Reference substances are recommended to evaluate the quality of laboratory test species and the reliability of ecotoxicity data. Boric acid (BA) has been recommended as reference substance in some standardized tests in OECD soil, but no data are available for Tropical Artificial Soil (TAS). For this purpose, avoidance tests with Eisenia andrei, lethality tests with E. andrei and Folsomia candida, and reproduction tests with E. andrei, Enchytraeus crypticus and F. candida were carried out in TAS (5% organic matter), following ISO guidelines, and compared between two laboratories. Collembolans were more sensitive than earthworms in lethality tests (LC = 342 and > 1000 mg kg, respectively). For both laboratories, the EC values were similar for reproduction of oligochaeta species (165 mg kg for E. crypticus; 242 and 281 mg kg for E. andrei), but significantly different for reproduction of F. candida (96 and 198 mg kg). Present results suggest that boric acid could replace the current pesticides recommended by ISO guidelines as reference substances on reproduction tests with soil invertebrates in TAS. Concerning avoidance tests, additional investigations should be performed with other substances that cause no neurotoxic effects on soil organisms.