Understanding the role of species traits in chemical exposure is crucial for bioaccumulation and toxicity assessment of chemicals. We measured and modelled bioaccumulation of polychlorinated biphenyls (PCBs) in Chironomus riparius, Hyalella azteca, Lumbriculus variegatus and Sphaerium corneum. We used a battery test procedure with multiple enclosures in one aquarium, which maximized uniformity of exposure for the different species, such that the remaining variability was due mostly to species traits. The relative importance of uptake from either pore water or sediment ingestion was manipulated by using 28 d aged standard OECD sediment with low (1%) and medium (5%) OM content and 13 months aged sediment with medium OM (5%) content. Survival was ≥76% and wet weight increased for all species. Reproduction of H. azteca and weight gain of H. azteca and S. corneum were significantly higher in the medium OM aged sediments than in other sediments, perhaps due to a more developed microbial community (i.e., increase in food resources). Biota-sediment accumulation factors (BSAF) ranged from 3 to 114, depending on species and PCB congener, with C. riparius (3–10) < S. corneum (10–17) ≤ L. variegatus (7–61) ≤ H. Azteca (5–114), thus challenging the presumed value of 1–2 typically employed in ecological risk assessment schemes. BSAFs for freshwater taxonomic groups were compared with their marine counterparts and showed overlapping values. The dynamic bioaccumulation model with species-specific bioaccumulation parameters fitted well to the experimental data and showed that bioaccumulation parameters were depended on species traits. Enclosure-based battery tests and mechanistic BSAF models are expected to improve the quality of the exposure assessment in whole sediment toxicity tests.