28-Day sediment-spiked laboratory toxicity tests with eight benthic macroinvertebrates and the lipophilic fungicide fludioxonil were conducted to verify the proposed tiered sediment effect assessment procedure as recommended by the European Food Safety Authority (EFSA). The test species were the oligochaetes Lumbriculus variegatus and Tubifex tubifex, the insects Chironomus riparius and Caenis horaria, the crustaceans Hyalella azteca and Asellus aquaticus and the bivalves Corbicula fluminalis and Pisidium amnicum. Toxicity estimates were expressed in terms of total concentration of dry sediment as well as in pore water concentration. Field-collected sediment, also used in a previously performed sediment-spiked microcosm experiment, was used in tests with all species. L. variegatus and C. riparius had similar lowest 28d-L(E)C values when expressed in terms of total sediment concentration, but in terms of pore water concentration L. variegatus was more sensitive. Three of the six additional benthic test species (A. aquaticus, C. horaria, C. fluminalis) had 28d-EC values a factor of 2-6 lower than that of L. variegatus. Comparing different effect assessment tiers for sediment organisms, i.e. Tier-0 (Modified Equilibrium Partitioning approach), Tier-1 (Standard Test Species approach), Tier-2 (Species Sensitivity Distribution (SSD) approach) and Tier-3 (Model Ecosystem approach), it is concluded that the tiers based on sediment-spiked laboratory toxicity tests provide sufficient protection when compared with the Tier-3 Regulatory Acceptable Concentration (RAC). Differences between Tier-1 and Tier-2 RACs, however, appear to be relatively small and not always consistent, irrespective of expressing the RAC in terms of total sediment or pore water concentration. Derivation of RACs by means of the SSD approach may be a challenge, because it is difficult obtaining a sufficient number of valid chronic EC values with appropriate 95% confidence bands for sediment-dwelling macroinvertebrates. Therefore, this paper proposes a Tier-2 Weight-of-Evidence approach to be used in case an insufficient number of valid additional toxicity data is made available. Similar studies with pesticides that differ in fate properties and toxic mode-of-action are necessary for further validation of the tiered effect assessment approach for sediment organisms.