Ecotoxicological studies predominantly rely on artificially contaminated soils and fieldwork on contaminated soils remains scarce. This study focuses on the Kargaly site in the Orenburg region near the southern Urals, where a rare instance of monometallic soil pollution has occurred with copper (Cu). We established Cu toxicity thresholds for Dendrobaena veneta, a European nightcrawler, using soils collected along a Cu toxicity gradient (total Cu content of 121–10,200mg kg−1) in a chernozem (Mollisol) agricultural field. Earthworm survival in the reproduction bioassay was an unreliable predictor of Cu toxicity. However, the number of juveniles in the reproduction bioassay and earthworm avoidance behavior were sensitive indicators of Cu toxicity. While total soil Cu strongly predicted earthworm responses, the effect of soluble (0.01 M CaCl