Micronized particles released from car tires have been found to contribute substantially to microplastic pollution, triggering the need to evaluate their effects on biota. In the present study, four freshwater benthic macroinvertebrates were exposed for 28 days to tread particles (TP; 10-586 µm) made from used car tires at concentrations of 0, 0.1, 0.3, 1, 3 and 10% sediment dry weight. No adverse effects were found on the survival, growth and feeding rate of Gammarus pulex and Asellus aquaticus, the survival and growth of Tubifex spp., and the number of worms and growth of Lumbriculus variegatus. A method to quantify TP numbers inside biota was developed and here applied to G. pulex. In bodies and faces of G. pulex exposed to 10% car tire TP, averages of 2.5 and 4 tread particles per organism were found, respectively. Chemical analysis showed that, although car tire TP had a high intrinsic zinc content, only small fractions of the heavy metals present were bioavailable. PAHs in the TP-sediment mixtures also remained below existing toxicity thresholds. This combination of results suggests that real in situ effects of TP and TP-associated contaminants when dispersed in sediments are probably lower than those reported after forced leaching of contaminants from car tire particles.