The toxic effect of glycerol-Cu(II) counterions on the free-living stages of Aedes aegypti

Abstract

New insecticidal products are needed to control mosquito populations reducing vector-borne diseases (VBD). Ovicidal control is essential to disease control because the egg bank replaces the population at each reproductive cycle in breeding grounds. The use of carriers and bioactive metals has already been proposed. In the present work, toxic glycerol mixtures with Cu(II) counterions (acetate, chloride, nitrate, and sulfate) were tested in different combinations, showing toxicity against eggs and larvae of <italic>A. aegypti</italic>. Glycerol is an efficient Cu(II) transporter to the larval gut’s intracellular environment. When using the glycerol-Cu(II) acetate, chloride, and nitrate samples, the larval incubation rate was greater than 95%, and the larval mortality rate ranged from 17% to 65%. The glycerol-Cu(II) sulfate was more toxic to eggs, and the sample glycerol-Cu(II) acetate was more toxic to the larvae among the tested counterions. The toxic action of Cu(II) would occur through the rupture and disintegration of the peritrophic matrix, permeation, formation of complexes, and the induction of oxidative stress in the region of the midgut (mesentery) of larvae and eggs of <italic>A. aegypti</italic> in alkaline pH. Using counterions may prevent the outbreak of mosquito populations in endemic areas, positively impacting regions that suffer from VBD.

Key words
Aedes aegypti; copper; glycerol; industrial waste; metal-insecticide

INTRODUCTION

Vector-borne diseases (VBD) such as dengue, yellow fever, chikungunya, and zika pose significant social, economic, and environmental threats (Kumar et al. 2023). Aedes aegypti, the primary vector, remains uncontrolled, with increasing populations and expanding areas. This has greatly impacted the welfare of affected communities (Maciel-de-Freitas et al. 2012). Traditional mosquito control methods using synthetic, bacterial, and hormonal insecticides face challenges due to efficacy, genetic resistance, and high development costs. Thus, research into simple, low-cost, and environmentally friendly control strategies is crucial to improving health, socioeconomic, and environmental conditions in affected areas (Zara et al. 2016).

Biofuels, derived from animal fats and vegetable oils, produce biodiesel through transesterification, resulting in about 10% glycerol as a byproduct (Delmondes et al. 2013). Whether pure or impure, glycerol can serve as a chemical intermediate in various industrial processes. Its renewable nature makes it beneficial, and it has shown potential as an ingredient in insecticide formulations to control vector borne diseases (VBD) (Delmondes et al. 2013, Gaban et al. 2015).

Transition metals like Cu(II) are essential micronutrients but are toxic in narrow concentrations (Gaban et al. 2015). They inhibit metabolic activities, block cell division, alter potassium ion concentrations, and disrupt insect functions. Cu(II) ions can cause cellular damage through oxidative DNA processes (Fenton or Haber-Weiss reactions) (Gaban et al. 2015), leading to lipid peroxidation, free radical production, and membrane alteration, ultimately limiting nutrient transport (Delmondes et al. 2013, Gaban et al. 2015, Reddy & Bhagyalakshmi 1994, Schock et al. 1995).

Glycerol can bind water, act as a carrier, and coordinate with Cu(II) ions in alkaline conditions (Corena et al. 2005, Norkus et al. 1995). Cu(II) complexes and copper particles form over a wide pH range (Cuppett et al. 2006). Glycerol-Cu(II) blends have several advantages over conventional insecticides: they are low-cost, renewable, biodegradable (Zhou et al. 2008), and have low relative toxicity (Ortolani 2011). Cu(II) is essential but toxic to mosquito eggs and larvae (da Silva et al. 2011, Arruda et al. 2010, Nardeli et al. 2014, Lima et al. 2015). Studies show Cu(II) solutions are lethal to Aedes aegypti larvae in concentration-dependent effects, with 100% mortality observed at a specific concentration (Miranda et al. 2022). This indicates the potential for developing metallo-insecticides using Cu(II) ions (Li 2020, Li & Li 2020, Beaty et al. 2002, Boudko et al. 2001, Miranda et al. 2022).

Copper sulfate (CuSO₄), widely used in agriculture, can also control insects, cancer diseases, parasites and others (Vivekanandhan et al. 2021, Rahman et al. 2022, Li 2020, Li & Li 2020). A study on Aedes aegypti larvae exposed to CuSO₄ showed decreased survival rates at concentrations of 1.5 and 15 mg L-1. Treated larvae developed into adults with reduced life expectancy and impaired development. Females from treated larvae laid significantly fewer eggs, and egg-hatching rates were affected. The midgut of treated larvae and pupae exhibited epithelial disorganization and increased cleaved caspase-3 cells. These findings indicate that CuSO₄ has insecticidal activity against A. aegypti, affecting midgut metamorphosis, stem cell proliferation, fertility, and fecundity.

The number of cleaved caspase-3 cells increased in the midgut of exposed pupae compared to the control (Beaty et al. 2002). The results showed reduced proliferating cells in the treated larvae and pupae. These studies concluded that the exposure to CuSO₄ presented insecticidal activity against A. aegypti, and the damage related to the impairment of midgut metamorphosis and reduction of stem cell proliferation compromised the insect’s fertility and fecundity (Boudko et al. 2001).

The present work intends to investigate the toxicity of mixtures of glycerol-Cu(II) counterions to eggs and larvae of A. aegypti.

MATERIALS AND METHODS

Preparation of mixtures based on glycerol with Cu(II) counterions

Mixtures of glycerol-Cu(II) salt as acetate (GCA), chloride (GCC), nitrate (GCN), or sulfate (GCS) were prepared using 0.2 M glycerol and 0.2 M of the metal salt (1:1 ratio) and water (final concentration 0.1 M).

Assessment of samples of glycerol-Cu(II) counterions against eggs and larvae of A. aegypti

Eggs were placed by female A. aegypti mosquitos that were kept on boxes and fed with mouse blood, and then, after 5 days, they were obtained on filter paper substrates. Papers with eggs were stored for one week to embryo the larvae. Four concentrations (from 8.53 to 136.5 mg L-1) of each mixture were prepared fresh before testing. Approximately 100 eggs were exposed to each glycerol-Cu(II) mixture concentration in triplicate.

After applying the sample as a spray solution (0.32 mL), the group of eggs was kept at room temperature (RT) for 15 min for drying. After that, the eggs on filter paper were placed in a glass container with 200 mL of distilled water for hatching and larval development. After 24 h, each container received crushed fish feed daily and ad libitum to feed the larvae. As a control, the same number of eggs were treated only with glycerol 0.2 M, equally to the other samples, applying the same experimental design described above. The hatched larvae were monitored until development at the pupal stage or until they were all dead.

Statistical analysis

The larval hatching block rate, the rate of larvae that hatched, and the larval mortality rate were analyzed for the calculation of the 50% effective concentrations (EC₅₀) and the lethal concentrations of 50% (LC₅₀) using the Probit analysis at a 95% confidence interval. Probit analysis is a specialized form of regression analysis that is applied to binomial response variables, i.e., variables with only one of two possible outcomes (positive/negative). The procedure transforms a concentration-response curve into a straight line that can then be analyzed by least squares or maximum likelihood regression. The maximum likelihood method is a general method for estimating parameters, especially in the case of normal distributions. (Alotaibi et al. 2024, Salje et al. 2021). The larval incubation block and mortality rates were compared using Student’s t-test (α = 0.05).

The differences between LD₅₀ and ED₅₀ can be described as follows. The LD₅₀, or lethal dose 50, is the dose that is lethal to 50% of the individuals tested and is an essential measure of the toxicity of the product. ED₅₀ stands for Effective Dose 50%, which indicates the median effective dose of a substance or the dose required to achieve 50% of the desired response in 50% of the population (Batool et al. 2024, Robert et al. 2022, Sittichok et al. 2024).

RESULTS

During the bioassay, the temperature varied from 23 to 30 °C. More than 90% of hatching and larval development occurred in the controls. For the GCA, GCC, and GCN samples, the 1st instar larvae (L1) were observed after the 7th day post-treatment (p.t.). The development of the larvae that survived to the 4th instar (L4) occurred on the 7th day when all the larvae changed to the pupa stage, starting the adult phase in 2 to 3 days. The effect of GCS on the larvae was different since the L1 occurred only after 15 days p.t. Larval development occurred until L4 and lasted 30 days. There was no development to pupa for the GCS sample.

Table I shows the larval hatching block rate, larval hatching rate, and larval mortality rate for all tested samples. Analyzing the results from the ovicidal effect, GCN was shown to be more effective, having the lowest hatching and larvicidal effect. GCA caused an ovicidal effect like GCN but with a slightly higher larvicidal effect. The ovicidal effect of GCC was similar to GCA and GCN, but the larvicidal activity was higher than 50%. Analyzing GCS, the ovicidal effect was statistically lower when compared to the other samples. However, it is noteworthy that among the larvae that hatched, GCS was 100% effective in killing them after 30 days of treatment. Statistical analysis showed that, for the ovicidal effect, there was a statistical difference (p < 0.05) between all samples except GCS and GCC. About the larvicidal effect, there was a statistical difference (p < 0.05) between all samples, except between GCS and GCN and between GCA and GCS.

Table I
Mean percentage and standard deviation of larval hatching block rate (% ovicide), larval hatching rate (% hatching), and larval mortality (% larvicidal) effect of the glycerol-Cu(II) counterions samples (GCA, GCC, GCN, and GCS) applied to Aedes aegypti eggs.

	Ovicide (%)	Hatching (%)	Larvicidal (%)
Acetate (GCA)	97.2 ± 2.1a,ac,ad	2.8 ± 1.6	17.1 ± 0.4a,ab,ad
Chloride (GCC)	96.6 ± 3.6b,bc,bd	3.5 ± 3.9	65.9 ± 3.3b,bc
Nitrate (GCN)	98.7 ± 2.8c,cd	1.3 ± 0.8	8.8 ± 0.9c,cd
Sulphate (GCS)	84.1 ± 6.2d	15.9 ± 5.8	100d

• Values followed by letters in the same column represent a significant difference ( p <0.05).

The larvae that hatched were affected by all the samples. Notably, the papers impregnated in concentrations of 8.53, 17.06, 34.12, and 136.5 mg L-1 for all samples were first inserted into 200 mL of water, thus constituting a new concentration. The theoretical concentrations were calculated considering 10 times dilution (0.0853, 0.170, 0.340, and 1.365 mg L-1).

The GCS sample showed a lower effective concentration for the ovicidal effect to prevent 50% of the eggs from becoming unviable. Analyzing the confidence intervals of the samples it is considered that there were no statistically significant differences between GCA and GCS, which did not happen for GCC. This means that GCA and GCS could cause 50% of unviable eggs with similar EC₅₀. The GCA sample showed a lower effective concentration for the larvicidal effect to kill 50% of the larvae. However, the confidence intervals were broader, demonstrating the variability in the response over the larval population. Only the GCA confidence interval did not overlap with the other intervals, meaning that this mixture’s toxic action seemed more efficient than the others. Calculating the LC₅₀ for the GCN was impossible because of its low larval mortality (Table II).

Table II
The effective concentration of 50% (EC₅₀) and lethal concentration of 50% (LC₅₀) (mg L-1) after 30 days of application of the samples of glycerol-Cu(II) counterions (GCA, GCC, GCN, and GCS) tested in eggs and larvae of Aedes aegypti.

Eggs	EC50 (CI0.05)	b ± SE	X2	DF
Acetate (GCA)	2.933 (1.770–4.899)	0.702 ± 0.837	18.193	13
Chloride (GCC)	5.744 (4.546–6.763)	0.055 ± 0.260	18.208	13
Nitrate (GCN)	15.559 (3.626–50.390)	26.878 ± 5.184	54.254	13
Sulphate (GCS)	1.864 (1.550–2.460)	0.390 ± 0.624	47.153	13
Larvae	LC50 (CI0.05)	b ± SE	X 2	DF
Acetate (GCA)	2.472 (0.658–11.386)	0.207 ± 0.455	7.180	13
Chloride (GCC)	7.834 (6.253–27.787)	0.124 ± 0.353	16.063	11
Nitrate (GCN)	n.t.	n.t.	n.t.	v
Sulphate (GCS)	11.876 (7.551 – 59.680)	0.384 ± 0.619	187.158	13

• b = slope; CI = confidence limit; DF = degrees of freedom; n.t. – not tested; SE = standard error.

The glycerol concentration for the complex was very low and stated in previous tests that it did not cause mortality in larvae or non-viable eggs, and therefore, it was not considered as a control in toxicity experiments. Toxicity and safety can be reported for glycerol, which presents very low toxicity when ingested; the oral LD₅₀ dose for rats is 12,600 mg kg-1 and 8,700 mg kg-1 for mice; thus, it does not appear to cause toxicity when inhaled, although changes in cell maturity have occurred in small sections of the lung in animals under the highest dose measured (Konstantinos 2021, Coulson et al. 2022).

DISCUSSION

Glycerol can be an excellent substrate for colonizing microorganisms (bacteria, fungi), and Cu(II) is a terrific bactericide and fungicide. Glycerol imparts properties to metal salts (counterions) mixtures, such as reduced volatility, surface adhesion, intermolecular interaction, electronic interaction, coordination, and easy dispersion on natural surfaces (Gaban et al. 2015). Its continuous contact can induce water stress in mosquito eggs due to unavailability and the blockage of water entry by the glycerol film (Gaban et al. 2015, Norkus et al. 1995).

Relative low humidity affects the survival of mosquito eggs and adults. The newly laid eggs are desiccated, and the adults undergo moisture-related reductions throughout their lifetimes. Glycerol is chemically similar to sugars and can be absorbed by the cell as a carrier for Cu(II) ions (Norkus et al. 1995).

In our systems, the presence of Cu(II) ions and counterions of copper salts can alter all molecular dynamics between water-glycerol molecules through competition. Cu(II) ions and glycerol molecules have different affinities when compared to the interactions that occur between water-glycerol. In a complementary way, the counterions can stabilize the new products of interactions between the species, resulting in clusters of these interactions between ions and molecules with different biological response and/or biological activities in the insecticide system (Nakagawa & Oyama 2019).

The temperature range during the experiments must be set to ideal conditions for the normal development of the insects. After the embryonic development of the larva within the egg, larval hatching occurs 24 h after their contact with water. The life cycle of A. aegypti lasts 7 to 10 days (Boudko et al. 2001). In the present experiment, larval hatching was postponed since L1 was between the 7th and the 15th days p.t. The results suggest that glycerol adhered to the surface of the eggs and prevented the crossing of the microbial cell wall, causing difficulty or even preventing larval hatching. The samples were applied to the eggs by spray-drying, which could have provided an uneven coverage of the solution over the eggs. A portion of the eggs was blocked, and another portion was partially blocked, allowing larval hatching for a percentage of the larvae (Table I) (Lima et al. 2015).

After application on the filter papers, the toxic effect of glycerol samples with solubilized Cu(II) counterions showed that the larvae came in contact with the mixtures when they hatched, probably when they left the micropyle. This initial contact caused mortality and/or retardation in larval development. The toxicity of CuSO₄ might injure its fitness and ability as a vector, affecting the development, lipid reserves, and peritrophic matrix formation in the midgut (Miranda et al. 2022).

Larvae that have developed into pupae also made into adults. The explanatory hypothesis could be: i) the samples of glycerol with Cu(II) counterions did not have a cumulative effect on the larvae over the pupae; probably the larvae that developed until L4 were able to expel the mixtures, allowing their development into adults; or ii) the mixtures were accumulated in the pupae and also in the adults. Further studies are required to determine if there was any alteration in the physiological development of adults, such as alteration of a lifetime, and oviposition rate, among others. The results suggest that there was damage to the larvae’s digestive system, interfering with the microbiota, feeding, and absorption of nutrients essential for the continuity of larval development.

The toxic effect of the Cu(II) ions occurs along the insect’s digestive system, mainly in the mesenteron (midgut), by cellular oxidative stress. CuSO₄ affects larval gut microbiota, thus causing gut dysfunction and influencing nutrient absorption, reducing the energy stored for molt, metamorphosis, and adult development (Beaty et al. 2002, Boudko et al. 2001). This action was slower and complementary to an insecticide with neurological effects as it depends on the digestive process, permeation, and transposition by the plasma membrane to cause extra and intracellular toxicity (Rayms-Keller et al. 1998, Miranda et al. 2022, Beaty et al. 2002, Boudko et al. 2001, Cabrini 2013). Histopathological studies have shown that the metal complex [Cu(EDTA)]2- caused cell damage to the midgut (alkaline medium) of A. aegypti with the occurrence of cellular residues in the lumen of the digestive system with destructive vacuolization of columnar and regenerative intestinal cells. In the apical surfaces of the columnar cells, there are projections oriented into the lumen, suggesting that these cells are involved in the process of apoptosis and/or necrosis dependent on Cu(II) (Gaban et al. 2015, Miranda et al. 2022, Beaty et al. 2002, Cabrini 2013).

The pH in mosquitoes’ larval intestines varies from 5-6 (buccal parts) to 10-11 (excretory system) (Gaban et al. 2015, Boudko et al. 2001). At pH 10-11, the Cu(II) mixture can be absorbed, adsorbed, permeated, and transformed into glycerol-Cu(II) complex throughout the digestive system, causing toxicity primarily to the insect midgut by carrying molecules with hydroxyl groups (Norkus et al. 1995). Sulfate ions (SO₄-2) can interfere, along with metals, in the metabolism of the insect, mainly in the balance of calcium (Ca(II)). As pH increases in the insect’s digestive system, the metal concentration will also increase due to SO₄-2 ions. The counterions (acetate, chloride, and nitrate) are quite soluble and have little effect on the availability of calcium. However, chloride ions may alter the permeability of the cells, increasing the absorption of intracellular Cu(II) (Reza & Ilmiawati 2020, Arduino & de Ávila 2015, Godoy 2015).

The pH of a glycerol solution can vary depending on the type of glycerol and its concentration in the solution with metal salts. Glycerol may have traces of acid, which causes a slight pH reduction in unbuffered solutions. In this case, glycerol has been mixed with different copper salts (acetate, chloride, nitrate, and sulfate). The glycerol solution (1% solution in distilled water) in water has a pH of 4.26–6.48.

Due to the influence of deprotonation of functional groups that participate in the process of making electron pairs available in the coordination of metal ions, i.e., Cu(II), the pH is important to be considered. The analysis at 20°C of 1% (m/v) Cu(II) salt solutions was performed with acetate at pH 6.48, chloride at pH 4.26, nitrate at pH 5.04, and sulfate at pH 4.87. The measurements with 1% glycerol for the previous solutions were: acetate/glycerol pH, chloride/glycerol pH, nitrate/glycerol pH, and sulfate/glycerol pH. The dilutions were performed for the Cu(II)/glycerol salt solutions at 10x, 100x, and 1000x. From the dilutions onwards, the concentration of Cu(II) ions and glycerol decreases proportionally, and the pH tends towards neutrality and proximity to pH 7.

The pH for the glycerol-Cu(II) mixture may allow coordination of the Cu(II) ion at pH > 7, especially at strongly alkaline pH. However, at pHs < 7, there is possibly an equilibrium mixture between glycerol, Cu(II) ions, and glycerol-Cu(II) complexes. Arruda et al. (2024) analyzed glycerol-Cu(II) interactions and/or coordination by EPR spectra in different aqueous solutions at 1:2 and 1:10 at pH 5 and 11. The EPR spectra of aqueous solutions of CuSO₄.5H₂O and the mixture of glycerol with CuSO₄.5H₂O in the ratio of 1:2 (molar) were obtained at pH 5, and their comparisons showed that at pH 5, adding glycerol in the ratio of 1:2 did not significantly alter the EPR spectral profile. However, there is an indication of additional interaction that can be perceived in the lower field region. The computational simulation of the spectra allowed the identification of 2 species in the CuSO₄.5H₂O and glycerol solution (1:2), characterized by the spin-Hamiltonian parameters. The presence of a species with different spin-Hamiltonian parameter values after adding glycerol at pH 5 suggests a strong interaction of the ligand with the Cu(II) center. However, at strongly alkaline pH (pH 11), the situation was identical, in which the spectra showed that the addition of glycerol promoted the formation of a different species in the solution. The spin-Hamiltonian parameter values calculated by computer simulation suggest that glycerol interacted with the Cu(II) center at pH 11. The influence of excess glycerol on the formation of Cu(II):glycerol complexes can also be evaluated by titration with a CuSO₄.5H₂O solution at pH 5 and glycerol in increasing amounts from 1:0.5 to 1:10. The spectra obtained by molar ratios of 1:2 and 1:10 were performed, and comparisons of the two spectra indicated that no significant change occurred after this procedure. The spin-Hamiltonian parameter values of all species were characteristic of oxygen coordination spheres; the values obtained by simulation are characteristic of the coordination of Cu(II) by 2 oxygen ligands, possibly oxygens 1 and 2 of glycerol (Rangel et al. 2014).

Bacillus thuringiensis var. israelensis (BTI) produces protein crystal toxins that are solubilized at alkaline intestinal pH (around 10), releasing pro-toxins into the lumen of the digestive system (Othman et al. 2016, Baumann et al. 1991). Glycerol-Cu(II) complexes exhibit toxic effects at an alkaline pH range of 10-11, following their surface deposition and cell membrane permeabilization. This mechanism is comparable to that of BTI and certain plant extracts. A new study evaluated the effect of Cu(II) (32, 3.2, and 0.32 mg L-1) in sets of 30 to 50 eggs of A. aegypti placed in plastic containers with tap water (Rayms-Keller 1998). The eggs remained exposed for 16 days and were then placed in containers with water free of metal to follow the larvae hatching. At the lower concentrations (3.2 and 0.32 mg L-1), there was a larval hatching blockage rate between 80% and 23%, respectively. In the present work, to obtain a larval blockage rate above 95%, it was necessary that eggs received the application of the sprayed samples and remained dry for only 15 min. The reduction in exposure time is due to the adhesion of the mixtures to the eggs. In addition, considering the theoretical concentrations (Table II), it was observed that the samples of glycerol-Cu(II) counterions were more toxic once it was necessary to apply concentrations of up to 1.365 mg L-1 to obtain a higher ovicidal effect (Rayms-Keller 1998). This is because the capacity of intracellular permeation has been facilitated by glycerol molecules.

One factor that suggests the potential for developing a substrate with slow-release characteristics is the solubilization from a substrate, such as a paper filter. This could be achieved using a cellulosic or polymeric material. Impregnating metal complexes/mixtures in polymers is a promising strategy for the long-term control of A. aegypti in breeding sites.

In the oviposition process for the continuity of the reproductive cycle, females of A. aegypti deposit their eggs on the walls of the breeding sites, above the aquatic surface, mainly in places where food is available for the survival of the larvae (Scolari et al. 2019). Food is the main motivation for oviposition, even if competitors or predators are in the media. Host-associated microbes (i.e., microbiota) recently emerged as a promising field for exploring novel environment-friendly vector control strategies. In particular, the gut microbiota has revealed its impact on multiple aspects of the biology of Aedes spp., including vector competence, thus being a promising target for manipulation (Scolari et al. 2019). Reducing bacteria, fungi, protozoa, and other microorganisms in the local microbiota can prevent or decrease the attractiveness of the females to the breeding sites. This is due to reduced microbiota emission of semiochemicals or volatile substances that appeal to the breeding sites. Consequently, a decline in food availability and attractiveness to the breeding sites suggest that population control may be achieved from the food chain and/or microenvironment of breeding sites (Beltrão & Silva-Filha 2007). Ponnusamy et al. (2015) and Gnanasoundari et al. (2017) indicated the importance of the local microbiota and sulfate content in Aedes species larvae breeding habitats as higher than other species. This points to the preference of the A. aegypti females for oviposition in these habitats, which favors using the GCS sample as an attraction for females and consequent toxic action on the larvae.

The literature describes the ability of polyols to dissolve copper (II) hydroxide in alkaline media; however, little is known about the composition and stability of copper (II) complexes with glycerol. Norkus et al. (1995) show the formation of copper (II) complexes with sucrose; in addition, the literature proposes that the geometry of copper (II) complexes is tetrahedral, formed only by ionized and non-ionized hydroxyl groups of polyols.

Additional discussions can be made and described that show the affinity between glycerol and copper ions (Cu(II) or metallic copper surfaces (Cu0) and that involve interaction with similar molecules, e.g., interactions with water molecules (Chi-Ucán et al. 2014).

Nakagawa & Oyama (2019) showed that intermolecular interactions between water and glycerol molecules affect their shape, geometry, and availability. The physical properties of water in glycerol-water mixtures at different concentrations were analyzed to explain behaviors of water-glycerol interactions in terms of water activity (Aw) or availability of water molecules that can have different repercussions and/or applications. Water activity (Aw) values can be correlated with the thermodynamics, interactive behavior, and molecular dynamics of water in solutions. Water agglomeration, network formation (percolation of hydrogen bonds in water molecules), as well as the interaction of water with glycerol, determine its availability (Aw). Glycerol-water mixtures can thus control equilibrium relative humidity (RH). The Aw of the solutions herein ranged from 0.0 to 1.0, and depending on the glycerol fraction and the hydrogen bond interactions between water and glycerol molecules, the behaviors can be explained. The Aw values of glycerol-water mixtures can be explained from the hydrogen bonding network and molecular dynamics of water molecules in the solution at the molecular scale.

The molecular scale interpretation of Aw in glycerol-water mixtures considers that in water-glycerol mixtures at high glycerol concentrations, water is hydrated in glycerol, and water molecules are isolated or present as small water clusters in these solutions, and thus the hydrogen bonds in water with glycerol are intense and strong; the diffusive movement of water is slowed down by the interaction with glycerol; the strong interaction of water with glycerol leads to lower availability expressed in Aw and less freedom of water molecules. It can be shown that at high Aw values (> 0.8), water molecules have their own dynamics and are faster than free water.

The changes in the dynamics of water molecules are probably coupled to water domains in the form of aggregates. In the case of dilute glycerol-water solutions, the weak interactions of water molecules with glycerol allow greater freedom for the water molecule, leading to lower dynamic water restrictions at the molecular scale and, consequently, greater availability (Aw).

Acknowledgements

The authors thank the support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Programa de Cooperação Acadêmica - Segurança Pública e Democracia (CAPES\PROCAD); National Postdoctoral Program (PNPD) (process number: 313367/2019-1); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); UNICAMP/FEQCAPES\FCT; FEUP\i3S-INEB.

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Publication Dates

• Publication in this collection
  18 Aug 2025
• Date of issue
  2025

History

• Received
  28 Aug 2024
• Accepted
  8 Apr 2025