Development of a method using Euglena gracilis to evaluate toxicity of free cyanide and strong metal cyanide complexes related to mining

Abstract

Cyanide has been used worldwide for gold extraction in mining industry. Gold particles in the ore exist in low concentration within other minerals. Cyanide also reacts with other metals and metalloids in the ore to form various compounds and complexes which are discharged in the tailing storage facilities (TSF) for long term protection of the environment. However episodes of bird’s mortality upon consumption of cyanide-bearing mining waste water have been reported. The toxicity is mainly attributed to the free cyanide released from the weak acid dissociable metalcyanide complexes (WAD). According to the International Cyanide Management Code (ICMI), 50 mg/L of WAD cyanide released into TSFs is deemed safe for wildlife protection. However the 50 mg/L value set by ICMI is based on observed bird’s carcasses and a lack of knowledge exists on the toxicity of free cyanide, weak acid dissociable metal-cyanide complexes (WAD) and strong metalcyanide complexes to birds and other biota. In this project the toxicities of free cyanide (HCN + CN-), nickel-cyanide (K2Ni(CN)4) as a moderately strong metal-cyanide complexes and cobaltcyanide (K3Co(CN)6) as a strong metal-cyanide complex were evaluated and compared in Euglena gracilis. The unicellular eukaryote, Euglena gracilis Z and SMZ was chosen as a test organism. Euglena is a unique organism since it possesses both, the plant-like (Z strain) and animal-like (SMZ strain) characteristics. E. gracilis has been widely used in assessing the toxicity of heavy metals and has been recommended as a sensitive and suitable model in environmental toxicity assessment. Furthermore E. gracilis can tolerate acidic pH (pH=3.5) which is relevant to acid mine drainage conditions and also corresponds to the pH of the upper gastrointestinal (G.I.) track in birds. The toxicity of cyanide-metal complexes was assessed using three methods including optical density measurement, CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS assay) and BacTiter-Glo™ Microbial Cell Viability Assay (ATP assay). Amongst these assays, the MTS assay provided more robust and reproducible results. However, optimization of the assay for Euglena gracilis was required due to a limitation in the formazan (reduced form of MTS reagent) colour development. This issue would have arisen from the medium interferences including oxidising agents, the lower pH of KH medium used in this project than pKa of formazan (6.0-6.5) and the lower temperature applied than formazan’s optimum temperature. Consequently the modified MTS assay was used to measure the toxicity of metal-cyanide complexes to E. gracilis. Euglena gracilis Z and SMZ cells were exposed to various concentrations of the selected toxicants for 24 h and 48 h. The dose-response curve was plotted and the 50% inhibition of growth rate related to each toxicant was calculated. The 48 h exposure results obtained from modified MTS assay indicates that nickel-cyanide is the most toxic to Z (IC50=126 μM) and SMZ (IC50=116 μM) Page 3 of 81 and cobalt-cyanide was the least toxic compound to both Z and SMZ with no significant toxicity up to the maximum concentration (5 mM) used. Sodium cyanide induced toxicity to both Z (IC50=182 μM) and SMZ (IC50=214 μM) which was comparable to nickel-cyanide’s toxicity. Nickel sulfate induced toxicity to Z (IC50=2491 μM) and SMZ (IC50=3598 μM) which was 20 times lower than nickel-cyanide. This indicates that nickel-cyanide’s toxicity derives from the free cyanide rather than nickel ion. Cobalt sulfate did not induce significant toxicity to E. gracilis cells. The results confirm the hypothesis that nickel-cyanide complex as moderately strong acid dissociable metal-cyanide complex releases cyanide under acidic pH of the KH medium used in the E. gracilis bioassay. Also cobalt-cyanide complex as a strong cyanide-metal does not seem to dissociate under highly acidic pH. Euglena gracilis was found to be a sensitive screening tool to assess the toxicity of metal-cyanide complexes related to mining. However further calibration of the results using an animal model could validate the potential of this bioassay organism for being used for cyanide toxicity protection in gold mining industry.