Structural and functional differentiation of bacterial communities in post-coal mining reclamation soils of South Africa: bioindicators of soil ecosystem restoration.

Soil microbial communities are suitable soil ecosystem health indicators due to their sensitivity to management practices and role in soil ecosystem processes. Presently, information on structural and functional differentiation of bacterial communities in post-coal mining reclamation soils of South Africa is sparse. Here, bacterial communities in three post-coal mining reclamation soils were investigated using community-level physiological profiling (CLPP), enzyme activities, and next-generation sequencing of 16S rRNA gene. Inferences were drawn in reference to adjacent unmined soils. CLPP-based species diversity and proportionality did not differ significantly (P > 0.05) whereas activities of ß-glucosidase, urease and phosphatases were significantly (P < 0.05) influenced by site and soil history (reclaimed vs unmined). Bacterial communities were influenced (PERMANOVA, P < 0.05) by soil history and site differences, with several phylotypes differentially abundant between soils. Contrastingly, predicted functional capabilities of bacterial communities were not different (PERMANOVA, P > 0.05), suggesting redundancy in bacterial community functions between reclamation and unmined soils. Silt content, bulk density, pH, electrical conductivity, Na and Ca significantly influenced soil bacterial communities. Overall, results indicate that bacterial community structure reflects underlying differences between soil ecosystems, and suggest the restoration of bacterial diversity and functions over chronological age in reclamation soils.

Effect of Bacillus cereus on the ecotoxicity of metal-based fungicide spiked soils: Earthworm bioassay.

Soil microorganisms exhibit varying levels of metal tolerance across a diverse range of environmental conditions. The use of metal-based fungicides such as mancozeb and copper oxychloride could potentially result in increased levels of manganese, zinc and copper which may adversely affect soil mesofauna. Under standardized earthworm bioassay conditions (temperature, pH, soil type and water content), we investigated the effect of Bacillus cereus on mancozeb and copper oxychloride ecotoxicity towards Eisenia andrei. A metal-tolerant Bacillus cereus strain previously isolated from a gold mining site was introduced into fungicide spiked soils. Earthworms were exposed to bacterial inoculated and non-inoculated substrates of mancozeb (8, 44, 800 and 1250 mg kg-1) and copper oxychloride (200, 450, 675 and 1000 mg kg-1). Experimental trials assessed avoidance-behavior, growth and reproduction utilizing standardized protocols (ISO and OECD). In the avoidance-behavior, E. andrei showed significant (p< 0.05) preference for inoculated substrates. Further, significant (p< 0.05) increases in biomass, survival, cocoons, juveniles and lower soil and tissue Mn, Cu and Zn contents were recorded at 8 and 44 mg kg-1 mancozeb and copper oxychloride 200 and 450 mg kg-1 inoculated soils compared to non-inoculated. However, at 800 and 1250 mg kg-1 mancozeb and 675 and 1000 mg kg-1 copper oxychloride concentrations, reproductive success in both inoculated and non-inoculated treatments was negatively (p< 0.05) affected. In conclusion, Bacillus cereus decreased the ecotoxicity of metal-based fungicides towards Eisenia andrei at 8 and 44 mg kg-1 mancozeb and 200 and 450 mg kg-1 copper oxychloride concentrations. The outcome observed with the inoculated substrates at elevated fungicides concentrations maybe as a result of the environmental conditions (pH and temperature).

Preliminary evidence of differences in cadmium tolerance in metal-free stocks of the standard earthworm test species Eisenia andrei (Oligochaeta).

To test whether metal-tolerant and metal-sensitive earthworm specimens could be an inherent part of metal-free earthworm populations, (i) we used DNA barcoding to identify and categorize earthworms from 8 populations of the standard test species Eisenia andrei, and (ii) the earthworms carrying three of the identified COI haplotypes (named Hap1, hap3 and Hap3) were paired up and exposed to Cd in order to assess the difference in Cd sensitivity between the breeding pairs. A total of six breeding pairs were exposed to 0, 25, 50 and 100 mg Cd/kg for 4 weeks at 20 °C. The survival of the breeding pairs, their change in biomass and cocoon production were assessed. For all of the endpoints assessed, the results indicated that couple 6 (Hap3 × Hap3) was the most sensitive breeding pair whereas couple 4 (Hap1 × Hap3) was the least sensitive one. The analysis of Cd tissue contents revealed that with increasing Cd concentration, Cp6 (Hap3 × Hap3) could accumulate significantly more Cd than any other breeding pair (p ≤ 0.01). Our findings indicate that E. andrei may harbour intrinsically Cd-tolerant and Cd-sensitive individuals and that this may be due to individual differences in Cd accumulation kinetics. In the context of ecotoxicological testing, our results underline the importance of using genetically diverse populations in laboratory testing to prevent generating flawed data from genetically homogeneous laboratory stocks. Although we do not regard the present mitochondrial haplotypes as proxy for possibly nuclear encoded traits, we discuss the necessity of a standardised earthworm barcoding protocol that could help not only to confirm the taxonomy of laboratory earthworm stocks but also to select genetically diverse stocks suitable for laboratory testing.

The enchytraeid reproduction test (ERT): A potentially quick and affordable tool for the assessment of metal contaminated soils in emerging economies.

The enchytraeid reproduction test (ERT) was used to assess the ecotoxicity of selected mine tailings and agricultural soils from South Africa. The mine tailings had higher cumulative metal concentrations than agricultural soils. The most contaminated mine tailings significantly reduced the survival of the oligochaete Enchytraeus doerjesi whose reproduction was suppressed in all mine waste substrates. Because it reliably singled out the most contaminated substrate and was found easy to perform, we suggest that the ERT could be a quick and affordable tool for assigning intervention values for soil remediation in emerging economies such as South Africa.

Utilizing Eisenia andrei to assess the ecotoxicity of platinum mine tailings disposal facilities.

South Africa is an important platinum mining country which results in environmental impacts due to the construction of tailing disposal facilities (TDFs). It is unclear what the effects of ageing are on the ecotoxicity of TDFs and whether it increases or decreases over time. The aim of this study was to determine the ecotoxicity of differently aged TDFs by investigating earthworm (Eisenia andrei) responses viz. growth, reproduction, neutral red retention times (NRRT) and tissue metal concentrations. Further, to evaluate the status of these in terms of a geoaccumulation index (I(geo)), pollution index and integrated pollution index. Results indicated that earthworms showed reduced reproductive success (hatchlings per cocoon) and decreased NRRT in all the sites. Juveniles per cocoon between all of the different treatment groups were; control (2.83 ± 0.54) > site 2 (20 years old; 1.83 ± 0.27) > sites 1 and 3 (40 years old; 1.06 ± 0.15 and 6 years old; 0.88 ± 0.39). This might be ascribed to the elevated levels of Cr (±200 to 1,166 µg g(-1)) and Ni (±100 to 316 µg g(-1)) in all of the sites. Earthworms did not bioaccumulate metals with bioconcentration factors for all the different treatments <0.01. Studies like these could be useful when establishing a ranking of TDFs in the future to provide legislative institutions with an indication of the environmental liabilities of platinum mines.