Colau indicated that there was a threat to fundamental rights and freedoms in Catalonia, where Spanish authorities were shutting down Catalan government's websites, organizations of civil society and arresting regional government officials.
Thousands of striking Catalan university students, many carrying pro-independence flags, marched in Barcelona to protest the central government crackdown on the ballot.
Catalonia's regional police force, the Mossos d'Esquadra, warned Wednesday that there was a risk of a "disruption of public order" if police sealed polling stations as they have been instructed to do. All police members are being told to close any schools and planned polling stations over the weekend to prevent the vote, along with prohibiting voting boxes within 100 meters of where Catalan official plan to have a polling station.
Colau reiterated in her letter that the Catalan issue was no longer a problem relating only to Spain's internal affairs, as the developments in Barcelona had "direct effects on Madrid, Paris, London and Brussels". "It's about standing up for our basic principles and rights".
Spanish Prime Minister Mariano Rajoy vowed to block the referendum, calling on the mayors to not "participate in an illegal referendum". Regional leaders have said they are ready to declare Catalonia's independence two days after the "yes" wins regardless the turnout.
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Senior Spanish government officials said authorities had done enough to prevent a meaningful referendum as Catalonia lacked an election commission, ballot boxes, ballot papers, a transparent census and election material.
A report by Reporters Without Borders on Thursday said the regional government's drive to impose its side of the story in local, Spanish and worldwide media has "crossed the red lines".
Spokeswoman Pauline Ades-Mevel called on Catalan authorities to come out against the stigmatization of Spanish media, saying it smacked of electoral campaigns such as those of Donald Trump and other "reactionary movements".
Speaking in Brussels on Thursday, Raul Romeva said European Union institutions need to "understand that (the referendum) is a big issue".
He said it has "generated an unprecedented level of shock". "However, given the political and legal barriers to Catalan secession, a compromise that simply grants the region more autonomy appears far more likely than independence", Jones said.