1A 1B 1A 1B 1B 1B 1A 1B 1B 1A 1A 1B 1A 1A 1B 1B 1B 1A 1B 1B 1B 1B 1B 1B

2A 2A 2B 2A 2A 2A 2B 2A 2B 2A 2A 2A 2B 2A 2B 2A 2A 2A 2A 2A 2B 2A 2A 2A 2B 2A 2B 2A 2B 2A 2A 2B 2B 2A 2A 2A 2A 2A 2B 2A 2A 2A 2B 2B 2A 2A 2A 2A

Dinitramine Dioxacarb Diphenamid Dipropetryn Disulfoton sulfone Ditalimfosa Edifenfos Endosulfan Ether Endosulfan,alpha Endrin Aldehydec EPN Eprinomectin Etaconazole Ethidimuron Ethiofencarb sulfone Ethion Ethofumesate Ethoxyquin Etoxazole Etrimfos Fenamidone Fenamiphos sulfone Fenarimol Fenbuconazol Fenchlorphos oxon Fenhexamid Fenobucarb Fenoxycarb Fenpropathrin Fenproximate Fensulfothion Fenthion Fenvalerate / Esfenvalerate Fipronil Sulfide Flamprop-methyl Fluazifop-p-butyl Flucythrinate Flufenacet Fluometuron Fluoxastrobin Fluridone Fluroxypyr-meptyl Flusilazole Flutriafol Folpet Fonofos Fosthiazate Furalaxyl


1B 1B 1A 1B 1B 1B 1B 1A 1A 1A 1A 1B 1B 1B 1B 1A 1A 1A 1B 1B 1B 1B 1A 1B 1A 1B 1B 1A 1A 1B 1B 1A 1A 1A 1A 1A 1B 1B 1B 1B 1B 1A 1B 1B 1A 1A 1A 1B

2A 2A 2A 2A 2A --2A 2A 2A 2B 2A 2A 2A 2A 2A 2A 2A 2B 2A 2B 2A 2A 2A 2A 2B 2A 2A 2A 2A 2A 2A 2B 2A 2A 2A 2A 2A 2B 2B 2A 2A 2A 2A 2A 2B 2A 2A 2B


Furathiocarb Halofenozide Haloxyfop-2-ethoxy ethyl Heptachlor epoxide Hexachlorobenzene Hexaflumuron Hexythiazox Iodofenphos Imazamethabenz-methyl Indoxacarb Iprobenfos Iprovalicarb Isocarbamid Isoprocarb Isoprothiolane Isoxaben Isoxaflutole Kresoxim-methyl Leptophos Linuron Malaoxon Mandipropamid Mefancet Mepanipyrim Mepronil Metalaxyl Metconazole Methacrifos Methidathionb Methomyl Methoxychlor Methoxyfenozide Metobromuron Metolcarb Metrafenone Mevinphos MGK 264 Molinate Monolinuron

1B 1B 1B 1A 1A 1B 1B 1A 1B 1B 1B 1B 1A 1B 1B 1B 1A 1B 1A 1B 1B 1B 1A 1B 1A 1B 1B 1B 1B 1B 1A 1B 1B 1B 1B 1A 1B 1B 1B

2A 2B 2A 2A 2A 2A 2A 2B 2A 2A 2A 2A 2A 2A 2A 2A 2B 2A 2B 2B 2B 2A 2A 2A 2A 2A 2A 2A 2A 2B 2A 2A 2B 2A 2B 2B 2B 2A 2B

Myclobutanil

1B

2A

Naphthalene acetamide Neburon Nitralin Nitrofen Nonachlor, transNorflurazon Nuarimol

1B 1B 1B 1A 1A 1B 1B

2A 2B 2A 2A 2A 2A 2A

Halfenprox Haloxyfop-methyl Heptachlor Heptenophos Hexaconazole Hexazinone Hydramethylnon Imazalil Imidacloprid Ipconazole Iprodione Isazophos Isofenphos Isopropalin Isoproturon Isoxadifen-ethyl Isoxathion Lactofen Lindane Lufenuron Malathion MCPA (as methyl ester) Mefepyr-diethyl Mephosfolan Merphos Metazachlor Methabenzthiazuron Methamidophos Methiocarb Methoprotryne Methoxychlor olefin, p, p′ Methyl paraoxon Metolachlor Metoxuron Metribuzin Mexacarbatec Mirex Monocrotophos Monuron N,N-Diallyl dichloroacetamide Napropamide Nitempyrama Nitrapyrin Nonachlor, cisNorea Novaluron Omethoate


Page 30 of 51

1A 1B 1A 1B 1B 1B 1B 1B 1B 1B 1B 1A 1A 1A 1B 1A 1B 1B 1A 1B 1B 1A 1A 1B 1B 1B 1B 1B 1B 1B 1A 1B 1B 1B 1A 1A 1A 1B 1B

2A 2B 2A 2B 2A 2A 2B 2A 2A 2A 2A 2B 2A 2A 2B 2A 2B 2A 2A 2A 2B 2B 2A 2B 2B 2B 2B 2A 2A 2B 2A 2B 2A 2B 2A 2B 2A 2A 2B

1B

2A

1B 1B 1A 1A 1B 1B 1B

2A --2A 2A 2B 2A 2A

Page 31 of 51


O-Phenylphenol Oxadixyl Oxycarboxin Oxyfluorfen Paraoxon Parathion-methyl PCB 18 Pebulate Pencycuron Pentachloroaniline Pentachlorobenzne Pentachlorophenyl methyl sulfide Perthane Phenothrin Phorate Phorate sulfoxide Phosmet Phoxim Picoxystrobin Piperalin Piperophos Pirimiphos-ethyl Prallethrin Prochloraz Procymidone Profluralin Prometon Propachlorc Propaquizafop Propazine Propham Propoxur Prosulfocarb Pymetrozine Pyraclofos Pyraflufen Ethyl Pyridaben Pyrifenoxc Pyriproxyfen Quinclomine Quizalfop ethyl Rotenone Sebuthylazine-desethyl Sethoxydim Silthiopham Simetryn Spinosad A + D Spiromefesin

1A 1B 1B 1A 1B 1A 1A 1B 1B 1A 1A 1A 1A 1A 1A 1B 1B 1B 1B 1B 1B 1A 1B 1B 1A 1A 1B 1A 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1A 1B 1B 1B 1B 1A 1B 1B 1B

2A 2B 2A 2A 2A 2B 2A 2A 2B 2A 2A 2A 2A 2A 2A 2A 2B 2A 2B 2A 2B 2A 2A 2A 2A 2A 2A 2B 2A 2A 2A 2A 2B 2A 2A 2B 2A 2B 2A 2A 2A 2B 2A 2B 2B 2A 2A 2B

Oxadiazon Oxamyl Oxydemeton-methyl Paclobutrazol Parathion PCB 153 PCB 52 Penconazol Pendimethalin Pentachloroanisole Pentachloronitrobenzene Permethrin Phenmedipham Phenthoate Phorate sulfone Phosalone Phosphamidon Picolinafen Pinoxaden Piperonyl butoxide Pirimicarb Pirimiphos-methyl Pretilachlor Procyazine Profenofos Promecarb Prometryn Propanil Propargite Propetamphos Propiconazole Propyzamide Prothiofos Pyracarbolid Pyraclostrobin Pyrazophos Pyridaphenthion Pyrimethanil Quinalphosc Quinoxyfen Resmethrinb Sebuthylazine Secbumeton Siduron Simazine Spinetoram Spirodiclofen Sulfallate


1A 1B 1B 1B 1A 1A 1A 1B 1A 1A 1A 1B 1B 1A 1A 1B 1B 1B 1B 1B 1A 1A 1B 1B 1B 1B 1B 1B 1A 1A 1B 1A 1B 1B 1B 1B 1B 1B 1A 1B 1B 1B 1B 1B 1B 1B 1B 1A

2A 2A 2B 2A 2A 2A 2A 2A 2A 2A 2A 2A 2B 2B 2A 2B 2B 2B 2B 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2B 2A 2A 2A 2A 2A 2A 2B 2B 2A


Sulfenone Sulprofos TCMTB Tebufenozide Tebupirimfos Tebuthiuron Teflubenzuron Temephos Tepraloxydim Terbufos Terbufos sulfoxide Terbuthlazine-desethyl Tetrachlorvinphos Tetradifon Tetrasul Thiacloprid Thidiazuron Thiodicarb Thiofanox sulfoxide Thiophanate-methyl Tolylfluanid Transfluthrin Triadimenol Triazophosc Trichlornat Trietazine

1A 1A 1B 1B 1B 1B 1B 1B 1B 1A 1B 1B 1B 1A 1A 1B 1B 1B 1B 1B 1B 1A 1B 1B 1A 1A

2A 2B 2A 2A 2A 2A 2B 2B 2B 2A 2B 2A 2A 2A 2A 2B 2A 2B 2A 2B 2B 2A 2A 2B 2A 2A

Trifluralin

1A

2A

Triticonazole Uniconazole-P Vinclozolin

1A 1B 1A

2A 2A 2A

a

c

Sulfotep Sulprofos sulfoxide Tebuconazole Tebufenpyrad Tebutam Tecnazene Tefluthrin TEPP Terbacil Terbufos sulfone Terbumeton Terbuthylazine Tetraconazole Tetramethrin Thiabendazole Thiazopyr Thiobencarb Thiofanox Thionazin Tolclofos-methyl Tralkoxydim Triadimefon Tri-allate Trichlorfon Tricyclazole Trifloxystrobin Tris-(1,3-dichloro-isopropyl) phosphate Tycor Venolate Zoxamide

Page 32 of 51

1B 1A 1B 1B 1A 1A 1A 1B 1A 1A 1B 1B 1B 1B 1B 1A 1B 1B 1A 1A 1B 1A 1A 1B 1B 1B

2B 2A 2A 2A 2A 2A 2A 2B 2A 2A 2A 2A 2A 2A 2A 2A 2A 2A 2B 2B 2B 2A 2A 2B 2A 2A

1B

2A

1B 1B 1A

2B 2A 2A

Analyte not included in second study. bAnalysis conducted on two analytical instruments.

Analyte not acquired in the first study. dAnalyzed using metabolite.


Page 33 of 51


Table 2. Storage times of ampoules at 50°°C for accelerated aging of standards. Ampoule Numbers 1-3 4-6 7-9 10 - 12

Treatment Group 0m 6m 12m 24m

Duration in days at 50°°C 0 1.5 3 6

Simulated Time Point (Month) Zero Six Twelve Twenty-Four



Page 34 of 51

Table 3. Analytes reported to have statistically significant changes in response with 95% confidence using accelerated aging protocols to simulate six, twelve, and twenty-four month storage at -20°°C.

Analyte Name

Mix

1-Naphthol

1Aa

2,6-Diethylaniline

1Bc

Percent Change versus Control in SSP #1 Trend 6m 12m 24m p-value slope -53% -68% -17% -2.9E-03 5.4E-05 -10%

-59%

-36%

-1.7E-02

2Ba

Percent Change versus Control in SSP #2 Trend 6m 12m 24m p-value slope -36% -52% -53% -2.0E-02 1.2E-09

9.3E-08

2Ba

-46%

-71%

-60%

-2.3E-02

4.7E-12

d

Mix

3,4-Dichloroaniline 3-Methyl-4-Nitrophenol Aclonifen Aldicarb

a

1A 1Ad 1Bg 1Bc

-42% 38% 8% -8%

-63% 52% 4% -15%

-87% 91% -1% -23%

8.1E+00 1.1E+01 1.6E+00 2.8E+00

8.1E-15 1.0E-05 9.5E-02 3.8E-10

2B 2Ae 2Ac 2Bc

25% -10% -7% -4.1%

34% -10% -9% -10.9%

52% -10% -8% -23.8%

2.1E-02 -3.4E-03 -3.0E-03 -1.0E-02

5.2E-06 3.3E-01 4.4E-02 2.4E-10

Aldicarb sulfone Aldicarb sulfoxide

1Be 1Ba

-2% -23%

-6% -38%

-10% -56%

1.4E+00 6.1E+00

1.8E-01 1.3E-19

2Ac 2Ba

-6% -29%

-11% -46%

-11% -69%

-4.3E-03 -2.8E-02

8.4E-03 3.2E-27

Aldrin Aminocarb Amitraz

1Ac 1Bc 1Ba

-2% -4% -70%

-8% -11% -66%

-9% -7% -97%

1.2E+00 1.7E-01 6.4E+00

7.7E-03 2.5E-03 1.2E-11

2Ag 2Bc 2Ba

1% -5.1% -84%

-1% -5.7% -98%

-4% -9.8% -100%

-1.9E-03 -3.7E-03 -3.6E-02

6.2E-01 8.0E-04 2.3E-40

Atrazine Azaconazole Azamethiphos Benfuracarb Bentazon-methyli Benzoximate Bifenazate Bioallethrini

1Be 1Bf 1Bc 1Ba --1Bc 1Ba ---

-1% 1% -5% -100% ---4% -59% ---

1% 2% -8% -100% ---5% -94% ---

-8% 4% -15% -100% ---10% -96% ---

1.6E+00 -5.2E-01 2.1E+00 -7.2E-04 --1.2E+00 5.1E+00 ---

1.4E-01 2.6E-01 1.1E-04 2.6E-35 --4.1E-08 6.6E-11 ---

2Ac 2Bd 2Bc 2Ba 2Bh 2Bb 2Ba 2Be

-4% 3.7% -6.7% -99% -3% -12.4% -98% -3%

-9% 5.2% -11.2% -99% 15% -13.5% -99% -1%

-11% 5.2% -18.4% -99% -5% -16.1% -99% -8%

-4.5E-03 1.9E-03 -7.5E-03 -3.3E-02 -9.6E-04 -5.7E-03 -3.3E-02 1.2

5.0E-03 2.8E-03 2.4E-12 1.4E-30 7.9E-03 1.4E-12 1.7E-40 1.1E-01

Bioresmethrin Bromophos

1Ac 1Aa

-6% -35%

-12% -52%

-15% -81%

1.4E+00 8.8E+00

3.5E-03 1.8E-21

2Bc 2Bb

-4.1% -12.0%

-5.9% -22.7%

-15.2% -41.0%

-6.2E-03 -1.7E-02

7.7E-03 2.7E-12


Page 35 of 51


Bupirimate Butocarboxim-sulfoxide Buturon

1Be 1Bh 1Ba

-3% 4% -89%

-2% 5% -92%

-3% -3% -93%

6.8E-02 1.7E+00 6.1E-01

3.1E-01 4.1E-08 3.9E-43

2Bc 2Bh 2Ba

-0.7% 14.0% -89%

-2.0% 8.8% -91%

-5.9% -4.0% -93%

-2.5E-03 -3.3E-03 -3.1E-02

1.2E-03 3.2E-09 2.1E-47

Captan Carbendazim Carbetamide Carbofuran

1Ah 1Bd 1Bd 1Bd

13% 24% 5% 28%

7% 26% 7% 20%

-7% 5% 8% 39%

3.8E+00 4.6E+00 -4.9E-01 -3.2E+00

3.4E-03 7.7E-09 1.5E-06 1.3E-09

2Bb 2Bd 2Bd 2Bd

-11.0% 35% 2.7% 8.8%

-14.6% 26% 4.5% 8.4%

-16.3% 6% 6.0% 10.0%

-6.1E-03 -1.2E-03 2.4E-03 3.4E-03

3.5E-02 3.5E-12 4.0E-02 4.1E-03

Carbophenothion Carbosulfan

1Ac 1Ba

0% -100%

-4% -100%

-13% -100%

2.5E+00 1.5E-03

4.8E-02 5.6E-25

2Bg 2Ba

-1% -55%

-1% -16%

-1% 76%

-4.0E-04 4.0E-02

9.6E-01 6.6E-10

Carboxin Chlorantraniliprole Chlorbromuron Chlordimeform

1Bc 1Bb 1Ba 1Ac

-2% -15% -23% -8%

-8% -18% -36% -14%

-4% -31% -48% -13%

-6.4E-04 3.3E+00 4.4E+00 6.9E-01

2.3E-02 2.3E-04 2.0E-24 2.4E-05

2Bg 2Bb 2Ba 2Bb

3% -9.9% -33% -10.8%

4% -12.3% -46% -14.0%

1% -16.3% -62% -20.9%

3.6E-04 -6.2E-03 -2.4E-02 -8.1E-03

3.0E-01 1.6E-10 4.3E-26 1.4E-06

Chlorflurazon Chlorothalonil

1Bc 1Ab

-4% -15%

-2% -29%

-8% -26%

1.1E+00 1.3E+00

4.2E-02 7.2E-10

2Af 2Ba

7% -34%

5% -41%

7% -46%

2.3E-03 -1.7E-02

8.1E-01 5.9E-12

Chlorotoluron Chloroxuron Chlorpyrifos oxon Chlorpyrifos-methyl

1Ba 1Ba 1Bc 1Aa

-46% -49% -5% -28%

-65% -67% -6% -43%

-83% -83% -10% -70%

6.4E+00 6.0E+00 1.1E+00 7.9E+00

1.5E-33 1.6E-33 1.2E-07 8.3E-20

2Ba 2Ba 2Bc 2Bc

-55% -60% -7.7% -8.6%

-74% -77% -6.6% -18.7%

-91% -93% -8.5% -34.3%

-3.5E-02 -3.5E-02 -2.9E-03 -1.4E-02

3.1E-42 8.9E-34 2.1E-05 3.1E-10

Clethodim Crotoxyphos

1Bc 1Bc

-6% -2%

-12% -3%

-17% -7%

2.0E+00 9.3E-01

4.6E-04 1.4E-06

2Be 2Bc

-4.2% -2.0%

-2.9% -4.0%

-8.8% -9.6%

-3.3E-03 -4.0E-03

8.9E-02 4.9E-08

Cyanazine Cyanophos Cyazofamid Cycloxydim Cycluron Cyprazine

1Be 1Ab 1Bb 1Bc 1Bc 1Bc

-6% -19% -16% -5% -7% -5%

-7% -31% -27% -12% -12% -3%

-7% -55% -47% -19% -24% -7%

1.8E-01 5.2E+00 6.0E+00 2.4E+00 3.3E+00 6.3E-01

7.1E-01 6.3E-15 4.9E-29 1.5E-03 1.9E-12 4.4E-04

2Ac 2Bc 2Bb 2Bc 2Bc 2Bc

-4% -8.5% -12.3% -0.8% -7.7% -2.5%

-3% -13.1% -21.0% 0.0% -12.8% -4.7%

-10% -27.9% -40.1% 0.0% -24.6% -11.2%

-3.7E-03 -8.3E-03 -1.6E-02 1.2E-04 -1.0E-02 -4.7E-03

4.3E-03 4.0E-04 9.7E-18 2.3E-02 2.0E-12 1.5E-06

Cyromazine Demeton-O

1Bd 1Aa

4% -42%

6% -72%

3% -90%

3.6E-01 7.9E+00

1.2E-05 4.8E-29

2Bh 2Ba

6.7% -49%

6.4% -73%

-0.7% -92%

-9.7E-04 -3.6E-02

5.6E-03 2.7E-27



Page 36 of 51

Demeton-S-methyl sulfone Desmedipham Diazinon

1Bc 1Bc 1Bc

-4% -7% -4%

-5% -8% -4%

-11% -16% -8%

1.3E+00 2.0E+00 8.2E-01

1.5E-10 1.3E-08 8.0E-05

2Bc 2Bc 2Bc

-6.1% -2.8% -4.3%

-8.4% -3.4% -5.7%

-14.1% -4.1% -13.0%

-5.6E-03 -1.5E-03 -5.2E-03

4.9E-10 2.1E-02 3.5E-06

Diazinon oxon Dicapthon Dichlofluanid-DMSA metabolite Dichlorvos Difenoxurone

1Bc 1Aa

-4% -49%

-6% -68%

-10% -94%

1.3E+00 8.2E+00

2.2E-03 2.3E-19

2Bc 2Ba

-4.4% -22%

-8.6% -40%

-18.7% -63%

-7.8E-03 -2.6E-02

9.2E-13 1.6E-15

1Ad

9%

10%

21%

-2.5E+00

4.2E-03

2Bd

27%

34%

39%

1.5E-02

2.5E-06

1Bc 1Ba

-9% -49%

-16% -67%

-30% -84%

4.0E+00 6.1E+00

1.8E-18 2.6E-31

2Bb 2Ba

-9.6% -61%

-15.5% -78%

-31.1% -94%

-1.3E-02 -3.5E-02

1.7E-15 7.6E-51

Diflubenzuron Dimefuron Dimethoate

1Bc 1Ba 1Bc

-2% -34% -5%

-3% -55% -8%

-9% -75% -14%

1.5E+00 7.3E+00 1.8E+00

2.3E-05 9.7E-23 4.4E-02

2Ae 2Ba 2Ac

0% -39% -7%

-1% -59% -4%

-6% -81% -11%

-2.8E-03 -3.2E-02 -3.9E-03

8.2E-02 3.8E-40 2.6E-02

Dioxathion Disulfoton sulfoxide Ditalimfosj Diuron

1Ad 1Bg 1Bf 1Ba

111% 0% 14% -46%

70% 1% 13% -33%

198% 1% 18% -47%

-2.3E+01 -9.2E-02 -9.3E-01 1.3E+00

3.3E-11 6.7E-01 8.1E-01 1.4E-02

2Bf 2Ad --2Ba

6.7% 10% ---35%

9.4% 24% ---49%

22.7% 25% ---71%

9.2E-03 1.0E-02 ---2.8E-02

4.4E-01 1.9E-10 --4.5E-12

Edifenfos Endosulfan sulfate

1Bc 1Ac

-4% -2%

-3% -8%

-7% -15%

7.3E-01 2.3E+00

2.7E-02 1.1E-03

2Ag 2Bg

1% 2%

0% 4%

1% 1%

1.7E-04 2.3E-04

9.2E-01 9.5E-01

Ethiofencarb Ethoxyquin

1Bg 1Aa

1% -80%

1% < LOQ

1% < LOQ

-1.2E-01 N/A

9.0E-01 1.6E-04

2Bd 2Ba

5.3% -91%

6.8% -99%

6.7% -100%

2.4E-03 -3.5E-02

1.4E-03 5.4E-37

Etoxazole Etrimfos Famphur Fenchlorphos

1Be 1Bb 1Bc 1Aa

-2% -12% -7% -35%

-1% -8% -12% -51%

-3% -18% -22% -81%

2.4E-01 1.6E+00 2.9E+00 8.8E+00

1.1E-01 6.4E-03 3.5E-13 2.2E-20

2Ac 2Bc 2Bc 2Bb

-2% -3.8% -5.5% -9.8%

-13% 0.0% -11.7% -22.0%

-34% 0.0% -21.4% -39.7%

-1.5E-02 5.5E-04 -9.0E-03 -1.7E-02

9.8E-12 1.2E-12 1.6E-14 8.4E-12

Fenchlorphos oxon

1Aa

-22%

-15%

-33%

3.0E+00

7.5E-04

2Bc

-9.2%

-14.1%

-30.2%

-1.2E-02

1.6E-07

c

Fenfuram Fenitrothion Fenthiocarbi

g

1B 1Ab ---

-1% -19% ---

1% -31% ---

0% -58% ---

-7.8E-02 7.5E+00 ---

9.2E-01 9.4E-17 7.5E-04

2A 2Bc 2Bd

-4% -7.3% 12.1%

-11% -13.4% 11.3%

-12% -25.8% 7.3%

-5.1E-03 -1.1E-02 2.0E-03

1.6E-08 6.7E-07 3.8E-06

Fenthion

1Ac

-5%

-10%

-19%

2.7E+00

1.0E-07

2Bg

1%

-1%

-6%

-2.8E-03

4.7E-01


Page 37 of 51


Fenuron Flufenacet

1Ba 1Bc

-51% -4%

-68% -7%

-85% -13%

7.6E+00 1.7E+00

6.4E-29 3.5E-12

2Ba 2Bc

-62% -3.8%

-79% -6.1%

-94% -13.5%

-3.5E-02 -5.5E-03

2.6E-35 2.7E-07

Flufenoxuron Fluometuron

1Bc 1Ba

-5% -39%

-5% -58%

-8% -78%

7.2E-01 6.9E+00

4.3E-07 1.7E-32

2Be 2Ba

-7% -27%

0% -41%

-13% -65%

-4.7E-03 -2.6E-02

2.4E-01 2.3E-34

Fluroxypyr meptyl Flurtamone Folpet Forchlorfenuron Fuberidazole Halofenozide Haloxyfop-methyl Heptenophos Hydramethylnon Idofenphos

1Ah 1Bg 1Ac 1Bf 1Be 1Bd 1Bc 1Bc 1Bb 1Aa

-1% -1% 8% 6% -3% 7% -5% -3% -14% -31%

5% -1% 2% 4% -2% 11% -5% -3% -56% -48%

4% 0% -7% 9% -4% 12% -6% -9% -40% -78%

-5.4E-01 -7.9E-02 2.8E+00 -7.3E-01 3.3E-01 -8.1E-01 3.2E-01 1.5E+00 2.2E+00 9.1E+00

5.0E-02 9.0E-01 4.0E-02 4.7E-01 5.3E-01 1.4E-02 3.6E-04 2.0E-08 7.0E-09 1.1E-18

2Ae 2Bb 2Be 2Ac 2Ab 2Bd 2Bc 2Bh 2Bc 2Bb

-4% -11.2% -6% -2% -10% 15.4% -8.4% 0.8% -4.9% -12.1%

-4% -18.4% -8% -3% -11% 18.6% -8.0% -0.9% -8.8% -24.4%

-4% -29.2% -10% -10% -15% 23.2% -10.8% -6.7% -12.9% -44.4%

-1.4E-03 -1.2E-02 -3.7E-03 -4.1E-03 -5.6E-03 8.6E-03 -3.8E-03 -3.0E-03 -5.2E-03 -1.8E-02

8.7E-01 2.2E-22 2.8E-01 7.2E-03 1.5E-10 3.4E-08 7.0E-06 1.2E-04 8.2E-09 1.1E-10

Imazalil

1Be

-2%

-4%

-6%

6.0E-01

9.1E-02

2Ac

-9%

-11%

-12%

-4.3E-03

5.6E-07

e

Isazophos Isoproturon

c

1A 1Ba

-4% -50%

-14% -68%

-10% -84%

5.8E-01 6.1E+00

3.1E-02 3.0E-41

2B 2Ba

0% -62%

-4% -79%

-5% -94%

-2.4E-03 -3.5E-02

4.3E-01 1.1E-40

Isoxaflutole Isoxathion Leptophos Linuron

1Ac 1Be 1Ac 1Bb

-9% -4% -2% -20%

-15% -4% -9% -29%

-19% -4% -19% -39%

1.6E+00 7.4E-02 3.1E+00 3.5E+00

3.9E-02 1.0E-01 2.5E-04 5.8E-12

2Be 2Bc 2Be 2Bb

-4% -5.5% -5% -15.1%

-6% -5.6% -6% -29.4%

-9% -7.3% -7% -43.5%

-3.8E-03 -2.6E-03 -2.8E-03 -1.8E-02

7.6E-01 5.4E-05 1.7E-01 1.6E-18

Malaoxon Malathion

1Bc 1Bb

-5% -12%

-7% -13%

-14% -27%

2.0E+00 3.1E+00

7.4E-15 1.2E-06

2Bh 2Bc

3.0% -6.5%

0.1% -12.8%

-7.9% -25.0%

-3.8E-03 -1.0E-02

3.2E-07 2.0E-08

MCPA Merphos Metazachlor

1Ad 1Bg 1Bc

92% 0% -3%

147% 1% -1%

152% 1% -4%

-8.6E+00 -1.3E-01 3.8E-01

1.4E-07 8.0E-01 3.2E-02

2Bg 2Bd 2Bc

5% 25% -4.1%

9% 34% -5.6%

3% 26% -9.9%

9.3E-04 9.4E-03 -3.9E-03

5.7E-01 5.4E-08 6.8E-05

Methabenzthiazuron

1Bd

12%

9%

16%

-1.2E+00

2.0E-03

2Bg

3%

4%

0%

-3.7E-05

3.3E-01

Methacrifos Methomyl

1Bg 1Bd

-1% 6%

1% 8%

0% 16%

-5.0E-02 -2.0E+00

9.5E-01 9.0E-06

2Ac 2Bd

-2% 43%

-9% 47%

-22% 48%

-9.5E-03 1.7E-02

6.7E-10 5.7E-16



Page 38 of 51

Methyl paraoxon Metobromuron Metoxuron

1Bc 1Ba 1Ba

-5% -23% -48%

-8% -35% -67%

-16% -51% -84%

2.1E+00 5.2E+00 6.3E+00

1.3E-06 4.0E-17 4.9E-37

2Bc 2Ba 2Ba

-4.6% -27% -59%

-6.7% -41% -77%

-14.0% -57% -93%

-5.6E-03 -2.3E-02 -3.5E-02

9.9E-07 3.3E-27 7.0E-43

Metrafenone Mevinphos Mexacarbatei MGK 264 Monolinuron Monuron

1Bg 1Ac --1Bd 1Bd 1Bb

-3% -4% --5% 20% -13%

-1% -10% --5% 30% -31%

-2% -18% --8% 23% -56%

4.1E-02 2.5E+00 ---5.7E-01 2.0E-01 8.0E+00

2.9E-01 6.2E-03 --2.4E-02 2.1E-08 4.1E-22

2Bc 2Bg 2Bb 2Bc 2Bd 2Ba

-5.9% 4% -17.2% -4.7% 24% -33%

-4.3% -2% -19.3% -5.8% 29% -52%

-9.5% -6% -29.3% -12.8% 24% -79%

-3.5E-03 -3.2E-03 -1.1E-02 -5.1E-03 8.0E-03 -3.2E-02

1.1E-02 5.2E-01 3.1E-03 2.8E-04 1.1E-10 3.0E-31

Neburon Nitempyramj

1Ba 1Ba

-41% -28%

-56% -75%

-67% -76%

4.6E+00 6.4E+00

1.1E-35 1.7E-12

2Ba 2Ba

-43% ---

-59% ---

-76% ---

-2.9E-02 ---

5.6E-39 ---

Nitrofen Norea Omethoate Oxydemeton-methyl

1Ah 1Bc 1Bc 1Bd

-4% -5% -5% 65%

1% -9% -7% 202%

5% -18% -10% 132%

-1.6E+00 2.6E+00 9.7E-01 -2.8E+00

4.9E-02 7.3E-12 8.8E-05 1.3E-09

2Ag 2Bc 2Ac 2Bd

-6% -2.9% -2% 117%

-4% -6.1% -8% 175%

-4% -16.4% -7% 237%

-9.8E-04 -6.9E-03 -3.0E-03 9.3E-02

5.3E-01 1.9E-12 3.5E-02 1.1E-23

Parathion-methyl Pencycuron Phenmedipham Phenthoate Phosalone Phosmet

1Aa 1Ba 1Ba 1Aa 1Bc 1Bc

-27% -92% -56% -22% -4% -5%

-41% -93% -90% -20% -5% -12%

-70% -96% -93% -43% -6% -16%

8.2E+00 6.8E-01 5.2E+00 4.9E+00 3.4E-01 1.7E+00

2.1E-20 1.1E-34 3.6E-10 1.1E-07 1.1E-02 5.4E-13

2Bb 2Ba 2Ba 2Bc 2Bb 2Bc

-9.5% -91% -88% -5.7% -10.3% -6.1%

-17.0% -93% -90% -10.0% -5.6% -7.7%

-31.3% -96% -89% -22.0% -12.5% -14.9%

-1.3E-02 -3.2E-02 -3.0E-02 -9.1E-03 -4.1E-03 -5.9E-03

7.7E-09 7.5E-54 2.3E-06 1.4E-06 1.4E-02 2.4E-07

Phosphamidon

1Bc

-2%

-3%

-6%

7.8E-01

1.9E-02

2Bc

-0.2%

-1.6%

-6.1%

-2.6E-03

1.6E-03

Picoxystrobin Pinoxaden

g

1B 1Ba

-2% -22%

-1% -28%

-2% -49%

1.2E-01 5.5E+00

5.5E-01 1.9E-26

c

2B 2Bc

-2.0% -1.1%

-3.0% -3.1%

-4.8% -8.6%

-1.9E-03 -3.7E-03

1.4E-02 9.6E-06

Piperophos Pirimiphos-methyl Prochloraz

1Be 1Ac 1Bf

-2% -7% 1%

-2% -25% 7%

-3% -34% 11%

2.4E-01 4.5E+00 -1.8E+00

4.9E-01 3.1E-02 9.0E-01

2Bc 2Ae 2Ab

-2.8% -1% -12%

-3.6% -1% -16%

-4.6% -13% -17%

-1.7E-03 -5.5E-03 -6.3E-03

3.6E-02 1.3E-01 4.2E-11

Procyazine Propazine

1Bg 1Bg

1% 0%

3% 1%

0% -1%

2.6E-01 2.1E-01

5.8E-01 9.4E-01

2Ac 2Ac

-1% -2%

-2% -3%

-6% -8%

-2.6E-03 -3.3E-03

1.6E-02 2.6E-03


Page 39 of 51


Pymetrozine Pyracarbolid Pyraflufen-ethyl

1Bg 1Be 1Bc

0% -2% -6%

2% -2% -4%

1% -5% -4%

-1.5E-01 7.9E-01 -1.7E-01

9.2E-01 2.1E-01 2.3E-02

2Ac 2Ac 2Bb

-3% -4% -12.8%

-3% -11% -7.8%

-8% -6% -8.1%

-3.2E-03 -2.5E-03 -2.0E-03

1.3E-03 1.8E-05 5.3E-03

Pyrazophos Pyrifenoxi

1Bg ---

-2% ---

-1% ---

1% ---

-6.4E-01 ---

8.5E-01 9.4E-01

2Ac 2Bc

-2% -5.1%

-4% -6.6%

-4% -10.0%

-1.6E-03 -3.9E-03

1.6E-02 1.0E-06

Resmethrin Rotenone

1Bd 1Bb

5% -15%

2% -23%

9% -34%

-1.1E+00 3.6E+00

1.2E-01 3.3E-17

2Ab 2Bc

-20% -2.7%

-30% -8.4%

-37% -18.9%

-1.4E-02 -8.1E-03

2.8E-05 1.6E-08

Sebuthylazine Sebuthylazine-desethyl Sethoxydim

1Bg 1Bg 1Bc

0% -1% -5%

1% 1% -11%

0% -4% -18%

2.6E-02 8.2E-01 2.4E+00

9.5E-01 2.8E-01 2.4E-03

2Ac 2Ac 2Bc

-2% -2% -2.0%

-5% -5% -3.0%

-7% -8% -6.9%

-3.0E-03 -3.6E-03 -2.8E-03

1.1E-03 2.0E-03 2.3E-03

Siduron Silthiopham Simazine

1Be 1Aa 1Bc

-1% 2% -4%

-2% -1% -3%

-4% -7% -5%

5.3E-01 1.7E+00 3.3E-01

6.6E-01 6.5E-04 1.9E-03

2Ac 2Be 2Ac

-1% -2% -5%

-4% -2% -9%

-6% -10% -11%

-2.8E-03 -4.1E-03 -4.4E-03

1.1E-05 1.3E-01 2.0E-07

Spinetoram Spinosad A Spirodiclofen Spiromefesin Sulfotep

1Bc 1Bc 1Bd 1Bc 1Ba

-1% -1% 37% -9% -52%

-4% -6% 25% -16% -57%

-3% -4% 41% -29% -86%

8.2E-02 2.9E-01 -2.0E+00 3.9E+00 7.1E+00

2.0E-04 1.6E-04 8.5E-07 4.4E-04 4.4E-17

2Be 2Ag 2Bd 2Bc 2Ba

0% -1% 17.6% -6.9% -22%

0% 0% 33.1% -12.2% -34%

-3% -2% 20.1% -19.6% -47%

-1.3E-03 -6.3E-04 7.7E-03 -8.0E-03 -1.9E-02

3.3E-01 5.4E-01 1.2E-03 3.6E-10 2.2E-19

Sulprofos Teflubenzuron Temephos

1Ad 1Bf 1Bc

2% 1% -7%

0% 2% -11%

11% 6% -22%

-2.2E+00 -1.1E+00 3.1E+00

3.2E-02 1.1E-01 9.2E-10

2Bg 2Bd 2Bb

1% 13.3% -11.1%

-1% 28.2% -10.4%

-1% 35.5% -21.5%

-7.2E-04 1.5E-02 -8.1E-03

8.4E-01 1.1E-11 3.2E-05

TEPP Tepraloxydim Terbufos sulfoxide

1Bc 1Bc 1Bc

-9% -4% -2%

-10% -8% -2%

-21% -11% -5%

2.6E+00 1.3E+00 5.3E-01

1.6E-17 4.8E-02 4.1E-04

2Bf 2Bc 2Bc

0.1% -9.2% -1.2%

2.4% -12.9% -2.0%

3.7% -16.7% -5.7%

1.7E-03 -6.5E-03 -2.4E-03

9.7E-02 8.5E-12 4.3E-03

Terbuthylazine Terbuthlazine-desethyl Thiabendazole Thiodicarb

1Bg 1Be 1Bg 1Bc

0% -4% -1% -5%

1% -1% -2% -5%

0% -6% -1% -9%

-2.4E-02 6.8E-01 -1.2E-01 8.6E-01

9.9E-01 8.8E-02 9.2E-01 1.4E-03

2Ac 2Ac 2Ab 2Ba

0% -2% -10% -28%

-3% -4% -10% -37%

-6% -8% -15% -43%

-2.7E-03 -3.2E-03 -5.6E-03 -1.6E-02

2.6E-02 4.3E-03 1.3E-07 2.0E-28

Thiofanox

1Be

-13%

-5%

-8%

2.4E+00

1.9E-01

2Ac

-7%

-13%

-20%

-7.2E-03

2.8E-02



Page 40 of 51

Thiofanox sulfoxide Thionazin Thiophanate methyl

1Be 1Ad 1Ba

-3% 3% -71%

-2% 11% -99%

-13% 13% -100%

-5.9E-01 -1.6E+00 4.0E+00

3.2E-01 4.3E-02 2.6E-20

2Ac 2Bg 2Ba

0% 7% -100%

-3% 2% -100%

-17% 2% -100%

-8.0E-03 1.2E-04 -3.3E-02

3.7E-02 7.0E-01 7.3E-45

Tolclofos methyl Tolylfluanid

1Ac 1Ba

-4% -36%

-10% -24%

-19% -64%

3.0E+00 7.3E+00

2.1E-12 9.9E-06

2Bc 2Ba

-3.5% -22%

-4.2% -28%

-13.9% -42%

-5.7E-03 -1.6E-02

1.3E-02 4.8E-17

Tralkoxydim Trichlorfon

1Bc 1Ba

-5% -31%

-11% -37%

-20% -62%

2.8E+00 6.4E+00

4.4E-03 1.2E-18

2Be 2Bc

-2% -6.7%

-1% -10.2%

-6% -17.1%

-2.4E-03 -6.9E-03

2.0E-01 4.2E-07

Tricyclazole Tycor

1Bg 1Ba

-2% -25%

-1% 2%

-3% 0%

2.8E-01 -3.2E+00

6.7E-01 3.5E-02

2Ac 2Bf

-3% 2.8%

-4% 4.0%

-4% 3.0%

-1.4E-03 1.1E-03

7.4E-03 1.1E-01

Analyte with a reported decrease in response aexceeding 20%, bbetween 10 and 20%, cbetween 0 and 10%. dAnalyte with a reported increase in response. Insignificant (p-value > 0.05) enegative trending observed; fpositive trending observed; g no change observed. h

Analyte produced a statistically significant change at 95% confidence with no observed trending. Not included in: iSSP #1, jSSP#2.

Rows in bold indicate a change in apparent stability between both protocols.


Page 41 of 51


Table 4. Enhancement of select pesticides due to degradation of precursors. Analyte Carbendazim Carbofuran Methomyl Monolinuron Oxydemeton-methyl 3,4-Dichloroaniline

Precursor Thiophanate-methyl24 Benfuracarb25 Thiodicarb26 Linuron27 Demeton-S-methyl28 Diuron, Linuron, and Neburon20


Study affected 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 2 only


Figure 1. a.


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Page 43 of 51


b.



c.


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Page 45 of 51


Figure 2.



Figure 3.


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Page 47 of 51


Figure 4.



Figure 5.


Page 48 of 51

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Figure 6.



Figure 7.


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Page 51 of 51


For Table of Contents Only


Determination of Stability from Multicomponent ... - ACS Publications

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