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

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

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

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

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)

Case Discussions Oleander Paraquat Zinc phosphide Anatoxin-a

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

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

Oleandrin in Human Blood – 9 ppb ESI (+) LC/MS-MS-MS Ion Chromatogram (m/z 355+373) and Product Ion Mass Spectrum

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

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 1990-1991 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 1977. These are included in most but not all formulations depending on cultural acceptance and product registration.

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

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

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.

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!

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

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

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.

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

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

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

TOXICOLOGISTS Dr. Birgit Puschner Dr. Robert Poppenga Dr. Asheesh Tiwary PHONE: (530) 752-6322 http://cahfs.ucdavis.edu/