Interlaboratory Validation of Toxicity Testing Using the Duckweed Lemna minor Root-Regrowth Test

SIMPLE SUMMARY: Duckweed (Lemna minor) is commonly used as a phytotoxicity test organism, adopted by the main international standardization organizations (ISO, OECD, USEPA, ASTM). For duckweed tests, measurements of fronds or biomass are usually preferred with a standard exposure period of at least 7 days. The proposed root- regrowth test differs from other internationally standardized methods in several important aspects: (a) the test can be performed within 72 h; (b) the test vessel was a 24-well cell plate; (c) the required volume of test water samples was 3 mL; (d) roots were excised before exposure and newly developed roots then measured. The validation of the new test method by interlaboratory comparison tests confirmed that the Lemna root bioassay is valid and reliable. The root growth test is therefore a valuable tool for rapid toxicity screening of wastewater effluents and hazardous pollutants in natural waters because it is simple to perform, quick to conduct, cost-effective to operate, and can have operational benefits for testing time, since management decisions need to be made promptly in the event of unpredictable pollution events. ABSTRACT: The common duckweed (Lemna minor), a freshwater monocot that floats on the surfaces of slow-moving streams and ponds, is commonly used in toxicity testing. The novel Lemna root- regrowth test is a toxicity test performed in replicate test vessels (24-well plates), each containing 3 mL test solution and a 2–3 frond colony. Prior to exposure, roots are excised from the plant, and newly developed roots are measured after 3 days of regrowth. Compared to the three internationally standardized methods, this bioassay is faster (72 h), simpler, more convenient (requiring only a 3-mL) and cheaper. The sensitivity of root regrowth to 3,5-dichlorophenol was statistically the same as using the conventional ISO test method. The results of interlaboratory comparison tests conducted by 10 international institutes showed 21.3% repeatability and 27.2% reproducibility for CuSO(4) and 21.28% repeatability and 18.6% reproducibility for wastewater. These validity criteria are well within the generally accepted levels of <30% to 40%, confirming that this test method is acceptable as a standardized biological test and can be used as a regulatory tool. The Lemna root regrowth test complements the lengthier conventional protocols and is suitable for rapid screening of wastewater and priority substances spikes in natural waters.