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Shortly after 11:00 a.m. local time on September 4, 2006, Irwin was fatally pierced in the chest by a short-tail stingray barb while diving in Batt Reef (part of the Great Barrier Reef), off the coast of Port Douglas in Queensland. Irwin was in the area filming his own documentary, to be called The Ocean's Deadliest, but weather had stalled filming. Irwin decided to take the opportunity to film some shallow water shots for a segment in the television program his daughter Bindi was hosting,[25] when, according to his friend and colleague John Stainton, he swam too close to one of the animals. "He came on top of the stingray and the stingray's barb went up and into his chest and put a hole into his heart," said Stainton, who was on board Irwin's boat, called the Croc I, at the time.

The events were caught on camera, and the footage was handed to the Queensland Police.[26] After reviewing the footage of the incident and speaking to the cameraman who recorded it, marine documentary filmmaker and fisherman Ben Cropp speculated that the stingray "felt threatened because Steve was alongside and there was the cameraman ahead.:"[26] In such a case, the stingray responds by automatically flexing the serrated barb on its tail up to a maximum of 25 cm (10 in) of length.

"He came over the top of a stingray that was buried in the sand, and the barb came up and hit him in the chest," Stainton said. Cropp said Irwin had accidentally boxed the animal in. "It stopped and twisted and threw up its tail with the spike, and it caught him in the chest. It's a defensive thing. It's like being stabbed with a dirty dagger." The stinging of Irwin by the Bull Ray was "a one-in-a-million thing," Cropp told Time magazine. "I have swum with many rays, and I have only had one do that to me."[27] In this case, the motion struck Irwin's chest and pierced his heart.[26] Crewmembers aboard his boat called emergency services in the nearest city of Cairns and administered CPR as they rushed the boat to nearby Lowe Isle to meet a rescue helicopter. Medical staff pronounced Irwin dead when they arrived a short time later.[28] The barb lodged in his chest and the toxins of the barb caused Irwin to die of cardiac arrest.[29]

This was only the third known fatality in Australian history from a stingray attack. As of 1996, only seventeen worldwide fatalities had been recorded,[30], and it is believed to be the only fatal attack ever captured on film.[31]

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