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Council bosses in Gravesend say they remain "steadfastly opposed" to the new Lower Thames Crossing.
National Highways has recently revised plans to build the £8.2bn new road, which will link Kent with Essex via a 14-mile route forged under the River Thames.
It hopes the tunnel will be the “greenest ever built in the UK” when it opens by either 2029 or 2030 and help ease congestion at the Dartford Crossing.
But campaigners have criticised its impact on “irreplaceable” ancient woodland and wildlife, and have questioned its traffic calming credentials.
The tunnel project, which would be built east of Gravesend, has been in discussion for more than a decade and the final planned stage of consultation concluded last month.
It has frequently come under fire from local councillors who have labelled the scheme "unacceptable" and "frankly appalling".
Gravesham council has once again reiterated its opposition to the proposals with leader Cllr John Burden claiming towns and villages would be dealing with harmful impacts "for generations to come".
The Labour leader said: “Our detailed response to this latest Lower Thames Crossing consultation stresses that there are still many fundamental environment-related issues which are still far from clear.
“Our borough will be dealing with the environmental effects of this development for generations to come, and uncertainty and ambiguity around these important issues are not acceptable.
“We remain steadfastly opposed to a Lower Thames Crossing to the east of Gravesend, but will continue to lobby and campaign for the best outcomes for our residents and the wider borough should the development go ahead.”
In its full response to National Highways' consultation the council also remarked that the original route choice was made in 2017.
As such it argues much has moved on since then and that therefore the basis of any decision made then has to be "seriously questioned".
It added that costs have also increased as the scope and scale of the issues require significant additional compensation and mitigation.
“Our Borough will be dealing with the environmental effects of this development for generations to come, and uncertainty and ambiguity around these important issues are not acceptable."
The council’s full response states: “The major comment has to be that a whole series of incremental changes have been made over the years, but it is very unclear that there is any understanding of the cumulative impact of these and how they integrate together in terms of the landscape, ecology, management access and any other factors that may be relevant.
“This overview needs to be integral to the process and stating that it will be dealt with the Environmental Statement is not helpful.
“It is also highly desirable for National Highways to facilitate (though it cannot make happen) an overall management plan across the Cobham/Shorne area involving such parties as Plantlife, the National Trust, Rochester and Cobham Park Golf Club, Cobham Hall School, Gravesham council, the Woodland Trust, Forest England, and Kent County Council.
It adds: “All these, through their landholdings, have different roles to play in obtaining the best landscape, ecological, recreational and historic environment plans for the area.
“These are matters of principal that cannot be left to detail to be sorted out post the DCO [Development Consent Order] being granted, though there will be elements of design that can be.
“It needs to be much clearer what is mitigation for what, and what is compensation for what.”
It comes just weeks after neighbouring Thurrock council penned a letter to the Treasury setting out its "significant concerns" about the viability of the project.
A golf club in Gravesend has also announced it will close next month due to uncertainty surrounding the project which runs straight through its 18-hole course.
For more than 50 years, the Dartford Crossing has provided the only road crossing of the Thames Estuary, east of London
Highways England says there is "an urgent need to address the challenges" faced by tens of thousands of people using that crucial travel artery every day.
It believes a new crossing would provide better journeys for millions of people and would unlock economic growth, supporting the creation of new homes and jobs.
An eleventh hour decision to pull an earlier bid was made in November 2020 after the government told highways bosses it would be refused.
But transport bosses say they have worked to provide sufficient mitigation and address local concerns since going back to the drawing board.
This includes using large swathes of farmland to soak up pollution from the new tunnel.
Last month it was announced a million trees would be planted as part of mitigation for the scheme.
Speaking at the time, Matt Palmer, executive director for the Lower Thames Crossing, said: “The Lower Thames Crossing will tackle the daily frustration caused by the congestion at Dartford, improve journeys and bring exciting opportunities for new jobs and businesses across the region, but we are determined that this will not come at the expense of the environment.
“We have planned its route and how we build it to not only reduce its impact but leave a legacy of bigger, better-connected and well-managed habitats that would give local wildlife and plant life the chance to thrive long into the future.”
A new planning application, known as a development consent order, is expected to be made later in the year.