Online supporting information for the following article published in Indoor Air
DOI: 10.1111/ina.12093
Title:
A Test House Study of Pesticides and Pesticide Degradation Products
Following an Indoor Application
Running Title: Pesticides and Degradation Products after an Indoor Pesticide Application.
James M. StarraII, Anthony A. Gemmab, Stephen E. Grahamc, Daniel M. Stout IIa
a
Human Exposure and Atmospheric Sciences Division, National Exposure Research Laboratory,
Office of Research and Development, U.S. Environmental Protection Agency, Research
Triangle Park, NC
b
c
National Caucus and Center for Black Aged SEE Program, Washington, D.C.
Health and Environmental Impacts Division, Office of Air Quality Planning and Standards,
U.S. Environmental Protection Agency, Research Triangle Park, NC
II
Corresponding Author:
James Starr
U.S. EPA MD D205-05
109 T.W. Alexander Drive, RTP, NC 27711
Email: starr.james@epa.gov
Tele: (919)541-4608
Fax: (919)541-3527
Running title: Pesticide Movement and Degradation Following an Indoor Application
Online Supplementary Material
1.
Sample Extraction and Clean-up.
Each sample (consisting of two wipes), was placed in a 33 mL Pressurized Fluid Extraction
(PFE) cell and spiked with 100 μL methanol containing 100 ng of each analyte. Samples were
extracted in a static cycle lasting 5 minutes with hexane:acetone (25:75, V:V) at 75 oC and
pressurized to 1500 psi. The solvent in the cell was then flushed to collection by addition of
hexane:acetone, (25:75, V:V) at 50% of volume required to initially fill the cell. The static and
flushing/collection cycles were repeated two additional times and pressure and temperature were
maintained throughout the process. After the final flush and collection the cell was purged with
nitrogen for two minutes. Following this, the cells were filled with methanol and the static and
flushing/collection cycles were performed twice, again followed by a two minute nitrogen purge.
The temperature, pressure, and flushing volume were the same as was used for the
hexane:acetone extraction. The hexane:acetone and methanol flushes were collected in separate
containers. After extraction the solvents containing the extracted analytes were transferred to
separate 125 mL flat bottom flasks. The methanol fraction was evaporated under reduced
pressure and elevated temperature to approximately 2-4 mL. To this, two mL of 0.01N HCl was
added and the volume was further reduced to 1-2 mL. This volume was transferred to a
graduated centrifuge tube and the 125 mL flask rinsed with additional 0.01N HCl to make the
final volume in the graduated centrifuge tubes 4 mL. The flat bottom flask was rinsed with 4 mL
of EtOAc which was also added to the centrifuge tube. To this, approximately 500 μL of water,
saturated with sodium sulfate (Na2SO4), was added to prevent formation of emulsions. The
aqueous layer was partitioned three times against equal volumes of EtOAc and after each
partition the organic layer was removed and added to the flask containing the hexane:acetone
extraction. The combined extract was evaporated under low pressure and elevated temperature
to a volume of 1-2 mL. Five mL of acetonitrile was added and the volume was reduced, again,
to 1-2 mL. The solvent was transferred to a graduated centrifuge tube and the volume was
adjusted with acetonitrile, as needed, to a volume of 2 mL. The acetonitrile was partitioned four
times against an equal volume of hexane. After each partition the acetonitrile layer was
transferred to a culture tube and fresh acetonitrile added. After partitioning, the hexane layer
was discarded and the acetonitrile was reduced to 1 mL under nitrogen.
Extracts were further cleaned using cartridges packed with 500 mg of C18 sorbent material. Prior
to sample processing, the C18 was conditioned with 4 mL methanol, followed by 4 mL
methanol:acetonitrile (1:9, V:V) then 4 mL acetonitrile. The extracts, were loaded onto the C18,
then eluted with 3 mL acetonitrile followed by 4 mL methanol:acetonitrile (1:9, V:V).
Following C18 clean-up 200 µL of 0.01N HCl were added to the eluant. The volume was
reduced to 100-200 µL under nitrogen at ambient temperature and the internal standards were
added. Finally, methanol, and 0.01N HCl were added to make a 1 mL solution with a
methanol:0.01N HCl ratio of 8:2 (V:V). This was transferred to an autosampler vial and stored
at -20 °C until analysis.
2.
Optimization of LC/MS/MS Conditions.
Mass spectrometer settings of the LC/MS/MS were optimized by first infusing individual
analytes (dissolved in 7:3, methanol:5mM ammonium acetate), into the source where an
electrospray (ES) probe was used to produce charged species. For analytes where the ES probe
was ineffective, Atmospheric Pressure Chemical Ionization (APCI) was used. The relative
abundance of each analyte was determined for M+1, M-1 and M+18 (ammonium adduct). At
this time other mass spectrometer voltages settings were also optimized to produce the most
abundant precursor and product ions.
The HPLC mobile phase proportions and flow rate were optimized using a C18 column (3.0 ×
150 mm, 3.5 μm). The final conditions were determined with achieving the highest signal to
noise ratio for each analyte as the most important parameter. Forming a charged IPP species
required APCI but all other analytes were ionized effectively using the ES probe. Table 1 in the
supplemental information lists all analytes, their retention times, ion pairs, and internal standards.
13
C6 trans-permethrin was used as a surrogate recovery standard for all parent pyrethroids listed.
4-F-3-PBA was used as the surrogate for all degradates, chlorpyrifos, propoxur, and fipronil.
The composition of mobile phase for all analyses was methanol:5 mM ammonium acetate in
water. However, the relative proportions of organic and aqueous were adjusted, dependent upon
the analytes. The ratio of organic:aqueous used for the pyrethroids was 98:2 while the
organic:aqueous ratio for IPP was 7:3. A gradient was used for the pyrethroid degradates,
chlorpyrifos, TCP, fipronil, its degradates, and propoxur. The initial ratios were 6:4
organic:aqueous for three minutes; ramp at 12.5% per minute to 85:15 organic:aqueous and hold
for ten minutes. The mobile phase was allowed to equilibrate for five minutes prior to each
injection. The identities of all analytes were verified using retention times and transition ion
pairs. The mobile phase conditions used for each group provided temporal separation of the cistrans isomers of both permethrin and DCCA. The cis-trans isomers of cypermethrin were not
resolved.
Multiple runs for each sample were used to achieve the best chromatography and maximum
sensitivity. Figure 1 in the supplemental information shows chromatograms of the pesticides and
degradates. Charged species of all analytes except IPP were formed using electrospray
ionization (ESI). All pyrethroids were ionized in the positive mode by forming pyrethroidammonium adducts. The pyrethroid degradates, chlorpyrifos, TCP, fipronil, it’s degradates, and
propoxur were ionized in the negative ion mode. IPP ions were generated using APCI in the
negative ion mode.
3.
Method Evaluation: Percent Recovery and Limits of Detection/Quantitation.
Baseline extraction recoveries from spiked “clean” wipes were established by spiking precleaned wipes then processing them according to the optimized method. Potential interferences
were assessed using pre-cleaned wipes wetted with isoproponyl then wiped across one of three
surfaces. The surfaces were: a laboratory bench top, tiled and waxed flooring, and laminate
flooring identical to that in the research Test House. Although laminate flooring was the only
floor surface in the Test House, the other two surfaces were used to demonstrate the ability of the
cleanup method to remove additional background interferences that could be encountered in the
Test House.
Each area to be wiped was delimited by a flat aluminum template with an interior area of 929
cm2. Two sequential wipe samples were taken from each area using an overlapping pattern that
covered the entire area inside the template, one time per wipe. Both wipes used for each
sampling area were stored together in amber jars at -20o C until processing.
After wiping, each set of two cotton wipes was spiked with one of three concentrations of
pesticides and degradates, then processed and analyzed to evaluate interferences and determine
percent recovery. Five replicate samples at each of the three spiking levels were taken from each
surface type. Each degradate (including summed cis/trans DCCA) were spiked at 25, 100, and
500 ng (n=5 each level). Pesticides were spiked at 25, 100, and 1000 ng (n=5 each level). Blank
wipes (wipes not spiked after wiping) of each surface were also taken.
In estimating the method limits of detection (MDL) and quantitation (MQL) for each analyte, the
same preparation, wiping, and spiking procedure as described for the method validation. After
wiping the surface, the cotton was spiked with either: 2, 5, 10, or 20 ng of each analyte. Wiping
and spiking were repeated four times at all concentrations for each of the three surfaces, resulting
in 12 samples per concentration. The samples were processed according to the final extraction
and clean-up procedure described above and then analyzed by LC/MS/MS. Each MDL/MQL
sample was injected and run on the LC/MS/MS four times. Analyte concentrations in the
replicates at each theoretical concentration were determined using a calibration curve ranging
from 1-50 ng/mL, then pooled to calculate group standard deviations of the estimated
concentrations. The standard deviations were set as the dependent variable while the theoretical
concentrations were independent. Least squares regression was used to estimate the y-intercept.
The intercept of the regression equation was defined as S0 with the MDL approximated by 3 ×
S0, and the MQL approximated by 10 × S0 (Taylor, 1987).
Reference List
Taylor, J. K. (1987). Quality Assurance of Chemical Measurements. Lewis Publishers, New
York.
Supplementary Table 1. LC/MS/MS Chromatographic Parameters.
Compound
Ionization
Retention Time Q1a
(min.)
Q2a
Pesticides
cis-permethrin
trans-permethrin
cypermethrin
deltamethrin
propoxur
chlorpyrifos
fipronil
M+18
M+18
M+18
M+18
M-1
M-1
M-1
3.2
3.5
2.7
2.7
2.0
2.8
8.8
408
408
433
521
210
350
435
183
183
191
279
93
198
330
Degradates
TCP
fipronil sulfide
fipronil sulfone
fipronil desulfinyl
3-PBA
DBCA
cis-DCCA
trans-DCCA
IPP
M-1
M-1
M-1
M-1
M-1
M-1
M-1
M-1
M-1
2.6
8.9
9.2
8.6
3.4
6.1
5.5
4.1
4.0
196
419
451
387
213
295
207
207
151
35
383
282
351
93
79
35
35
109
Surrogates
13
C6 trans-permethrin
M+18
3.2
414
4-F-3-PBA
M-1
3.2
231
a. Q1 and Q2 – first and second ion mass filters, respectively.
189
93
Internal Standard
13
C6 cis-permethrin
13
C6 cypermethrin
propoxur D3
chlorpyrifos D10
fipronil des F3
fipronil des F3
13
C6 3-PBA
13
C6 trans DCCA
fipronil des F3
13
C6 cis-permethrin
C6 3-PBA
13
Supplementary Table 2. Concentrations of Study Pesticides and their Degradation Products in Den and Living Room.
Number of Days After Pesticide Application
PreApplication
1
2
cis-permethrin
7
Concentration in
Pesticides Applied
cypermethrin
3
NDb
37 ± 19
(11)c
15 ± 11 (6)
Dena
14
21
28
35
(pg/cm2)
17 ± 7 (5)
17 ± 7 (6)
24 ± 13 (5)
33 ± 23 (5)
17 ± 0 (1)
NA
13 ± 0 (1)
40 ± 18 (6)
31 ± 2 (5)
34 ± 13 (6)
70 ± 50 (5)
53 ± 23 (5)
9 ± 0 (1)
NA
83 ± 0 (1)
trans- permethrin
6 ± 6 (11)
37 ± 16 (6)
35 ± 10 (5)
35 ± 12 (6)
72 ± 49 (5)
58 ± 29 (5)
9 ± 0 (1)
NA
75 ± 0 (1)
fipronil
11 ± 11 (8)
3 ± 2 (5)
11 ± 7 (5)
8 ± 7 (6)
10 ± 2 (5)
16 ± 9 (5)
8 ± 0 (1)
NA
3 ± 0 (1)
propoxur
4 ± 2 (10)
213 ± 103 (6)
290 ± 20 (5)
281 ± 121 (6)
282 ± 102 (5)
198 ± 56 (5)
53 ± 0 (1)
NA
98 ± 0 (1)
3-PBA
NAd
ND
ND
ND
1 ± 1 (1)
ND
ND
NA
2 ± 0 (1)
cis- DCCA
NA
ND
0 ± 1 (1)
0 ± 0 (1)
0 ± 1 (1)
1 ± 1(2)
ND
NA
1 ± 0 (1)
trans- DCCA
IPP
fipronil desulfinyl
NA
NA
NA
1 ± 1 (3)
ND
ND
2 ± 0 (4)
ND
ND
2 ± 2 (2)
ND
ND
3 ± 1 (4)
ND
ND
2 ± 1(4)
ND
ND
ND
ND
ND
NA
NA
NA
2 ± 0 (1)
ND
ND
fipronil sulfone
NA
4 ± 3 (5)
5 ± 2 (5)
5 ± 3 (6)
4 ± 3 (4)
6 ± 2 (5)
4 ± 0 (1)
NA
13 ± 0 (1)
fipronil sulfide
NA
ND
ND
ND
ND
ND
ND
NA
ND
chlorpyrifos
5 ± 4 (10)
5 ± 2 (6)
4 ± 1 (5)
4 ± 1 (6)
6 ± 1 (5)
5 ± 1 (5)
2 ± 0 (1)
NA
6.2 ± 0 (1)
deltamethrin
NA
11 ± 4 (6)
12 ± 4 (5)
10 ± 4 (6)
11 ± 4 (5)
10 ± 4 (5)
ND
NA
16 ± 0 (1)
TCP
NA
30 ± 12 (6)
24 ± 2 (5)
9 ± 10 (6)
24 ± 5 (5)
29 ± 13 (5)
17 ± 0 (1)
NA
37 ± 0 (1)
DBCA
NA
ND
ND
ND
ND
ND
ND
NA
ND
Degradates of Applied
Pesticides
Pesticides Not Applied
Degradates of
Pesticides Not Applied
Concentration in Living Room (pg/cm2)
Pesticides Applied
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
30 ± 11 (5)
29 ± 10 (5)
102 ± 83 (3)
(11)c
40 ± 11 (5)
41 ± 9 (5)
69 ± 31 (3)
50 ± 28 (11)
NA
NA
NA
NA
NA
39 ± 10 (5)
44 ± 9 (5)
94 ± 52 (3)
6 ± 1 (11)
NA
NA
NA
NA
NA
18 ± 3 (5)
12 ± 8 (5)
3 ± 1 (3)
ND
NA
NA
NA
NA
NA
163 ± 41 (5)
119 ± 31 (5)
98 ± 26 (3)
3-PBA
NAd
NA
NA
NA
NA
NA
ND
ND
2 ± 0 (3)
cis- DCCA
NA
NA
NA
NA
NA
NA
0 ± 1 (1)
1 ± 1 (2)
1 ± 1 (2)
trans- DCCA
IPP
fipronil desulfinyl
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
5 ± 2 (5)
ND
ND
2 ± 1 (4)
ND
ND
2 ± 1 (3)
ND
ND
fipronil sulfone
NA
NA
NA
NA
NA
NA
7 ± 4 (5)
7 ± 3 (5)
4 ± 4 (3)
fipronil sulfide
NA
NA
NA
NA
NA
NA
ND
ND
ND
3 ± 1 (10)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
5 ± 1 (5)
8 ± 3 (5)
4 ± 1 (5)
7 ± 2 (5)
5 ± 1 (3)
12 ± 1 (3)
TCP
NA
NA
NA
NA
NA
NA
22 ± 2 (5)
23 ± 5 (5)
23 ± 5 (3)
DBCA
NA
NA
NA
NA
NA
NA
ND
ND
ND
cypermethrin
cis-permethrin
trans- permethrin
fipronil
propoxur
NDb
48 ± 34
Degradates of Applied
Pesticides
Pesticides Not Applied
chlorpyrifos
deltamethrin
Degradates of
Pesticides Not Applied
a. Pre-Application; n = 11den, 11 living room. Days 2, 7, 14; n=5 den, 0 living room. Days 1and 3; n = 6 den, 0 living room. Day 21; n= 1 den, 5 living room.
Day 28; n= 0 den, 5 living room. Day 35; n= 1 den, 3 living room (5 living room results for pesticides and degradates of day 35 samples with pesticide
overspray of permethrin, cypermethrin and propoxur have been removed).
b. ND = Analyte not detected in any sample.
c. (n) = number of samples where analyte detected
d. NA = No samples taken.
Supplementary Figure 1. Example Chromatograms of Study Pesticides, Degradates, Surrogates,
and Internal Standards.
a
b
b
c
a. Organic:aqueous 98:2. Positive LC/MS/MS ESI.
b. Organic:aqueous 6:4 for three minutes; ramp at 12.5% per minute to organic:aqueous 85:15. Negative
LC/MS/MS ESI.
c. Organic:aqueous 7:3. Negative LC/MS/MS APCI.