Ecotoxicological study of glucose:choline chloride and sorbitol:choline chloride at different contents of water

1 Ecotoxicological study of Glucose:Choline chloride and Sorbitol:Choline chloride at different 1 content of water 2 Authors: 3 Laura Lomba. 4 Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 5 50830. Villanueva de Gállego Zaragoza. Spain 6 Diego Errazquin. 7 Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 8 50830. Villanueva de Gállego Zaragoza. Spain 9 Pilar Garralaga. 10 Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 11 50830. Villanueva de Gállego Zaragoza. Spain 12 Noelia López. 13 Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 14 50830. Villanueva de Gállego Zaragoza. Spain 15 Corresponding autor: 16 Beatriz Giner. 17 Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 18 50830. Villanueva de Gállego Zaragoza. Spain 19 e-mail: [email protected], phone: 0034 976 060 100, fax 0034 976 077 584 20 2 Abstract: The search of new solvents is currently focused on Deep Eutectic Solvents (DES). However, 21 there are not many ecotoxicological studies in different biomodels of DES that allow knowing how these 22 chemicals affect to the environment along the trophic chain. In this manuscript, two DES at different 23 proportion of water have been prepared and characterized from the ecotoxicological point of view. These 24 solvents are: glucose:chloride choline (2:5) and sorbitol:chloride choline (3:2) at different content of water. 25 To carry out the ecotoxicological study, three biomodels have been used: bacteria Allivibrio fisheri (A. 26 fisheri), crustacean Daphnia magna (D. magna) and algae Raphidocelis subcapitata (R. subcapitata). The 27 obtained results show that the ecotoxicity of these chemicals depends on the biomodel used and the amount 28 of water, being toxicity values lower for chemicals with higher water content. However, it is important to 29 highlight that the ecotoxicity for all chemicals is quite low with effective concentrations, EC50 values above 30 1000 mg/L in all the studied cases. 31 32 Keywords: ecotoxicity, DES, glucose sorbitol, Aliivibrio fischeri, Daphnia magna, Raphidocelis 33 subcapitata. 34 35 3 1. Introduction 36 Deep Eutectic Solvents (DES) and Natural Deep Eutectic Solvents (NADES) are mixtures formed 37 by two or more chemicals at solid state at room temperature but, when combined in a specific molar ratio, 38 form a liquid solution. This is mainly due the strong and complex network of intermolecular forces 39 stablished that cause a sharp diminution of the melting point (Laura Lomba 2021a). 40 DES can be considered as “design solvents” since the possibility of combinations of different 41 components and compositions is almost unlimited. The goal is setting the right mixture for obtaining the 42 physicochemical properties required for a specific chemical processes(Mitar et al. 2019). 43 DES are formed by a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) and the 44 typical components are natural chemicals such as: organic acids, alcohols, sugars, amino acids, urea, small 45 hydrophilic molecules or natural metabolites among others(Nystedt et al. 2021). 46 DES are usually low cost, are also easy to prepare (Benvenutti et al. 2020, Benvenutti et al. 2019), 47 present low volatility, are biodegradable (Wils et al. 2021) and normally non-reactive with water. The label 48 of non-toxic mixtures has to be reviewed and lately there is some controversy in the scientific community 49 in this regard; there are an important number of studies that show low toxicity of eutectic mixtures for the 50 environment or human both in vitro and in vivo studies (Laura Lomba 2021b), while some others studies 51 demonstrate the toxicity of DES above a certain limit of concentration (Torregrosa-Crespo et al. 2020). 52 Nevertheless, the toxicity of DES depends on several factors that include not only the nature of the 53 components of the mixture but also the organisms on which toxicity is studied, their pre-adaptation periods 54 or even the sterilization processes carried out during the measurement of toxicity. This may be caused by 55 the acidification of the medium caused by the DES hydrolysis (Torregrosa-Crespo et al. 2020). Thus, it is 56 required more specific studies about the effect of DES on different endpoints, covering several trophic 57 levels, organisms, cells or tissues and the use of more appropriate toxicological techniques (Marchel et al. 58 2022b, Ruesgas-Ramon et al. 2017). 59 It is also said that present good physicochemical properties for several chemical processes thanks 60 to their wide polarity range and low vapour pressure or chemical stability, among others(Benvenutti et al. 61 2020, Laura Lomba 2021a, Laura Lomba 2021b, Silva et al. 2019). For these reasons DES can be used as 62 stationary phases in chromatography (Momotko et al. 2021, 2022) and membranes (Castro-Munoz et al. 63 2022, Khajavian et al. 2022) and additionally, they are attractive for several industrial processes such as 64 biocatalysis (Zhou et al. 2021), cryopreservation(Craveiro et al. 2021), biochemical applications(Silva et 65 al. 2019, Yang 2019), extraction medium(Ruesgas-Ramon et al. 2017, Rukavina et al. 2021), biological 66 assays (Liu et al. 2018), pharmaceutical industry(Liu et al. 2021, Morrison et al. 2009) or cosmetic 67 industry(Benoit et al. 2021). 68 However, the main barrier to the widespread use of DES is the lack of information regarding 69 various physiochemical and toxic properties. This limitation arises from the very nature of the DES; the 70 multiple combinations between components and compositions (AlOmar et al. 2016, Lapena et al. 2020, 71 Lapena et al. 2019b, a) makes difficult to have a complete set of properties that allow knowing the specific 72 characteristics of a mixture (components and concentration) (Li et al. 2022) or even to predict the behaviour. 73 However, it is important notice that the thermal stability of DES can be diminished at high 74 temperature. Most of the studies about this issue analyse the thermal stability of DES using the onset 75 4 decomposition temperatures (Tonset). These temperatures, that were normally obtained from dynamic 76 thermogravimetry (TGA) under different experimental conditions that, in general, lead to the 77 overestimation of the onset decomposition temperature. It is important to bear in mind that there are some 78 scientific studies related to the thermal stability of DES that have found, in some cases, that DES can 79 decompose into HBA and HBD due to the weaking of the hydrogen bonds. This process can occur in two 80 steps: the first one, the DES component with a lower boiling point or little stability undergoes volatilization 81 or decomposition (normally HBD) and the second one, the decomposition of the other DES at a higher 82 temperature (normally the HBA). Thus, the role of the hydrogen bonds in the thermal stability seems to be 83 determinant. (Gutierrez et al. 2010, Marchel et al. 2022a). Additionally, it is important to note that the 84 toxicity of DES may vary when the hydrogen bond net is modified; new supramolecular structures can be 85 created, and thus, the toxicological activity may be also different.(Gutierrez et al. 2010, Marchel et al. 86 2022b, a) 87 With the aim of increasing the information available on these substances, we have previously 88 explored the ecotoxicity of some of them: reline, ethaline and glyceline towards several aquatic biomodels 89 (algae, crustaceans and bacteria)(Lapena et al. 2021). We founded that the ecotoxic effect clearly depended 90 on the studied biomodel and the type of test used as a tool to monitor toxicity. Furthermore, the water 91 content did not follow the expected trend and studied DES behaved erratically with regards to the water 92 composition(Lapena et al. 2021). 93 Thus, with the objective of deepening the knowledge of the ecotoxic behaviour of DES, in this 94 work we have studied the ecotoxicity of several eutectic mixtures containing sugars and chloride choline 95 at different concentrations of water: glucose:chloride choline (2:5) and sorbitol:chloride choline (3:2) with 96 different water content. Concretely, we have obtained the toxic effect that these mixtures produce in various 97 aquatic organisms, covering the trophic chain: bacteria Allivibrio fisheri (A. fisheri), crustacean Daphnia 98 magna (D. magna) and algae Raphidocelis subcapitata (R. subcapitata). 99 Besides, it has been previously reported that viscosity is an important properties key to explain 100 toxicity in some cells. An increase of toxicity is normally associated with higher cell lethality rates(Hayyan 101 et al. 2016). For that reason, we have also measured the kinematic viscosity of the studied DES. Toxicity 102 results have first been analysed in an independent way, considering both the DES and the biomodel. They 103 were then considered together and the final conclusions about the ecotoxic behavior have been reached. 104 105 2. Material and methods 106 2.1 Chemicals and preparation of Deep Eutectic Solvents 107 In Table 1, the used chemicals for the DES preparation are gathered. Choline chloride has been 108 dried under vacuum for 24 h prior to use. Mixtures with water have been prepared considering the previous 109 amount of water of each of the components of the mixtures with Milli-Q water (resistivity less than 18.2 110 M·cm) using a Sartorius Entris 5201-1S balance (uncertainty ±10-1 g). Once each substance has been 111 weighed, has been transferred to a closed flask (250 ml) with a magnetic nucleus inside. The flask has been 112 placed on a heating plate and heated to a temperature of approximately 80ºC with continuous stirring. The 113 process has been completed within one hour and a homogeneous liquid has been observed. In Table 2, 114 studies DES, composition, and abbreviation are shown. 115 5 Table 1. Characteristics of the used chemicals 116 Name CAS Supplier Mass fraction purity Formula Molar mass (g/mol) Glucose anhydrous 50-99-77 Panreac 0.975 C6H12O6 180.16 Sorbitol 50-70-4 Sigma-Adrich 0.980 C6H14O6 182.17 Choline Chloride 67-48-1 Sigma-Aldrich 0.993 C5H14ClNO 139.63 117 Table 2. Studied DES: composition and abbreviation 118 HBD HBA Add-on Molar ratio Abbreviation Glucose Chloline chloride Water 5:2:5 Glu5 5:2:7.5 Glu7.5 5:2:10 Glu10 Sorbitol Chloline chloride water 2:3:5 Sor5 2:3:7.5 Sor7.5 2:3:10 Sor10 119 2.2 Viscosity measurements 120 Kinematic viscosities, ν, have been obtained using a Schoot-GeräteAVS-440 automatic measuring 121 unit along with several Ubbelohde capillary viscosimeters. The uncertainty of the time flow measurements 122 is 0.01 s, and kinetic energy corrections have been applied to the experimental data. The temperature has 123 been controlled at 25ºC by means of a Schoot-Geräte CT 1150/2 thermostat, with a temperature uncertainty 124 of 0.01K. 125 126 2.3 A. fischeri ecotoxicity test 127 The lyophilized A. fischeri (strain NRRL-B-11177 and reference 945006) have been supplied by 128 Macharey-Nagel. This test has been based on UNE-11348-3(UNE-11348-3 2009). All solutions have been 129 prepared with 2% of NaCl and the pH has been adjusted using 0.1 M HCl or 0.1 M NaOH in 2 % NaCl. 130 Additionally, positive controls such as phenol and zinc sulfate have been used(Jennings et al. 2001) and 131 negative control has been culturing medium (Biofix Lumi medium for freeze-dried luminous bacteria by 132 DIN EN USO 11348–3, Macherey-Nagel, Duren, Germany). 133 Luminescence has been measured in acute mode (Biotox B) using a Biofix Lumi-10 luminometer 134 (MachereyNagel) after 30 minutes of exposure and at 15ºC. Three replicates for each control have been 135 tested. More details of this test can be found in our previous works(Garcia et al. 2015, Lomba et al. 2014). 136 137 2.4 D. magna ecotoxicity test 138 D. magna ecotoxicity test have been based on the guidelines OECD 202(202 2004). The D. magna 139 ephippia (Toxkit, Daphtokit F Magna, ref. DM090812) have been stored at 4ºC and purchased from 140 Vidrafoc. The eggs have been incubated with culturing medium considering the specifications of the 141 supplier, during 72 h at 22ºC with 6000 lux in a Toxkit CH-0120D-AC/DC incubator (supplied by Ecotest) 142 6 and fed with Spirulina algae 2 h before starting the experiment. The pH has been adjusted 7-7.5 before 143 exposure. Furthermore, positive control (sodium dichromate) and negative controls have been tested. 144 A total of 20 newborn daphnids (aged less than 24 h) have been exposed to the different dilutions 145 of the DES in dark for 24 h at 20ºC per compound and concentration. The crustaceans have been separated 146 into four groups of five organisms, four replicates per concentration exposure. The test has been repeated 147 in triplicate. Daphnids have been considered immobilized if they have been not able to swim for 15 s after 148 agitation. More details of this protocol can be found in bibliography(Perales et al. 2017). 149 150 2.5 R. subcapitata ecotoxicity test 151 R. subcapitata test have been carried out according to OECD 201(OECD 1984). Algae have been 152 purchased from ECOTEST (SC2B1214). All the experiments have been repeated in triplicate. Moreover, 153 culturing medium and de-mobilization of algae have been prepared using the supplier specifications. 154 The algae cells have been incubated at 23ºC in 100 mL beaker containing 50 mL of culturing 155 medium with 10000 lux of illumination. Cells densities of have been correlated with optical density (OD) 156 at 670 nm basing on the supplier specifications. The algae initial concentration has been 3·105 cells/ml. 157 The pH of the solutions has been adjusted in the range of 7.9 and 8.2 using 0.1 M NaOH or 0.1 M 158 HCl solutions. The assay has been carried out in a 96 well plate. To minimize evaporation, the outer wells 159 of the plate have been filled with 0.3 mL of distilled water and only the inner wells have been filled with 160 controls and test solutions. The initial OD has been measured at 670 nm. The well plate has been incubated 161 at 23ºC for 72 hours. Next, all plates have been re-suspended to avoid the settling of algae and the final OD 162 has been measured. 163 164 2.6 Statistics treatment 165 Data have been analysed using a non-linear regression-representing log (inhibitor) versus 166 inhibition. 167 To obtain the EC50 values of each compound, the experimental results have been fitted to the 168 following function: 169 ( ) ( ) bCa log 101 100 I% − + = (4) 170 where %I denotes % inhibition of luminescence for A. fischeri, % of immobilization for D. magna and % 171 of inhibition of growth for R. subcapitata; C is the concentration in mg/L and Log EC50 and a are the 172 adjustable parameters. 173 174 3. Results and discussion 175 In this section, the obtained results for the studies compounds are shown. Regarding to the 176 prepared DES, it is important to note that all DES formed by sorbitol (Sor5, Sor7.5 and Sor10) and glucose 177 (Glu10) have been obtained in a unique a clear phase at room temperature (25ºC), however, in the case of 178 glucose DES (Glu5, Glu7) it has been necessary to slightly heat (30ºC) to obtain that liquid phase before 179 the tests. 180 7 In this section, results have been analyzed from the perspective of the DES (or DES + water 181 mixtures) itself, as a whole. However, it is important to revise the nature of the components of the eutectic 182 mixtures from the toxicological point of view. For instance, choline chloride is a known component of 183 Vitamin B, and plays important metabolic functions; choline is usually the chosen cellular raw material for 184 the synthesis of cellular phospholipidic membranes (phosphatidylcholine and sphingomyelin) (Hayyan et 185 al. 2016). On the other hand, from a cellular perspective glucose and sorbitol are essential carbohydrates; 186 their metabolism provides the required energy for different cellular functions. After adsorption, glucose 187 undergo glycolysis when energy is required in the cell, otherwise is stored as glycogen. The sorbitol 188 metabolism is an accessory pathway in the glucose route. Through a chain of reactions, the sorbitol is an 189 intermediate that converts glucose into fructose. The glycolytic pathway for glucose and fructose leads to 190 synthesis of nucleic acids through the pentose phosphate pathway, the energy production in the 191 mitochondria through tricarboxylic acid pathway and the fatty acid synthesis through the lipogenesis 192 process. For these reasons, it is expected high cellular tolerance of the studied DES formed by choline and 193 these sugars. However, during the eutectic mixture formation process and the establishment of the hydrogen 194 bond network, an important modification of the chemical and physical properties is made, and this includes 195 the toxicological behavior of the eutectic mixtures. 196 Returning to the analysis of the results obtained by the DES studied in the biomodels, values of 197 EC50 towards A. fischeri, D. magna and R. subcapitata are shown in Table 3. For all the studied chemicals, 198 there is a dependence relationship between the toxicity and the concentration; i.e, the toxic effect increases 199 as the concentration does. The general toxicity trend observed for all biomodels is the following: Glu 5 > 200 Glu 7.5 > Glu10 > Sor5 > Sor 7.5 > Sor10. In Figure 1, the toxicity of obtained values for all the studied 201 biomodels are presented. The results indicate that the most sensitive species was D. magna followed by R. 202 subcapitata and A. fischeri. 203 It is important to highlight that none of the studies mixtures can be considered toxic to the 204 environment (values of EC50 do not exceed the general limit of 1000 mg/L). The Globally Harmonized 205 System of classification And labelling of Chemicals (GHS) (Nations 2021) has been used to categorize the 206 toxicity for substances hazardous to the aquatic environment, Short-term (acute) as follow: Acute 1 (EC50 207 ≤ 0.1 mg/L), acute 2 ( EC50 > 1 but ≤ 10 mg/L), acute 3 ( EC50 > 10 but ≤ 100 mg/L). For this GHS 208 classification, algae, crustacea and fish, are considered surrogate species. However, data on other organisms 209 may also be considered and the classification can be applied too. The ecotoxicity results obtained int this 210 work indicates that for all studied mixtures there is no need to classify for short-term (acute) hazard. It is 211 worth mentioning the case of A. fischeri where some mixtures exhibited values of EC50 higher than 100000 212 mg/L indicating very low toxicity to this biomodel (Figure 1). 213 8 214 Figure 1. Comparison of the obtained results for each biomodel. The solid line at Log EC50 = 3 215 mg/L correspond to the general toxicity limit 1000 mg/L. 216 217 The lipophilia is one of the key properties traditionally used to explain the toxic behavior: the 218 higher lipophilic character, the higher ability of the molecules to cross through the biological membranes 219 and produce a whatever effect on the activity. In this case, focusing of the sugars forming part of the DES, 220 glucose and sorbitol, our toxicity results are in close agreement with lipophilic trend described by their 221 corresponding octanol-water partition coefficients, log Kow = -3,17 and -4.67 respectively (Chemspider, 222 accessed at April 13rd 2022). 223 In Table 3, values of kinematic viscosity are also gathered. For these mixtures, the viscosity has 224 been higher than the viscosity of some typical molecular liquids such as water or some other low molecular 225 weight organic solvents. In the case of glucose mixtures, the higher viscosity has been obtained for Glu5 226 followed by Glu7 and Glu10. In the case of DES containing sorbitol, the trend is the same as for glucose 227 mixtures, being the most viscous Sor5 followed by Sor7.5 and Sor10. This property is clearly affected by 228 the nature of its components, temperature, or molar ratio. The content of water is important too, not only in 229 physicochemical properties but also in the integrity of DES changing their supramolecular interactions (El 230 Achkar et al. 2021). As mentioned before, viscosity and content of water may explain the obtained results; 231 it has been observed that high viscosity are associated with increased lethality(Hayyan et al. 2016). 232 233 Table 3. Values of EC50 (mg/L) for A. fischeri, D. magna and R. subcapitata and values of kinematic 234 viscosity at 25ºC. 235 DES A. fischeri D. magna R. subcapitata Kinematic Viscosities 30 min 24 h 72 h ν, (mm/s) Glu5 34196 ± 3965 2433 ± 31 1021 ± 122 787.69 Glu7.5 39955 ± 2455 2527 ± 22 1305 ±114 375.22 Glu10 69591 ± 3073 3048 ± 24 7544 ± 475 185.02 Sor5 149748 ± 1279 5153 ± 85 12496 ± 663 3333.9 Sor7.5 161857 ± 812 5942 ± 283 17545 ± 295 613.01 Sor10 171209 ± 789 6557 ± 242 31325 ± 131 503.51 236 Glu5 Glu7.5 Glu10 Sor5 Sor7.5 Sor10 0 1 2 3 4 5 6 Log EC50 (mg/L) A. fischeri D. magna R. subcapitata 9 A. fischeri bacteria is Gram-negative, flagellated, non-pathogenic and rod-shaped bacterium, 237 which is distributed in marine environments(Abbas et al. 2018). The bioluminescence mechanism is based 238 in two substrates: luciferin and a long chain fatty aldehyde. Exogenous agents (reduced) produce the 239 reduction of FMN (flavin mononucleotide oxidated) to FMNH2 (flavin mononucleotide reduced) through 240 luciferase (flavin mono-oxygenase oxidoreductase) action which reacts with oxygen forming an 241 intermediate chemical called 4a-peroxy-flavin. This compound oxidizes the fatty aldehyde forming its 242 corresponding acid and a luciferase–hydroxyflavin complex. This intermediate is decomposed slowly and 243 emitting a blue-green light with its highest intensity at 490 nm(Meighen 1991). This process is completely 244 linked to respiration, through the electron transport chain, and gives an idea about the metabolic status as a 245 chemical toxicity. The presence of toxic substances diminishes the resultant luminescence. The inhibition 246 of bacterial metabolism is manifested by attenuation of light emittance which corresponds to the toxicity 247 level of the tested substance (Abbas et al. 2018, Bulich 1982). 248 For the studied mixtures, the curve dose-response for A. fischeri at 30 min are shown in Figure 2. 249 For this biomodel, the values of EC50 are very high varying between 30000-172000 mg/L, indicating a low 250 toxicity of the studied mixtures towards this biomodel; however, DES containing glucose are more toxic 251 than DES containing sorbitol, concretely between 4.1 and 2.5 times more toxic, depending on the DES. In 252 all cases, toxicity decreases with water content. In the case of DES with sorbitol, this decrease of toxicity 253 is almost linear while for glucose DES, the the decrease is more pronounced if the water content increases. 254 255 256 Figure 2. Curve dose-response in A. fischeri for Glu and Sor at 30 min. Content of: 5 of water ( ), 7.5 of 257 water ( ) and 10 of water ( ). 258 259 D. magna is one of the oldest organisms used in biological research (Tkaczyk et al. 2021). They 260 can filter bacteria, algae, protozoans or other small aqueous particles. Daphnia are quite important for the 261 freshwater aquatic food chain, as a primary consumer for predatory invertebrates and fish. Additionally, 262 daphnids consume algae and thus, improve water quality (Dietrich et al. 2010, Tkaczyk et al. 2021). The 263 Daphnia motility is easy to observe and therefore, this feature is commonly used in immobilization, 264 lethality and reproduction tests(Bownik 2020). In addition, swimming behavior parameters (swimming 265 activity, swimming time, swimming speed, behavioral strength and hopping frequency) can be used as 266 sensitive endpoints (Bownik 2017). However, it is important to control the assay conditions because 267 2 3 4 5 6 7 0 25 50 75 100 Log C (mg/L) % Inhibition of luminescence Glucose:choline chloride:water 4.0 4.5 5.0 5.5 6.0 0 25 50 75 100 Log C (mg/L) % Inhibition of luminescence Sorbitol:choline chloride: water