Humic acids aggregates as microheterogeneus reaction media: Alkaline hydrolysis reactions

The influence of humic aggregates in water solution upon the chemical stability under basic conditions of different substrates has been reviewed. The kinetic behavior of each substrate has been modelized in terms of micellar pseudophase model.


Introduction
It is well known that colloidal aggregates, in their different forms, constitute true chemical nano-reactors. These reaction media, by concentrating the reactants in restricted areas or separating them between the different domains of the system, can cause both important catalytic phenomena and dramatic inhibitions of chemical processes [1][2][3] .
One such reaction medium is micellar aggregates. In fact, it has long been known that aqueous micelles significantly influence both the equilibrium and the rate of chemical reactions, as evidenced by the extensive literature on this topic [4][5][6] . Micellar catalysis on organic and inorganic reactions has been extensively studied and it is well known that it is related to the ability of micelles to absorb all types of molecules [7][8][9] . The incorporation of the reagents into the micelle is driven by both hydrophilic / hydrophobic interactions and electrostatic interactions between the micellar surface and the reagents. In this way, the interaction between the reagents and the micellar aggregate can be modeled by applying the pseudophase model 10 . In this model, bulk water can be considered as one phase and micellar aggregate as other. In this case, the reagents would be distributed between both phases or they would be left behind from one of them. In the same way, the chemical reaction could also take place in both phases or in one of them.
We have introduced the kinetic behavior in the presence of micelles because humic acids have a behavior in aqueous solution like micellar aggregates 11,12 .
Humic substances (HSs) represent a significant part of the organic matter that we can find in soils and in natural environments. These have a high specific surface area 13,14 , with a negative surface charge due to the presence of numerous deprotonable carboxylic and phenolic groups. Furthermore, due to the presence of amino groups that can be protonated, it enables the presence of a positive surface charge. That is why the surface charge has a large dependence on pH. Full ionized charge capacity (CEC) is 0.3-0.6 mol(+)kg -1 .

Scheme 1:
Model of Humic Acid chemical structure 15 In aqueous solution [16][17][18][19] , in which HS acts as micellar-like aggregates, they can adsorb different substrates 20-26 that can act as environmental pollutants increasing or reducing their bioavailability. This is the case of different xenobiotics 27,28 that can be persistent in the aquatic environment or in soils 29,30 . The role of humates and humic acids in different organic substrates mobility was conveniently illustrated by Ramus et al. 31,32 Thus, it was shown that when the content of humic substances in solution increases, significant decreases are observed in the mass transfer values at the gas-liquid interface. Furthermore, they can modify pollutants destination in the environment and, also, act as catalysts in the chemical breakdown of some pollutants 27,33,34 . Previous studies show the effect of HS in solution on hydrolysis reactions (the main transformation pathway for many xenobiotics in the environment 34,35 . These substrates are normally composed of a marked hydrophobic character that show a high affinity for HS aggregates 33-36 . Humic substances are also able to "kidnap" heavy metals [37][38][39][40][41][42][43][44] and interact with minerals [45][46][47] , modifying the absorption and toxicity of these compounds. There are few studies in the literature in which the catalytic activity of HS has been compared 23,[33][34][35][36]48,49 . These studies can be very important, considering their possible in the biogeochemical cycle of elements and the high concentrations of HS in some natural environments.
In this paper we will review some of the contributions of our research group 50-54 that tried to shed light on the role of humic acids and humates in aqueous solution, as colloidal aggregates, in the catalysis of different basic hydrolysis processes.

Hydrolysis of carbocations 51
The first group of reactions that we decided to analyze in the presence of humic substances was the alkaline fading of stable triaryl methyl carbocations (Scheme 2). This is because it is a reaction that has alredy been studied and that these carbocations entangled with different nucleophiles was used for the construction of the nucleophilia scale of the Ritchie N + family 55,56 which it was considered as a challenge to reactivity-selectivity principle 57,58 . These reasons made it an instrument for studying reactivity in other microorganized environments. In fact, one of the first published studies on honeydew catalysis dealt precisely with the basic hydrolysis of crystal violet 59,60 . Subsequently, they were widely used in studies on the catalytic or inhibitory effects of normal micelles 61 , reverse micelles or microemulsions [62][63][64] and other microhetoerogeneus systems 65 . In this way we understood that it could be an excellent chemical probe to obtain valuable information on the role of the different factors that affect the general reactivity in HS micelles, such as the compartmentalization of the reagents and the characteristics of the HS aggregates as reaction medium.

Scheme 2: Crystal Violet (CV) and Malachite Green (MG)
In this way, the basic hydrolysis of CV and MG in the presence of different amounts of HSs has been studied under conditions in which all of the humic acid is in the form of humate. The presence of HSs has been observed to inhibit the hydrolysis reaction as shown in table 1. An inhibition of 4.5 times was observed for the hydrolysis of CV and 24 times for the case of MG.
This behavior is justified based on the distribution of the reagents and the different reaction loci in the microeterogeneous system. Thus, the reaction can take place inside the aggregate, on the surface of the aggregate (in the Stern layer) or in the bulk solvent. It is evident that the interior of the aggregate will have a hydrophobic nature, however, the CV or MG that are housed there could not be exposed to HO-and therefore would not participate in the reaction. The same would happen on the surface of the aggregate, since the HO-would also be excluded due to electrostatic repulsions between the HO-and the negatively charged groups located on the surface of the aggregate 66,67 . In this way, the only take place in the bulk water, where the HO-and a part of the carbocations non-associated with the aggregate will be found. Scheme 3 shows the mechanism that takes place in this microheterogeneous system.

Scheme 3.-Pseudophase model for basic hydrolysis of CV/MG in presence of HSs
The observed results 51 would be equivalent to those observed in anionic micelles 68 . The observed kinetic behavior complies with the kinetic equation obtained from Scheme 3 (eq 1), were the subscripts s and w denote the pseudophase micellar and the bulk water respectively and Ks (eq 2.) is the carbocation association constant to the micellar aggregate. [3] From this equation, we were able to obtain values of the substrate association constants to HSS aggregate. The vales obtained were Ks = (0.13001) mg -1 L and Ks = (0.650.05) mg -1 L respectively for the CV and the MG. This higher value of Ks observed for the VM versus the CV is due to the lower polarity of the first versus the second, with which it would be expected that it would penetrate more deeply into the HSs 51 . Figure 1 shows the fit of experimental results to linearized eq 1 (eq 3). Table 1 shows the kinetic parameter for these reactions.   54 .

Hydrolysis of N-methyl-N-nitroso-p-toluene sulfonamide
Another molecule of interest whose study was approached in our laboratory was Nmethyl-N-nitroso-p-toluenesulfonamide (MNTS), which has biological interest because it is well known that nitrosulfonamides are very effective nitrosating agents 69 . Other advantage of this molecule the detailed research of our team of the mechanisms of its hydrolysis reactions (both acidic and basic) together with the mechanisms of the transnitrosation processes that involve it, both in homogeneous and microheterogeneous media 1-3,5,70-73 . That is why we consider it as a suitable chemical probe for its study in aggregated HSs since it could be a valuable tool for deepening the knowledge of the chemical reactivity in the presence of HS micelles, complementing the previous observations acquired from the study of the processes of hydrolysis of CV and MG (vide supra).  In the previous results, it is shown that the comparability presented by the HSs aggregates have a kinetic behavior similar to the conventional micellar aggregates. With the study of the basic hydrolysis of both ionic substances (MG and CV) 51 and with non-ionic substances (MNTS 54 ) the validity of the micellar pseudophase model was demonstrated beyond any doubt. For this reason, the stability of different xenobiotics in basic media and in the presence of HSs in aqueous solution was discussed below. In this way, the basic hydrolysis of carbofuran (CF) and two derivatives of carbofuran -3-hydroxy-carbofuran (HCF) and 3-keto-carbofuran (KCF) -, as well as iprodione (IP) and vinclozolin (VI) in the presence of HSs, was analyzed and their behavior was compared with similar results in ionic and non-ionic micelles. Scheme 5 shows the xenobiotics under study.

Scheme 5: Carbofuran and carbofuran derivatives, iprodione and vinclozolin
In the case of carbamates, a curious behavior was observed, since we found an inhibition on the basic hydrolysis of HCF 53 and KCF 53 , justifiable, as in the previous cases, based on the association of the substrates to the aggregate and the exclusion of OH-from their vicinity based on electrostatic considerations (vide supra). When the experimental data is fitted to a mechanism similar to that used for the case of MNTS and ionic compounds, (Scheme 3) (eq. 1 and eq. 2) we obtain values of the constant in water of kw = (1.860.06)x10 2 M -1 min -1 and kw = (11.40.6)x10 3 M -1 min -1 and some values of association constants of Ks = (1.00.1)x10 -2 mg -1 L and Ks = (51)x10 -3 mg-1L respectively for HCF and KCF. However, no effect is found on the reaction rate for CF 53 hydrolysis, so we must assume that in this case CF absorption does not occur inside the HSs aggregate and all the CF remains in the bulk water. Table 2 shows the kinetic results obtained and Figure 3, as example, shows the pseudophase model fit of experimental results for basic hydrolysis of carbofuran and carbofuran-derivatives.  When the basic hydrolysis of iprodione (IP) 52 was analyzed, an inhibition was also observed. This behavior indicates that the mechanistic model applied in the case of carbofuran and carbofuran-derivatives is still valid. In this case a slightly higher inhibition was observed than of the previous cases. In the case of carbofuran-derivatives, an inhibition of -1.7 and 1.5 times-fold respectively had been found for the case of HCF and KCF (we must remember that we had not found an effect of the presence of HSs aggregates on the basic hydrolysis of CF) while in the case of the IP, the decrease in speed was 2 times-fold.
As in the previous cases, from Eq 1 we have obtained the corresponding kinetic parameters. A rate constant for the hydrolysis process in bulk water of kw = (1.878 0.006)x10 3 M-1min-1 and an association constant to the addition of Ks = (1.40 0.1)10 -2 mg -1 L (table 2). The higher value of the observed Ks would justify the greater inhibition observed on the hydrolysis of the PI compared to the carbofuran-derivatives.
Regarding the results obtained for the basic hydrolysis of vinclozolin (VI) 50 , the inhibition observed due to the presence of HSs aggregates was much more dramatic, obtaining a decrease in the reaction rate of 9 times-fold. When the basic hydrolysis of iprodione (IP) was analyzed, an inhibition was also observed. By applying the pseudophase model and adjusting the experimental data to Eq 1, we obtain an association constant to the aggregate significantly higher than in the previous cases, Ks = (9.70.1)x10 -2 mg -1 L (table 2). The greater affinity of the VI towards the HSs aggregates would be the cause of this greater inhibition observed on the basic hydrolysis in this compound.

Binding constants and hydrophobicity of HSs core
Because of the units of Ks, it is not possible to apply a direct comparison of this value with the corresponding ones for normal micelles in order to evaluate the hydrophobicity of the HS core.
However, with the data obtained for the different hydrolysis reactions discussed in the previous sections, we can compare the ratios between the association constants obtained for both aggregated HSs and normal micelles.
In the literature there are abundant studies that obtain the association constants of some of these compounds (MNTS 72,74-76 and CV 65,77 ) to a large number of micelle aggregates, finding a linear correlation between Ks ( MNTS) / Ks (CV) and the length of the hydrocarbon chain of traditional surfactants. Thus, the Ks (MNTS) / Ks (CV) ratio in the presence of HSs aggregates is 192. For the OTACl we find that Ks (MNTS) / Ks (CV) has a value of 10, that is, 19 times less than that found for HSs. In the case of SDS, the value of Ks (MNTS) / Ks (CV) would have a value of 4.3, in this case 45 times less than the corresponding one for HSs. Figure 4 shows the ratio value between MNTS and CV binding constants to different chain length surfactants. It is therefore evident that the core of aggregate HSs is drastically more hydrophobic than that of traditional micelles, both anionic and cationic. Furthermore, it is evident that the hydrophilic / hydrophobic interactions are the main driving force behind the association of the different substrates to the aggregate, as evidenced by the evident correlation between the logP values of the substrates and the association constants found from the adjustment of the experimental data to the micellar pseudophase model (see figure 5).

Conclusions.
In summary, the effect of the presence of micelles formed by HSs on the basic hydrolysis reactions results in an inhibition due to the exclusion of HOfrom the Stern layer of the aggregate due to the electrostatic interactions between the negative surface charge of the HSs and the HOion. In this way, the inhibitions would be due to the inclusion of the different substrates inside micelles protecting them from nucleophilic attack by HO -.
The fact that the hydrophilic / hydrophobic interactions are the main driving force behind the association of the different substrates to the aggregate has been demonstrate due to the evident correlation between the logP values of the different substrates and their binding constants.
Therefore, when comparing the constants obtained in the presence of HSs with the constants of association to other micellar aggregates, it can be concluded that the core of the aggregates of HSs has a hydrophobic character significantly greater than that of "usual" micelles of sodium alkylsulfate, alkyl trimethyl ammonium chloride. or alkyl pryridium chloride, even for hydrocarbon chains of more than 18 carbon atoms.

Conflicts of Interest:
The authors declare no conflict of interest and the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.