Ecotoxicological Evaluation of Ethylammonium Nitrate and Aluminium Salt Mixture †

: The ecotoxicity of a mixture of the ionic liquid ethylammonium nitrate (EAN) and aluminum nitrate salt (Al(NO 3 ) 3 ) as well as the corresponding to pure components was studied in this work. This mixture has singular interest as electrolyte in electrochemical applications and data about the effects of mixture and components on the environment can hardly be found in literature. Changes on the bioluminescence of the Aliivibrio fischeri (Beijerinck) Urbanczyk bacteria through Microtox ® test and on microbial activity, measured by microcalorimetry, of two soils with different organic matter content, when exposed to solutions of different concentrations of these compounds, were analyzed.


Introduction
Ionic liquids (ILs) are salts with an organic cation and an organic or inorganic anion with melting points lower than 100 °C. ILs are a group of synthetic compounds with unique properties (low inflammability, low vapor pressure, variable solubility, low viscosity, high thermal and chemical stability, high ionic conductivity and a wide electrochemical window) that make then attractive for their use on numerous industrial applications as advanced materials for smart electrochemical devices as supercapacitors, dye-sensitized solar cells or lithium and aluminum batteries [1][2][3][4]. ILs are considered as a good alternative to the conventional volatile organic solvents due to their, above mentioned, negligible vapor pressure reducing the possible risk of atmospheric contamination. Romero et al. [5]. have stated that some imidazolium ILs can show higher toxicity than common organic solvents. This statement can be explained taking into account their water solubility and biodegradability resistance that could make them strong pollutants and persistent in the environment, and therefore they could pose a danger for the aquatic organisms, plants and soil microorganisms considering probable spills into the environment [6-8]. Despite this fact, and although the applicability of ILs spreads rapidly, toxicity studies of these compounds are very unusual. Thus, ILs could enter into commercial mass production without fully understanding of their ecotoxicity [9].
The IL used in this work, ethylammonium nitrate (EAN), is considered as RTIL (roomtemperature IL); this compound presents water like properties [10] and mainly due to its ability to form hydrogen bounds, some studies as an electrolyte, additive, detergent or precipitating agent among other applications can be found in recent literature [11][12][13]. But, in spite of their applicability and the high probably of spills and filtration into terrestrial and aquatic environments, studies about their toxicity on these environments are very scarce.
In this work, the ecotoxicity of ethylamonium nitrate, the aluminum nitrate salt (Al(NO3)3·9H2O) and the saturated mixture of both components was studied using two different tests. The first one is a standardized method based on the variation of the bioluminescence generated by the bacteria Aliivibrio fischeri (Beijerinck) Urbanczyk through Microtox ® test. Moreover, a good way to analyze the impact of a substance on soil microorganisms or even isolated organisms [14], is through the variation of their metabolic activity, and microcalorimetric techniques with high power sensibility can be used for this propose; in particular, Isothermal Microcalorimetry is an extremely general technique as it measures heat production rate, which accompanies nearly all physical, chemical and biological processes. The use of this technique has been increased during recent years in the microbiological field due to its simplicity, versatility and fast analysis [15]. In this study, changes on the heat released by microorganism of two soils with different organic matter content as a consequence of the application of different amounts of the pure compounds (EAN, Al) and of aluminum-saturated EAN were analyzed.

Chemicals
The main characteristics, name, CAS registration number, structure, molecular mass, purity and provenance of the selected ionic liquid and salt are indicated in Table 1. Further purification of the pure IL with high vacuum procedure was performed until water content below 100 ppm was obtained.

Microtox Test
The toxicity was assessed by the Microtox ® Toxicity Test kit (Microbics Corporation, 1992) using the luminescence production of the marine bacteria Aliivibrio fischeri (Beijerinck) Urbanczyk understood as a metabolic effect to test treatments which comprise a range of aqueous solutions (0-81.9% of a 2 molal stock solution) of each compound. The light output of the luminescent bacteria following 5, 15 and 30 min of exposure was read and compared to that of a blank control. Concentrations promoting 50, 20 and 10% of luminescence inhibition (EC50, EC20 and EC10), and the corresponding 95% confidence intervals were estimated through a non-linear regression, using the least-squares method to fit the data to the logistic equation [17].

Microcalorimetric Test
These experiments were performed using an isothermal microcalorimeter Thermal Activity Monitor (TAM III) Thermometric AB. The effect of different solutions (0, 1, 10, 25, 50 and 75%) in distilled water, of the pure compounds and their mixture, on the heat released by microorganisms of two soils with different organic matter content (OM) and similar pH was studied, in order to determine the influence of the OM on the effects of the contaminants. The measurements were carried out at 25 °C, with a quantity of field-moist soil equivalent to 2 g of air-dried soil spiked with 0.2 mL of the 0 to 75% solutions above indicated. Immediately before the measurements, 0.2 mL of a glucose solution in water with a concentration of 6.25 g L −1 were added to activate the metabolism of soil microorganisms. The heat released by soil microorganisms until the total consumption of glucose was measured [14]. Table 2 shows the main characteristics of the two soils used for the study. The first soil corresponds to a corn field collected in A Pedra (A Coruña) on 17/07/2018, the second one is a forest soil collected in Negreira on 25/10/2018. When they were collected their humidity was 13.54% and 27.72%, respectively.

Microtox Test
The results of mean effective concentration values to obtain a reduction of 10, 20 and 50% of bioluminescence of the bacteria after 5, 15 and 30 min of exposition, determined by Microtox ® test are shown in Table 3. As it can be expected, these values decrease with the time of exposition indicating that toxicity increases. The highest EC values were found for EAN, and the lowest for Al(NO3)3, whereas the mixture showed slightly higher values than the pure salt. Table 3. Mean effective concentration values (EC10, EC20, EC50) in mg l −1 and the respective 95% confidence intervals (CI), obtained after exposure of the marine bacteria Vibrio fischeri to the studied compounds during 5, 10 and 30 min. Taking into account that higher values of EC indicate lower toxicity, pure Al(NO3)3 and the mixture (EAN+Al) can be considered slightly toxic (10 mg L −1 < EC50 < 100 mg L −1 ) and EAN can be classified as relatively harmless (greater than 1000 mg L −1 ) using the Passino and Smith classification [18]. Other works also highlighted the harmless, or moderately toxic, effect of different ILs towards A. fischeri, and detected the influence on the toxicity of the chemical elements attached to the IL, like the increase of the toxicity when an alkyl side chain increases, that is not the case of EAN. In Table 1 it can be seen that this IL is no so bulky as pyrrolidinium or imidazolium can be, and this can be the reason of its low toxicity [19]   Chen et al. [20] suggested the division of the power-time curves in four phases: lag, log, stationary and decline phases. Similar shapes can be observed in our experiments where the increase of the lag phase happened in the two soils for all the compounds together with a slower and progressive log phase for the highest doses. The stationary phase stays similar for all the doses and compounds, and the same happened with the decline phase, with just a few exceptions in both cases.

Microcalorimetric Test
The main remark is that pure EAN and the mixture present the highest toxicity in both soils, where no signal was obtained beyond 25% for both compounds in the soil with low organic matter, and a delay on the heat released peak is observed for 50 and 75% doses on the soil with high organic matter content. By the contrary, the aluminum salt is the least toxic compound, showing heat release signal for all the studied concentrations. This does not agree with the toxicity of these compounds shown by Microtox analysis. Ucha et al. [21] detected a very high EAN toxicity, either alone or mixed with Al, on the germination of three tree species. The toxicity was already significant for doses of 2.5% of EAN or EAN+Al in water. However, it must be considered that the seeds were sown in a Petri dish without adding any amount of soil. This would explain why the toxicity was even higher than that obtained in the present work by microcalorimetry for the two studied soils, reinforcing the idea that organic matter could partially buffer the toxicity of this ionic liquid. Also suggests that Microtox analysis is less sensitive, although faster, than microcalorimetry and seed germination analysis to show the toxicity of compounds like those investigated in this study.
This study also demonstrates the inverse relationship between organic matter content and ILs toxicity exposed by Salgado et al. [14] and Sixto et al. [22], in agreement with a similar inverse relationship observed for other organic compounds toxic for soil microorganisms [23].

Conclusions
In this work ecotoxicity of the ionic liquid ethylammonium nitrate (EAN) and aluminum nitrate salt (Al(NO3)3) as well as the mixture of both compounds were analyzed in terms of the bioluminescence of the marine bacteria A. fischeri and the microbial growth of two soils after the addition of several concentrations of these complexes. Ecotoxicity studies showed that EAN results very toxic for the soil microorganisms, unlike for the marine bacteria A. fischeri. By the contrary, Al(NO3)3 showed higher toxicity for the marine bacteria than for the soil microorganisms. Beside this, the toxicity for the mixture of both is always similar to that of the more toxic compound in each case.