Helle Würtz

Endotoxin potency in the A549 lung epithelial cell bioassay and the limulus amebocyte lysate assay

The purpose of this study was to elucidate to what extent the potency of endotoxins measured by the limulus amebocyte lysate (LAL) assay is reflected in the potency in an in vitro assay based on release of interleukin-8 (IL-8) from a lung epithelial cell line, A549. Lipopolysaccharides (LPS) from Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis and detoxified LPS from E. coli were applied in serial dilutions in the LAL assay and in the A549 bioassay. Also 19 organic dust samples from waste recycling plants were tested. The A549 cells were incubated for 24 h with LPS or dust, and the IL-8 secretion was determined by ELISA. The method for evaluation of the LAL assay showed linearity for the four endotoxins. Using the slope as a measure of the potency factor (PF), LPS from E. coli and S. enteritidis was about four times more potent than that for LPS from K. pneumoniae and P. aeruginosa. In the A549 bioassay each of the different types of endotoxin had characteristic and very different dose-response curves. The potency of the LPS, in the A549 bioassay, ranked as follows K. pneumoniae > P. aeruginosa > E. coli > or = S. enteritidis. The content of endotoxin in the dust samples did not correlate with their potency in the A549 bioassay. The present study indicates a poor correlation between the potency of endotoxin in the LAL assay compared with the A549 bioassay. The lack of correlation when organic dust samples are tested may reflect the fact that these samples contain biological active compounds, which are non-reactive in the LAL-assay but stimulate IL-8 secretion from epithelial cells.

Microorganisms and endotoxin in stored biowaste percolate and aerosols

Source separated biowaste was stored in containers at temper atures ranging from 16°C to 29°C in a climate chamber for two weeks, simulating outdoor storage in a domestic waste collec tion system. Samples of exuded percolate were collected after 3, 4, 6, 8, 10, 12 and 14 days and analyzed for content of micro organisms and endotoxin. Throughout the storage period, the mean concentrations (GM) of total microorganisms ranged from 5.0 to 12 × 109 cells ml-1 and concentrations of endotoxin were between 0.54 and 1.5 × 106 EU ml-1 (45 to 130 ug ml-1). The maximum levels of microorganisms and endotoxin in the percolate were stable during storage and no significant differ ence was found between storage times of one or two weeks, which corresponds to common Danish collection frequencies of biowaste. Analyses of the microflora indicated dominance of bacteria as demonstrated by almost equal concentrations obtained by aerobic and anaerobic cultivation (2.8 to 9.0 × 108 and 3.1 to 12 × 10 8 cfu ml-1, respectively). Yeasts formed a minor part of the microflora (below 0.5% of the total number of microorganisms) and molds were only detected sporadically at concentrations close to the limit of detection. For percolate keeping a pH below 5 during the first week of storage, a ten dency (p = 0.08) was observed towards lower concentrations of aerobic Gram-negative bacteria and yeasts as compared to per colate exceeding a pH of 5. In two weeks, a mass of 17 kg of bio waste exuded approximately 1.31 of percolate (range: 0.7 to 2.1 1), and handling experiments demonstrated that bioaerosols generated from splashing percolate may cause exposure risks of endotoxin and microorganisms. Bioaerosols above stored bio waste contained fungal spores up to 1.8 × 104 cfu m-3 but no detectable bacteria and endotoxin. Headspace measurements of gases showed maximum emission of hydrogen sulfide and methyl mercaptan after one week of storage, while concentra tions of ammonia increased throughout the two week storage period.