Forensic scientists solved the mystery of what killed a large portion the the wild mustang herd in southern Nevada using a technique called stable isotope ratio mass spectrometry.

In July 2007, 71 horses of the 250-head herd of wild horses were found dead near Main Lake depression in southern Nevada. Testing performed by the California Animal Health and Food Safety Laboratory system found high levels of nitrate (NO3-) and nitrite (NO2-). The horses were negative for botulin, anatoxin-a, microcystins, and testing for organic compounds was also negative. The lake itself--which the herd commonly used--had high nitrate levels. Other ions such as chloride and sulfate were also markedly high.

After catching wind of the fallen horses, a handful of public groups speculated that the horses had suffered a repeat of the 1988 dumping of nitrate-containing deicing fluids which resulted in the accidental poisoning of 61 wild horses near Cactus Flat, Nev.

Nitrate is formed naturally via biological transformation of ammonia (NH4+) in soil, sediments and water. It is also synthetically produced by humans in large quantities used in such products as fertilizers and homemade explosives.

Since different isotopes or "forms" of nitrogen (N) and oxygen (O) exist, determining the type of N and O in the nitrates found in Main Lake could indicate the source of the nitrates that killed the horses.

Based on the different isotopes found in Main Lake, the scientists concluded that accidental (or intentional) poisoning of the horses was not the problem.

The cause of death was the horses' own manure and urine.

"The isotopic data suggested that the most probably source of the nitrate was nitrification of nitrogen from horse manure and urine that had leached into the pond. The (results) suggested that extreme evaporative concentration had occurred, resulting in toxic levels of nitrate accumulating in the Main Lake depression," the team wrote.

The mathematical models used to uncover the source of the poisoning are provided in detail in the study, "Multiple isotope forensics of nitrate in a wild horse poisoning incident," will be published in an coming edition of the journal Forensic Science International.