SUPPLEMENTAL DATA
INTER- AND INTRA-SPECIES CHEMICAL SENSITIVITY: A CASE STUDY USING 2,4DINITROANISOLE
Alan J Kennedy†, Jennifer G. Laird†, Chris Lounds‡, Ping Gong‡, Natalie D. Barker‡, Sandra
M. Brasfield†, Amber L. Russell‡, Mark S. Johnson§
† U.S. Army Engineer Research and Development Center, Environmental Laboratory, 3909
Halls Ferry Road, Vicksburg, Mississippi 39180
‡ Badger Technical Services, LLC, 12500 San Pedro Avenue, Suite 450, San Antonio, TX 78216
§ U.S. Army Institute of Public Health, Toxicology Portfolio, Aberdeen Proving Ground, MD
Total pages: 12
Figures: 3
Tables: 3
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Analytical
The GC method involved collection of 40 mL water samples sent to AIPH for analysis.
The samples were maintained at 4 ºC in dark conditions at all times. A 10-mL water aliquot was
extracted from each sample using 2 mL of isoamyl acetate while shaking on a flatbed shaker for
1 hr. After separation, the isoamy acetate layer was placed in an auto-sampler vial and stored at 4
ºC until analysis. Samples were extracted the day after receipt. DNAN (Lot # BAE10H281-008)
used for calibration and quantification was obtained from BAE Systems (Ordnance Systems,
4509 West Stone Drive, Kingsport, TN 37660). DNAN used for quality control samples (LCSLCS Duplicate) was obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, 63103). The
standards used to calibrate the analytical analysis system were extracted with the same water
volume (10 mL) as the samples to compensate for potential differences in extraction efficiency.
The samples were diluted as necessary to bring the sample concentrations within the range of the
instrument calibration. Analysis was performed using an Agilent 6890 gas chromatography (GC)
fitted with an electron capture detector (ECD; Santa Clara, CA). A J&W DB-17 column was
used for the primary analytical column and DB-1 was used for confirmation. The samples were
reported in units of µg/mL. Laboratory control samples (LCS) and duplicates (LCSD) were
analyzed with test samples and percent recoveries were within the current acceptable limit.
Genotyping
Genomic DNA (gDNA) was extracted from individual D. pulex using Qiagen DNeasy® Tissue
kits (Valencia, CA). The yield and quality of gDNA were determined using NanoDrop® ND1000 (Wilmington, DE). Only good quality gDNA (A260/A280 > 1.8 and A260/A230 > 2.0)
was used for subsequent PCR amplification. A 10-µl nested PCR reaction was performed for
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each individual gDNA sample using a universal M13 primer carrying a fluorescent dye label [1].
The reaction mix consisted of 5-PRIME Taq buffer with Mg2+ (1×), MgCl2 (2.0 mM), a forward
primer with M13 tag (0.025 µM), a reverse primer (0.25 µM), a FAM-labeled M13 primer (0.25
µM), dNTP mix (0.2 mM), 5-PRIME Taq DNA polymerase (0.5 U), and D. pulex gDNA (10-20
ng). See Table S1 for the sequences of sequence-specific primer sets and the universal M13
primer. The following thermal cycling program was employed: 94 °C for 5 min, followed by 30
cycles of 94 °C for 30 s, 56 °C for 45 s, and 68 °C for 45 s, then 8 cycles of 94 °C for 30 s, 53 °C
for 45 s, and 68 °C for 45 s, and a final extension at 68 °C for 10 min. Amplicon size was
analyzed by running a 12.5-µl mix of PCR product (2 µl), GeneScan 500 ROX size standard (0.5
µl), and Hi-Di Formamide (10 µl) on an ABI 3100 Prism Genetic Analyzer (Applied
Biosystems, Foster City, CA).
Data analysis:
The scored SSR results were arranged in an Excel spreadsheet and saved as a “Tab Delimited”
file. This file was imported as the input file for analysis using a command line software program
called Microsatellite Analyzer (MSA) v.4.05 [2]. Nei’s chord distance Da was calculated as
follows and used to estimate genetic distances between different populations [3].
Da = 1 - ΣkΣa sqrt( fa,i * fa,j )/D
Sum over all Loci and all Alleles (k)
fa,i/a,j = Frequency of allele a in Pop i/j
D = Number of Loci
Chronic toxicity of methanol
We conducted a three-brood Ceriodaphnia dubia chronic toxicity test according to USEPA [4]
with methanol (HPLC grade, Honeywell Burdick & Jackson, Muskegon, MI, USA) that resulted
in an inhibition concentration causing a 25% reduction in reproduction (IC25) of 0.02% (v/v)
3
methanol in MHRW. Similarly, the derived 21-day chronic value for acetonitrile for Daphnia
magna is 230 mg/L [5], or 0.02% v/v. Unfortunately, at least 0.1% methanol was required for
spiking 50 mg/L DNAN into the toxicity test treatments.
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Table S 1. Summary of different populations and source of Daphnia pulex.
Population
Code
Type
Original Source
POC
Pop-A
Lab
EPA-ORD
Steve Poirier
Pop-B
Field
St. Louis, MO
Howard Webb
Pop-C
Field
UK
Andrew Beckerman
Institution, location
5
ECTesting, Superior, WI,
USA
Saint Louis, MO
University of Sheffield,
South Yorkshire, United
Kingdom
Table S 2. Genotyping results expressed as the pair-wise genetic distance between two D. pulex
populations that was determined by Nei’s chord distance Da. Amplicon size of 20 microsatellite
SSR markers was collected from 30 individual animals per population and used for Da inference.
Similarity indices indicate large differences between the Pop-A, Pop-B and Pop-B populations,
indicated they are from distinct geneotypes. However, Pop-A was similar to a population
acquired from Aquatic Biosystems (ABS), Fort Collins, CO, USA, and was likely the same
genotype.
ABS
Pop-A
Pop-B
Pop-C
ABS
Pop-A Pop-B
0.03
0.46
0.95
0.32
0.95
6
0.97
Pop-C
Table S 3. Primer sequence and flanking region length of the 20 validated microsatellite SSR markers. MSATCOMMANDER v0.8.2
[6] was use to design the primer sets. Green highlighted are sequence-specific primers fused with a universal M13 sequence (in red)
[7]. The last row shows a universal FAM-labeled M13 primer.
PRIMER_RIGHT
Flanking
length (nt)
(AAC)^5 AAGGACGACATCTGGCAATC
GGAAACAGCTATGACCATAAAGACAAACCTGAAGGGAACG
153
DQ249365
(GGT)^6 GGAAACAGCTATGACCATCTTCTTCAACGACGGCAG
CGTTGTGTCCCTTTGACCC
209
DQ249412
(AC)^6
GGAAACAGCTATGACCATGTTTGCCTCAATCGACCGC
CCTTGCTGATGGACTCACG
206
DQ249424
(CT)^8
TGTTTGTAGCTTCCTTATATCCCTTG
GGAAACAGCTATGACCATGAGAAACGGAATCGTGCGG
164
DQ249446
(GTT)^5 GGAAACAGCTATGACCATCGACTAATGGCGTTGACGG
TCCACAAGTGTCCGGCTTG
198
AF233359
(AC)^6
GGAAACAGCTATGACCATTTCCGGATGGGTGACGAG
AGGGAAACGGATAAACGGG
387
DQ249366
(AC)^7
GGAAACAGCTATGACCATAAAGGGATTATAGACAATGTGGAG CCTCCTGGTCGGATTACAG
DQ249425
(AAC)^6 GTCGACGAGATGGGAATGTTTAG
GGAAACAGCTATGACCATCCCAGGAAATCAAGAGATTATTACAAG
173
AY619553
(AG)^12 GGAAACAGCTATGACCATCGACTCCGGTCGGGC
GCACCGTGGAATGTCTCAAG
195
DQ249352
(AGC)^4 GGAAACAGCTATGACCATTAACCGAGAAACTGCGCC
AGTGCGCTGTTCCACTACG
263
DQ249355
(GT)^7
ATGACTTGCCACCCGAGAC
161
DQ249368
(CTT)^4 GGTGAGAAAGTGCGGTTGG
GGAAACAGCTATGACCATTGGGTGGCGTCGTAGAAC
168
DQ249377
(CCT)^7 GGAAACAGCTATGACCATCACCCTCCCTTCCAGAGC
CGGAGAGTATTGAGGGTTGC
148
DQ249387
(AG)^9
GGAAACAGCTATGACCATCTTGTGCACCAACTGAAATCC
AGGACACGCAAACGATGAG
206
DQ249396
(GT)^8
TGGACAGTCTGCGAACGAG
GGAAACAGCTATGACCATCAATCCAAGCGCGACATTC
188
DQ249403
(GT)^10 CGCTTGTACAGCCGAAACG
GGAAACAGCTATGACCATCCTATCCGCGATTCACCC
228
DQ249406
(CT)^6
CCTAGTGGGTTCTCCGCAC
248
DQ249417
(ATC)^4 ATGATAATGGAGCCTAGTCCG
GGAAACAGCTATGACCATCGCAGAAAGAGCATTCAAACC
208
DQ249442
(CTT)^7 TCGGTTGTGTAGGTTTAGAGG
GGAAACAGCTATGACCATGTTAACCGGCGGCCAAAG
255
DQ249465
(GCT)^4 TCTCAGACATTGTGTACTATTTCCC
GGAAACAGCTATGACCATCGAGCGAATTAAGCGGTCTG
264
Locus name
Repeat
DQ249360
PRIMER_LEFT
GGAAACAGCTATGACCATGTTAAATATAGAAGGGCGGGAC
GGAAACAGCTATGACCATCACCATCCGGCCATTTCAC
M13_FAM_labelled_tag (primer)
[5'-FAM]GGAAACAGCTATGACCAT
7
177
Figure S 1. Results of the methanol toxicity test using Ceriodaphnia dubia.
100
40
80
30
IC25: 0.02%
60
20
40
20
10
Survival
Neonates/survivor
0
0
0.001
0.01
0.1
Percent methanol (v/v)
8
1
Mean neonates/survivor
Mean percent survival
50
Figure S2. Chronic toxicity test conducted to compare the toxicity of sonicated versus
magnetically stirred DNAN preparation methods using Daphnia pulex (Pop-B population). Both
survival (a) and reproduction (b) data are presented as means (± 1 standard deviation).
Proportion surviving indicates the decimal fraction of the surviving animals (0.9 = 90%
survival).
A
B
9
Figure S 3. Acute (a) and chronic (b) toxicity reference values for 2,4-dinitroanisole (DNAN)
relative to other munitions. Toxicity reference values are effect endpoints (e.g., LC50, IC50,
LOEC) for fish, Daphnia and Ceriodaphnia species obtained from the literature [8,9] and
USEPA’s ECOTOX database (http://cfpub.epa.gov/ecotox/; queried May 2012) using measured
values based on measured concentrations. DNAN data were generated in the present study.
Silver and lead are included as examples of chemicals in other toxicity categories. Box margins
represent the 25th and 75th percentiles, error bars represent 10th and 90th percentiles (single
points represent outlier data), and lines within the boxes represent the median. General toxicity
severity ranges are from USFWS [10]. NQ = nitroguanidine, NTO = 3-nitro-1,2,4-triazol-5-one,
2,4-DNP = 2,4 dinitrophenol, RDX = royal demolition explosive, DNB = dinitrobenzene, TNT =
trinitrotoluene, TNB = trinitrobenzene, Pb = lead.
A
B
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