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The latest, from a manhole in Stanley St on Tuesday afternoon, was caused by a build-up of congealed fat which blocked a pipe and caused overflow.
A statement released by the Queenstown Lakes District Council said an estimated 2000 litres of wastewater subsequently entered Horne Creek and signs were placed along a short section of beach where the creek enters Lake Wakatipu.
Water testing on Tuesday night confirmed E. coli was present and the council was waiting for the result of yesterday's testing, the council's infrastructure and assets general manager, Erik Barnes, said. It was likely that the fat came from a restaurant but the council has not been able to find where it entered the sewerage system, Mr Barnes said.
In December, raw sewage flowed on to Athol St and last month the Otago Daily Times reported that in the past year there had been nine unauthorised sewage spills into Lake Wakatipu in the Queenstown, Frankton and Fernhill areas.
There had also been two last month, one at Kelvin Heights and the other at Frankton.
The council was looking at developing a trade waste bylaw which would make it easier to prosecute people found putting inappropriate material into the sewers and stormwater drains.
''We want to do better,'' Mr Barnes said, but added, ''It's one of those tough things.''
An education programme had also been launched to inform the public about the consequences of tipping fat, oil or grease into drains either at home or work.
On average, it costs the council $1000 to have contractors clean up spills.