Dozens of small fish have died after slurry was released into the River Brue in Somerset.
Environment Agency officers have carried out a rescue operation along a four kilometre stretch of the river between Bruton and Cole.
About 20 brown trout and a small number of crayfish were also killed by high levels of ammonia in the river.
An investigation into the cause of the incident has begun. The agency has confirmed the source of the pollution.
More than 100 fish have been relocated to a safe area upstream of Bruton and Environment Agency ecologists are surveying the river to discover the full impact of the spill.
"This was a serious pollution incident," said Brian Payne, from the Environment Agency.
"We have had teams of officers on site monitoring the spread of the pollution downstream and investigating the spill."
Last year the Environment Agency had to re-stock a section of the River Brue after the local fish population was wiped out following an even bigger pollution spill.