Surface soil was collected in the upwind of a mercury-related enterprises located in the southwest China (Xiushan County, Chongqing) by random sampling method. The fresh soil sample was collected into sterilized plastic bags and immediately stored in a freezer at 4°C. After on-site sampling, soil samples were sent to the laboratory immediately to conduct the following analysis. Mercury content was analyzed by CV-AAS (Shimadzu AA-6300C, Japan) and methylmercury was determined by GC (Shimadzu GC-2010PLUS, with an alkyl mercury special chromatography column from Sino Chemical Co., Ltd., Japan) with a solvent extraction technique. The water, soil and containers used in the experiment were all sterilized at 121°C. The basic physical and chemical properties of soil sample were shown in Table 1:

Abiotic methylation is ubiquitous in nature, and most chemical reactions are solution reactions, so the research on the factors to abiotic methylation was applied in liquid phase. Oscillation balance method was applied to simulate the process of abiotic methylation. Acetic acid, methanol, humic acid, and methylcobalamin (MeB12) were selected as methyl donors and added to the mercury solution (CHg = 500mg/L), and the concentration of methylation donors was 200mg/L. The effects of solution pH on the abiotic methylation process were conducted under the condition of the pH at 3–10, 500 mg/L Hg²⁺ and 200 mg/L methyl donors. Besides, the effects of reaction temperatures on mercury abiotic methylation were performed at 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C, respectively.

Initial mercury concentrations (100, 300 and 500 mg/L), pH (3, 5 and 7) and reaction temperatures (25, 35 and 45℃) were selected as the three main factors of influencing methylation to perform orthogonal experiments.

Methylcobalamin was added to 500 mg/L HgCl₂ solution with a concentration of 200 mg/L. The mixed solution was magnetically stirred at 25°C, and 10 ml of which were taken at 1, 5, 10, 20, 30, 45, 60, 90, 120, 180 and 240 minutes to measure the methylmercury content, and the abiotic methylation reaction rate were calculated by kinetic equation fitting.

Five grams of the sterilized soil were placed in a centrifuge tube, and 5ml of methylcobalamin in HgCl₂ solution was added to make the mercury concentration in the soil at 500mg/kg and methylcobalamin concentration at 200mg/kg. The prepared soils were placed in a 25°C constant temperature incubator for light and dark cultivation respectively in order to simulate the effect of surface soil and deep soil on mercury abiotic methylation. Samples were taken at 1, 2, 4, 6, 8, and 10 days to measure the methylmercury content in the soil after freeze-drying.

Orthogonal experiments and range analysis were applied to determine the degree of influence of different factors on abiotic methylation reaction. The kinetic reaction equation fitting and cartography were applied by software version Origin 2018.

The yields of methylmercury produced under different methyl donor conditions are shown in Table 2.

Acetic acid, methanol, humic acid, and methylcobalamin (MeB12) were selected as methyl donors for abiotic methylation of inorganic mercury. The methylation ability of methylcobalamin was obviously higher than the other methyl donors due to having the most yield of methylmercury which reached 447.62µg. By contrast, the humic acid had the lowest methylation ability, and the content of methylmercury production was only 0.22µg. Among the four ethyl donors, the order of their methylation ability was: methylcobalamin > methanol > acetic acid > humic acid.

Methylcobalamin is a corrin-like coenzyme and ubiquitous in soil. It could be assimilated by methanogens in methane synthesis (Qin et al. 2018), which plays a vital role in soil ecosystems. In the molecular structure of methylcobalamin, cobalt is in the active center of methylcobalamin, the fifth ligand is DMBI (dimethylbenzimidazole) nucleotides, and the sixth ligand is -CH₃, thereby forming a Co-C coordination bond (Kumar and Kozlowski 2017).In the corrin ring structure of methylcobalamin, all atoms are not in the same plane, so its rigidity is weak and easy to change the conformation. Thus, the Co-C coordination bond is unstable, and its methyl groups easily split and separate. The methyl group derived from methylcobalamin combined with Hg²⁺ to form methylmercury. The C-C bonds of humic acid, acetic acid, and methanol belong to covalent bonds compared with methylcobalamin, thus the bond energy exceeded the Co-C bond with low capacity of -CH₃ separation so that the methylation efficiency was much less than methylcobalamin. In addition to the methyl group, humic acid also contains a large number of carboxyl, hydroxyl, and methoxy groups. The ability to bind mercury was stronger than methanol and acetic acid. In addition, the C-C bond energy was slightly higher than the C-O bond, resulting in the methylation capacity of methanol was slightly higher than acetic acid.

pH not only affected the solubility of metal ions in solution, but also the ion activities. The effect of pH on the abiotic methylation of mercury is shown in Figture 1:

Figture 1 Effect to abiotic methylation by pH

The yields of methylmercury were kept constant at pH ≤ 4, and the methylation efficiency increased at pH 4 ~ 5. At pH = 5, the methylmercury generation was the most, and the methylation production reached 47.4 mg/L. When pH > 6, the concentration of methylmercury decreased sharply, and the methylation rate decreased to 0.16% at pH = 10. Under acidic conditions, the mercury ion is highly active so that it easily reacted with the methyl group dissociated from methylcobalamin to form methylmercury. However, under strong acid conditions, methylcobalamin lied in the Base-off state, the Co-N bond made the Co-C bond more stable, which made no obvious effect on the methylation when the pH ≤ 4. Whereas, methylcobalamin is most stable in a weak acid environment (pH = 4.5 ~ 5.0). If the pH is too high or too low, it can decompose and reduce the methylation efficiency. Therefore, at pH = 5, methylmercury in solution was the highest concentration. It was worth noting that red methylcobalamin crystals precipitated in the solution under basic conditions, and as the pH increased, the precipitated crystals increased, thus the methyl donors effectively participating in the methylation reaction in the solution decreased. Therefore, the concentration of methylmercury decreased sharply under alkaline conditions. Meanwhile, the amount of OH- in the solution increased, which caused Hg(OH)₂ precipitation and reduced the activity of Hg²⁺ in the solution. This is also the main reason for the decrease of methylcobalamin methyl capacity under alkaline conditions.

The methylmercury yields in the solution under different temperature conditions are shown in Figture 2:

Figture 2 Effect to methylation by tempreture

As can be seen from Figture 2, the yields of methylmercury increased before and then decreased with reaction temperatures. The methylmercury generation gradually increased from 25 to 35°C. As mentioned earlier, the efficiency of methylation in the soil in summer was higher than that in winter, and the temperature had a significant effect on the methylation process. However, when the temperature was above 35°C, the changing trend of methylmercury generation was opposite, which may be due to the evaporation of mercury in the solution at high temperature causing the decrease of methylation efficiency.

Orthogonal experiments were applied to select the optimal parameters for abiotic methylation, and find the most unfavorable conditions for yielding methylmercury. The orthogonal data obtained are shown in Table 3 and Figture 3:

Figture 3 Trend chart of concentration of methylmercury in different condition in orthogonal

Range analysis shows that R values of the three factors were above the control group (3.03), indicating that the initial mercury concentration, pH, and temperature affected the abiotic methylation process. Among these, the R value of pH was the largest (8.31), revealing that compared with the initial mercury concentration and temperature, the effect pH on the abiotic methylation process was the most remarkable. According to the k and R values of these three factors, the optimal parameters for methylmercury yield were the initial mercury concentration of 500mg / L, pH = 5 and temperature of 35°C, respectively. Thus, the most unfavorable condition to the ecological environment should avoid the above reaction parameters during daily production and life in order to reduce the abiotic methylation reaction in the environment. The screening of the three influencing conditions is consistent with the results obtained by single-factor experiments.

The kinetic of the abiotic methylation reaction of mercury with methylcobalamin in solution over time is shown in Figture 4:

Figture 4 Effect to methylation process by time

The methylmercury generation rate increased sharply before 15 min, and the reaction rate gradually decreased at 15–20 minutes, then the methylation reached dynamic equilibrium. The methylation rate was 3.09% at 1 minute, and the methylation rate increased rapidly to 4.37% at 15 minutes. The kinetic experiment measured the equilibrium concentration of methylmercury in the abiotic methylation reaction at 44.05mg/L.

In previous literatures, the methylation of mercury belong to pseudo first-order kinetic reaction (Hu et al. 2013; Imla Syafiqah and Yussof 2018), and the reaction formula is as follows:

Integrating formula (1), the linear expression is:

Where CMeB12 is the initial methylcobalamin concentration (mg/L); CMeHg is the methylmercury concentration (mg/L); k and a are the reaction constant. Since CMeHg increases with time, (CMeB12-CMeHg)/CMeB12 is 0–1 and gradually decreases with time, the slope of the linear fitting curve should be negative. The linear expression obtained by linear fitting of the data is , and the correlation coefficient is 0.8130.

The second-order reaction kinetic equation was used to further simulate the experimental data. The second-order reaction kinetic equation is as follows:

Where k is the reaction rate constant in mg/(L*min); Ce is the maximum concentration of methylmercury generated by abiotic methylation reaction (mg/L). Integrating formula (3), the linear expression is:

From formula (4), the second-order reaction kinetic model can be transformed into a linear equation of t/C against time. The second-order reaction kinetic equation of mercury methylation is t/C = 0.022t⁺0.005, and the correlation coefficient is as high as 0.9999. The maximum amount of generated methylmercury Ce in the reaction was determined to be 45.45 mg/L, and the reaction rate constant k was at 0.097 mg/(L*min). Therefore, the second-order reaction kinetic model rather than the pseudo-first-order reaction kinetic model can better fit the abiotic methylation process of mercury in solution, and the correlation coefficient was 0.9999.

Figture 5 shows the amount of soil methylmercury generation from simulated topsoil and deep soil under constant light and dark conditions.

Figture 5 Production of methylmercury in light and darkness in soil

Experts thought that the methylation reactions mostly occur in anaerobic environment (Kaschak et al. 2014), but in this work, simulate experiments proved that abiotic methylation in soil can be under aerobic conditions. Under the light and dark conditions, the yields of methylmercury in the soil gradually increased with time. The amount of methylmercury in the soil added with methylcobalamin was significantly higher than that in the soil without methylcobalamin. The yield of methylmercury was 8.56 times that of the soil without methylcobalamin under light condition, indicating that methylcobalamin was an efficient abiotic methylation donor. Regardless that, the presence of organic matter such as humus in the soil combined with Hg²⁺ to form methylmercury, but the highest methylation rate was only 0.082%, which was far lower than the biological methylation efficiency. By contrast, the methylmercury yield was 5.47 times that of the soil without methylcobalamin under dark condition, which was below the yield of light condition, revealing that light can promote abiotic methylation to produce methylmercury.

The amount of methylmercury produced in the soil was significantly higher than that in the dark culture and its yields in the light was 1.61 times that in the dark at 10 days. Under the condition of ultraviolet irradiation, small molecules aldehydes, ketones and acids can complete abiotic methylation reaction with Hg²⁺ through intra-molecular alkyl transfer (Diao et al. 2018). Research found that the amount of methylmercury generated in aqueous solutions under sunlight was higher than in non-irradiated conditions, and the conclusions of this study are similar to the above results (Pacyna 2020).

The rate of change of methylmercury under light and dark conditions gradually decreased with time. According to the two-point model, when a high-concentration mercury solution enters the soil environment, Hg²⁺ was firstly adsorbed by the soil colloid with a high binding site. Point-adsorbed mercury ions were difficult to desorb. However, mercury ions bound to low binding sites and free mercury ions can undergo abiotic methylation reactions with methylcobalamin. The highest abiotic methylation rate in the soil was only 0.70%, which is far lower than in aqueous solution. Furthermore, the abiotic methylation time in the soil was longer than that of the solution reaction, indicating that the soil environment had a lagging effect on the abiotic methylation reaction of mercury. Therefore, the adsorption of mercury on soil significantly affects the abiotic methylation reaction of mercury in the soil, which will reduce the abiotic methylation rate in the soil and prolong the reaction process.

Inorganic mercury generated methylmercury through abiotic methylation reaction with methyl donors (eg. methylcobalamin). The molecular structure of methyl donor methylcobalamin is shown in Figture 6.

Figture 6 Illustrative structural diagrams of methylcobalamin

Methylcobalamin is an octahedral structure with a cobalt (III) center, containing a corrin ring, a fifth ligand DMBI (dimethylbenzimidazole) and a sixth ligand which is methyl. Therefore, methylcobalamin is a rare metal-alkyl bond-containing compound that occurs naturally. All atoms of its corrin ring are not in the same plane so that its structure is fragile and its conformation is easy to change. The stability of methylcobalamin is affected not only by the sixth ligand but also by the fifth ligand DMBI. The existence of DMBI accelerates the separation of Co-C bond. Crystallographic data shows that the interaction of DMBI and the corrin ring results in an upward distortion of the corrin ring, creating a spatially unnatural corrin ring. When another ligand presented, the opposite repulsion caused the corrin ring deformation from the unstable state to the stable state and the corrin ring generated a force that splits the Co-C bond. The structural change of the corrin ring transfer the tension to the Co-C bond and trigger the subsequent bond elongation and fracture. After the Co-C bond is broken, the relaxed state of the corrin ring disappears, and then returns to the "bent up" structure. The overall structure of the alkyl ligand is unstable because of its simple structure and no negatively charged oxygen atoms. The activation of methylcobalamin was reflected in the change of the sixth axial ligand.

The effect of the sixth ligand on the structural transformation of the fifth ligand is to reduce the spatial compression of the alpha carbon, thus the Co-N bond under the condition of strong acidification (Base-off) will make the Co-C bond more stable. In the natural environment, methylcobalamin is often on the "Base-on" state, and its structural strength is weaker than under strong acid conditions. Hence, it can explain that methylcobalamin has the highest methylation efficiency at pH = 5. Carbon-cobalt binds were in the form of coordination bonds. Hg²⁺ electrophilically attacked the carbon-cobalt bond to cause it to split and generate carbanion and hydroxycobalamin. Carbanion combined with Hg²⁺ to form CH₃Hg⁺. Based on electron distribution and hybrid theory, Co is d2sp3 hybrid, thus methylcobalamin is an outer orbital complex. The outermost electron pair of Co and the outermost electron pair of C formed a coordination bond, and Hg²⁺ was used as an electrophile. When attacking methylcobalamin, mercury ions first combined with the Co-C bond to form a π complex, and then transitions to an intermediate (σ ligand). When the electrophilic performance of the electrophile is greater than that of the corrin ring, it will compete for carbanions (CH₃⁻) to form methylmercury. In terms of electronegativity, five of the six ligands of Co in methylcobalamin were connected by Co-N bonds and one is connected by Co-C bonds. The electronegativity of N is higher than C, and Co-N bonds is higher than that of Co-C bonds, so that the binding with Co is more stable. The first thing that Hg²⁺ electro-aggressively attacks methylcobalamin is Co-C bonds.

Another possible abiotic methylation pathway is methyl radical transfer. The Co-C bond of methylcobalamin is unstable and can generate methyl radicals under conditions such as light and heat. Methyl radicals are sp2 hybrids, and it can form a near-plane configuration with an unpaired single electron which is located in the p orbit with a dissociation energy of 104 kcal/mol, that is very active and easily reacts with other substances. Hg²⁺ is the loss of two electrons in the 6s orbit of the Hg atom. It has strong electrophilic properties and can combine with methyl radicals to form methylmercury. The above experiments proved that carbanion transfer and methyl radical reaction should occur simultaneously in the abiotic methylation reaction of mercury.