Analysis of Pesticides in African Wildlife Samples
Meaghan P. Drumm*, MSFS, Stephen F. Donovan, Ph.D., Alana J. Balogh, BFA, Barry K. Logan, Ph.D.
1 Disclosure
• No external funding provided to support the costs of the project
2 BACKGROUND
3 Background
• Extermination • Targeted animals mentality – Lions • Targeted carnivores – Hyaenas and crop raiding – Antelope animals – Moles – Threats to human and – Porcupines food production – Wild dogs • Poisons favored – Cheetahs method of eradication – Jackals – Vultures
4 Reasons for Poisonings
• Control damage causing animals • Harvest food • Traditional medicine – Lions, Crocodiles, Vultures – Increased need for medicine due to increase in human population • Wildlife products – Ivory tusks – Rhinoceros horns 5 Methods to Poison Wildlife
• Baited carcasses • Fruit • Snails • Rice and other grains • Bushmeat −Poison mixed with salt • Poisoned waterholes
6 Secondary Poisonings
• Non target animals – Vultures – Scavengers
7 Classes of Pesticides
• Alkaloids – Strychnine • Organochlorides • Organophosphates – Monocrotophos • Carbamates – Carbofuran – Aldicarb
8 THE PROJECT
9 Objective
• Develop a qualitative method – Collection and extraction of samples in-field conditions • Confirm the presence of select pesticides • Provide forensic evidence – Wildlife advocates – Local authorities
10 Samples
• Southern Africa • Collected samples – Baited fruit – Stomach – Intestinal and crop contents – Muscle – Urine – Liver – Serum – Tissue
11 In-Field Extraction
• Extracted in the field • Items purchased at local hardware stores – Acetone – Funnel – Paper towel – Glass jar • Dried • Extracted blank control • Shipped to USA
12 METHODS
13 Extraction Method
1. 2x2 inch square 4. Reconstitute in 0.5 cutting mL of mobile phase 2. Passively extract for 5. Centrifuge at 3800 at least 10 minutes in rpm for 10 min ethyl acetate 6. Perform dilution (if 3. Evaporate to dryness needed) for 30 mins at 40℃
14 LC/MS/MS Method Information
Time (Min) Flow (mL/Min) %A %B Initial 0.45 90 10 0.5 0.45 90 10 4 0.45 20 80 4.1 0.45 90 10 5 0.45 90 10
Instrument: Waters Acquity UPLC, Waters Xevo TQD MS
Mobile Phase A: Ammonium Formate (pH 4) Mobile Phase B: 0.1% FA in MeOH
Column: Acquity UPLC® BEH C18 column (2.1 mm x 100 mm x 1.7 mm) 15 LC/MS/MS Method Information
Daughter 1 Daughter 2 Collison 1 Collison 2 Analyte Parent (m/z) Cone (V) (m/z) (m/z) (V) (V)
3-Hydroxycarbofuran H+ 237.9 162.7 180.8 24 16 12
3-Hydroxycarbofuran NH4+ 255 162.7 180.7/219.8 12 20 18/10
Aldicarb Na+ 212.8 88.8 115.8 28 16 12
Aldicarb Sulfone H+ 223 76 85.9 10 6 14
Aldicarb Sulfone NH4+ 239.9 85.9 147.8 10 18 14
Aldicarb Sulfoxide NH4+ 223.9 88.6 104.6/131.6 12 18 16/10
Carbaryl H+ 201.9 126.8 144.9 22 26 12
Carbaryl NH4+ 218.9 126.8 144.9 22 26 12
Carbofuran H+ 221.9 122.9 164.9 23 18 12
Methomyl H+ 163 88 106 15 9 10
Monocrotophos H+ 223.9 97.7 126.6/192.7 20 12 18/20
Monocrotophos NH4+ 240.9 97.7 126.7/192.7 12 20 22/14
Strychnine H+ 335.1 155.7 183.7 40 70 1666 Chromatography
Monocrotophos 3-Hydroxycarbofuran Aldicarb Carbaryl 2.30 2.72 3.19 3.70 10 ng on Methomyl Strychnine column 2.12 2.32
Aldicarb Aldicarb Carbofuran Sulfoxide Sulfone 3.56 1.74 1.89
17 Validation
• Fit for purpose – Limit of detection – Recovery – Stability on paper towel • 3 temperatures
18 Limit of Detection
Limit of Detection Analyte (amount on column) 3-Hydroxycarbofuran H+ 0.1 pg 3-Hydroxycarbofuran NH4+ 0.1 pg Carbofuran H+ 0.1 pg Neat standard injected 5 times Aldicarb Sulfone NH4+ 0.5 pg Aldicarb Sulfoxide NH4+ 0.5 pg Carbaryl H+ 0.5 pg Monocrotophos H+ 0.5 pg Aldicarb Na+ 5 pg Aldicarb Sulfone H+ 5 pg Methomyl H+ 5 pg Monocrotophos NH4+ 5 pg Carbaryl NH4+ 10 pg Strychnine H+ 10 pg 19 Recovery
Analyte Recovery (%) Strychnine H+ 17.4 Aldicarb Na+ 52.2 Carbaryl NH4+ 64.9 3-Hydroxycarbofuran NH4+ 68.5 3-Hydroxycarbofuran H+ 68.8 Aldicarb Sulfoxide NH4+ 73.0 Aldicarb Sulfone NH4+ 80.3 Methomyl H+ 81.7 Carbaryl H+ 83.0 Aldicarb Sulfone H+ 86.3 Monocrotophos NH4+ 86.7 Monocrotophos H+ 86.8 Carbofuran H+ 107.4 20 Room Temperature (23°C) Stability
100000 Percentage lost 90000 79-100% 80000
70000
60000
50000
Peak Area Peak 40000
30000
20000
10000
0 Day 0 Day 7 Day 14 Time
Aldicarb Na+ 3-Hydroxycarbofuran NH4+ 3-Hydroxycarbofuran H+
Carbofuran H+ Aldicarb Sulfone H+ Aldicarb Sulfone NH4+
Aldicarb Sulfoxide NH4+ Monocrotophos H+ Monocrotophos NH4+ 21 Heat (33.9°C) Stability
100000 Percentage lost 90000 91-100% 80000
70000
60000
50000
Peak Area Peak 40000
30000
20000
10000
0 Day 0 Time Day 7 Day 14
Aldicarb Na+ 3-Hydroxycarbofuran NH4+ 3-Hydroxycarbofuran H+ Carbofuran H+ Aldicarb Sulfone H+ Aldicarb Sulfone NH4+
Aldicarb Sulfoxide NH4+ Monocrotophos H+ Monocrotophos NH4+ 22 Freezer (-80°C) Stability
100000 Percentage lost 90000 82-100% 80000
70000
60000
50000 Peak Area Peak 40000
30000
20000
10000
0 Day 0 Day 7 Day 14 Time
Aldicarb Na+ 3-Hydroxycarbofuran NH4+ 3-Hydroxycarbofuran H+ Carbofuran H+ Aldicarb Sulfone H+ Aldicarb Sulfone NH4+ Aldicarb Sulfoxide NH4+ Monocrotophos H+ Monocrotophos NH4+ 23 RESULTS AND CONCLUSION
24 Case Samples
• 16 Different Animals – Knob-billed goose (n=1) – Warthog (n=1) – Elephant (n=9) – Vulture (n=2) – Hippopotamus (n=2) – Buffalo (n=1) • 3 Bait Samples – Watermelon (n=1) – Marula Fruit (n=2) • 16 Controls
25 Results
3-Hydroxy Aldicarb Aldicarb Aldicarb Carbofuran Monocrotophos carbofuran Sulfone Sulfoxide
Buffalo Liver X Buffalo Stomach X X X X Elephant Stomach X X X X Hippo Stomach X X
Knob-billed Goose Crop X
Knob-billed Goose Liver X
Marula Fruit X X X Vulture Liver X X X X Vulture Stomach X X X X X Warthog Liver X Warthog Serum X
Watermelon X 26 Conclusion
• In-field extraction shows promising results – Way to export for analysis that would not be performed due to lack of laboratory facilities • Analytical method is sensitive within a wide range of matrices • Results can be used to make connections to aid in the apprehension of individuals • Provides forensic evidence to help deregister the legal use of these compounds
27 Future Work
• Continue to receive more samples – Current collaborators – New collaborators • Funding opportunities – Help expand the scope – Provide funding for shipping costs and other materials – Produce educational video
28 Questions?
29