At least 5,000 fish are now thought to have been killed over the last 24 hours in a tributary of the River Nore in Co Laois.
Officials of Laois County Council say they have not yet been able to detect what caused the deaths of the brown trout in the River Gully near Durrow.
The Durrow and District Anglers Club say it will take the stream up to five years to recover.
A club spokesman, Michael Walsh, said it was essential that whoever was responsible for the fish kill is identified.
