The Ocean Viking, which is operated by the Doctors without Borders and SOS Mediterranee aid groups, has been on standby since it completed rescues of 356 men, women and children in the central Mediterranean Sea nine days ago.
The ship is currently in international waters, about 32 nautical miles from European shores between Malta and the Italian island of Linosa. Both countries have refused it permission to disembark. The situation aboard the ship was under control, but SOS Mediterranee said that people are sleeping on the floor, with few showers and a limited water capacity.
"These people have suffered enormously, most of them have gone through detention centers in Libya," the group said on Twitter. "They need to disembark as soon as possible." France has pledged to take some of the migrants, repeating the model of an agreement earlier this week for some European Union members to accept a separate group rescued by the Open Arms, a vessel run by a Spanish aid group.
European countries have been at odds over how to handle the steady flow of economic migrants and asylum seekers who take the perilous journey across the Mediterranean, often putting themselves in the hands of trafficking mafias.
Despite the number of sea arrivals dropping sharply from 2015, Italy's hard-line interior minister has become a symbol for Europeans who reject migration. Matteo Salvini has closed his country's ports to humanitarian boats and has accused them of colluding with human traffickers.
That has boosted Salvini's popularity at home, emboldening him to pull the plug on the uneasy governing coalition in a bid for new elections. More broadly, it has exposed the EU's disunity in dealing with the sea arrivals of migrants.
On Wednesday, Doctors without Borders and Amnesty International separately called on Europe to urgently find a solution to the repeated stand-offs with humanitarian rescue ships. The crisis is "a result of the fracas of European migratory policies, said Maribel Tellado, campaign director for Amnesty International Spain.
An Italian prosecutor is now probing possible kidnapping and other charges over Salvini's refusal to allow dozens of migrants to get off the Open Arms, as well as the failure of officials to assign a safe port and provide hygienic conditions and health care once the boat was in Italian waters.
The aid group had repeatedly warned of an emergency situation on board, which resulted in the evacuation of nearly 70 of the 163 people it had rescued. After 19 days at sea, most of them at a short distance from the shores of Lampedusa, Sicilian prosecutor Luigi Patronaggio ordered the seizure of the ship late Tuesday, as well as the immediate evacuation of its 83 remaining passengers.
Salvini, who has staved off previous similar investigations, had refused to open the port of Italy's southernmost island even after Spain, Portugal, Germany, France and Luxembourg agreed to take the migrants.
The EU's executive branch said Wednesday that it would supervise the relocations. The EU's border and asylum agencies are also in charge of pre-screening them to begin establishing whether they are eligible for international protection.
The migrants had undergone medical screenings before being transported to a temporary facility known as a "hotspot," where they are typically identified. None had serious health issues, Italian news agency ANSA reported.
Meanwhile, the boat that brought them ashore arrived at the port of Empedocle, in Sicily, following orders of the prosecutor, said the Open Arms' head of mission, Riccardo Gati. Gati said the aid group would cooperate with the Italian judicial probe to find "those responsible for creating psychological suffering that has forced people to take extreme measures" that include suicide attempts and desperate efforts to reach shore. On Tuesday, a dozen migrants jumped overboard trying to swim to Lampedusa before they were plucked by the Italian Coast Guard.
Spanish Deputy Prime Minister Carmen Calvo said that a navy ship dispatched to the area will stay close to Lampedusa ready to take the country's agreed shared of migrants. She didn't specify how many. France says it will take 40 migrants and Portugal has pledged to take in 10.
The Spanish government has threatened Open Arms with fines of up to 900,000 euros (nearly $1 million) if it actively conducts "search and rescue" operations. But vowing to return to the Central Mediterranean as soon as the Open Arms is released, Gati said that rescuing people in need is everybody's obligation.
Parra reported from Madrid. Lorne Cook in Brussels contributed to this story.