The combined upper layers containing the pigments were evaporated to dryness under nitrogen and then redissolved in 20 mL n-hexane. The concentration of astaxanthin in the extracts was determined by measuring the absorbance at 477 nm, using a molar extinction coefficient of 2172 in a 1 cm cell. Astaxanthin standard was obtained from Sigma Chemical Co. St Louis, MO, USA and
lutein standard from Extrasynthese, Genay, France. From earlier analyses we knew that astaxanthin and its esters accounted for about 95% of the total carotenoids and therefore the lesser ones such as lutein were not specified but would have contributed to the total. All
samples were stored in the dark, under nitrogen at−20°C until required for further analysis