Discharge of metals and their mineral flotation collectors into the soil environment causes severe ecological and health impacts, which is still not fully understood. This is of great concern, particularly with regards to their effect on the soil microorganisms whose functions determine not only the soil quality and function but also influence the air and water quality. This study aimed to analyze and compare, microcalorimetrically, the single chemical toxic effect with the combined effect of copper (Cu) and two of its main flotation collectors, potassium amyl xanthate (PAX) and sodium isoamyl xanthate (SIAX), on soil microbial community. All chemicals, individually and as a binary mixture of copper and each of its flotation collectors, exhibited a significant dose-effect relationship, and the highest and lowest microbial activity inhibition being associated with SIAX and Cu, respectively (e.g. IC 50 of 447.5, 158.3 and 83.9 μg·g −1 soil for copper, PAX and SIAX, respectively). For all cases, the microbial activity was more affected by the mixture than by the individual mixture components. Increasing the xanthates dose (from 25 to 100 μg·g −1 soil) in the mixture with a copper dose of 200 μg·g −1 soil led to the increase of the microbial activity inhibition rate, from 23.08 % to 53.85% in case of PAX and from 26.92% to 57.69% in case of SIAX). Similarly, the toxicity level of the mixture of equitoxic components doses increased with the increased mixture doses. Since the observed activity level can be attributed to the surviving microbes, capable of adapting to both chemical and their mixture, a genetically based analysis should be conducted to allow identifying and characterizing the potentially resistant strains that can be useful for the remediation of the pollution by copper and xanthates and for the sustainability of copper mining and flotation, and for all soil, water, and air quality and function interest.
Soil microorganisms constitute the most soil active constituent, mediating almost all processes within the soil [
Copper (Cu) is widely distributed in the environment, ranking 26th in abundance in the lithosphere and is one of the most extensively mined and processed metals worldwide [
On the other hand, flotation reagents constitute a non-replaceable part of mineral beneficiation through flotation, a process of extracting valuable minerals from the rest of the gangue [
With regard to their toxicity, both copper and xanthates are known for their adversity for the living system. However, the sensitivity of different organisms to pollutants depends on chemical species [
Exposure to structurally and toxicologically different substances can lead to different toxicological types of reactions, including additivity (usually upon dose addition), synergism (greater than additive action) or antagonism (less than additive toxicity) [
Soil from farmland, in Beijing (39˚59' - 116˚21'), was selected for this study. After the removal of the uppermost soil layer, soils samples were collected at a depth of 5 - 15 cm ground surface soil, the most populated soil layer by microorganisms [
Cu was used as CuSO4・5H2O compound. Copper sulfate (CuSO4) is one of the most used copper compounds and one of the most contributors to environmental pollution by this element. Xanthates, PAX and SIAX, were provided in the solid and analytical form from Beijing General Research Institute of Mining and Metallurgy. Both copper and collectors stock solutions were prepared using distilled water and were kept at 4˚C during the experimental time.
The soil samples treatment was performed as follows: 1) for the individual toxicity analysis, the soil samples were amended with different doses of Cu (100, 200, 300, 400, and 600 μg・g−1 soil) and xanthates (25, 50, 100, and 200 μg・g−1 soil). The obtained results allowed determining different inhibitory concentrations, IC 12.5, IC 25 and IC 50, corresponding to the chemical concentrations that can inhibit 12.5%, 25% and 50% of the microbial growth, respectively. Comparison between chemicals was made based on the inhibitory concentrations. 2) to analyze the level of the mixture toxicity, soil samples were treated with the mixture of the equitoxic doses of Cu and xanthates, and 3) to allow understanding the effect of the variation of xanthates concentration in the mixture, the soil samples were spiked with an increasing dose of xanthates (25, 50, and 100 μg・g−1 soil) and a fixed dose of Cu (200 μg・g−1 soil).
The measurement of soil microbial activity in the absence of pollutants (control sample) and under the exposure to the individual and mixture of copper and each of the studied flotation collectors were conducted using a multi-channel thermal activity monitor, TAM III (TA Instruments, Delware, USA). Soil samples were firstly conditioned at the temperature of microcalorimetric measurement (28˚C). The experiments were conducted in cleaned, sterilized, and hermetically sealed 4-mL steel ampoules containing 1 g of soil samples. To stimulate the soil microbial growth, all samples were supplemented with 0.2 mL of a nutrient solution made of 5.0 mg glucose and 5.0 mg ammonium sulfate that can provide enough nutrients (i.e. C, N, S) for the microbial growth [
Data analysis was conducted using the Origin 8.5 (OriginLab, MA, USA) software, Excel, and Spss Statistics 17.0 software packages. Comparison between results was performed through the Pearson correlation coefficient analysis, with a statistical significance of P < 0.05 or P < 0.01. All treated samples were compared to the control sample. The power-time curves allowed obtaining the peak-time (Tpeak) values of the microbial growth and the corresponding peak-power (Ppeak) values. The microbial growth rate constant (k) was calculated through linear fitting equation using the semilogarithmic conversion of the heat flow rate data [
P t = P 0 exp ( k t ) or ln P t = n P 0 + k t (1)
I ( % ) = K 0 − K c K 0 × 100 (2)
where P0 and Pt are, respectively, the heat output at time 0 and time t; I (%) is the inhibitory ratio and K0 and Kc are the growth rate constant of the control and the growth rate constant of soil microbes under c dose of toxicant, respectively. K and I are the most used parameters for a quantitative evaluation of the microbial response to a given stressful condition [
R = M I ∑ n = 1 i I i (3)
where R represents the ratio between the observed mixture inhibitory ratio (MI) and the additive inhibitory ratios of the mixture components, Ii represents the inhibitory ratio of i-mixture components alone. R = 1 indicates a sample additivity; R < 1 and R > 1 correspond to an antagonistic action and a synergistic action between the mixture components, respectively.
The power-time curves corresponding to the soil microbial activity in the control sample and treated samples with different doses of Cu, PAX, and SIAX are shown in Figures 1(a)-(c). As it was reported in the previous similar studies, samples exhibited differences in the curves profiles and duration of the microbial growth phases (lag, exponential, stationary and death phases) indicating a dose-effect relationship [
The lag and stationary phases were relatively extended in relation to the increase in the dose of pollutants. Different samples exhibited an interruption of the decline phase or death phase by reactivation and increase of the soil microbial growth, particularly for the samples treated with xanthates, individually or in the mixtures. Such a situation may be due to the growth of the surviving microbes that grow slowly (e.g. Fungi) [
The thermokinetic parameters and the corresponding statistics are presented in
Different reasons may explain the observed lower toxicity level for copper than for xanthates.
In contrast to xanthates which are manmade organic chemicals recently produced, mainly for the mineral flotation purposes [
wide range of use as a biocide and its application in medicine. With respect to xanthates, it was reported that even a very low dose, less than 1 mg・L−1, is harmful to aquatic organisms [
Treatments | Dose (µg∙g−1) | Tpeak (h) | Ppeak (µW∙g−1) | Ppeak r (%) | K (h-1) | I (%) | IC 12.5 | IC 25 | IC 50 |
---|---|---|---|---|---|---|---|---|---|
control | 0 | 14.88 | 1113.10 | 100.00 | 0.26 | 0.00 | 106.25 | 220.00 | 447.5 |
Cu | 100 | 14.28 | 832.23 | 74.77 | 0.23 | 11.54 | |||
200 | 14.19 | 676.01 | 60.73 | 0.20 | 23.08 | ||||
300 | 15.31 | 530.52 | 47.66 | 0.17 | 34.62 | ||||
400 | 25.03 | 411.28 | 36.95 | 0.13 | 50.00 | ||||
600 | 16.17 | 264.13 | 23.73 | 0.09 | 65.38 | ||||
PAX | 25 | 13.80 | 847.27 | 76.12 | 0.23 | 10.77 | 28.05 | 54.15 | 158.3 |
50 | 15.75 | 677.34 | 60.85 | 0.20 | 23.08 | ||||
100 | 13.79 | 563.09 | 50.59 | 0.16 | 38.46 | ||||
200 | 13.85 | 436.55 | 39.22 | 0.12 | 53.85 | ||||
SIAX | 25 (Peak I) | 12.62 | 725.51 | 65.18 | 0.23 | 10.00 | 35.83 | 41.34 | 83.95 |
(Peak II) | 25.18 | 467.72 | 42.02 | ||||||
50 (Peak I) | 13.36 | 554.89 | 49.85 | 0.18 | 30.77 | ||||
(Peak II) | 34.17 | 502.03 | 45.10 | ||||||
100 (Peak I) | 13.04 | 372.75 | 33.49 | 0.11 | 57.69 | ||||
(Peak II) | 34.15 | 401.84 | 36.10 | ||||||
200 | 31.32 | 310.45 | 27.89 | 0.04 | 84.62 |
Tpeak = peak time; Ppeak = peak power; K = microbial growth rate constant; I = Inhibitory ration; IC = Inhibitory concentration. In case of SIAX, the peak time and peak power are given for both first peak (Peak I) and second peak (Peak II) of the power time curves, respectively.
processes, the most important being the microbial-mediated decomposition [
Dose | Tpeak | Ppeak | K | I | |
---|---|---|---|---|---|
Copper | |||||
Dose | 1 | ||||
Tpeak | 0.431 | 1 | |||
Ppeak | −0.969** | −0.466 | 1 | ||
K | −0.995** | −0.509 | 0.978** | 1 | |
I | 0.995** | 0.509 | −0.978** | −1.000** | 1 |
Potassium amyl xanthate | |||||
Dose | 1 | ||||
Tpeak | −0.445 | 1 | |||
Ppeak | −0.890* | 0.331 | 1 | ||
K | −0.969** | 0.400 | 0.966** | 1 | |
I | 0.969** | −0.400 | −0.966** | −1.000** | 1 |
Sodium isoamyl xanthate | |||||
Dose | 1 | ||||
Tpeak | 0.851 | 1 | |||
Ppeak | −0.839 | −0.450 | 1 | ||
K | −0.978** | −0.737 | 0.905* | 1 | |
I | 0.978** | 0.737 | −0.905* | −1.000** | 1 |
**Correlation is significant at the 0.01 level (2-tailed). *Correlation is significant at the 0.05 level (2-tailed).
[
O 2 + H 2 S → S o + H 2 O 2 (4)
H2O2 is an oxidative stress inducer and can lead to bacterial cell damage and death [
The power-time curves corresponding to microbial activity under the binary mixtures of the equitoxic doses of copper and xanthates, PAX and SIAX, are shown in
The corresponding thermokinetic parameters and the related statistical analysis are shown in
For all treated samples the peak-time (Tpeak) was higher than that of the control sample (
For the same dose of copper (200 μg・g−1 soil), increasing the xanthates dose (from 25 to 100 μg・g−1 soil) in the mixture was followed by a decrease in the peak-power (Ppeak) and microbial growth constant (K). Conversely, the inhibitory ratio (I) increased in the same treatment conditions. For all cases, the observed mixture inhibitory ratio (MI) was higher than that of the corresponding individual mixture components, meaning that the microbial communities are more affected by the simultaneous exposure to copper and xanthates than by the exposure to the single chemicals. For a mixture containing a copper dose of 200 µg・g−1 and a xanthates dose of 100 µg・g−1, the corresponding inhibitory ratio was 53.85% compared to 38.5% for xanthates and 23.1% for copper. For example, in case of the mixture containing 200 µg・Cu・g−1 and a xanthates dose of 25, 50 and 100 µg・g−1, the ratio (R) between the mixture inhibitory ratio and the sum of the individual components inhibitory ratios was less than 1, corresponding to an antagonistic mixture-type reaction. However, with respect to the mixture of the equitoxic amounts, synergism was observed in soil treated with the lower chemicals doses (R < 1).
From these results, the microbial activity was not completely suppressed by any of the applied doses, including by the components whose sum of the individual inhibitory ratios equals 100% (
can adapt to all applied individual and combined chemicals doses, as well as to the formed complex copper/xanthates and to the xanthates associated decomposition products. It is well known that metals are mostly toxic in their free ionic form, mainly Cu2+ in case of copper, which can interact with the polar group (S=C-S−) of xanthates [
Mixtures components | Peak-time (h) | Peak-power (Ppeak) | K | AI (Ia+Ib) | Obs. MI | R (MI/AI) | Mixture-type reaction | |||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Copper | Xanthates | |||||||||||
Dose (µg∙g−1) | Ia (%) | Dose (µg∙g−1) | Ib (%) | |||||||||
Increased xanthates dose in the mixture | Cu + PAX | 200.00 | 23.07 | 25.00 | 10.77 | 15.75 | 711.68 | 0.20 | 33.85 | 23.08 | 0.68 | Antagonism |
50.00 | 23.08 | 17.93 | 777.25 | 0.14 | 46.16 | 46.15 | 1.00 | Additivity | ||||
100.00 | 38.46 | 21.5 | 684.84 | 0.12 | 61.54 | 53.85 | 0.87 | Antagonism | ||||
Cu + SIAX | 25.00 | 10.00 | 16.20 | 675.88 | 0.19 | 33.08 | 26.92 | 0.81 | Antagonism | |||
50.00 | 30.77 | 22.41 | 637.05 | 0.15 | 53.85 | 42.31 | 0.79 | Antagonism | ||||
100.00 | 57.70 | 30.22 | 530.23 | 0.11 | 80.77 | 57.69 | 0.71 | Antagonism | ||||
Mixture of equitoxic Cu and xanthates doses | Cu + PAX | 106.25 | 12.50 | 28.05 | 12.5 | 15.64 | 741.03 | 0.19 | 25.00 | 28.46 | 1.14 | Synergism |
220.00 | 25.00 | 54.15 | 25.00 | 20.09 | 688.13 | 0.15 | 50.00 | 44.23 | 0.88 | Antagonism | ||
447.50 | 50.00 | 158.3 | 50.00 | 33.95 | 454.16 | 0.05 | 100.00 | 81.92 | 0.82 | Antagonism | ||
Cu + SIAX | 106.25 | 12.50 | 13.59 | 12.50 | 19.65 | 669.78 | 0.16 | 25.00 | 38.08 | 1.52 | Synergism | |
220.00 | 25.00 | 26.90 | 25.00 | 26.48 | 568.91 | 0.11 | 50.00 | 57.54 | 1.15 | Synergism | ||
447.50 | 50.00 | 68.61 | 50.00 | 32.08 | 347.71 | 0.03 | 100.00 | 88.08 | 0.88 | Antagonism |
Obs.MI = observed mixture inhibitory ratio; AI = additive inhibitory ratio; K = microbial growth rate constant; I = Inhibitory ratio; Ia = inhibitory ratio of copper alone; Ib = Inhibitory ratio of xanthates alone; R = ratio between the observed mixture inhibitory ratio and the additive inhibitory ratio of the mixture components. For all cases, the observed mixture inhibitory ratio (MI) is higher than the individual copper and xanthates inhibitory ratios.
family Thiobacteriaceae [
The analysis of single and mixture toxicity of copper, potassium amyl xanthates and sodium amyl xanthates illustrated that all chemicals negatively affected the soil microbial activity. The highest and lowest inhibitory ratios were associated with SIAX and copper, respectively. Exposure to the binary mixtures of copper and potassium amyl xanthate or sodium amyl xanthate led to an increase in the toxicity level compared to individual chemicals. Increasing the xanthates doses (from 25 to 100 µg・g−1 soil) in the mixture with a copper dose of 200 µg・g−1 soil resulted in the increase of the mixture toxicity. Compared to additive individual chemical effects, exposure to a mixture of equitoxic amounts of copper and xanthates resulted either in synergistic mixture-type reaction or antagonistic mixture-type reaction. Therefore, in case of simultaneous application of copper and PAX or SIAX, and for the sustainability of the flotation process, optimizing the chemicals dose is still of a scientific and biotechnological challenge. This is not only interesting regarding the soil quality and function issues, but also for air and water quality issues that are influenced by the soil microbial mediated functions. Since the observed activity can be attributed to the activity of the resistant or less affected microbes, genetically based studies can allow a better understanding of the interactions between the soil microbial community and the chemicals mixture in presence. Resistance microbial strains can, therefore, be identified and can serve for the remediation of the pollution by copper and xanthates.
This study was supported by the Fundamental Research Funds for the Central Universities under the contract number FRF-OT-16-026.
Authors declare that there no potential conflict of interest associated with this work.
Bararunyeretse, P., Beckford, H.O. and Ji, H.B. (2019) Interactive Effect of Copper and Its Mineral Collectors on Soil Microbial Activity―A Microcalorimetric Analysis. Open Journal of Soil Science, 9, 47-64. https://doi.org/10.4236/ojss.2019.93003