Fresh doubts have been raised about the future of the freight ferry service between Rosyth and Zeebrugge.
Forth Ports chief executive Charles Hammond has written to the Scottish government voicing his concerns about European environmental legislation.
He said rules coming into force at the start of 2015 will increase costs disproportionately.
The Scottish government said it has already provided "significant support" to the service.
It added that it would look at any further proposals brought forward by the industry.
The new concerns have been prompted by the EU Sulphur Directive.
It will force shipping operators to switch from high-sulphur marine fuel oil to more expensive marine gas oil.
North Sea and English Channel operators are allowed to fit exhaust scrubber systems as an alternative, although these are expensive and not suitable for all existing vessels.
Mr Hammond believes the cost on the longer Scottish route will be much greater than its rivals.
For example, he suggests that the additional cost for the Rosyth service will be twice as great as that for Teeside.
He wrote: "This has the potential to severely impact on the financial viability of existing freight ferry service into and out of Rosyth.
"Indications are that the disproportionate costs increases for the Scottish ferry service will not be acceptable to the current users of the service and that alternative freight routes will be used, utilising southern UK port and transferring the traffic to road."
DFDS currently sails three times a week from Rosyth to Zeebrugge.
A Scottish government spokesman said: "We understand DFDS has been consulting customers on the options to deal with the impacts of the EU Sulphur Directive, which comes into effect next year.
"The Scottish government has provided significant support to the service, within EU State Aid rules, and will continue to support any proposals for EU funding brought forward from the industry that could help mitigate the impact of the EU Sulphur Directive."