Investigations into Lead (Pb) Accumulation in Symphytum officinale L.: A Phytoremediation Study

Abstract

Lead (Pb) is the number one heavy metal pollutant in the environment. The high cost and environmental concerns of conventional remediation technologies has led to an emerging alternative technology for heavy metal remediation: phytoremediation. This study was set out to advance Pb phytoremediation by investigating plant-associated factors (e.g. polyphenol levels, Pb-tannin chelation, and superoxide dismutase activity) and chemical-based factors (e.g. concentration of Pb, and the type and dosage of chelating agents in treatments) that may affect Pb accumulation. Using a hydroponic system, sand-grown Symphytum officinale L. plants were exposed to nutrient solutions with or without lead nitrate (Pb(NO₃)₂) and ethylenediamine tetraacetic acid (EDTA). Using flame atomic absorption spectroscopy (to measure Pb content) and bovine serum albumin-protein precipitation (to measure polyphenol and tannin levels), a significant in vivo correlation between tannin level and Pb accumulation level was observed in roots of plants exposed to all Pb treatments. Higher tannin containing-lateral roots accumulated significantly more Pb than lowertannin main roots. Transmission electron micrographs of unchelated Pb-treated plants supported these findings, whilst dialysis-based in vitro Pb-chelation studies with crude S. officinale root polyphenol extracts did not. The dialysis method was likely to be subject to fructan interference. A new, more accurate and simple method based on tannin immobilisation was consequently developed. Results using this method supported the hydroponic trends. This new method was also verified with purified tannic acid (from Sigma). Together, these findings demonstrate that S. officinale root tannins have the ability to chelate Pb. This may be a mechanism to cope with Pb stress (adaptive tolerance). Despite the typical signs of Pb stress at root level (e.g. root growth inhibition, and degraded cytoplasms), shoots showed no signs of stress under any Pb treatments. Most importantly, since this chelation-based tolerance mechanism also influences the accumulation levels, the phytochemical composition of plants should also be considered when screening plants for phytoremediation. The level of Pb accumulated in the shoots depended on the concentration of Pb(NO₃)₂ and presence of chelating agents (EDTA or N-[2 acetamido] iminodiacetic acid (ADA)) in the nutrient solution. The highest level of Pb in shoots was between 0.05-0.06% (d.w. on average) using EDTA or ADA, well short of the 1% (d.w.) shoot accumulation target for Pb phytoextraction. The highest level of Pb in the roots (and of all measurements) was with unchelated 500 µM Pb(NO₃)₂; on average 2% (d.w.) accumulated in root. Overall, since S. officinale accumulated Pb predominately in the roots, it is most suited for rhizofiltration and phytostabilisation. Whilst chelating agents enhanced Pb accumulation in shoots, root levels were unexpectedly reduced compared to unchelated Pb treatments. The level of Pb translocated did not completely account for this loss. Minor factors relating to EDTA desorption of roots, EDTA specificity, and charge repulsion of the PbEDTA complex may account for some of the loss, but the main cause remains unclear. In vitro S. officinale cultures were developed and somaclonal variation (involving Pb pre-treatment of petioles) was used as a tool to further investigate, and attempt to improve its Pb phytoremediation potential. The shoots and roots of plants produced from petioles pre-treated with Pb(NO₃)₂ appeared more stressed than those without Pb pre-treatment. After re-treatment with Pb (Pb(NO₃)₂ or PbADA), plants developed from most Pb pre-treated petioles appeared to have reduced Pb accumulation and polyphenol levels, and increased superoxide dismutase activity in roots (although no statistically significant trends were found). Overall, plants produced from Pb pre-treated petioles in this study may have less phytoremediation potential.