The fate of a proposed 50m sq miles sanctuary for migratory whales was hanging in the balance on Monday night after objections from enough delegates on the conference floor of the International Whaling Commission (IWC)'s biennial conference to block its creation.

Japan has led the opposition to establishing a marine reserve in the South Atlantic – supported by Norway, Iceland and some Caribbean states – arguing that the IWC's remit should prioritise commercial over conservation considerations.

Environmentalists are concerned that a vote, which could come as early as Tuesday, may now turn the clocks back to a time before the IWC considered the environmental impacts of unfettered exploitation of the world's marine reserves.

"Given the objections raised in the plenary today from Japan and its supporters, I'm not sure that we will attain the three-quarters majority we need for the motion to pass," said Kitty Block, vice-president and head of the delegation for the Humane Society International.

"It is shocking that these nations do not want to allow a sanctuary protecting the breeding and calving area for whales, which face so many environmental threats."

As well as whalers' harpoons, whales must navigate the dangers of ship strikes, entanglements in shipping nets and other ocean apparatus, disorientating man-made ocean noises, habitat destruction, resource exploitation, climate change and pollution.

"Undoubtedly they're facing more man-made threats than at any other time in history," Block said. These may soon be amplified.

The sanctuary vote is being seen as a precursor to a touchstone debate at the IWC conference in Portoroz, Slovenia, over an International Court of Justice (ICJ) ruling last March that Japan's whaling in the Southern Ocean violated the IWC's moratorium on commercial whaling.

New Zealand is expected to propose that no further "illegal permits for scientific whaling" be issued. This dovetails with the ICJ's ruling that Japan's annual cull of up to 900 minkes and a smaller number of fin whales in the Antarctic was not primarily motivated by research purposes.

Japan ceased whaling in the Antarctic after the ruling, but has continued to hunt the sea mammals in the North Pacific, and now plans to propose a new round of 'scientific whaling' programmes at the IWC.

British Labour MEP David Martin called on the EU delegates to vote with their conscience.

"I have already written to (Environment) Commissioner Potocnik and the Italian presidency, urging them to ensure that EU officials go to the International Whaling Commission meeting determined to prevent any further scientific whaling activities by Japan," he told the Guardian. "All forms of commercial whaling are unacceptable, and we must not let this barbaric practice continue under the guide of scientific research."

The final vote may hinge on whether the EU takes a common position – which depends on consensus – or whether opposition materialises from states such as Denmark, which has traditionally defended the practice among aboriginals in its former colony of Greenland. In the absence of unanimity, the bloc is likely to abstain.

On Monday EU states voted en masse to support a motion raising the quota for aboriginal whale kills in Greenland to 207 per year by 2018. The vote passed by 46 to 11, with three abstentions.