Toxin exposure in wildlife is a vast field of study.  The forensic investigator may encounter cases that range from intentional poisoning of pest species and secondary exposure of non-target species, to accidental exposure of wildlife by labeled use of pesticides, to cases involving massive environmental pollution and major liability.  A popular misconception about toxicologic screening is that the technology exists to test a sample for a broad spectrum of different substances.  New equipment is being developed that greatly diminishes the labor involved in toxin screening and increases the spectrum of available tests; however, there is no such thing as a toxin panel or profile.  Investigators must a have an idea of likely suspect toxins based on history, previous cases for a given region, and post-mortem examination.  Also, identification of specific substances is relatively expensive.  In some cases, fines for poisoning wildlife may not be much greater (if at all) than the costs of analyses required to obtain a conviction.

We will concentrate our discussion on compounds used to intentionally poison wildlife rather than cases of industrial pollution.  Toxins most commonly used include carbamates, organophosphates (OP's), strychnine, compound 1080 (sodium fluoroacetate), anticoagulant rodenticides, thallium, and cyanide.  Carbamates and OP's comprise the greatest percentage of toxicosis cases.  Many of these chemicals are or have been used in agriculture and are available through legal distributors as well as illegal sources.

Several factors may clue investigators into a suspicion of toxicosis.  For example, a dead animal or localized group of dead animals in an apparently good nutritional state is highly suspicious of a toxin, especially if multiple species are involved.  In some cases, investigators may locate a poisoned bait or have a good indication of agricultural pesticides commonly used in a given area.  Whenever a toxin is suspected, necropsy staff, as well as field personnel, should take precautions to prevent accidental human exposure.  Samples should be taken in appropriate containers (acid-rinsed glassware) and toxin analyses should be conducted as soon as possible.  A thorough sampling of tissues in suspected toxicosis cases includes stomach contents, small intestine contents, liver, kidney, brain, and fat.

Now, we will look at a case to demonstrate some additional principles. 

This Bald eagle was found staggering along a country roadside.  She was reported to have been experiencing muscle tremors and severe hypersalivation when she was captured by a passing motorist.  The eagle was taken to a local veterinary clinic, where she died shortly thereafter.  The local U.S. Fish and Wildlife officer was called to pick up the carcass.  He returned to the area in which the eagle was found and discovered two more dead eagles and a partially eviscerated coyote.  In a nearby field, he observed a sheep carcass that appeared to have been scavenged or predated.  He presents the laboratory with the eagles, the coyote, and some samples from the sheep.  Grossly, the eagle appears to have been in excellent condition.

This case may be a little too easy; however, all of the facts given in the history and scenario are very common in these cases.  The hypersalivation and muscle tremors are highly suspicious of anticholinesterase toxicosis.  Carbamates and organophosphate compounds commonly are applied to kill predators, such as coyotes.  A common scenario is to find a poisoned coyote in the same area as affected scavengers that have fed either on the dead coyote or the baited carcass.  When animals directly are affected by the application of a substance to poisoned bait, this is referred to as primary toxicosis.  Secondary toxicosis occurs when scavengers feed on other animals affected by a original poison source.

Primary Poisonings vs. Secondary Poisoning

Identification of crop or stomach contents may help determine whether a case was caused by primary or secondary toxicosis. Gross comparison of ingested tissues with other carcasses found in the vicinity may help determine the sequence of toxicosis.  It may be necessary to separate the contents from different organs (i.e. crop from proventriculus) and analyze portions or food items separately.  There are many keys available for identification of animal hair, and tissues can be positively identified by serologic methods.  Also, with today's technology, animals can be positively linked by DNA analysis.

Toxicologic Analyses

As stated above, there is no effective broad-screen method for identifying toxins.  The range of possibilities should be narrowed by the history, physical signs, use of pesticides in a given region, and previous cases in an area.  Some compounds break down relatively quickly, even those stored in a frozen state.  Thus, chemical analyses should be conducted as quickly as possible.  Separation of gastrointestinal contents and individual analysis on each sub-component is preferred.  This measure will avoid diluting the toxin, which may occur if a composite sample is submitted.

Brain cholinesterase activities can be determined as a screening test for anticholinesterase toxins (carbamates and organophosphates).  Brain cholinesterase suppression of 50% or greater is considered diagnostic for death by anticholinesterase poisoning; however, levels frequently exceed 70% under experimental conditions.  Wide variation occurs in brain cholinesterase activities under field conditions.  This may be confounded by the absence of “normal” reference values for many species.  In addition, animals exposed to carbamate pesticides may undergo spontaneous reactivation of brain cholinesterase activities.  If you refer back to your toxicology, you will remember that carbamates are reversible inhibitors, while OP’s are non-reversible inhibitors.  Thus, animals that succumb to carbamate poisoning may have brain cholinesterase activity within reference limits.  This biochemical characteristic has been used to develop additional tests that help distinguish between carbamate and OP exposure.  These techniques employ various methods, such as thermal and 2-PAM reactivation, to reactivate cholinesterase enzymes.

Interpreting Results

Following exposure to a toxin, many processes may affect subsequent post-mortem analyses.  The amount of a toxin quantitated in the laboratory is what is left following absorption into tissues, dilution in ingesta, and decomposition of the compound.  Also, vomition or regurgitation will diminish the amount of a compound remaining in the carcass.  The lack of poison-laced ingested material does not preclude toxin exposure, as water and percutaneous exposure also may occur.  For these reasons, thorough evaluation of toxicosis cases includes ruling out other possible causes of death.

Precautions

Always remember that human exposure is a risk with any possible toxicosis case.  Adequate protective wear and disposal of tissues and materials is critical.  In addition, investigators must familiarize themselves with shipping regulations and follow standards set for hazardous materials.

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