Sensitivity of two procedures to estimate the impact of the ionic liquid 1,3-dimetilimidazolio dimetilfosfato [C1C1Im][DMP] on soil microbial activity

Ionic liquids (IL) represent an alternative to organic solvents in processes of synthesis compounds and in separation technologies due their tuneable physicochemical properties as they can be synthesised by choosing from an enormous amount of cations and anions or even by changes in the alkyl chains of these ions. One of the key properties of these compounds is their negligible vapour pressure, due to which they are non-contaminant to the atmosphere and play an increasingly relevant role in green chemistry. Nevertheless, before their use can be generalised, toxicity studies must be performed as they could be toxic for terrestrial ecosystems. However, studies of the effects on soil and vegetation, media that can be reached by these liquids as a result of accidental spills, are scarce so far. The 1,3 dimethylimidazolium dimethylphospate, [C1C1Im][DMP], is an imidazolium ionic liquid with potential use in several industrial applications, as for example increasing the rate of cellulose hydrolysis by cellulase enzyme in the bleaching process in paper and pulp industry. Thus, once its use will be widespread, the probability that reaches terrestrial ecosystems will be high and therefore estimating the toxicity of this compound to these systems is of priority. The main goals of this study were i) to estimate the toxicity of [C1C1Im][DMP] to the soil by analysing its effect on soil microbial activity, ii) to test the sensitivity of two procedures (microcalorimetry and the classical method of soil basal respiration determination) to estimate microbial activity in IL-polluted soils, and iii) to compare and combine the information provided by both procedures to obtain a more insightful picture of the impact of this compound on soil microbial activity. The microcalorimetry technique is based on the analysis of the heat released by soil microbiota, activated with a solution of glucose, against time. The second methodology (soil basal respiration) is the classic measurement of the CO2 emitted by the soil incubated under optimal laboratory conditions of moisture (60-80% of water holding capacity) and temperature (25-28 ºC) during a given period of time (10-28 days). Since microcalorimetry was first applied for studying soil microbial activity it has being increasingly applied in soil studies. However, to our knowledge comparative analysis of the information provided by both techniques has not been performed. Two acid soils with sandy-loam texture and different organic matter (OM) contents were selected: a forest soil with high OM (FOR) and a crop soil (CROP) with low OM. These soils were artificially contaminated with increasing doses of [C1C1Im][DMP] (0 to 123.48 g LI kg-1 air-dried soil) and analysed for microbial activity by the two methods. The heat released by soil microbiota was determined against time and dose (only up to 92.61 g de IL kg-1 soil) using an isothemal microcalorimeter (TAM III, TA Instrument). In addition, after three days of soil-IL contact time, all the polluted soil samples (0 to 123.48 g IL kg-1 soil) were incubated at 25 ºC and 80% of water holding capacity to measure soil basal respiration. In our laboratory the soils are generally incubated for 10 days. However, with IL contaminated soil this period had to be increased until the CO2 emitted by soil respiration in IL-polluted soils was stable or has reached the level of control soil (126 days). The power-time curves resulting from microcalorimetry, showed important differences on the response of both soils to the IL addition: the FOR soil, with the highest OM content, an increase in heat released during the first hours of the experience followed by the dead of the microorganism was observed for the lowest doses, in comparison with the control. Whereas the highest doses caused a delay in the growth phase of the activity. The CROP soil, with low OM content, showed an increasing delay on growth phase with the dose was observed. As expected, soil basal respiration was more intense in the soil with high OM content (FOR) than in the soil with low OM. In both soils, the lowest amount of IL (1.92 g IL kg-1) did not affect soil respiration. However, all the other doses of IL increased very strongly the soil respiration and showed a peak in CO2 emission. This peak was progressively increasing with the amount of IL, up to 61.74 g LI kg-1, and thereafter tended to decrease with respect to this maximum. In all cases, and similarly to what was observed by microcalorimetry, whit increasing amounts of Il the moment of the peak was progressively delayed. The amount of CO2 emitted during the 126-days of incubation (accumulated CO2) by the soil with the lowest dose of IL (1.92 g IL kg-1) was similar to that of the control both in FOR and CROP soils. However, highest amounts of IL strongly increased the amount of accumulated CO2, but this increase was not proportional to the dose. Both in FOR and in CROP soils the highest amount of accumulated CO2 was obtained for the soil with an intermediate amount of IL (61.74 g LI kg-1), while the amount of CO2 emitted decreased for the two highest doses of [C1C1Im][DMP], being this decrease especially important for the highest dose in the soil with the lowest OM. In summary, both microcalorimetry and the classical method for measurement of soil basal respiration probed to be similarly sensitive to the presence and the amount of [C1C1Im][DMP], both in soils with high and low OM content. In addition, both methodologies showed a similar pattern of CO2 emissions in the IL-amended soils with the amount of IL and with time (increasingly highest amount of CO2 emitted with increasing doses of IL up to reach a maximum at intermediate doses; delayed peak of CO2 emission; increasing delay in the peak appearance with increasing amount of IL), showing that both methods are sensitive to the temporal changes of microbial activity in soils amended with different amounts of C1C1Im][DMP].Peer reviewe