Effects of 5-chloro-2-methyl-4-isothiazolin-3-one and other candidate biodiesel biocides on rat alveolar macrophages and NR8383 cells

Biocides are added to biodiesels to inhibit and remove microbial growth. The effects of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), a candidate biodiesel biocide, were studied using freshly isolated rat alveolar macrophages (AM) and NR8383 cell line. CMIT markedly inhibited phagocytic oxidative burst as measured by zymosan-induced chemiluminescence, and cellular cytokine secretion as measured by zymosan-induced TNF-α secretion. The 50% inhibition concentration (LC50) for CMIT was 0.002–0.004 mM for both cellular functions. AM exposed to CMIT for as little as 2 min showed markedly inhibited functions that persisted for at least 5 h. Sodium metabisulfite was able to partially neutralize the inhibitory activity of CMIT. Cysteine and glutathione, when present at a molar ratio of 2–1 or higher against CMIT, were effective neutralizers, while serine, histidine, alanine, and albumin were without effect. When the AM testing system was used to compare the toxicity of CMIT against three other candidate biodiesel biocides, methylene dithiocyanate (MDC) was found to be of comparable toxicity to CMIT, 2-methyl-4-isothiazolin-3-one (MIT) was much less toxic, and dimethyl acetylenedicarboxylate (DMAD) was non-toxic. Because AM is among the first cell-type exposed to inhaled biodiesel aerosols, the result suggested that CMIT present in biodiesel may produce respiratory effects, and further investigations including animal studies are warranted.