An improved barnacle attachment inhibition assay

Abstract

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.

Correlation between surface chemistry and settlement behaviour in barnacle cyprids (Balanus improvisus)

“…As B. amphitrite became established as a laboratory test species, however, methods to address 'floating' were developed since this species is affected, albeit to a lesser degree. Qiu et al (2008) suggested that the size and relative strength of cyprids from the different species may explain why smaller cyprids of B. crenatus and B. amphitrite may be more prone to floating than those of S. balanoides or Megabalanus spp., having a proportionally larger surface area in contact with the air-water interface and reduced strength to break free. Although plausible, this explanation does not completely explain why there are such stark differences in the frequency of floating between species with similar-sized cyprids, such as Elminius modestus where floating is highly problematic and B. amphitrite where it is relatively less common.…”

“…The phenomenon of 'floating cyprids' in conventional 24-well plate settlement assays (Qiu et al 2008) is particularly problematic in assays involving B. improvisus. Observations in initial assays suggested that very few cyprids (< 10%) were able to actively explore the bottom of the wells, with most remaining trapped in the meniscus.…”

“…In recognition of the effect of this phenomenon on the results of cyprid assays, particularly those using species highly prone to floating, Qiu et al (2008) designed an assay format that used filled and hermetically sealed Petri dishes; essentially a modification of the method used by Pyefinch & Mott (1948). This method was adopted in this work with B. improvisus and demonstrated a marked improvement in the reliability of the assay result compared to more conventional approaches.…”

“…Furthermore, the effect of cyprid age on the settlement behaviour of B. improvisus was evaluated using sealed polystyrene Petri dishes to reduce the phenomenon of floating cyprids, as proposed by Qiu et al (2008). The results from this work have the potential to guide the development of novel environmentally-friendly antifouling coatings by repelling cyprids during the initial stages of surface colonization.…”

A novel geometry for a laboratory-based larval settlement assay

“…This novel geometry confines a volume of seawater between the apposing surfaces of interest and air, thus avoiding the artefact associated with a choice assay that is generated by adding an external bounding material. Finally, similarly to sessile drop assays, cyprids trapped at the air-water interface can come into contact with the surface of interest, thus mitigating the reduction in efficiency associated with cyprids confined to the upper liquid surface of a three-dimensional well assay (Qiu et al 2008).…”