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White paint flowed down the Kaikorai Stream at the Nairn St Reserve in Dunedin yesterday.
Otago Regional Council environmental monitoring and operations director Scott MacLean said the paint came from a stormwater outlet and was traced to a painting contractor working in the area.
The council would decide on what action would be taken next after it completed a report on the spill, he said.
The latest contamination comes after the stream was polluted with paint in November, with another incident about a year before.
At the time Mr MacLean said paint in waterways poisons fish and plants and also affects the coastal environment.
Last week Kaikorai Stream tributary in Frasers Gully glowed green, which the ORC believed was likely because it was contaminated with to unconsented non-toxic tracer dye.