The envelope was addressed to Justice Arthur Engoron, who imposed a nearly half-billion dollar judgment against Trump.
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The letter was received in the operations office of the courthouse at 60 Centre St. around 9:55 a.m. on Wednesday.
The operations office where the letter was opened was closed and the affected staff were isolated until Emergency Services tested the powder, officials said.
Preliminary testing showed the powder was negative for hazardous substances.
Justice Engoron had no exposure to the letter or the powdery substance.
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Two court officers were the only ones exposed.
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No one was injured and the court remained open.
The matter is under investigation by law enforcement
Justice Engoron received multiple threats before, during and after Trump's civil fraud trial, including a bomb threat at his Long Island home on the day of closing arguments.
Engoron imposed a limited gag order on Trump's statements to protect court staff. A similar gag order is being sought by Manhattan District Attorney Alvin Bragg for Trump's criminal trial next month at a courthouse two blocks away.
Separately, a white powder envelope was received on Tuesday at 1 Empire State Plaza, the Albany office of state Attorney General Letitia James
The letter tested negative, and no one was injured or reported any symptoms.