Optimisation of analytical methods for metal(loid)s in a lichen biomonitor

Abstract

Although a number of studies have examined metals in lichens, these studies are often limited to total metal analyses of a few target analytes. Moreover, there has not been a strong focus on the speciation of metal(loid)s in lichens, where these types of studies are highly relevant in providing information about bioavailability, source apportionment and toxicity. Furthermore, little attention has been paid to sample preparation, storage and the optimization of extractions for species characterizations where these aspects are essential in such evaluations. The use of the foliose lichen, Parmotrema austrosinense (Zahlbr.) Hale, as an appropriate biomonitor of air pollution in South Africa was investigated by evaluating the concentrations of various metal(loid)s in this lichen species growing at both an urban and mining impacted site. P. austrosinense was found to be reflective of the different sources of pollution at both of the sites chosen for this study, proving its fitness for use in future metal(loid) air pollution biomonitoring evaluations. Thereafter, P. austrosinense was evaluated for metal and metalloid content at sites with varying sources and magnitudes of anthropogenic impacts. In this study the importance of combining multi-element studies with meteorological data was emphasized, as this allows for correlations between the observed concentration in the lichen thallus and those present as a result of air pollution to be drawn. The outcomes from this study not only serve as a baseline for future air biomonitoring studies in South Africa, but was also the first study to evaluate the concentrations of a suite of metals in lichens at the Cape Point Global Atmospheric Watch (GAW) baseline reference station, providing essential information for comparative assessments between other GAW sites around the globe. After noting the combined use of multi-elemental and meteorological data as a useful tool for the assessment of lichen biomonitors, the use of a four-step sequential extraction scheme was employed for its novel application in the extraction of metals and metalloids from lichens. Here, greater attention was paid to sample preparation strategies and storage than had been done in studies by other researchers. Cryogenic freezing of samples using liquid nitrogen was found to be the most appropriate sample preparation strategy for total metal analysis, however this did not hold true for speciation analyses using sequential extractions, and a recommendation was made that the implementation thereof be revisited. A sample size of 10 g of lichen material was found to be adequate for providing a homogenous and representative sample of the lichen population for atmospheric pollution evaluations. Thereafter the focus shifted toward the targeted analyses of arsenic species in lichens, and some of the lesser researched aspects, such as the optimization of extraction methods to improve the recoveries of inorganic arsenic species from the lichen matrix. An isocratic, High Pressure Liquid Chromatography Inductively Coupled Plasma Mass Spectrometry (HPLC-ICP-MS) method, capable of baseline separating arsenobetaine (AsB) from arsenite (As III) using a Hamilton PRP X-100 anion exchange column was developed. The benefit of the developed chromatographic method is that five of the most toxicologically relevant arsenic species, namely As III, arsenate (As V), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and AsB could all be quantified using a single analytical column with a single mobile phase, saving on the cost and time of analysis. Moreover, the extraction procedure used only deionized water as an extraction solvent, and employed a low concentration ammonium nitrate (17.5 mmol) in 1% MeOH solution as the mobile phase, making the routine application of this method a more eco-friendly and “green” alternative to typical HPLC-ICP-MS methods for As speciation studies. Having observed the benefits of both the sequential extraction, which was able to identify arsenic compounds that become available using solvents with different properties and ionic strengths, and the optimized chromatographic method which was able to further separate the soluble or exchangeable fraction into different species, the water extraction step of the sequential extraction was, for the first time, replaced with a chromatographic separation and applied to lichens from an urban impacted and rural site in South Africa. The findings from this study were significant, resulting in the highest mass-balance yields for arsenic than had been previously reported in literature for either chromatographic techniques or sequential extractions, being 104 % and 111% for the urban and rural sites respectively. The chromatographic method was thereafter applied to a bulk sample of the lichen, P. austrosinense, over a period of 1 month with assessments performed every week to determine the short-term stability of arsenic species in the water-extractable (bioavailable) fraction. It was found that the species of arsenic in the bioavailable fraction changed week by week. Here, the perception that the biotransformation capabilities of lichens makes them unsuitable biomonitors of air pollution was challenged. Instead, it was proposed that the predictable timing of the biotransformation of chemical species is an essential component of biomonitoring studies, and can assist in elucidating information about the timing of the exposure and the scale of the exposure event. Furthermore, the results were able to provide new insight into the current understanding of lichen metabolism, and a plausible explanation for a previously observed increase in As V postulated by other researchers. This thesis contributes to the body of science related to the analytical practices employed in lichen biomonitoring of metal and metalloid air pollutants, with a strong focus on the carcinogenic species of the metalloid, arsenic. It is one of a handful of studies which evaluate As speciation in lichens, where the optimized extraction and novel combination of chromatographic and sequential extraction techniques are able to provide a substantial amount of information regarding source apportionment, and the timing and magnitude of pollution events. As such, this study provides additional insights into both the total analysis of a number of different metals and metalloids, and the optimized extraction and selective and sensitive separation of the various chemical forms of arsenic, making it of global interest. On a more local front, this is one of three published studies in South Africa which evaluated the concentration of heavy metals and metalloids in a lichen biomonitor, and is the first to address more than five elements, therefore the results will form a point of reference for future studies of this type.