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Assessing the fate and effects of an insecticidal formulation
Chloé de Perre,†, Karl W. J. Williard,‡, Jon E. Schoonover,‡, Bryan G. Young,§, Tracye M.
Murphy,†, Michael J. Lydy,*†
† Center for Fisheries, Aquaculture, and Aquatic Sciences and Department of Zoology, Southern
Illinois University, Carbondale, Illinois 62901, USA
‡ Department of Forestry, Center for Ecology, Southern Illinois University, Carbondale, Illinois
62901, USA
§ Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907,
USA
Materials and methods
Insecticide extractions
Water samples were extracted using liquid-liquid extraction of 200 mL of each sample and 3×20
mL of pesticide grade dichloromethane (Fisher Scientific). The dichloromethane extract was drained from
the separatory funnel and then concentrated to a volume of 0.5 mL using nitrogen gas and a Turbovap II
evaporator (Zymark, Hopkinton, MA, USA). After extraction of the unfiltered runoff water, settled
sediments were freeze-dried (benchtop FreeZone 2.5 freeze-dryer, Labconco, Kansas City, MO, USA),
and then extracted following the soil protocol. For soils and sediments, 5g of each sample were weighed
and 20 mL of a 50/50 (v/v) acetone/dichloromethane solution were added, before the sample was rotated
for 1h on a tube rotator (BBL, division of BioQuest, Cockeysville, MD, USA). To assess potential
bioaccumulation, surviving worms from the bioassays (dead ones disintegrated during the test) were
extracted after 14 days of exposure following a similar protocol to the one used for sediments. Worms,
between 0.5 and 2g of freeze-dried tissue, depending on availability, were extracted with the same solvent
mixture, sonicated for 30 min before being rotated for 1h. Samples were finely ground (PowerGen 700
tissue grinder, Fisher Scientific), then centrifuged for 10 min at 20°C and 4400 rpm (Centrifuge 5702R,
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Eppendorf, Germany). All samples from solid matrices were then stored in the freezer for a few hours to
allow the solids to settle and potential moisture to freeze. Making sure ice pellets were not pulled, 10 mL
of the solution were transferred into a test tube and the solution was concentrated to 0.5 mL using a
Rapidvap evaporator (Reacti-Therm III, Pierce, Rockford, IL). Additional steps were introduced to clean up
worm samples from the lipid molecules, including filtration and gel permeation chromatography (GPC).
Samples were solvent exchanged to dichloromethane, evaporated down to 1.5 mL, then filtered through
PTFE 0.2 µm GD/X syringe filters (Whatman, GE Healthcare Life Sciences, USA), and brought back to a
final volume of 1 mL in dichloromethane. The GPC procedure was performed on 500 µL of the sample
using an Agilent Technologies (Santa Clara, CA, USA) 1100 Series Liquid Chromatograph (LC) equipped
with an Envirogel GPC 15 µm 4.6×30 mm guard column, an Envirogel GPC cleanup 15 µm 19×300 mm
column (both columns from Waters, Taunton, MA, USA), and a Foxy Jr. fraction collector (ISCO, Lincoln,
NE, USA). Dichloromethane was used as the mobile phase at 5 mL/min and the fraction from 7 to 11 min
was collected for analysis. After extraction, all samples from aqueous and solid matrices were cleaned up
using 1000 mg/6 mL Solid Phase Extraction (SPE) Hypersep Florisil cartridges (Thermo Scientific,
Bellefonte, PA, USA). Cartridges were pre-conditioned using 5 mL of pesticide grade hexane (Fisher
Scientific), the samples were loaded onto the cartridge, and test tubes were rinsed using 3×0.5 mL of a
hexane:ethyl acetate (pesticide residue analysis grade, Acros, NJ, USA) (80:20,v/v) solution, which was
then added to the cartridge. The samples were eluted from the cartridge using 7 mL of a hexane/ethyl
acetate (20:80,v/v) solution. Samples were then concentrated, transferred to injection vials, and
evaporated down to close to dryness. A volume of 475 µL of a 0.6% (v/v) acetic acid (99.5%, Mallinckrodt)
in hexane solution and 25 µL of a 1 µg/mL internal standard solution in hexane were added.
Decachlorobiphenyl and fenpropathrin (not used in Illinois) were used as internal standards to quantify
phostebupirim and cyfluthrin, respectively. Bifenthrin or p-terphenyl were used as surrogates and were
added to each sample right before extraction, for samples analyzed by Gas Chromatography-Mass
Spectrometry (GC-MS) in negative chemical ionization (NCI) or electron impact (EI) mode, respectively.
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Method detection limits
Method detection limits (MDLs) were calculated from seven samples of either runoff water or
agricultural soil from an adjacent field, spiked with low concentrations of phostebupirim and cyfluthrin, i.e.,
just above the limit of detection. One blank of each matrix was analyzed to check that they were free of
these insecticides. The measured concentrations in the seven samples were then statistically processed
to determine the MDLs of each matrix; the standard deviation was multiplied by the Students’ t-value at
99% confidence level, i.e., 3.143. The operation was repeated for each mode of analysis, i.e., NCI and EI,
since the insecticides had to be spiked at different levels to satisfy the conditions MDLs < spiking levels <
10 MDLs.
Quality analysis, quality controls
Quality control was conducted with each batch of 20 soil samples or 18 water samples using a
laboratory blank, i.e., laboratory sand or moderately hard reconstituted water, a laboratory spike , which
was the same as the blank but spiked with phostebupirim and cyfluthrin, a matrix spike and a matrix spike
duplicate, both on the same sample randomly chosen from the batch, and both spiked with the same
amount of insecticides as in the laboratory spike. The surrogate was added to each sample in the same
amount. Quality controls were considered satisfactory if the analytes and surrogates had recoveries
between 70 and 130%, and the matrix spike and matrix spike duplicate were within 20%. Samples with
surrogates recoveries below 70% were re-extracted until satisfactory recoveries were obtained.
During GC-MS analysis, one of the middle standards of the calibration was chosen every eight
samples to be re-run to monitor the stability of the signal and the validity of the calibration during the whole
sequence. Samples were re-injected with a new calibration curve if the recoveries of the standards were
not within 80-120%.
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Figure S1. Comparison of cyfluthrin chromatograms extracted for the ion m/z = 207 from soil samples collected right
after planting (top) and after five months (bottom). The retention times differed due to a column change but the same
type of column was installed. The proportions of cyfluthrin isomers was different for the two sampling events: right
after planting the peaks had equivalent heights and the second peak was a little higher; five months later, the second
and fourth peak were much smaller than the others. This trend was observed for most samples of 2013, and some in
2012 and 2011.
50000
45000
Abundance
40000
35000
30000
25000
20000
15000
10000
5000
13.5
13.4
13.2
13.1
13.0
12.9
12.7
0
Time (min)
12000
10000
6000
4000
2000
Time (min)
15.1
15.0
14.9
14.7
14.6
14.5
14.4
0
14.3
Abundance
8000
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For clarity, a flow chart of this study is provided in Figure S2.
Figure S2. Flow chart of the current study steps.
Field Data
Surface Soil
Toxicity Data
Runoff Water
Terrestrial
RISK
Aquatic