Evaluation of five extraction protocols for quantification of endotoxin in metalworking fluid aerosol.

OBJECTIVES: Occupational exposures to endotoxin-contaminated, water-based metalworking fluids (MWFs) are thought to contribute to cases of respiratory illness. Before occupational exposure limits for endotoxin can be proposed, accuracy and reproducibility of laboratory measurements must be established. The method most commonly used to quantify endotoxin is the Limulus amebocyte lysate (LAL) assay and this is the basis for the American Society for Testing and Materials (ASTM) method E2144-01. This study was conducted to generate multiple samples with similar mass and endotoxin loading in order to compare four alternative extraction methods with the ASTM method. METHODS: Using an exposure chamber system that provides a uniform distribution of MWF mist, aerosols with three concentrations of endotoxins (4.5, 350 and 1141 EU/m(3)) were collected simultaneously on multiple filter samples. The filters were examined for endotoxin concentration using five different extraction protocols: extraction with 1 h shaking at 25 degrees C in 30 ml pyrogen-free water (PFW) (protocol 1) or in PFW with 0.05% Tween-20 (protocol 2); or shaking at 68 degrees C in 30 ml PFW (protocol 3) or PFW with Tween-20 (protocol 4); or extraction into 20 ml PFW with sonication at 25 degrees C and pH adjustment to 7.5 (ASTM protocol). RESULTS: The uniformity of the aerosol mass yielded coefficients of variation of 12.7, 7.7 and 1.4% for the low, medium and high exposure groups, respectively. The variance in the endotoxin extraction protocols was highest for the ASTM method for the low, medium and high concentration trials. Low, medium and high endotoxin groups were statistically different (P < 0.001), but there were no statistical differences between extraction protocols within these exposure levels. CONCLUSIONS: ASTM method E2144-01 yielded comparable estimations of MWF endotoxin aerosol concentrations but with higher variability than the four other extraction methods. This study shows that extraction into PFW at 25 degrees C with or without Tween-20 was an improvement over the ASTM method in that the estimation was more precise and the method is simpler.