You do not have $io in the sub you need to pass this to it or open the file within that sub.

use strict;
use warnings;

use Text::CSV;

open my $fh, "<:encoding(utf8)", "test.csv" or die "test.csv: $!";
my @rows = column_segregation( $fh, 2 );
print "$_\n" for @rows;
close $fh;


sub column_segregation
{
        my ( $io, $o ) = @_;

        my $csv = Text::CSV->new ({ binary => 1 });
        my @array_A2;
        while (my $row = $csv->getline($io))
        {
                push @array_A2, $row->[$o];
        }

        return (@array_A2);
}
[download]

Input
1,2,3,4
5,6,7,8
a,b,c,d

Output
3
7
c
[download]