The resultant buy SKI-606 mixture was briefly shaken and maintained at room temperature, in the dark for 30 min. At the end of this period, the absorbance of the mixture was measured at 517 nm, using an SLT Spectral Rainbow microtiter plate reader. Brine shrimps (Artemia salina) is a simple convenient general bioassay and also indicative for cytotoxicity. 6 The brine shrimp eggs were
hatched in artificial seawater (ASW). 40 mg/L of the eggs were supplemented with 6 mg/L dried yeast and oxygenated with aquarium pump for 48 h in room temperature (22–25 °C). 100 μL of the sample solution (1 mg/mL) were transferred into sterile microtiter plate. The plate was left until evaporated over night. Then 150 μL of the A. salina culture medium together with a few A. salina larvae was added, followed by 150 μL water. For each sample, four replicates were performed. After 24 and 48 h the plates were examined under a binocular microscope and the numbers of dead (non-motile) nauplii in each well were counted against the negative control. Cytotoxic assay was conducted using MTT [3-(4,5-dimethylthiazole -2-il)-2,5-diphenyltetrazoliumbromide] BIBW2992 chemical structure in a 96-wheel plate on the cell cultures that had been treated with the specimen compounds in a variety of concentrations. The cells
had a density of 2 × 104 cells/well. The absorbance was read using ELISA reader with a wavelength of 550 nm. The results of absorbance measurements were used to determine the life percentage (%) with the formula = (1−absorbancy of treated cells/absorbancy of untreated cells) × 100 followed by the determination of death percentage (%) and IC50 using probit analysis. Pecaron Bay
Situbondo is one of the regencies in the East Java Province. It has a line of coastal area where coral reef ecosystem can be found. Other flora and fauna found in the coral reef ecosystem include alga, sponge animals and soft reef; meanwhile biotic factors that contribute to the coral reef ecosystem include sands, stones, and reef fragments with a coverage capacity of 57.41% to 62.638%. Pecaron Bay is located at Situbondo East Java (Fig. 1) This bay has reef structure which consists of Poriferan and Coelenterata. It has been Oxalosuccinic acid known that poriferan or marine sponge has several roles such as an impacts on substrate (including bioerosion, reef creation, and substrate stabilization, consolidation and regeneration), benthospelagic coupling (including carbon inhibitors cycling, silicon cycling, oxygen depletion and nitrogen cycling) and associations with other organisms (facilitating primary production, secondary production, provision of microhabitat, enhanced predation protection, survival success, range expansions and camouflage though association with sponges, sponges as a settlement substrate, disrupting near-boundary and reef level flow regimes, sponges as agents of biological disturbance, sponges as releasers of chemicals and sponges as tools for other organisms).