Toxicity evaluation of copper and zinc cyanide complexes using Euglena gracilis Z and SMZ as an in-vivo model

Abstract

Gold cyanidation is the most used technique worldwide despite cyanide being highly toxic. The reaction of cyanide with metals such as copper and zinc during or after gold recovery builds up moderate and weak metal–cyanide complexes in tailings storage facilities (TSFs) which if not managed may find their way into the environment. Migratory birds and other waterfowl that ingest with the tailings water or waste discharged from cyanidation operations have the potential to cause adverse effects mainly due to the highly toxic free cyanide (HCN + CN-) and free cyanide released from metal-cyanide complexes particularly under acid condition. The International Cyanide Management Code (ICMC) has set 50 mg/L of weak acid dissociable (WAD) cyanide discharge into tailings dam as a protective guideline for birds and biota. However, this guideline value was established based on field observations and is not based directly on scientific toxicological data as identified by NICNAS and this remains a knowledge gap. The toxicity of cyanide and metal-cyanide complexes was assessed using Euglena gracilis strains Z and SMZ. The unique plant- and animal-like properties of Euglena gracilis, and its tolerance to a wide range of pH values including acid pH which is similar to that of the upper gastrointestinal tract of birds where dissociation of metal-cyanide complexes take place, offer a distinct advantage over other algal cells to be selected for this project. Euglena gracilis Z and SMZ strains were exposed to copper sulfate, zinc sulfate, sodium cyanide, copper- and zinc-cyanide complexes mixed species solutions and one tailings sample for 24 h and 48 h. Two methods for the assessment of inhibitory concentration at 50% (IC50) were applied namely an optical density measurement at the absorbance wavelength 610 nm and a modified resazurin assay. The modified resazurin assay provided more robust and reproducible results. The 48 h exposure showed that CuSO4 induced toxicity to Z (IC50= 20 mg Cu/L) and SMZ (IC50= 116 mg Cu/L). Zinc sulfate showed limited toxicity to both strains of E. gracilis with high IC50 values (IC50=565 mg Zn/L) for Z and (IC50=1490 mg Zn/L) for SMZ. Based on a WAD CN molar basis the cytotoxicity data (IC50) for E. gracilis Z in the order of: NaCN (1.24 mg CN/L, 95% CI: 1.03 to 1.5) ~ zinc-cyanide complex mixed species (1.61 mg CN/L, 95% CI:1.3 to 2) > copper-cyanide complex mixed species (4.17 mg CN/L, 95% CI:3.7 to 4.7) and for SMZ cells in the order of NaCN (1.02 mg CN/L, 95% CI:0.91 to 1.1) > zinc-cyanide complex mixed species (2.13 mg CN/L, 95% CI:1.7 to 2.7) > copper-cyanide complex mixed species (5.54 mg CN/L, 95% CI:5 to 6.1), respectively. Further, the chicken was used as a surrogated model for the migratory birds to determine the effects of short-term exposure to sub-lethal doses of sodium cyanide, copper-cyanide complex mixed species, zinc-cyanide complex mixed species and gold mine tailings sample contains less than the ICMC guideline (50 mg WAD CN/L). Based on the CN released it has been found that the toxicity (in chickens) is in the order of: NaCN > zinc-cyanide complex mixed species > copper-cyanide complex mixed species. Calibration of toxicity of NaCN to E. gracilis against chicken showed a good relationship (correlation coefficient R2= 0.9858 and 0.9663 for Z and SMZ, respectively) between the two models, giving a predictive equation for the safe CN level in the water that migratory birds may consume if accessing TSFs at gold mines. No clinical signs were observed in chickens treated with tailings water contains 22 mg/L WAD CN. However, from the predictive equations it was concluded that the current ICMC guideline of 50 mg WAD CN/L may not be totally safe to some bird species such as Mallard duck Anas platyrhynchos, American kestrel (AK) and Black vulture (BV) based on comparison with existing toxicity data for these species. In addition, this study provides a practical guidance for the risk assessment of biota from cyanide exposure using Euglena gracilis that can potentially replace the conventional toxicity testing of a bird species (or chicken) and can reduce routine chemical cyanide speciation measurements. The latter is restricted by the availability of only a few analytical laboratories in the country and requirement for sample preservation. This study confirmed that Euglena gracilis Z and SMZ can be used as a screening tool to monitor toxicity of copper- and zinc-cyanide complexes that are present in gold mining tailings. In order to use this bioassay organism for cyanide and metal-cyanide complexes assessment, further laboratory and fieldwork may be required to calibrate the toxicity of other metal-cyanide complexes with the chicken model and evaluated the toxicity of cyanide and WAD CN considering multiple-doses. It is worthy of notice that the use of wild birds for toxicity testing is ethically impossible. Therefore the chicken model is a good alternative.