Combining Analytical Chemistry and DNA Barcoding techniques to
identify chemicals and species involved in the poisoning of vertebrate
wildlife in Scotland
Results & Discussion
SASA operates the Wildlife Incident Investigation Scheme (WIIS) in Scotland and
conducts analytical investigations in order to identify and quantify the chemicals (mainly
pesticides) involved in the accidental or deliberate poisoning of wildlife, beneficial
insects, livestock and domestic animals. The principal techniques employed are Gas or
Liquid Chromatography combined with tandem mass spectromentry i.e. GCMSMS and
LCMSMS.
Extracts collected from the eagle’s gullet, mouth, stomach contents and liver tissue all
tested positive for the presence of carbofuran, a carbamate insecticide, following analysis
by GCMSMS. This pesticide is not approved for use in the UK and approval expired
in 2001. It is also illegal to possess substances that contain carbofuran as an active
ingredient [Possession of Pesticides (Scotland) Order 2005].
Figures 2a and 2b show the corresponding ‘sample screen’ ion chromatograms from a
reagent blank, gullet content extract and proximate matrix standard and associated production (MSMS) mass spectra, indicating a positive result for the presence of carbofuran.
Chromatogram Plots
BP: 192.9 (11=100%), r blank_8-10-2007_3-40-56 pm.sms
R Blank_8-10-2007_3-40-56 PM.SMS
kCounts
10.103 min, Scans: 2423-2427, Merged, Ion: 65000 us, RIC: 25, BC
192.9
11
100%
50%
164.9
4
25%
2.5
136.0
1
162.9
1
100
std 4ng_8-14-2007_2-00-13 AM.SMS
164.0>110:160 [0.60V]
75
165.9
2
179.9
1
125
150
175
200
m/z
10.105 min, Scans: 1858-1860, 164.0>110:160 [0.60V], Ion: 4012 us, RIC: 79231,
148.9
35541
BP: 148.9 (35541=100%), std 4ng_8-14-2007_2-00-13 am.sms
100%
50%
146.2
12707
131.2
10545
25%
25
118.2
2483
145.3
4950
123.2
2231
147.2
2681
0%
0
kCounts
110
07103-1 GullC_8-14-2007_3-52-09 AM.SMS
164.0>110:160 [0.60V]
120
130
140
150
160
m/z
10.117 min, Scans: 1862-1864, 164.0>110:160 [0.60V], Ion: 4317 us, RIC: 75103,
148.9
34120
BP: 148.9 (34120=100%), 07103-1 gullc_8-14-2007_3-52-09 am.sms
100%
75
75%
Although the deliberate poisoning of wildlife in Scotland is a criminal offence, SASA deals
with numerous cases each year. These cases involve a variety of vertebrate species,
mainly birds of prey and the identity and quantity of the chemical used as poison is crucial
in support of police investigations of wildlife crime. In 2007, a Golden Eagle was found
dead in Peeblesshire, Scotland (Figure 1). The bird (a protected species) was well known
to local conservationists as it was the female half of a pair of birds that had been breeding
successfully for several years. The bird was found in perfect bodily condition with no
obvious signs of disease or trauma and pesticide poisoning was suspected.
SASA, Roddinglaw Road
Edinburgh, EH12 9FJ
Scotland
75%
50
Introduction
193.9
4
0%
0.0
kCounts
Authors:
E. Sharp
A. Giela
V. Mulholland
M. J. Taylor
75%
7.5
5.0
This poster illustrates how molecular biology (DNA barcoding) and analytical chemistry
techniques (GC-MSMS) were used in combination to provide detailed and complementary
information concerning the death of a Golden Eagle (aquila chrysaetos) exposed to prey
contaminated with deadly pesticides.
Rural Payments and Inspections Directorate
Chemisty
Section
Abstract
Occasionally, the identity of the species involved by visual inspection alone can be
extremely difficult due to the poor condition and ambiguity of some specimens submitted
for analysis. However, tissues of various types, including feathers, can be subjected to a
molecular biology technique, usually termed “DNA Barcoding”. This technique provides
information on the provenance of tissue samples, which can be vital to the identification
of poisoned baits, victims or ingested food.
Science and Advice for Scottish Agriculture
T: +44 (0) 131 244 8874
F: +44 (0) 131 244 8940
E: [email protected]
www.sasa.gov.uk
50
50%
25
25%
118.2
2257
0
146.2
11512
131.2
9623
121.2
1869
145.3
5142
123.2
2323
147.2
2507
0%
7
8
9
10
11
110
12
minutes
120
130
140
150
160
m/z
OCONHCH 3
O
CH 3
CH 3
Figure 2a and 2b
Confirmation of this screen result was
achieved by matching the full-scan
electron impact (EI) mass spectra
obtained following analysis of the
extract and a carbofuran standard.
Figure 3 illustrates the similarity of the
relevant mass spectra.
BP: 164.0 (216365=100%), std 4ng_8-14-2007_8-11-11 am.sms
10.114 min, Scans: 1340-1342, 50:520, Ion: 1057 us, RIC: 788329
164.0
216365
100%
75%
149.2
118925
50%
25%
77.3
11365
91.4
17651
103.3
16516
121.4
24786
131.4
33270
165.0
21760
146.5
17011
0%
50
75
BP: 164.0 (208819=100%), 07103-1 gullc_8-14-2007_10-01-15 am.sms
100
125
150
100%
175
m/z
10.113 min, Scans: 1346-1348, 50:520, Ion: 1176 us, RIC: 764805
164.0
208819
75%
149.3
109513
50%
25%
77.3
11403
91.4
16790
103.4
15756
122.4
23449
131.4
30819
165.0
20913
146.5
16580
0%
50
75
100
125
150
175
m/z
Figure 3
Figure 1. Golden Eagle found dead in Peeblesshire, Scotland
SASA has been using GCMS and LCMS techniques for several years to detect and
identify residues of poison present in various specimens taken from poisoned animals
or baits e.g. animal tissue, liver, digestive tract material or stomach contents. Gas
chromatography (GC) combined with tandem mass spectrometry (MSMS) provides a
powerful technique able to separate components present in complex mixtures (i.e. sample
extracts) and screen these mixtures for the presence of pesticide residues with a highly
selective and sensitive detector.
The use of of DNA barcoding, a molecular biology-based method which uses a 648 bp
region of the mitochondrial cytochrome c oxidase I gene (COI) to identify samples to the
species level, presents a complementary technique capable of the identification of the
type of species involved e.g. remains of bait/food in digestive tract or stomach, particularly
in circumstances where visual inspection is inadequate.
Methods & Materials
GCMSMS
Ion pair
(m/z)
Ion Ratio
(sample)
Ion Ratio
(standard)
Variance
(%)
164:149
0.543
0.551
1.5
164:146
0.065
0.077
18.5
164:131
0.114
0.141
23.7
164:122
0.164
0.149
10.1
Table 1. Ion ratios used to confirm positive result for carbofuran.
DNA barcoding experiments were performed on feathers taken from the mouth, gullet
and stomach. The mean size of the DNA sequence was 599 bp and all sequences were
analysed using the BOLD-IDS system and the Reference Barcode Database. Fifteen
matches were at the species-level, identified with a 100% probability as red grouse
(Lagopus lagopus), whilst the remaining two were identified a Lagopus sp. to the genuslevel. It was therefore suspected that a red grouse (contaminated with deadly carbofuran)
was used as a bait to lure the eagle.
Conclusions
Gas Chromatography Mass Spectrometer
●Varian 3800 ● Column: VF-5MS 30m x 0.25mm x 0.25μm
● Inj. Temp: 250 ºC
● Total Flow: 40cmsec-1
● Column flow: 1mLmin-1
● Split ratio: 1:20
● Inj. Volume: 1μL
● Oven Temp:
40 ºC/hold 2min then
200 ºC at 25 ºCmin-1 then
250 ºC at 10 ºCmin-1/hold 17 min
This similarity is ultimately qualified by ensuring that the ion ratios of the ‘actual’ mass
spectral data and ‘reference’ mass spectral data comply with laboratory AQC requirements
and are within ±25% variance (Table 1).
● Varian: 4000 ITMS
● Ionisation mode: EI+
● Transfer Line Temp: 180 ºC
● Source Temp: 180 ºC
● Precursor ion: m/z 164
● Product ion mass range: m/z 110-174
● Excitation storage level: 104 m/z
● Excitation amplitude: 0.8V
● Emission current: 25 μAmps
Chemical and biological analysis of wildlife specimens can reveal important information
about the cause and circumstance of their death or intoxication. The golden eagle involved
in this case undoubtedly died following lethal exposure to carbofuran a pesticide banned
for use in the UK since 2001. The probable source of the exposure was red grouse laid
out as bait to attract wildlife.
The combination of GCMSMS and DNA barcoding can be extremely useful in the generation
of information that can be used by wildlife crime investigators. This particular case
remains open as the investigation of wildlife crime in Scotland continues.
Sample preparation
Residues are extracted from samples by maceration or tumbling in solvent. Crude
extracts are then cleaned-up by gel permeation chromatography (GPC) before analysis
by GCMSMS.
DNA Barcoding
1. Collect Specimens
Be careful not to
cross-contaminate
2. Extract DNA
Use methods developed
for forensics
3. DNA Amplification
Standardised and
robust methods
CAGCCTCACCTGGCATCAAATT
120
130
6. Use the on-line DNA Barcode database
Gives an identification and confidence level
5. Sequence
Get the DNA Barcode
4. Screen PCR Products
Check on a gel
This poster was
prepared by the SASA
Photography Unit.
All photographs and
text are SASA © Crown
Copyright except where
otherwise indicated.