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Photograph: HINA/ Damir SENČAR /ds

A significant majority of Dutch voters on Wednesday rejected an agreement to strengthen ties between the European Union and Ukraine, according to Dutch broadcaster projections based on exit polls.

Sixty-four per cent of Dutch voters opposed the referendum, according to the exit polls. The result sends a negative sign to the EU on closer integration.

It was still unclear whether voter turnout would reach 30 per cent - the amount required in order for the result to be valid. Initial estimates indicated 32 per cent of the electorate cast ballots. A more reliable result is expected overnight.

The EU association agreement is meant to lead to closer economic and political cooperation with Ukraine and has already been ratified by the other 27 EU member states.

Around 13 million Dutch people had been called upon to vote on the referendum, which was seen as a signal to the bloc amid growing eurosceptic sentiment.

The vote is non-binding, but a rejection of its outcome would be an embarrassment to the Netherlands, which currently holds the EU presidency.

The so-called Association Agreement went into effect in part on January 1 after both houses of the Dutch parliament passed the necessary acts by large majorities. It has already been ratified by the other 27 EU member states.

Prime Minister Mark Rutte on Wednesday morning called on the electorate to vote 'yes,' saying the agreement would provide "more stability at the EU's external borders."

But a eurosceptic initiative drummed up more than 400,000 signatures to force the Netherlands into its first application of the Advisory Referendum Act, which went into force in mid-2015.

The US government also encouraged a yes vote. State Department spokesman Mark Toner reiterated the US view that the agreement would help ensure that Ukraine becomes a democratic and economically stable country.

But it appeared the no-campaign was more convincing.

"Today, NL [The Netherlands] can win back a piece of its sovereignty from the Brussels and Hague elite," right-wing populist Geert Wilders wrote on Twitter.

No-campaigners also expressed concerns that the agreement is seen as the first step towards EU membership for Ukraine and cited the conflict pitting pro-Russian rebels against pro-Western forces.

For the yes-side, Dutch business is looking to increase exports to Ukraine, which last year totalled 650 million euros (725 million dollars).

The agreement also aims to buttress democracy in the former Soviet republic Ukraine, reduce its dependence on Russia and fight endemic corruption.

However, memories of the Dutch rejection of the EU constitution in 2005 are still fresh.

If the government bows to a no-vote, the agreement will collapse. "A no may open the door to a major continental crisis," European Commission President Jean-Claude Juncker has warned.

Meanwhile, the anti-EU UK Independence Party (UKIP) was watching the vote closely ahead of Britain's in-out referendum on EU membership, set for June 23.

"Today's Dutch referendum on the EU's expansionist agreement with Ukraine really is the people's referendum," UKIP leader Nigel Farage, who is a member of the European Parliament, wrote in the Daily Telegraph.

Farage praised the growth of the Dutch eurosceptic movement GeenPeil and its role in forcing the referendum as "an extraordinary achievement that highlights how western democracy is evolving."

Steven Woolfe, another UKIP member of the European Parliament, said the Dutch referendum showed that "euroscepticism is rife throughout Europe."

Woolfe said on Twitter that he hoped Dutch people would "give the EU elite a kick in the teeth and vote no" in the referendum.

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