Pollution-induced community tolerance (PICT) approaches involve comparing tolerance levels of natural communities to a particular contaminant or a contaminant mixture using short-term toxicity tests performed under controlled conditions. However, results from toxicity tests can be modulated by various environmental and experimental conditions, raising questions about their reproducibility and comparability. In this context, the present study aimed to determine the influence of exposure duration, periphyton suspension concentration and periphyton maturation stage on the measurement of short-term effects of copper on phototrophic periphyton communities. Our results showed the very weak influence of exposure duration in the tested range (2-6 h) on toxicity level, whereas periphyton biomass in the tested suspension (in terms of both chlorophyll a concentrations and dry weight), proved a crucial determinant in toxicity assessment. Results also highlighted the potential tolerance increase with the periphyton maturation stage. This parameter conditioned the positive linear relationship between tolerance level and periphyton suspension concentration, leading to an increase in the linear regression slope with the maturation stage. This suggests that such a relationship is probably highly periphyton-dependent. Consequently, to enable data toxicity comparisons, an a priori normalization of the periphyton suspension biomass is necessary, and PICT approaches require the use, as much of possible, of periphyton with similar maturation stage. Finally, the present study clearly shows that a better standardization of PICT approaches could help to improve reproducibility. It could thus facilitate the comparison of tolerance levels measured in the same study (e.g. spatial and/or temporal and/or inter-treatment comparison) as well as the comparison obtained from different experimental and in situ research.