Acta Oceanol. Sin., 2011, Vol. 30, No. 4, P. 33-42 DOI: 10.1007/s13131-011-0131-6 http://www.hyxb.org.cn E-mail: hyxbe@263.net Orthogonal design for optimization of pigment extraction from surface sediments of the Changjiang River Estuary ZHAO Jun 1,2† , YAO Peng 3,4 , YU Zhigang 3,4∗ , Thomas S Bianchi 2 1 Key Laboratory of Marine Environment and Ecology, Ministry of Education, Ocean University of China, Qingdao 266100, China 2 Department of Oceanography, Texas A & M University, College Station, TX 77843, USA 3 Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China 4 Institute of Marine Organic Geochemistry, College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China Received 7 July 2010; accepted 20 October 2010 ©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2011 Abstract Using a suitable solvent for extracting pigments from sediment for high performance liquid chro- matography (HPLC) analysis is critical for obtaining qualitative and quantitative estimates of phytoplanktonic and benthic algal biomass, as well as community composition. Five methodolog- ical factors (sample dehydration, extraction solvent, extraction duration, number of extractions, and ratio of solvent volume: sample weight) were studied using an L 9 (3 4 ) orthogonal design in a sedimentary pigment extraction experiment on samples collected from the Changjiang large-river delta-front estuary (LDE), using HPLC analysis. The results show that the optimal extraction method for sedimentary pigments should include freeze-drying samples prior to extraction. The effects of different factors on sedimentary pigment extraction were separated by the L 9 (3 4 ) or- thogonal design experiments and showed that the extraction solvent was the most important, with extraction duration the second most important, and numbers of extraction and ratio of solvent vol- ume: sample weight was the least important. The mixed solvent treatment comprised of acetone, methanol and water (80:15:5, by volume) was best for polar pigment extraction, with 100% acetone better for apolar pigments. For most pigments employed in this study (i.e., peridinin, fucoxanthin, alloxanthin, diatoxanthin, zeaxanthin, pheophytin-a and β-carotene), 3 h was found to be enough time for extraction from these deltaic sediments. However, for chlorophyll-a, the most important pigment used for estimating algal biomass, 12 h was needed. A small amount of solvent (3 ml) with duplicate extractions obtained the greatest amount and diversity of pigments. Unfortunately, no extraction method was found to be suitable for all pigments in sediments. The choice of extraction procedure should be made in accordance with the objective of each study, taking into consideration the properties of sediments and pigments in question. Key words: sedimentary pigments, extraction, orthogonal design, HPLC, Changjiang River Est- uary 1 Introduction Sedimentary pigments have been shown to be useful biomarkers of algal and bacterial communi- ties in contemporary estuarine and marine ecosystems (Bianchi, 2007; Jeffrey et al., 1997, and references therein). Pigments have also been used as fossil in- dicators of past primary productivity (Harris et al., 1996; Repeta, 1993) and historical reconstruction of environmental change (Dahl et al., 2004; Chen et al., 2001; Bianchi et al., 2000; Schaeffer et al., 1993). In estuarine and coastal zones, which are commonly im- pacted by anthropogenic activities, sedimentary pig- ments can be used to reconstruct historical changes in phytoplankton community composition (Bianchi et al., 2002; Chen et al., 2001), effects of eutrophication (Kowalewska, 2005; Bianchi et al., 2000), and to trace Foundation item: The National Natural Science Foundation of China (NSFC) under contract Nos 40920164004, 40676068 and 40806029; China Scholarship Council under contract No. 2009633049. † Present address: Key Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, State Oceanic Administration of China, Hangzhou 310012, China ∗ Corresponding author, E-mail: zhigangyu@ouc.edu.cn 1