Legal action could be pursued unless the government can 'prove it acted lawfully' in granting an emergency authorisation of a product containing a banned neonicotinoid.
Earlier this month, the government issued an emergency authorisation for use of Syngenta's Cruiser SB on sugar beet seed in the UK.
Defra said it was in recognition of the potential danger posed to this year's crop from virus yellows.
The department issued a statement on 8 January on the decision to issue – 'with strict conditions' – the product's emergency authorisation.
The NFU had warned that the disease was having an 'unprecedented harmful impact' on the UK's sugar beet crop.
Some growers had reported yield losses of up to 80 percent, according to the union.
However, the Wildlife Trusts, which has sent a legal letter to Defra Secretary George Eustice, said the emergency authorisation was 'flawed'.
The neonicotinoid is banned for use outdoors in the European Union because of the risk it poses to bees and wild pollinators.
Use of the pesticide in the UK is also banned for widespread use under EU law retained by the EU Withdrawal Act 2018.
Although Defra's emergency authorisation proposes to control and strictly limit its use, Wildlife Trusts believes it will still cause harm to the environment.
Represented by Leigh Day solicitors, the charity has written to Defra with their challenge, questioning whether Mr Eustice's move was 'legally sustainable'.
Craig Bennett, chief executive of the Wildlife Trusts said: “We are preparing to take legal action unless the government can prove it acted lawfully.
"The government refused a request for emergency authorisation in 2018 and we want to know what’s changed. Where’s the new evidence that it’s ok to use this extremely harmful pesticide?
“Using neonicotinoids not only threatens bees but is also extremely harmful to aquatic wildlife because the majority of the pesticide leaches into soil and then into waterways."
He added: "Worse still, farmers are being recommended to use weedkiller to kill wildflowers in and around sugar beet crops in a misguided attempt to prevent harm to bees in the surrounding area.”
Leigh Day solicitor Tom Short added that there was a possibility of the matter being pursued in the courts if Mr Eustice's response 'falls short'.
“Our client is deeply concerned that the Secretary of State has granted this authorisation despite the serious risks that use of neonicotinoids outdoors presents to the natural world.
"The Secretary of State’s announcement provides no new evidence of a genuine need to resort to the use neonicotinoids justifying an emergency authorisation or that the risks of such use can be adequately controlled."