A standardisation of Ciona intestinalis (Chordata, Ascidiacea) embryo-larval bioassay for ecotoxicological studies

A standardisation of the ascidian Ciona intestinalis embryo-larval bioassay for marine pollution assessment has been developed. The minimum percentage of embryogenesis success was established to assess the quality of the biological material used; minimum sample size and number of replicates per treatment were also estimated. The suitability of artificial and natural seawater for the incubation of ascidian embryos and larvae was compared, and the optimum conditions of temperature, salinity, pH, density of embryos in the vials and the sperm/egg ratio were investigated. On the basis of the 10th percentile of the distribution of larval abnormalities, we proposed a threshold of 50% normal larvae in the control in order to consider the test of acceptable biological quality. According to our results n=5 is a sufficiently high replication to detect 5% differences among treatment means with a power of P=90% and alpha=0.05, and a sampling size >/=222 allows a 95% confidence in the estimate with an error of 0.05. Egg density did not affect larval development within the range 1-20 eggs/ml, and the optimum sperm/egg ratio which fertilise 100% of the eggs was 3000-30,000 sperm/egg (i.e. 10(8)-10(7) sperm/ml). There were not significant differences between the two water types tested, and the optimum tolerance ranges were 18-23 degrees C temperature, 34-42 ppt salinity (42 ppt was the highest salinity tested), and 7.4-8.8 pH. The median effective concentration (EC(50)) of copper (Cu) causing a 50% reduction of normal hatched larvae was 54.2 microg/l (0.85 microM), which shows a sensitivity of this species similar to the commonly used bivalve and sea-urchin tests. The ascidian embryo-larval bioassay is an accurate, reliable, simple and rapid method that can be used in ecotoxicological studies.