You need to open the file handle for each column. You can ensure that by including the code in the sub like this ;

#!perl
use strict;
use Text::CSV;
my @ar;

@ar = column_segregation('test.csv',0); 
print "@ar\n";

@ar = column_segregation('test.csv',3);
print "@ar\n";

sub column_segregation {
  my ($file,$col) = @_;
  my @array_A2 = ();
  open my $io,'<',$file or die "$!";
  my $csv = Text::CSV->new ({ binary => 1 });
  while (my $row = $csv->getline($io)) {
    push @array_A2, $row->[$col];
  }
  close $io;
  return (@array_A2);                                     
}

__DATA__
1,2,3,4,5
a,b,c,d,e
6,7,8,9,0
x,z,y,q.w
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