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Hundreds of dead fish have been found in the Swalecliffe Brook.
Around 300 sticklebacks and populations of eels, an endangered species, have died.
Investigations by the Environment Agency have revealed that the incident was caused by a sewage leak, and they have launched a major clean-up operation.
Contractors have been brought in to increase the levels of oxygen in the water with specialist equipment so any further harm to wildlife and the environment is minimised.
Colin Bowley of the Environment Agency said: “This is a serious incident which has affected a significant stretch of the brook.
“Our officers have taken action to use the tidal sluice to isolate the pollutant in the worst affected stretch and we are now concentrating our efforts to improve water quality in the brook.
"Environment officers are currently on site collecting evidence, taking further water samples from the river and monitoring the watercourse closely.
“In partnership with Canterbury City Council, we are investigating the causes and impact of what has clearly been a serious incident.
"Both organisations have been busy advising the public and putting up signage to warn people of the incident.”
The Environment Agency has traced the source of the pollution and will continue to oversee the clean up of the incident.
The investigation is ongoing and no further details can be released at this stage.
Members of the public can report pollution incidents by calling the Environment Agency’s emergency hotline on 0800 807060.