More 1,000 fish have been found dead in a river in what is being described by investigators as a "major fish-kill incident", prompting a criminal investigation.
The Northern Ireland Environment Agency said pollution in the water was being classified as "high severity" and samples have been taken.
Investigating officers have identified the potential source of the discharge into a stretch of the River Oona, near Dungannon, as being agricultural.
NIEA spokesman Brian Luke said 950 brown and rainbow trout as well as 300 sticklebacks and roach are among the dead species discovered in the polluted Co Tyrone water.
"We received a phone call around 4pm on Sunday from a member of public to say they saw dead fish and our experts were on site within an hour," Mr Luke said.
"We've categorised this as a major fish kill and a high severity pollution incident.
"It's probably the biggest incident of its kind so far this year.
"Investigators are in the process of carrying out a complete survey of the river and that is expected to continue until the end of the week.
"At this stage we can tell that mostly young fish have been killed."
Mr Luke said both the Northern Ireland Environment Agency (NIEA) and the Inland Fisheries agencies are investigating the water pollution.
Environment Minister Mark H Durkan has voiced his concerns.
"I am concerned about this and I am keeping a close eye on the situation along with NIEA officials," Mr Durkan said.
The River Oona runs through south Tyrone and is part of the Blackwater river system, understood to be an important fishing area.
Mr Luke said the results of the investigation would be known in due course.
"It's a criminal offence to cause water pollution.
"We've taken statutory samples and we've begun our enforcement process," he said.
"Water pollution can be done accidentally or it could be a case of negligence. Unfortunately both are crimes."
Last August, thousands of fish were killed following a severe water pollution incident in the Enler River near Comber, Co Down.
Local farmer Martin Hamilton admitted the pollution was from dirty water which came from fields on his property and he apologised.
The alarm was raised through an anonymous report from a member of the public to the Water Pollution Hotline after large numbers of dead fish were seen in the river upstream of Comber town.
Mr Hamilton said water used for washing crops had leaked into the Enler River, which is a tributary of Strangford Lough.
He estimated about or 25,000 litres of the dirty water, which kills the oxygen in the river, had left the farm.
"We have a massive run-off on one of the fields and we have got ourselves into a real mess," the farmer said.
Almost 5,000 dead brown and sea trout were found after a fish kill on the river Enler in Co Down in August 2013. Dirty water entered the river, near Comber, and removed the oxygen from it. A marine biologist said it could take decades before the river recovers. In January 2011, up to 500 fish, mainly trout and salmon, died in the Six Mile Water river in Co Antrim after it was hit by a fish kill for the second time in three years. In 2008 there was a major kill in the same river, when thousands of fish were wiped out.