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The increasing use of natural products from marine organisms and their synthetic analogs as antifouling agents is replacing chemical biocides due to their ecological compatibility. This study evaluated the antifouling potential of pyrrolo compound (PC) from the sponge-associated bacterium Halobacillus kuroshimensis against the barnacle Amphibalanus reticulatus larval survival, metamorphosis and settlement in the laboratory. The successful concentrations were also subjected to field based static immersion tests to study the efficacy of the PC extract on macrofouling settlement. Results showed decreased naupliar survival and affected larval metamorphosis in a dose-dependent manner, with varying sensitivity among developmental stages. Higher PC concentrations significantly reduced cyprid yield and settlement, with over 50% of cyprid larvae failing to settle at concentrations of 1300 μg/ml and above. Field experiments confirmed a steady decline in barnacle settlement on PC-treated coupons, with less than 50% settlement observed at concentrations of 500–2000 μg/ml over 50 days. The LC 50 /EC 50 ratio for nauplius N-II was 1.11, with higher ratios observed in later stages and for the cypris (1.66), suggesting moderate toxicity and a narrow margin of safety. These ratios suggest barnacle settlement inhibition occurring through a toxic mechanism. The results demonstrate the compound's ability to disrupt barnacle settlement, but also raise concerns about its environmental impact (long-term usage), particularly the potential risks to non-target species before using the compound in real-world antifouling applications. Despite higher concentrations used compared to previous reports, PC shows potential as an effective natural antifouling compound. • Pyrrolo from Halobacillus kuroshimensis was reported for its antifouling potential. • Pyrrolo decreased the naupliar survival and disrupted larval metamorphosis. • Higher Pyrrolo concentrations significantly reduced cyprid yield and settlement. • Field trials showed less than 50% barnacle settlement on Pyrrolo -treated coupons. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Antifouling potent "Pyrrolo" isolated from Marine sponge associated bacterium. • FT-IR, HPLC, GC MS and FT-NMR characterization identified the bioactive Pyrrolo. • Pyrrolo inhibited the growth of fouling bacterial strains. • Molecular docking studies confirmed the antifouling potential of the active Pyrrolo. • Antidiatom and Mussel byssal thread inhibition confirmed the potential of Pyrrolo. Abstract In the present study, an attempt has been made to explore the antifouling potential of bioactive compound isolated from sponge associated bacterium Halobacillus kuroshimensis SNSAB01. The crude extract of SNSAB01 strongly inhibited the growth of fouling bacterial strains with least minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The bioactive compound was characterized through FT-IR, HPLC, GC MS and NMR predicted as 'pyrrolo". From the mass spectral library, structure was elucidated as pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro. The in silico studies provided encouraging docking scores with two interactions by GLN 200 and GLU 304. The extract inhibited 89% diatom adhesion at 350 μg/ml concentration against Amphora sp. An EC 50 value of 150 μg/ml for 50% inhibition of byssal thread of Perna viridis and LC 50 was found to be 500 μg/ml. The LC 50 /EC 50 ratio of 3.0 indicated nontoxic to nature. The result suggested that pyrrolo[1,2- a ]pyrazine-1,4-dione can be used for antifouling coating. [ABSTRACT FROM AUTHOR]