The spill was reported in the waters of the River Teith as concerned locals looked for answers.
A clean-up is underway after another fuel spill was reported in the waters around rural Stirling.
The presence of fuel in the water was reported and captured by images in the River Teith near Callander last week – leading to concerns over a link between the discovery and the major leak just up the water in Killin.
A connection between the two leaks was dismissed by initial investigations as officials from Scottish Water and SEPA got to work in sorting out the leak in the Teith and advising locals.
The first advice from Scottish Water insisted there was no risk to the drinking water as a result of the incident, while nearby Deanston Distillery was also notified as a precaution.
A later update from SEPA said the leak was due to a “localised issue” which was being washed from the road into the river through surface drains.
The SEPA statement to Stirling MSP Evelyn Tweed added that there may be the presence of “visible oil sheens” for the following days due to some of the residue being trapped in mud, but there was no risk to the local wildlife population.
In a response to the Observer, a SEPA spokesman said: “SEPA received reports on Thursday March 5 of pollution to the River Teith via a Scottish Water surface water outlet.
“We are continuing to investigate this incident and are liaising with Scottish Water and Stirling Council throughout this process.
“We would like to thank those who reported the incident to SEPA and encourage anyone who witnesses a pollution incident to contact us immediately on 0800 807060 or by submitting an online report with as much information as possible.”
The spokesman added that clean-up work was also being carried out on site by officials from Stirling Council as part of keeping the area safe.
A Stirling Council spokesman said: “We can confirm that we have been in contact with SEPA regarding the oil spill into the River Teith.
“Officers from our Roads Team attended the site to help clean residual oil which had spilled onto the road surface.”
The Callander leak comes on the back of the major fuel spill in the River Dochart in Killin.
Locals in the village were forced to use bottled water and make use of services elsewhere – including in Callander – for daily essentials as SEPA, Scottish Water and the council came together to find a solution to that spill.
The spill was initially linked to a botched robbery at a petrol station on the edge of the village, but the exact cause of the fuel leak remains under investigation for the precise circumstances.