Scotland's 17 leading independent schools have a combined annual income of more than £160m, fuelling the debate over whether they should retain their charitable status.

Figures released for the first time yesterday show top private schools, including Fettes College and Merchiston Castle in Edinburgh, Hutchesons' Grammar in Glasgow and Dollar Academy in Clackmannanshire, are among the top 300 charities in the country for income - the main source of which in their case is fees.

Edinburgh Merchant Company Education, which runs Mary Erskine School, Stewart's Melville College and George Watson's College in the capital, tops the list of independent school trusts with an income of more than £39m - ahead of the National Trust for Scotland, the National Galleries and the Roslin Institute in the overall rankings.

Hutchesons' is in second place, with an income of £14.6m, Fettes third, with £12.3m, followed by Merchiston, at £11m. The total of £160m, from the list published yesterday by the Office of the Scottish Charities Regulator (OSCR), does not include returns from some 20 smaller independent schools.

The scale of the income generated by private schools through fees to parents - which in Scotland average some £7000 a year for a day pupil - prompted renewed concern over their charitable status. Politicians warned it raised serious issues about whether such institutions passed a test of public benefit, given that many parents cannot afford them.

Christine Grahame, SNP MSP for South of Scotland, said: "When you see the amount of fee income these schools have, while not wishing to pre-judge the decisions of the charities' regulator, it would be extraordinary if they could retain charitable status. Great swathes of the population could not possibly access these schools, which makes them exclusive."

Karen Whitefield, Labour MSP for Airdrie and Shotts, said: "These figures show why it is so important to keep the charitable status of these institutions under constant review. It is my hope schools should be able to demonstrate clearly what they do to benefit the wider community in order to retain charitable status, and not just through a handful of scholarships."

OSCR was set up to enforce legislation introduced by the Scottish Parliament in 2006 to tighten control of charities following a number of high-profile scandals.

Under new rules, charities must be able to demonstrate they have a charitable aim and benefit the public, while OSCR also checks on what proportion of money a charity uses to pay for administration and other costs.

Although the intention was to clamp down on bogus charities, private schools were drawn into the review because, as providers of education, they enjoy the benefits of being a charity - worth more than £4m to the sector every year.

There had been speculation some schools might lose charitable status because of high fees and selection of pupils by ability. However, OSCR ruled last summer, after the first round of the review, that the High School of Dundee does qualify for charitable status.

Jane Ryder, OSCR's chief executive, had stressed the decision was on an individual basis, adding, "it should not be assumed this establishes definitive benchmarks for the independent school sector".

Judith Sischy, director of the Scottish Council of Independent Schools (SCIS), said that apart from modest investment in facilities and buildings, "these are all very large schools with lots of pupils to educate, so although it sounds like a lot of money, every penny is needed to pay for the education of the children.

"If there was spare money around, we would be lowering fees. Private education is expensive, but parents are getting value for money," she said.

Michael Spens, headteacher of Fettes, said: "We run on a very small surplus once teaching costs, building costs and a substantial amount of scholarships and bursaries have been awarded."