Endotoxins

Bacterial endotoxins are lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria. They are invariably associated with Gram-negative bacteria whether or not the organisms are pathogenic. Endotoxins elicit a variety of inflammatory responses in animals and humans and are considered to be strong pyrogens or fever-inducing substances. Pyrogens can be detected and quantified in very low concentrations in water, dust or air samples via biochemical assays. One commonly used method utilizes Limulus Amebocyte Lysate (LAL) recovered from the blood of the Atlantic horseshoe crab Limulus polyphemus.

Why do I need to test for Endotoxins?

Inhalation of endotoxins at high concentrations has been associated with acute airway obstruction, hypersensitivity pneumonitis, chronic bronchitis and decreased lung function^†1. Occupational settings with high bacterial concentrations (e.g. paper mills, poultry farms, compost facilities, wastewater treatment plants) can pose a significant threat to worker’s health and should be monitored. A health based occupational exposure limit of 90 Endotoxin Units per m3 based on personal inhalable dust exposure - measured as an eight hour time weighted average - has been proposed by the Health Council of the Netherlands and is commonly referenced^†2. Another application for this assay is monitoring endotoxin concentrations in metal working fluids (MWF) and worker’s exposure in metal cutting or grinding operations^†3.

References

1. Flaherty DK, Deck FH, Cooper J, Bishop K, Winzenburger PA, Smith LR, Bynum L, Witmer WB: Bacterial endotoxin isolated from a water spray air humidification system as a putative agent of occupation-related lung disease. Infection and immunity 1984, 43(1):206-212.
2. Health Council of the Netherlands. Endotoxins 2010. Health-based recommended occupational exposure limit. The Hague: Health Council of the Netherlands; 2010
3. Simpson AT, Stear M, Groves JA, Piney M, Bradley SD, Stagg S, Crook B. Occupational exposure to metalworking fluid mist and sump fluid contaminants. Ann Occup Hyg. 2003 Jan; 47(1):17-30.
4. Rolph, C.A.; Gwyther, C.L.; Tyrrel, S.F.; Nasir, Z.A.; Drew, G.H.; Jackson, S.K.; Khera, S.; Hayes, E.T.; Williams, B.; Bennett, A.; Collins, S.; Walsh, K.; Kinnersley, R.; Gladding, T.L. Sources of Airborne Endotoxins in Ambient Air and Exposure of Nearby Communities—A Review. Atmosphere 2018, 9, 375.
5. Mendy A, Wilkerson J, Salo PM, Zeldin DC, Thorne PS. Endotoxin clustering with allergens in house dust and asthma outcomes in a U.S. national study. Environ Health. 2020 Mar 16;19(1):35.

Endotoxins in air samples

As a component of bioaerosols, endotoxins cause respiratory effects making air an important sample type to assess exposure risk. The Health Council of the Netherlands proposed occupational exposure limits of 90 Endotoxin Units (EU) per m3 and some have suggested diving the threshold level by 3 (30 EU per m3) to account for uncertainty. Most published studies on air sampling use air cassettes with polycarbonate, Teflon, glass fiber or other filter material at flow rates of 2 to 16 liters per minute^†4. Ideally, sampling is performed for several hours to obtain values for a time weighted average. Total collected sample volume should exceed 250 liters.

Limulus Amebocyte Lysate (LAL) Test:
The LAL test for endotoxins is a kinetic-colorimetric test that provides the most sensitive and most specific procedure for detection and quantification of pyrogens. The test is based on chemically-induced color change over time as the LAL substrate reacts with bacterial endotoxins. Concentrations are determined by comparing the reaction to an E. coli standard. Inhibitors are accounted for by performing parallel experiments with spike recoveries of the client samples. The test sensitivity is 0.025 EU per m3 for air samples, 0.005 EU per ml for liquids and 2.5 EU per gram for dust samples. Positive samples above the quantification range or samples with inhibitors are diluted accordingly to obtain quantitative results. It is important to use sampling and laboratory materials that are specifically produced and tested for pyrogen contaminations. For air sampling it is recommended to use endotoxin-free sampling cassettes.

Endotoxins in water and machining fluids

Gram-negative bacteria are commonly found in water and other liquids including metal working fluids (MWF). Because the LAL assay provides a very sensitive method for the detection and quantification of endotoxins and other pyrogens it is the method of choice to monitor contaminations in medical equipment such as dental water lines and hemodialysis equipment. This test is also commonly used to assess the risk of excessive bacterial exposure through aerosols produced in machining operations. Metal working fluids can grow bacteria in high concentrations. Exposure to fluid mist may cause irritation of the respiratory tract, impairment of lung function, bronchitis and asthma. Published studies provide typical endotoxin concentrations in MWF for comparison^†3.

Endotoxins in dust

Endotoxin can be detected and quantified in house dust or in dust from occupational settings. There have been a number studies correlating dampness and the presence of pets with an increase of endotoxins. The correlation of high endotoxin concentration in house dust with respiratory symptoms such as wheezing, cough and shortness of breath has been discusses but is controversial because endotoxins typically cluster with allergens, molds and pets^†5.