Development and characterization of chlorophyll-amended montmorillonite clays for the adsorption and detoxification of benzene

Abstract

After disasters, such as forest fires and oil spills, high levels of benzene (> 1 ppm) can be detected in the water, soil, and air surrounding the disaster site, which poses a significant health risk to human, animal, and plant populations in the area. While remediation methods with activated carbons have been employed, these strategies are limited in their effectiveness due to benzene's inherent stability and limited retention to most surfaces. To address this problem, calcium and sodium montmorillonite clays were amended with a mixture of chlorophyll (a) and (b); their binding profile and ability to detoxify benzene were characterized using in vitro, in silico, and well-established ecotoxicological (ecotox) bioassay methods. The results of in vitro isothermal analyses indicated that chlorophyll-amended clays showed an improved binding profile in terms of an increased binding affinity (K_f = 668 vs 67), increased binding percentage (52% vs 11%), and decreased rates of desorption (28% vs 100%), compared to the parent clay. In silico simulation studies elucidated the adsorption mechanism and validated that the addition of the chlorophyll to the clays increased the adsorption of benzene through Van der Waals forces (i.e., aromatic π-π stacking and alkyl-π interactions). The sorbents were also assessed for their safety and ability to protect sensitive ecotox organisms (Lemna minor and Caenorhabditis elegans) from the toxicity of benzene. The inclusion of chlorophyll-amended clays in the culture medium significantly reduced benzene toxicity to both organisms, protecting C. elegans by 98-100% from benzene-induced mortality and enhancing the growth rates of L. minor. Isothermal analyses, in silico modeling, and independent bioassays all validated our proof of concept that benzene can be sequestered, tightly bound, and stabilized by chlorophyll-amended montmorillonite clays. These novel sorbents can be utilized during disasters and emergencies to decrease unintentional exposures from contaminated water, soil, and air.

Keywords: Adsorption isotherm; Benzene; Chlorophyll; Molecular simulations; Montmorillonite; Remediation.

Fig. 1.

Increased hydrophobicity, measured by the absorption ratio of n-heptane/water, for CM-CH, SM-CH and collapsed derivatives compared to parent CM and SM (A). Absorbance of chlorophyll and CM-CH suspensions in pH 6.5 and 7.5 water after 96 h of light exposure (B). The absorbance reading at the starting point was adjusted to 1. The absorbance of chlorophyll decreased with time at both pH conditions and light exposures, while chlorophyll in the CM-CH clay resisted photodegradation.

Fig. 2.

Plots of adsorption isotherms of benzene with parent SM and SM-CH (A) and CM and CM-CH (B) clays at 25 °C for 2 h. Isotherm plots in the Freundlich model were depicted by an average of adsorption (mmol/kg) (solid shapes), line of best fit (solid line), and upper and lower 95% confidence bands of adsorption (dotted lines). SM-CH and CM-CH showed a short reaction time to reach adsorption equilibrium and an increased adsorption affinity (K_f = 74.75 and 665.60, respectively) compared to parent clays (K_f = 4.68 × 10⁻² and 4.78 for SM and CM, respectively).

Fig. 3.

Plots of benzene adsorption remaining on SM and SM-CH (A) and CM and CM-CH (B) clays after desorption at 24 °C for 24 h, plotted by lines of best fit (solid) and upper and lower 95% confidence intervals (dotted). SM-CH and CM-CH showed a decreased desorption percentage (25–28%) compared to unamended parent clays (100%), indicating an increased ability to retain benzene for extended time periods.

Fig. 4.

Comparison of benzene binding propensity (%) between CM and CM-CH clays (A). Red: binding to clay alone; dark blue: binding to clay through chlorophyll; light blue: binding to single or aggregated anchored chlorophyll. Corresponding examples are shown in cartoons, labeled and colored in boxes (B-E). Cartoons show examples of interactions and do not cover all possibilities of interactions within each category. Brown: clay, denoted as “CM”; black: benzene; green: chlorophyll, denoted as “CH”. Detailed descriptions of the interactions depicted in each case (B-E) is provided in the text.

Fig. 5.

Snapshots of simulations showing diverse interactions between benzene and anchored chlorophyll molecules (A). Several interactions between benzene molecules and single or aggregated chlorophyll molecules. Clay is shown in the vdW (van der Waals) representation, and chlorophyll and benzene molecules are shown in green and black, respectively. Snapshots of simulations showing a bound chlorophyll dimer interacting with three benzene molecules (B). From left to right, benzene interacts with hydrophobic tail (pink), tail and core simultanously (purple), and core (blue) of chlorophyll.

Fig. 6.

Dose-dependent toxicity of benzene exposure on L. minor surface area (A), frond number (B), chlorophyll content (C), inhibition percentage (D, bar graph), and growth rate (D, line). Data is depicted as the mean response ± standard deviation. * = p ≤ 0.05; ** = p ≤ 0.01, compared to blank medium control.

Fig. 7.

Protection of 1% or 2% CM-CH and SM-CH against benzene toxicity on plant surface area (A), frond number (B), chlorophyll content (C), inhibition percentage (D, bar graph), and growth rate (D, line) on day 7 of exposure. Data is depicted as the mean response ± standard deviation. * = p ≤ 0.05; ** = p ≤ 0.01, compared to blank medium control.

Fig. 8.

Dose- and time-dependent toxicity on C. elegans body length after 24 and 48 h of exposure (A) and survival rate after 48 h of exposure (B). Protection against benzene toxicity by SM-CH and CM-CH on body length after 24 and 48 h of exposure (C) and survival rate after 48 h of exposure (D). Data is depicted as the mean response ± standard deviation. * = p ≤ 0.05 ** = p ≤ 0.01, *** = p ≤ 0.001, compared to blank medium control.