An improved barnacle attachment inhibition assay.

The Balanus amphitrite attachment inhibition assay, developed by Rittschof et al. (1992), has been widely used for screening antifouling compounds. One shortcoming of this assay, however, is the low (often < 40%) attachment rate of cyprids, including in the controls that contain seawater only. In this study, trapping of cyprids at the air-water interface was found to be a main contributor to the low attachment rate. Procedures to eliminate the air-water interface were thus introduced. With the improved bioassay, a much higher cyprid attachment rate (>70%) was attained. To further illustrate the usefulness of the improved assay (ie eliminating the air-water interface), the effects of the length of cyprid storage and the effect of a reference biocide, tributyltin chloride, on the survival and attachment rate of the cyprids were examined. The length of cyprid storage was important, with newly molted cyprids, 3- to 9-day old cyprids and 12-day old cyprids having an attachment rate of 43%,>75% and 36%, respectively. The low attachment rate in the newly molted cyprids was due to a high percentage of cyprids that still swam at the end of exposure period, whereas the low attachment rate in the 12-day old cyprids was due to a high mortality rate. The cyprids showed an EC50 of 22 microg l(-1) for attachment inhibition and LC50 of 25 microg l(-1) for mortality. It is concluded that the air-water interface has an important confounding effect on cyprid attachment rate in the conventional B. amphitrite attachment assay. By eliminating the air-water interface more robust quantitative assay results were obtained.