At least 20 birds are confirmed dead south-west of Bendigo with reports of more as authorities try to establish a cause.
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Parks Victoria is urging people to stay away from Bells Swamp Nature Conservation Reserve until further notice as an investigation unfolds.
"Samples have been taken for testing and we expect to get the results early next week," district manager Suzanne Hughes said.
It is not yet clear what has killed the birds.
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One private volunteer wildlife rescuer says he has pulled more dead birds out of the shallows since Tuesday but a number are floating in the water.
The Conservation Regulator responded to reports of dead birds at the site on Wednesday and initial test results showed no signs of human involvement.
The reserve is a 30 minute drive from Bendigo in the rural area of Neereman, on the Bridgewater-Maldon Road.
Its wetland is fed by runoff from storms, which feed the red gums and other plant species, according to a draft management plan from 2015, which was commissioned for a group helping manage the site.
Ducks, coots, grebes and cormorants are all known to forage and rest in the area, along with wading birds like egrets, herons and spoonbills, among other wildlife.
Threatened birds like musk duck and brown treecreepers have been recorded breeding at the swamp in the past, according to the draft management plan.
The sort of mass death event that appears to be unfolding at the swamp is not necessarily unheard of in nature.
There are a number of conditions including neuromuscular illnesses caused by bacteria which can break out when weather and habitat conditions are right.
It is not yet clear whether such an outbreak has triggered this death event.
Parks Victoria planned to put up signs at Bells Swamp warning people of potential health risks, Ms Hughes said.
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