Isocyanates and Amines – Sampling and Analytical Procedures

This thesis covers sampling and analytical procedures for isocyanates (R-NCO) and amines (R-NH2), two kinds of chemicals frequently used in association with the polymeric material polyurethane (PUR). Exposure to isocyanates may result in respiratory disorders and dermal sensitisation, and they are one of the main causes of occupational asthma. Several of the aromatic diamines associated with PUR production are classified as suspected carcinogens. Hence, the presence of these chemicals in different exposure situations must be monitored. In the context of determining isocyanates in air, the methodologies included derivatisation with the reagent di-n-butylamine (DBA) upon collection and subsequent determination using liquid chromatography (LC) and mass spectrometric detection (MS). A user-friendly solvent-free sampler for collection of airborne isocyanates was developed as an alternative to a more cumbersome impinger-filter sampling technique. The combination of the DBA reagent together with MS detection techniques revealed several new exposure situations for isocyanates, such as isocyanic acid during thermal degradation of PUR and urea-based resins. Further, a method for characterising isocyanates in technical products used in the production of PUR was developed. This enabled determination of isocyanates in air for which pure analytical standards are missing. Tandem MS (MS/MS) determination of isocyanates in air below 10-6 of the threshold limit values was achieved. As for the determination of amines, the analytical methods included derivatisation into pentafluoropropionic amide or ethyl carbamate ester derivatives and subsequent MS analysis. Several amines in biological fluids, as markers of exposure for either the amines themselves or the corresponding isocyanates, were determined by LC-MS/MS at amol level. In aqueous extraction solutions of flexible PUR foam products, toluene diamine and related compounds were found. In conclusion, this thesis demonstrates the usefulness of well characterised analytical procedures and techniques for determination of hazardous compounds. Without reliable and robust methodologies there is a risk that exposure levels will be underestimated or, even worse, that relevant compounds will be completely missed.

The neurotoxin β-N-methylamino-L-alanine (BMAA) : Sources, bioaccumulation and extraction procedures

β-methylamino-L-alanine (BMAA) is a neurotoxin linked to neurodegeneration, which is manifested in the devastating human diseases amyotrophic lateral sclerosis, Alzheimer’s and Parkinson’s disease. This neurotoxin is known to be produced by almost all tested species within the cyanobacterial phylum including free living as well as the symbiotic strains. The global distribution of the BMAA producers ranges from a terrestrial ecosystem on the Island of Guam in the Pacific Ocean to an aquatic ecosystem in Northern Europe, the Baltic Sea, where annually massive surface blooms occur. BMAA had been shown to accumulate in the Baltic Sea food web, with highest levels in the bottom dwelling fish-species as well as in mollusks. One of the aims of this thesis was to test the bottom-dwelling bioaccumulation hypothesis by using a larger number of samples allowing a statistical evaluation. Hence, a large set of fish individuals from the lake Finjasjön, were caught and the BMAA concentrations in different tissues were related to the season of catching, fish gender, total weight and species. The results reveal that fish total weight and fish species were positively correlated with BMAA concentration in the fish brain. Therefore, significantly higher concentrations of BMAA in the brain were detected in plankti-benthivorous fish species and heavier (potentially older) individuals. Another goal was to investigate the potential production of BMAA by other phytoplankton organisms. Therefore, diatom cultures were investigated and confirmed to produce BMAA, even in higher concentrations than cyanobacteria. All diatom cultures studied during this thesis work were show to contain BMAA, as well as one dinoflagellate species. This might imply that the environmental spread of BMAA in aquatic ecosystems is even higher than previously thought. Earlier reports on the concentration of BMAA in different organisms have shown highly variable results and the methods used for quantification have been intensively discussed in the scientific community. In the most recent studies, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the instrument of choice, due to its high sensitivity and selectivity. Even so, different studies show quite variable concentrations of BMAA. In this thesis, three of the most common BMAA extraction protocols were evaluated in order to find out if the extraction could be one of the sources of variability. It was found that the method involving precipitation of proteins using trichloroacetic acid gave the best performance, complying with all in-house validation criteria. However, extractions of diatom and cyanobacteria cultures with this validated method and quantified using LC-MS/MS still resulted in variable BMAA concentrations, which suggest that also biological reasons contribute to the discrepancies. The current knowledge on the environmental factors that can induce or reduce BMAA production is still limited. In cyanobacteria, production of BMAA was earlier shown to be negative correlated with nitrogen availability – both in laboratory cultures as well as in natural populations. Based on this observation, it was suggested that in unicellular non-diazotrophic cyanobacteria, BMAA might take part in nitrogen metabolism. In order to find out if BMAA has a similar role in diatoms, BMAA was added to two diatom species in culture, in concentrations corresponding to those earlier found in the diatoms. The results suggest that BMAA might induce a nitrogen starvation signal in diatoms, as was earlier observed in cyanobacteria. However, diatoms recover shortly by the extracellular presence of excreted ammonia. Thus, also in diatoms, BMAA might be involved in the nitrogen balance in the cell.