Sick of Toxic Black Mould? Development of a Stable-Isotope Dilution Assay for the Quantification of Stachybotrys chartarum Trichothecenes in New Zealand’s Leaky Buildings

<p dir="ltr"><b>Living in damp, mouldy housing is linked to the development of respiratory disorders, and New Zealand’s leaky building crisis in the mid-1990’s was accompanied by both pervasive mould growth and many anecdotal reports of illness. The most common fungus identified in the rotting timber of these houses, </b><b><i>Stachybotrys chartarum</i></b><b>, has long been suspected of causing building-related illness, and can produce some of the most potent mycotoxins known — the macrocyclic trichothecenes (MCTs). A Government inquiry into the leaky building crisis recommended investigating the health impacts from exposure to </b><b><i>S. chartarum</i></b><b> in such buildings, and in response, a group of multi-disciplinary health researchers proposed an epidemiological study into the incidence of asthma- related symptoms in New Zealand’s leaky building occupants in relation to low-level chronic exposure to </b><b><i>Stachybotrys</i></b><b> trichothecenes. The present research aimed to quantify low-level trichothecenes in dust samples by developing two complementary analytical methods.</b></p><p dir="ltr">The gold standard in low-level mycotoxin analysis, stable-isotope dilution assay (SIDA), has not been applied to <i>S. chartarum</i>. Firstly, it was proposed that a SIDA-based gas chromatography-mass spectrometry (GCMS) method be developed to quantify verrucarol, the hydrolysis product of most <i>Stachybotrys</i> trichothecenes, as a proxy for total trichothecene content. Secondly, the development of a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method was proposed to quantify key trichothecenes (KTCs), including two of the most potent MCTs, satratoxins G and H, in samples where verrucarol was detected using the SIDA-GCMS method. To enable this method development, standards of both natural verrucarol and a tri-deuterated isotopologue required for the SIDA-GCMS analysis were to be prepared by semi-synthesis from a related trichothecene, diacetoxyscirpenol, while standards of the KTCs required for HPLC-MS analysis would be isolated from large scale rice cultures of <i>S. chartarum</i>.</p><p dir="ltr">Synthesis of verrucarol and a tri-deuterated isotopologue 15-²H₁-3-²H₂-verrucarol was successful, allowing development of the SIDA-GCMS method. In contrast, the HPLC-MS method was not developed due to a lack of KTC recovery from fungal cultures. The cultured <i>S. chartarum</i> strains were therefore subject to genomic analysis which revealed only two of five strains were potential MCT producers. The desired house dust samples could not be obtained. Instead, the SIDA-GCMS method was applied to extracts from the <i>S. chartarum</i> rice cultures and a selection of infested building materials provided by industry partners Biodet Services Ltd. Verrucarol was quantified in 15 of 101 hydrolysed extracts of the building materials and detected in a further five. Extracts of the cultured strains identified as MCT producers by genomic analysis yielded high levels of verrucarol upon hydrolysis, with low levels detected from a third isolate. These results strongly correlated with a qualitative analysis for the presence of KTCs (from an external provider), demonstrating the validity of SIDA-GCMS method. This work thus represents the first application of SIDA to the analysis of <i>Stachybotrys</i> trichothecenes and should provide a powerful tool for future investigation into their effect on human health.</p>