Pauline Hanson's parliamentary clout and credibility has been dealt a blow with the defection from her One Nation party of a senator just hours after he was sworn in.
Queensland business consultant Fraser Anning replaced Malcolm Roberts, who was disqualified by the High Court over his dual UK citizenship.
Senator Hanson had sought over recent weeks to convince Senator Anning to stand aside and allow Mr Roberts to return.
But the final straw was the One Nation leader's decision to exclude Senator Anning's staff - who formerly worked for Mr Roberts - from a party room meeting on Monday morning, just before the swearing in by Governor-General Sir Peter Cosgrove.
"I'm not going to be told who I can have and who I can't have," Senator Anning said, adding Senator Hanson had a history of appointing "poor advisers".
The decision made an immediate impact on the Queensland state election campaign, with Premier Annastacia Palaszczuk saying it showed the "chaos" wreaked by One Nation-coalition governments.
It leaves One Nation with three senators and the government having to conduct separate negotiations on key legislation with Senator Anning.
The chamber now comprises 29 coalition senators, 26 Labor, nine Greens, three One Nation, three Nick Xenophon Team, as well as Jacqui Lambie, Lucy Gichuhi, Derryn Hinch, David Leyonhjelm, Cory Bernardi and Senator Anning.
Prime Minister Malcolm Turnbull, whose coalition is running behind Labor 45-55 in two-party preferred terms according to the latest Newspoll, also faces a test of his lower house numbers with the starting gun fired on the December 16 Bennelong by-election.
Liberal MP John Alexander quit parliament on Sunday as he sought to clarify his dual British citizenship.
The one-seat majority government is already fighting a by-election in the seat of New England, held by former deputy prime minister Barnaby Joyce.
Labor and the coalition agreed on Monday to a December 1 deadline for all MPs to publicly disclose details of their Australian citizenship or renunciation of foreign ties.
Another victim of the fiasco, Stephen Parry, was replaced as Senate president by former special minister of state, Scott Ryan, on Monday.
Andrew Bartlett and Jordan Steele-John were sworn in as new Greens senators for Queensland and Western Australia respectively.
