The effects of iron (Fe) and spent mushroom substrate (SMS) arsenic (As) phytotoxicity towards lettuce in artificial soils were investigated to separate the adverse soil parameters relating to As toxicity using a response surface methodology. SMS induced the root elongation of lettuce in both control and As-treated soils. However, in phytotoxicity test using a median effective concentration (EC(50)) of As, Fe and the interaction between both parameters (Fe*SMS) significantly affected EC(50), which explained 71% ancl 23% of the response, respectively. The refined model was as follows: EC(50) of As (mg kg(-1)) = 10.99 + 60.03 x Fe - 10.50 x Fe*SMS. The results confirmed that the soil parameters relating to the As mobility in soils were important factors affecting its toxicity. In conclusion, Fe significantly reduced the As phytotoxicity. However, although SMS enhanced the root elongation, SMS in As-treated soils decreased ECso of As on the root growth via its interaction with Fe. Despite the limitations of the artificial soils and range of parameters studied, the application of this statistical tool can be considered a powerful and efficient technique for interpretation and prediction of the complicated results caused by the interactions between many factors within the soil environments. (C) 2011 Elsevier B.V. All rights reserved.