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The destruction of natural habitats and the associated loss of Ecosystem Services (ES) are rarely jointly assessed and quantified in Environmental Impact Assessment (EIA). Based on a terrestrial transport infrastructure project, the objective of this paper is to quantify the potential loss of ES associated with direct and indirect impacts, and illustrate their contribution to decision-making for route options. We first quantify how much of each type of ES is generated by different land units in the absence of the infrastructure (baseline conditions). We then estimate ES loss caused by infrastructure construction in a way that discriminates among different types of ES and losses because some ES, in addition to losses that are directly proportional to the surface impacted, can show additional indirect losses associated with landscape connectivity. In addition, we illustrate how the assessment of threshold effects in particular ecosystem types that may be most sensitive to their occurrence can affect the estimation of ES loss. We compare implementation options to provide an example of how choices can be improved by assessing ES loss associated with a combination of direct and indirect impacts. This kind of analysis could be used more generally to assess development projects simply by adapting the framework of analysis to the type of project and the ecosystems concerned. [ABSTRACT FROM AUTHOR]