Development of an ecotoxicological test procedure for soil microalgae.

Since the 80s, ISO and OECD organizations have been developing guidelines for assessing the toxicity of new and existing chemical substances to soil biota. Up to now, any of these guidelines had soil algae as test organisms. Nevertheless, microalgae are relevant components of soil microbial communities and soil biological crusts (BSC) with a great contribution to different soil functions and ecosystem services. In an attempt to bridge the gap, the present work aimed to develop, describe and validate a standard operating procedure for an ecotoxicological test with soil microalgae. Three phases were performed, each one with specific objectives. First, soil microalgae and cyanobacteria were isolated from BSC and then genetically and morphologically characterized. The green microalga Micractinium inermum was selected because it is a species with a wide geographic distribution. Secondly, M. inermum growth curves were obtained in liquid (BG and Woods-Hole MBL) and solid media (OECD artificial soil) to determine test duration. The growth curves were also used to analyze the reproducibility of the test's endpoint and to propose a validation criterion. Ultimately, a range of concentrations of two reference substances (glyphosate and copper) were tested, both in soil and liquid media, to assess procedure's reproducibility. The tests made in liquid medium followed the standard guideline for ecotoxicological tests with freshwater microalgae and cyanobacteria (OECD 201:2011). The results obtained prove that when the artificial soil is used, as a test substrate, the sensitivity of M. inermum increases. The tests performed with both reference substances demonstrate that the procedure described for testing in soil was reproducible. Additionally, it will be relevant to test with other reference substances and adjust the procedure for natural soils. It will be also interesting to validate the test procedure with soil cyanobacteria.