Clean Water Endotoxins


What are Endotoxins?In the world of microbiology, there is a strong focus on the pathogenic potential of certain bacterium. Yet the pathogenic potential of a specific bacteria is not the only factor that needs to be considered in the healthcare environment. Endotoxins, a microbial by-product, are an important risk factor in several diseases and reactions. In this article we will discuss the importance of endotoxins in water bioburden testing. 

What are Endotoxins?  

Endotoxins are a lipopolysaccharide (i.e LPS) that are a major component cellular wall of gram-negative bacteria. This compound is typically composed of a core polysaccharide, Lipid A and O-side chain. Endotoxins are not pathogenic per se, instead they are a component of a bacteria that when inhaled can produce an immune response in the host.

Why are Endotoxins a Concern?


      Endotoxins can trigger a potentially fatal inflammatory response

Endotoxins are a known pyrogen, or fever-inducing compound. This response is triggered by the Toll-like receptor 4 and molecule MD-2 interactions with the LPS (3, 4). Toll-like receptors (TLR) are primarily responsible for pathogen detection within the host, with accessory molecules like MD-2 conferring response to lipopolysaccharides (4). The TLRs then elect an innate immune response in the form of inflammation. While inflammation does cause damage to a pathogen, it often damages healthy tissue in the process (2). If this response is not carefully controlled, a host can develop sepsis and go into septic shock (5). The immune response varies depending on the host and the bacteria the endotoxin originated from, however even low levels of endotoxins can cause acute respiratory illness. 

Potable Water Legionella


Critical Water Testing and Methodology


      Endotoxin Testing is required by the TIR-34

The TIR-34 states that while the threshold of endotoxin levels that could elicit reaction in patient is difficult, that levels as low as 100 EU/mL could trigger an adverse reaction in a patient. This is especially true with devices that encounter critical body fluids, such as cerebrospinal fluid and blood. Dialysate, or dialysis water, falls under the stricter ANSI/AAMI/ISO 13959 recommendation with an allowable level of 0.25 EU/mL (6). With this in consideration, it is imperative that waters used for dialysis, sterilization of medical devices and implants be pyrogen-free (1, 6).

      Gel Clot LAL Assay

The gel clot Limulus amebocyte lysate (LAL) assay is the gold standard for testing the presence of endotoxins in water. The water sample is combined with lysed blood cells from the Atlantic horseshoe crab, which will react with any endotoxins present in the sample. At EST, our standard LAL assay has a high sensitivity of 10 EU/mL and 20 EU/mL, well within the TIR-34 recommendation. EST also features a specialized kit for the collection, processing, and interpretation of dialysis water as well.

Other Possible Roles of Endotoxins


      A potential factor in Legionellosis

It is well accepted that the Legionella genus, specifically Legionella pneumophila, is responsible for Legionnaires’ Disease and the less severe Pontiac Fever. However, determinants of Legionella virulence are not well understood. A growing body of evidence has suggested that endotoxins produced by the Legionella genus plays a significant role in the virulence of the bacteria.

One of the earliest studies of this hypothesis can be found in a 2001 article on a 1998 outbreak of Pontiac Fever in a Wisconsin hotel (Fields et al., 2001). The outbreak was eventually attributed to L. micdadei found in the swimming pool filter and spa water of the hotel. The endotoxin concentration was also tested at the time of the outbreak, where the water from the spa reported 14,400 endotoxin units/mL (EU/mL), well above the 0.2 EU/mL that has been linked to acute respiratory illness (1). The authors propose in the article that environmental endotoxins may play a part in the pathogenesis of Pontiac Fever.

Another article published in 2005 provides additional support for this hypothesis, which focused on a 2000 outbreak of Pontiac Fever in a Minnesota sugar-beet processing plant (Caster et al., 2005). The outbreak was eventually attributed to L. pneumophila that had possibly been contaminated in the local water treatment plant and made its way into the factory’s hot water tank that was used for high-pressure washing. The endotoxin concentration was also tested at the time of the outbreak, where the water from the pump house that fed the factory’s hot water tank reported at 22,200 EU/mL, and water from two power washers linked to the outbreak reported 20,400 EU/mL. At the time of the study, the authors discussed the possible implication of endotoxins being a factor for Legionellosis pathogenesis, they acknowledge that L. pneumophila LPS has a low endotoxic potential, a potential challenge to the hypothesis.

This low endotoxic potential however may behave more as an asset than hinderance to L. pneumophila potential in virulence. There is evidence that the weak endotoxin activity of L. pneumophila may assist in evading the immune system of the host, allowing it more time to proliferate in the host before an immune response occurs (7, 8).


  1. ATTACHMENT B AAMI TIR34 2014 r2017.pdf – GovTribe
  2. Toll-like Receptors, Infection | Learn Science at Scitable (
  3. Endotoxin recognition molecules, Toll-like receptor 4-MD-2 – PubMed (


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