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The coast guard ship is one of two Italian vessels that took on some of the Aquarius's passengers before escorting it to Spain, at the invitation of Prime Minister Pedro Sanchez.
The Aquarius became the source of a heated political row in the EU about the migrant crisis in the Mediterranean.
For the migrants, mainly from Africa, the arrival in Valencia marked the end of a week-long odyssey in the Mediterranean Sea.
European odyssey exposes extent of migrant crisis
Spain swooped to help the group of mainly sub-Saharan Africans on board the Aquarius last week, offering the charity-run ship a berth 700 nautical miles away after Italy and Malta refused to let it dock.
An Italian coast guard ship that took aboard some of the Aquarius's passengers to make the trip safer arrived soon after dawn in the eastern Spanish port of Valencia, where a staff of 2,320, including volunteers, translators and health officials, awaited.
“The first ship by the Italian coast guard, the Datillo, has just docked right behind me,” explained FRANCE 24’s Sarah Morris, reporting from the Valencia port shortly after dawn. “Members of the health department are already on board, carrying out the first process of identifying those passengers. There’s about 270 of them, some of them in need of urgent medical attention…We know that between seven to nine people on board those ships may be pregnant, there are about a hundred under-18-year-olds. It’s going to be a very long process.”
Uniformed police looked on as officials in white overalls and protective masks greeted the first migrants to step off the boat.
The ship's predicament gave Italy's new government the chance to assert its anti-migrant credentials, and Spanish Prime Minister Pedro Sanchez, in office for just over a week, took the opportunity to underline a more liberal stance.
But the plight of the Aquarius has highlighted the European Union's failure to agree on how to manage huge numbers of people fleeing poverty and conflict.
"People are coming to Europe seeking European values of solidarity and support," Red Cross Secretary General Elhadj As Sy told a news conference in Valencia on Saturday.
"Anything less than that is a betrayal (of) Europe itself."
Migrant arrivals increasing in Spain
Anti-migrant feeling has surged in Italy, where more than 600,000 people have arrived on its shores over the past five years, helping to propel the nationalist League into a coalition government.
Far fewer come to Spain, but the numbers are rising fast.
Most Spaniards support the idea of welcoming and helping to integrate refugees, pollsters say, allowing Sanchez, a socialist, to offer migrant-friendly policies to voters who feel previous governments did not do enough.
France, which chided Italy for turning away the Aquarius, has offered to take in any passengers who qualify for asylum and want to go there.