Submitted to: Chemosphere
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/23/2007
Publication Date: 8/1/2007
Citation: Cayuela, M.L., Millner, P.D., Slovin, J.P., Roig, A. 2007. Duckweed (lemna gibba) growth inhibition bioassays for evaluating the toxicity of olive mill wastes before and during composting. Chemosphere. 68(10):1985-1991. Interpretive Summary: Environmental problems associated with new, water-conserving methods of olive oil production (two-phase systems) have led to studies of possible remediation and treatment strategies for the oily, solid residual pulp generated. The solids are phytotoxic if not treated prior to land application. Composting with other agricultural by-products is one treatment for stabilizing the residue and reducing phytotoxicity. The most commonly used test for measuring compost phytotoxicity is the cress seed bioassay with Lepidium sativum. This plant is highly sensitive to ammonium and phenols, moderately sensitive to salinity and very fast growing. However, the cress assay is tedious with regard to counting of seeds and the high variability of results. This research report describes an alternative method that uses duckweed, Lemna gibba, and an image analysis system in a multi-well assay format to assess compost maturity and phytotoxicity of olive-mill residues. Results show that the toxic substances in two-phase olive mill residues not only influence frond reproduction, but also frond vigour and dimensions, which are better evaluated by measuring area than by counting the number of fronds produced. The duckweed assay precision is greater than that of the cress test in part because of the clonal (identical) nature of the duckweed in contrast to the different weight distribution and genetic make-up inherent in non-clonal, cress seed tests. This is the first report showing the suitability of the Lemna gibba bioassay with frond area measurement using image analysis to evaluate two-phase olive mill waste phytotoxicity before and during composting. Results show that the cress seed test is appropriate to estimate toxicity during composting of this type of olive mill residue, but not to make distinctions in toxicity among different types of two-phase olive mill residue samples. In the Lemna gibba test, the effects of toxic substances can be analysed with image analysis software that saves time, gives unbiased, reliable and significantly more precise results than does the cress test. When only frond counting, rather than frond area, is used there is no significant difference between variances of the two methods.
Technical Abstract: Two-phase olive mill waste (TPOMW) is considered a major problem confronting the modern oil extraction and processing industry. Composting has been recently proposed as a suitable method to treat TPOMW so that it is suitable for use in agriculture. In the work reported here, the Lemna gibba bioassay (a standard method that is used by many certification entities to evaluate phytotoxicity in a wide range of environmental samples) was tested to assess the toxicity of TPOMW before and during the composting process. The method was compared with the Lepidium sativum bioassay (the most widely used test for evaluating toxicity during composting) and with other chemical maturity indices traditionally reported in the literature. The Lemna gibba test proved to be a simple, sensitive, and accurate method to evaluate toxicity before and during the composting of TPOMW. Plant growth response was measured by two methods: counting the number of fronds (leaves) and measuring total frond area (TFA) with image analysis software. This is the first reported use of ‘ASSESS’ image analysis software with Lemna to evaluate compost extract effects. Compared to the tedious counting of fronds (Lemna) or seeds (Lepidium), the use of area-measuring software permitted a very rapid, unbiased and precise method of analysing the toxicity of TPOMW before and during composting. Although the accuracy of the frond count method was similar to the traditional cress seed test, data analysis showed that the TFA measurement method was statistically more accurate (significantly lower variance) than the frond count approach. Highly significant correlations were found between TFA and some important maturation indices commonly reported in literature indicating that the Lemna bioassay can be a useful tool to determine the degree of maturity of TPOMW composts.