1. What’s going on in a Veterinary Toxicology Laboratory?
Birgit Puschner
Veterinary Toxicologist
DVM, PhD, Dipl. ABVT

2. Toxicology Laboratory Capabilities
Providing information
Diagnosis of intoxications and nutritional imbalances
Prevention of intoxications
Ensuring food safety
Prevention/identification of chemical agroterrorism

3. Laboratory Set-up 2 ABVT certified toxicologists
1 resident/PhD student
1 Senior analytical chemist (PhD)
10 analytical chemists (M.S., B.S)
Sample receiving area

4. Toxicology Testing 450 to 500 case submissions per month
~35,000 tests per year
Chain-of-custody
QC/QA in place
Over 80 validated analytical methods
Established turn-around-times
Method development

5. Toxicology Instrumentation
Triple quadrupole LC-MS/MS (2)
Ion-trap LC-MS/MS (1)
HPLC and GC/MS
ECD, FPD, FID, NPD, UV, and fluorescence detectors
ICP-MS (1)
ICP/AES (1)
AA (1)
Elisa plate reader (2)

6. Case Discussions
Oleander
Paraquat
Zinc phosphide
Anatoxin-a

7. Oleander Highly toxic Dried and fresh
~7 leaves can kill a horse or cow
~1 leaf can kill a human

8. Oleander Method by LC-MS
Serum, urine and tissues
MDL (serum, urine): 1 ppb
MDL (liver): 5 ppb
Mass spec identification and quantitation
Presented himself to the emergency room
Diarrhea, salivation, vomiting
Cardiac arrhythmias, cardiac blocks
Died after several days of intensive treatment
Radioimmunoassay (RIA) for digoxin:
Positive
No history of digoxin medication

9. Oleandrin in Human Blood – 9 ppb
ESI (+) LC/MS-MS-MS Ion Chromatogram (m/z ) and Product Ion Mass Spectrum

10. Tests for Plant Toxins Cardiotoxin screen: oleandrin, gitoxin, GTXs
Strophanthidin
Alkaloids:
coniine, nicotine, anabasine, lupanine
atropine, scopolamine
taxines
senecionine, seneciophylline, retrorsine
Nitrate/nitrite
Cyanide
Gossypol

11. Paraquat History: Samples collected for toxicology testing:
OR in 2003 and 2004: more than 20 dogs become ill after visiting an off-leash park and a beach area. Most of them died.
Acute onset of vomiting and diarrhea after park or beach visit.
Many dogs were not presented until 2-3 days.
Inappetence and lethargy, renal and respiratory failure
Samples collected for toxicology testing:
Urine, lung, vomitus
Accidental or deliberate ingestion of paraquat has been responsible for a large number of pesticide-related deaths. It is a major suicide agent in many developing countries. In Sri Lanka a 1989 study of 669 poison incidents indicated that agrochemicals were responsible for 59% and paraquat was the commonest poisoning agent with a fatality rate of 68%(22). Paraquat poisonings are still common in the UK, where a study of pesticide poisoning between indicates 44 deaths with paraquat responsible for 75%(23). Many of these may be suicides. There is concern in developing countries that the easy availability of pesticides leads to suicides which might not otherwise occur. Most cases are self-poisoning, but not all intend to die(24).     To prevent accidental deaths, Zeneca added three alerting agents in the formulations. A stenching agent, was added to some formulations in 1975, an emetic to induce vomiting in 1977, and a blue dye to prevent confusion with cola, black coffee or other beverages was added from These are included in most but not all formulations depending on cultural acceptance and product registration.

12. Paraquat Colorimetric, SPE quick test
Development of a new LC/MS methodology
Sample
SPE
LC/MS
Urine #1
Positive
N/A
Urine #2
Negative
Positive (10-50 ppb)
Urine #3
Urine #4
Lung
Positive (0.5 ppm)
Vomitus
Positive (1-10%)
Sample (either lung extract or urine) is basified and applied to C18 SPE column
Sodium dithionite and NaOH are added
Positive sample is indicated by blue color of reduced paraquat

13. Paraquat LC/MS – Implications
Provides unambiguous identification
Very fast: analysis within 1-2 hours
Low detection limits:
Urine: 50 ppb
Lung: 500 ppb
Quantitation of paraquat in serum or plasma of poisoned animals or humans:
Used to assess the severity of the intoxication
Used as a prognostic indicator
In humans: plasma paraquat concentrations of > 0.3 ppm 15 hours after ingestion are predictive of a fatal outcome regardless of treatment

14. Zinc Phosphide History: Samples collected for toxicology testing:
Puppy presented in critical condition at vet clinic
Owners mentioned “gopher bait” in backyard
Vets and veterinary technicians developed severe headaches, itchy skin and sore throats
Samples collected for toxicology testing:
Stomach contents
The puppy presented to a veterinary hospital in critical condition. She had pale mucous membranes, had no palpable pulse and her breathing was shallow and agonal. She also vomited during the initial examination. She was intubated and started on oxygen ventilation. A jugular catheter was placed in IV fluids were started. The ECG showed arrhythmias. The owner elected euthanasia due to suffering and a grave prognosis. The owner mentioned that exterminators had recently been in the backyard to place “safe” gopher bait into gopher holes.
Veterinarians and technicians treating the animal developed severe headaches, itchy skin and sore throats. Two technicians presented themselves to the emergency room. One of them had to wait for 40 minutes and decided to go back to work. The other technician was treated for potential toxin exposure and was supposedly given steroids to treat the skin reaction. This technician has been off work for several days.

15. Zinc Phosphide Diagnostic testing: Phosphide-containing gopher baits:
SPME extraction, followed by GC/MS
 Confirmed presence of phosphine
Phosphide-containing gopher baits:
Grain bait, paste, tablets, tracking powder
Concentration from 0.5% to 10%
Minimum lethal dose for most species:12 – 50 mg/kg
Precaution when collecting samples!

16. Tests for Pesticides/Household Toxins
Insecticides:
Organophosphates (43)
Carbamates (11)
Organochlorines (22)
Pyrethrins (8)
Rodenticides:
Anticoagulant rodenticides (8)
Strychnine
Bromethalin
Zinc phosphide
Others:
Avitrol
Paraquat
Metaldehyde
Ethylene glycol
Cyanide
Drugs:
NSAIDs
Macrolide endectocides

17. Cyanotoxins History: Samples collected for toxicology testing:
3 dogs developed seizures and died after swimming in the Eel River
Samples collected for toxicology testing:
Stomach contents
Liver
Water
Initial testing: Strychnine, metaldehyde, Zinc/Al phosphide, OPs and carbamates

18. Water sample from Eel River
Algae Identification
Water sample from Eel River ?
Planktothrix sp.
Performed by Dr. Pia Moisander and Jennifer Jendro:  MOST LIKELY PLANKTOTHRIX SPECIES
Shape of cells: box-like; no sheath (sheath is typical trait for Lyngbya species!)
Cell height was too large compared to the width to be Lyngbya
Lyngbya have more disc-like cells aligned in a sheath much like pennies in a roll
Planktothrix have been shown to produce anatoxin-a!
Microscopic examination: dominant algal species = diatoms and filamentous, non-heterocyst forming cyanobacteria (Carmichael)
Initially identified as Lyngbya species as the dominant genera of filamentous cyanobacteria
Therefore, the water sample should be analyzed PSTs: Ridascreen Saxitoxin Plate Kit (commercially available ELISA kit) was applied. Detection limit for STX in this assay is 0.01 ppb. The sample was positive for STX, but at a very low concentration of approximately 1 ppb. Considered a low hazard since a dog would not drink enouhg to cause acute toxicity. But the concentration that the dog actually consumed might have been higher depending on the bloom concentration where the dogs were exposed.
Controversy over some species of Planktothrix, because some identical species have been called Oscillatoria as well as Plantothrix when referring to the same species!
Lyngbya sp.
Planktothrix spp.

19. Cyanotoxins LC/MS analysis
 Confirmed presence of anatoxin-a in stomach contents and water A B C
Stomach content (S42)from a dog positive for Anatoxin a, at 0.24 ppm (wet wt.) Chromatograms of m/z 166 (A), m/z 149 (B) and spectrum of Anatoxin a (C). Samples analyzed by Dr. Carmichael

20. Tests for Natural Toxins
Algal toxins:
Microcystins
Anatoxin-a
Domoic acid
Saxitoxins
Mushroom toxins
Amanitins
Mycotoxins
Aflatoxin
Tricothecenes
Fumonisin
Ochratoxin
Zearalenone
Penitrem A/Roquefortine

21. Tests for Metals and Minerals
Lead, arsenic, mercury, cadmium, manganese
Copper, zinc, selenium, molybdenum, iron
Thallium, barium, and many others
Minerals:
Calcium, magnesium, sodium, sulfur, potassium
Vitamins:
Vitamin A and E

22. TOXICOLOGISTS Dr. Birgit Puschner Dr. Robert Poppenga
Dr. Asheesh Tiwary
PHONE: (530)