Dr Thomas Holt has told Florida's Agriculture Commissioner Charles Bronson that the horses that died had "significantly increased selenium levels" in samples tested.
"Signs exhibited by the horses and their rapid deaths were consistent with toxic doses of selenium," Holt said.
Selenium is a trace mineral essential for normal cell function and health in animals, and is often included in small quantities in supplements and feed for horses. Large doses, however, can be fatal.
Dr Holt said that the findings obtained at the department's Animal Disease Diagnostic Laboratory in Kissimmee were confirmed by independent testing conducted at the University of Florida's College of Veterinary Medicine in Gainesville, the University of California Davis Animal Health and Food Safety laboratory, and at testing facilities at Cornell University in Ithaca, New York.
Bronson thanked the work done by the University of Florida, which conducted post mortem examinations on 15 of the horses and performed extensive toxicology testing. He also thanked the University of California Davis and Cornell University for testing conducted in their labs.
The commissioner said the deaths had triggered an investigation by several state agencies and he emphasised that those inquiries were ongoing.
He said that no further information on the investigation could be disclosed at present to prevent the investigation from being compromised.
The 21 ponies that died were all members of the Venezeulan-based Lechuza Caracas Polo team, which was in the United States to play in the US Polo Open.
The horses, collectively worth an estimated $US2 million, began dying shortly after they were trucked to the Palm Beach polo grounds where their match was to be played.
Following the deaths, a Florida pharmacy admitted there was an error in medication it prepared for the ponies.
Franck's Pharmacy chief operations officer Jennifer Beckett said the strength of an ingredient in the preparation was incorrect. The ingredient has not been named by the pharmacy.
Beckett said the preparation was made on an order from a veterinarian.
"As soon as we learned of the tragic incident, we conducted an internal investigation that was led by an outside lawyer and, upon its conclusion, we immediately alerted the state Department of Health and Board of Pharmacy," Beckett said.
"The report, which we are furnishing to these agencies, concluded that the strength of an ingredient in the medication was incorrect," she said.
"We will co-operate fully with the authorities as they continue their investigations.
"Because of the ongoing investigations, we cannot discuss further details about this matter at this time," Beckett added.
Lechuza Polo released a statement confirming that a Florida-licensed veterinarian wrote a prescription for a compounded vitamin supplement intended as a substitute for a branded medication which is made in France.
The compounded medication was to contain vitamin B, potassium, magnesium and selenium, it said.
"This compound was prepared in the State of Florida by a compounding pharmacy. Only the horses treated with the compound became sick and died within three hours of treatment. The horses that were not treated remain healthy and normal.
"Lechuza will continue co-operating with the authorities. We are committed to determining the actual cause and it is our hope that medical experts can do so in a timely and conclusive manner."