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A wildlife trust says a multi-billion pound crossing between Kent and Essex would "destroy" important habitats.
Kent Wildlife Trust has registered as an interested party to voice its concerns about the Lower Thames Crossing.
Plans for Britain's longest road tunnel, which would affect Gravesend and Dartford, have been discussed years.
Now, the wildlife charity wants to "give nature a voice" and joins a group of conservationists including Woodland Trust and Essex Wildlife Trust in voicing their concerns about the £9billion development.
The Trust says the proposals threaten part of Shorne and Ashenbank ancient woodland, which is a site of special scientific interest, and is home to rare wildlife including woodpeckers, great crested newts and dormice.
There is concerns about the increase in traffic, and the Co2 emissions it would create around the 14.3 mile road.
The groups are also asking that the impact on ancient woodland is significantly reduced and remain concerned about the pollution that would be created by the scheme.
They say while planting new trees is valuable, it will never compensate for the loss of ancient woodland.
Kent Wildlife Trust's planning and policy officer Emma Waller said: “Kent Wildlife Trust share the concerns of other organisations, that the Lower Thames Crossing still has a long way to go in achieving its claims of protecting and enhancing wildlife.
"A project at this scale, which would destroy nationally important habitats and release vast amounts of CO2, cannot have an overarching positive impact to wildlife, biodiversity and climate change.
“Ploughing ahead with the proposed crossing will result in the loss of irreplaceable habitats, including ancient woodlands and wetlands which are locking up significant levels of carbon, going against the government’s commitments to the environment.
“At a time when Kent is increasingly facing the pressure of development, and whilst we are tackling a biodiversity and climate crisis, to proceed with a project that will result in habitat loss within important wildlife areas and cause the displacement of species, shows a disregard for the important role that the impacted habitats play in protecting Kent, and its population, from the worst impacts of climate change."
People who share the charities concerns over the crossing can join almost 11,000 others who have signed Woodland Trust’s petition against the crossing.