Around 13 million Dutch people were called upon Wednesday to vote on an agreement to strengthen ties between the European Union and Ukraine, in a referendum set to send a signal to the bloc amid growing eurosceptic sentiment.

Polling stations opened at 8:30 am (0630 GMT), but commuters were able to vote at train stations from as early as 5 am. They were due to close at 9 pm.

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.

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 no-campaign, which had majority support in pre-referendum polls, is hoping for the Dutch people to send out an anti-EU signal.

"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 have also expressed concerns that the agreement is the first step towards EU membership for Ukraine and the cause of 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.

In order for the vote to be valid, at least 30 per cent of the Dutch electorate must take part.

Even though it is non-binding, the vote could have far-reaching repercussions. A valid no-vote would be a huge embarrassment for the Netherlands, coming in the middle of its six-month EU presidency.

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 will "give the EU elite a kick in the teeth and vote no" in the referendum.

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