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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article   Research Article

Determination of the toxicity of zinc sulphate hydrate solution using the Spirotox biotesting method

Elizaveta Evgenievna Kuzmenko*, Hoang Thi Ngoc Quynh, Alena Mikhailovna Koldina, Gleb Vladimirovich Petrov, Anton Vladimirovich Syroeshkin

Department of Pharmaceutical and Toxicological Chemistry, Peoples’ Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya St, Moscow, 117198, Russian Federation

 

 

INTRODUCTION

Zinc is one of the naturally occurring essential trace elements that is required by living organisms and is of biological importance for the health of the population. Zinc is essential for the normal functioning of over 300 enzymes. It plays an important role in the metabolism of monoamine oxidase, controls DNA synthesis, and is involved in normal growth, brain development, behavioural responses, bone formation, and other physiological functions ¹. Zinc has indirect antioxidant properties by stabilising cell membrane structures, being a structural component of the antioxidant Cu, Zn-containing superoxide dismutase, and maintaining metallothionein levels ^{2, 3}. Furthermore, zinc has been demonstrated to exert a direct influence on the immune system, stimulating the proliferation and activation of neutrophils and natural killer cells ^4,5.

Zinc deficiency represents the most prevalent cause of secondary immunodeficiency in humans ⁶. Zinc deficiency affects approximately two billion people globally and is a significant contributing factor to a multitude of diseases. In children, zinc deficiency results in stunted growth, increased susceptibility to infection and diarrhoea, and is responsible for approximately 800,000 child deaths globally each year. Excessive zinc intake can result in a range of adverse effects, including nausea, dizziness, headaches, abdominal discomfort, vomiting, and even coma. Additionally, it can lead to copper deficiency, altered iron metabolism, and reduced immune function ^7-9.

Zinc is less toxic than the majority of other heavy metals. The toxicity of zinc is primarily contingent upon the anion with which it is associated. The mean zinc concentration in adults is approximately 0.5 µmol/g, which equates to a total body zinc content of approximately 2 g ¹⁰. The physiological requirement for zinc in adult males is 1.4 mg/day, while that for females is 1.0 mg/day ¹¹. The maximum tolerable intake of zinc is set at 40 mg per day ¹².

Zinc sulphate (ZnSO₄) is a significant pharmaceutical substance containing zinc that is primarily absorbed by the human body in a dissolved state ¹³. Zinc sulphate forms a number of hydrates as a result of strong hydrogen bonds with water molecules. These include the heptahydrate (ZnSO₄·7H₂O), hexahydrate (ZnSO₄·6H₂O) and monohydrate (ZnSO₄·H₂O) ^{14, 15}. ZnSO₄·7H₂O is also referred to as Goslarite. It is a secondary mineral that is found in the oxidation zone and in old sphalerite mines. It is readily dehydrated and forms minerals.

The thermal dehydration process of ZnSO₄ ·7H₂O comprises several stages, which are outlined in detail in reference ¹⁶:

	39°C - 60°C

 

60°C - 100°C

ZnSO₄⋅7H₂O                             ZnSO₄⋅6H₂O + H₂O (1)

ZnSO₄⋅6H₂O                              ZnSO₄⋅H₂O + 5H₂O (2)

The above two chemical equations demonstrate that the stable hydrate in equilibrium with water within the range of room temperature to 39°C is ZnSO₄·7H₂O. ZnSO₄·6H₂O exists within the range of 39-60°C, while ZnSO₄·H₂O exists within the range of 60-100°C. The aforementioned chemical equations demonstrate that the stable hydrate in equilibrium with water within the temperature range of room temperature to 39°C is ZnSO₄·7H₂O. ZnSO₄·6H₂O is observed to exist within the temperature range of 39-60°C, while ZnSO₄·H₂O is found to exist within the temperature range of 60-100°C. Additionally, the water molecules within the hydrate are also coordinated with the zinc ions. The arrangement of water molecules around the zinc ions is a factor that affects the physical and chemical properties of the zinc ions, leading to significant differences in intermolecular interactions in different hydrates. The three hydrated forms of zinc exhibit distinct crystal structures. The crystal structures of ZnSO₄·H₂O and ZnSO₄·6H₂O are monoclinic, exhibiting distinct lattice parameters. ZnSO₄·7H₂O, on the other hand, is rhombic in its crystal structure ¹⁶. The relationship between the structure and activity of substances has also been the subject of recent research. This relationship has been demonstrated in numerous studies ¹⁷. Zinc sulphate is employed in the medical field as a food additive ¹⁸. It is employed in the treatment and prophylaxis of zinc deficiency. Despite zinc being an essential element, it is also toxic. High concentrations of free zinc ions in solution have been demonstrated to be toxic to both invertebrates and vertebrates, including fish.

Protozoa play a pivotal role in the functioning of aquatic ecosystems, facilitating the decomposition of organic matter and the conversion of energy to higher trophic levels ^{19, 20}. From the perspective of microbial ecotoxicology, infusoria lack a cell wall but possess a thin membrane that enables them to absorb chemicals, resulting in alterations to membrane structure. Consequently, infusoria may exhibit altered responses when exposed to metals ²¹. 

Spirostomum ambiguum is an active component of water and wastewater filtration systems. It is 2-3 mm in size and can be observed without the use of a microscope. S. ambiguum exhibits considerable resilience to a range of environmental factors, particularly fluctuations in pH levels and dissolved oxygen. Consequently, protozoa are frequently employed as biological indicators of chemical pollution, particularly in aquatic ecosystems ^{22, 23}. The aim of the study is to evaluate the biological activity of zinc sulphate API solutions with different degrees of wetting at the sample preparation stage using the Spirotox method, in order to see the relationship between the structure and activity of APIs.

MATERIALS AND METHODS

Substances and solutions 

In this study, we employed a range of pharmaceutical substances, including ZnSO₄·H₂O (produced by Acros Organics, Barcelona, Spain), ZnSO₄·6H₂O (produced by Fluka, Steinheim, Germany), and ZnSO₄·7H₂O (produced by Sigma-Aldrich, USA). All solutions were prepared at a concentration of 0.01M and 0.001M. The prepared solutions were filtered using a submicron filter with a pore size of 0.22 µm (Merck Millipore, USA). The pharmaceutical substance sodium chloride (NaCl, manufactured by Sigma-Aldrich, USA) was dissolved in water to a solution concentration of 0.01M.

The pH of the solution was calibrated through the utilisation of a solution of dilute sulphuric acid (H₂SO₄) and potassium hydroxide (KOH).

The deionised water was purified in a Milli-Q unit at 25 °C (Merck Millipore, USA) to a [D/H] ratio of 140 ppm.

pH measurement

In order to ascertain the pH value of the solution, a basic laboratory pH meter (PB-11, Sartorius, Germany) is utilised.

Spirotox test

A study was conducted to investigate the biological activity of zinc sulphate under different irrigation regimes. The cell biosensor Spirostomum ambigua, which is highly sensitive to heavy metals, was employed in this study. The temperature was maintained at a constant level using a 5-well thermoregulated plate, which enabled the temperature to be controlled from 22°C to 28°C in 2°C increments (Lauda Alpha 6, Germany). The behaviour of the infusoria was observed using a binocular microscope, and low-power lamps (10 W) were used. 

The introduction of a S. ambiguum infusoria into the thermostatically controlled cell of the analysed sample, followed by each of the wells, resulted in the occurrence of cell death at varying time intervals. The period of cellular viability was determined by measuring the time elapsed between the commencement of incubation and the occurrence of cell death. The determination of cell death was conducted through the disruption of cell wall integrity, which resulted in the release of cell contents, or through the immobilisation of cells that exhibited an absence of a contractile response to mechanical stimulation. This methodology is founded upon the principles of the dose-response diagram, as outlined in reference ²⁴. The activation energy is calculated using the Arrhenius thermodynamic equation ²⁵.

X-ray Fluorescence (XRF)

X-ray fluorescence spectroscopy (energy dispersive X-ray fluorescence spectrometer EDX-7000 (Shimadzu, Japan)) was employed to evaluate the concentration of impurities present in the native pharmaceutical substances under investigation. The range of elements analysed spanned from 11Na to 92U. The X-ray generator comprised a tube with a Rh-anode, with a current of 1-1000 µA. The method employed was based on the irradiation of the sample under study with X-rays, which resulted in the excitation of the atom. This caused electrons to move to a higher energy level, and upon the reverse transition, the emission of light quanta (fluorescence) was observed. The energy of the radiation emitted (in keV) is a characteristic value for each element.

Dynamic Light Scattering (DLS)

In order to ascertain the dimensions of the nanoparticles present in the aqueous medium, a series of Dynamic Light Scattering (DLS) experiments were conducted on a Zetasizer Nano ZS instrument, Malvern, UK. The instrument is capable of measuring particle sizes ranging from 0.1 to 1000 nm. Upon irradiation with a laser beam, the particles within the sample scatter the incident light. The movement of particles in a liquid is characterised by a high degree of chaotic scattering in all directions. The particles are in a state of constant motion and collision within the solution, according to the principles of Brownian motion. This results in fluctuations in the intensity of the light scattered.

Statistics

The results of the analyses are presented as mean ± standard deviation (n=5). All calculations and statistical processing were conducted using OriginPro 9.1 software (OriginLab, USA).

RESULTS

Determination of the purity and the absence of impurities

The results of the X-ray fluorescence analysis demonstrated that the APIs were chemically pure, as no other measurable impurities were present. The presence of zinc in the samples tested was demonstrated by the presence of a characteristic peak in the XRF spectra at the Kα and Kβ lines of zinc (8.63 and 9.57 keV, respectively). 

 

 

Figure 1: XRF spectrum at characteristic fluorescence energy of zinc (Kα=8.63 and Kβ=9.57 keV)

 

 

Determination of the Biological Activity of Solutions ZnSO₄·H₂O with different concentrations by Spirotox test.

The interaction of S. ambiguum with the studied solution was found to include the rapid formation of an intermediate state, which then underwent a slow transition to a stationary state. It has been demonstrated that the mortality of S. ambiguum exhibits an Arrhenius-type temperature dependence. The mortality rate of Spirostomum ambiguum exhibits variation at different zinc concentrations. At a concentration of 0.01 M zinc sulphate monohydrate solution, the mortality rate of the infusoria was higher than at a concentration of 0.001 M.

 

 

Table 1: The Biological Activity of Solutions ZnSO₄·H₂O with different concentrations by Spirotox test.

Temperature (K)	295	297	299	301
Concentration (mol/l)	Lifetime (s) of S.ambiuum (n=5, mean±SD)
0.01 М	185±3	120±8	106±7	84±4
0.001 М	246±4	185±5	147±3	107±2

 

It was observed that the lifespan of the infusoria exhibited a difference depending on the concentration of the sample under study. The exposure of infusoria to zinc ions results in their death through the transition state, in accordance with the Michaelis-Menten model of enzymatic kinetics ²⁶. At each equivalent concentration of zinc, the toxicity of zinc sulfate API solutions with varying degrees of dilution was evaluated in terms of its impact on infusoria.

Measurement of pH value of three zinc sulphate solutions of different forms of watering at different concentrations.

The pH of prepared solutions of ZnSO₄·H₂O, ZnSO₄·6H₂O and ZnSO₄·7H₂O was measured under room conditions (n=3). In accordance with the European Pharmacopoeia, the pH value of a ZnSO₄·H₂O solution is within the range of 4.0 to 5.6 (C=0.28 mol/l), while that of a ZnSO₄·6H₂O and ZnSO·7H₂O solution is within the range of 4.4 to 5.6 (C=0.18 mol/l). It can be observed that at a concentration of 0.01 M, the pH value attains its maximum value. In conjunction with Table 1, solutions at a concentration of 0.01 M were selected for further optimisation studies.

Determination of biological activity of ZnSO₄·H₂O, ZnSO₄·6H₂O and ZnSO₄·7H₂O solutions before and after filtration using the Spirotox test.

Figure 3 illustrates the activation energy value of ligand-induced death of Spirostomum ambiguum in solutions comprising ZnSO₄·H₂O, ZnSO₄·6H₂O, and ZnSO₄·7H₂O substances at a concentration of 0.001 M, both prior to and following filtration.

The data illustrated in the figure demonstrate that prior to filtration, the lowest activation energy is exhibited by the ZnSO₄·H₂O solution (68 ± 7 kJ/mol, n = 5), whereas the remaining ZnSO₄·6H₂O and ZnSO₄·7H₂O solutions display equivalent biological activity with corresponding E_a of 113 ± 5 kJ/mol (n = 5) and 119 ± 5 kJ/mol (n = 5), respectively. The results obtained permit a comparison of the biological activity and toxicity of the hydrated forms of zinc sulphate. The lower value of the activation energy and the shorter lifetime of S. ambigua (twofold) in the same temperature range indicate that the ZnSO₄·H₂O solution exhibits a higher biological activity than the other two solutions.

Figure 2: pH value of zinc sulphate solutions of different forms of watering at different concentrations

 

 

A

B

  

Figure 3: Activation energy of death of S. Ambiguum in 0.01M zinc sulphate hydrate solutions as a function of filtration. A – the solutions are prepared immediately prior to the filtration process; B – the solutions were subjected to filtration through a submicron filter.

 

 

Figure 3A illustrates the activation energy value of ligand-induced death of Spirostomum ambiguum in unfiltered solutions comprising ZnSO₄·H₂O, ZnSO₄·6H₂O, and ZnSO₄·7H₂O substances. A comparison of the activation energy of the solution prior to and following filtration is presented in Fig. 3a and 3b. It can be observed that the unfiltered monohydrate solution exhibits a distinct activation energy value when compared to the zinc sulfate hexa- and heptahydrate solutions. Following filtration (Fig. 3b), however, all solutions display an identical activation energy value. The discrepancy in the activation energy of the unfiltered solution indicates that the toxicity of the solution is influenced by the size of the clusters and their heterogeneous distribution within the solution. Submicron filtration has been demonstrated to destroy these density inhomogeneities ²⁷.

 

Determination of cluster size in the studied solutions by Dynamic Light Scattering (DLS) method.

Despite comparable concentrations, the three solutions of hydrated zinc sulfate exhibited disparate toxicity profiles. Consequently, we conducted a dynamic light scattering (DLS) analysis to ascertain the dimensions of the nanoparticles in an aqueous solution.

 

 

    

Figure 4: Results obtained by the DLS method, showing the intensity distribution of fractions of the dispersed phase. A, B, C: solutions before the filtering process; D, E, F: solutions filtered through a submicron filter.

 

Figure 4 illustrates that there is a discernible discrepancy in particle size among zinc sulphate solutions of varying forms of watering. Prior to filtration, the particle sizes of the ZnSO₄·H₂O solution were observed to be 295 nm, while the ZnSO₄·6H₂O solution exhibited a particle size of 615 nm and the ZnSO₄·7H₂O solution demonstrated a particle size of 531 nm. Following filtration, no particles were observed within the particle size measurement range of 0.1 to 1000 nm. The formation of hydrogen bonds between water molecules and ions is a consequence of the dissolution of zinc sulphate in water. From this, it can be observed that there is a difference in particle size within the solution.

 

Determination of the pH value dependence of the solution toxicity

Experiments were conducted on a solution of ZnSO₄-H₂O at a concentration of 0.01 M, at two pH levels: 5.2 and 6.5. A control solution of NaCl at the same concentration of 0.01 M was tested at pH values of 5.0, 5.8 and 6.5. As illustrated in Figure 5, an increase in pH from 5.2 to 6.5 was observed to result in a reduction in the lifespan of S. ambigua (from 118 ± 9 s to 67 ± 6 s at 297 K).

 

Figure 5: Dependence of Spirostomum ambiguum lifetime on temperature at different pH values in ZnSO4-H2O 0.01M solution (n=5)

The control solution of NaCl 0.01M was observed for 30 minutes, during which no mortality of infusoria was noted (Tab. 2). It is established that S. ambiguum is stable within a pH range of 5.0 to 6.5.

Table 2: Effect of pH value on the lifetime of S.ambigua on 0.01M NaCl background solution

NaCl 0.01M
pH	5.0	5.8	6.5
t, min	> 30 min	> 30 min	> 30 min

 

 

The investigation of the toxicity of zinc sulphate monohydrate solution at two different pH levels revealed a correlation between the pH values and the toxicity of the solution. This hypothesis has been corroborated by numerous studies ^{28, 29}. As illustrated in Figure 4, the toxicity of the zinc sulphate hydrate solution towards the S. ambiguum organism exhibited a gradual increase with rising pH levels and approaching neutrality. The influence of the initial pharmaceutical substance's watering on the preparation of solutions was demonstrated.

DISCUSSION

The investigation of the bioactivity of zinc sulphate in three different hydrate forms at a constant concentration revealed that the hexa- and heptahydrate exhibited comparable biological activity. The solution obtained from the pharmaceutical substance ZnSO4-H2O demonstrated the most pronounced biological activity. In the case of the heptahydrate, six molecules of crystallisation water combine with the zinc ion in a coordination complex. However, the remaining water molecule is weakly bonded to the sulfate ion. Consequently, the water molecule can be readily removed by drying at a constant temperature (at what specific temperature) to form hexahydrate. This may be the reason for the similar energy of the two solutions. Zinc is capable of forming complexes with small molecules, exhibiting coordination numbers 4, 5 and 6: [Zn(H₂O)₆]²⁺, [Zn(H₂O)₅]H₂O, [Zn(H₂O)₄]²⁺₂H₂O ³⁰. It is hypothesised that the number of water molecules in the first and second hydrate shells forms molecular clusters of varying sizes, a conclusion supported by the DLS results.

The results obtained lead us to the assumption that crystals of zinc sulfate hydrate of different watering forms will form different giant clusters with water molecules. The formation of giant heterophase water clusters is facilitated by the formation of aquacomplexes comprising metal cations and various anions, which act as 'centres'. The formation of hydrogen bonds between water molecules and ZnSO₄, as well as with zinc ions, results in alterations to the bonding grid space, the molecular arrangement of ZnSO₄, and the intermolecular forces. These changes give rise to notable distinctions in the intermolecular interactions observed in the different hydrates. The dimensions of molecular clusters and their distribution within a solution are factors that influence their biological activity. The particle size, as determined by the DLS method, indicates that the particles in the zinc sulphate monohydrate solution are the smallest. This enables them to be evenly distributed and to exhibit the greatest toxicity to infusoria when compared to zinc sulphate hexa- and heptahydrate solutions. The formation of clusters of water molecules, comprising a minimum of six molecules, is a consequence of hydrogen bonding. A substantial body of literature exists on the formation of water clusters in solution ³¹. These clusters are in a constant state of creation and destruction ^{32, 33}. The size spectrum of these clusters is contingent upon not only the chemical composition but also the history of sample preparation ³⁴. Submicron filtration represents a means of obviating the necessity for the sample preparation history to be committed to memory, with the consequence that the structures will cease to exist. Following filtration, no particles in the size range of 0.1 to 1000 nm were observed in the solutions under study. It is postulated that clusters may form below 0.1 nm in size or that homogeneous giant clusters may be re-established during the infusoria experiment, resulting in comparable toxicity outcomes. 

In water clusters, the interaction between covalent and hydrogen bonds between oxygen atoms and hydrogen atoms enables the migration of protons (H+) by relay mechanism, thereby stabilising the latter. As the size of the clusters increases, their stability also increases up to a certain critical size. Additionally, giant water clusters have been observed to exhibit biological activity ³⁵.

The objective of this study was to further substantiate the correlation between pH and the toxicity of zinc solutions, with a particular focus on drug substances in general. This contributes to the expansion of the research map for infusoria, as there have been numerous studies on the relationship between metal toxicity and environmental pH for vertebrates and microalgae ^{36, 37}, but no such studies have been conducted for infusoria. As illustrated in the Pourbaix diagram of zinc ion properties in water ³⁸, the majority of free zinc ions are present at pH values below 4, with a gradual decline observed from pH 5 to 8. In slightly alkaline conditions, the appearance of Zn(OH)₂ and ZnO is observed at pH values between 8 and 10.5. At pH values exceeding 11, the formation of  ions is noted. Obviously, as the pH is gradually increased to neutral, the amount of free zinc ions gradually decreases. However, for some species of fish and infusoria, an increase in pH will lead to an increase in the toxicity of the solution ³⁹. Therefore, the toxic impact on infusoria is not attributable to a modification in the acidity index of the medium. Rather, it is a consequence of xenobiotics affecting their cell walls, a factor of considerable significance in the context of Ea and its correlation with LD50 ⁴⁰, both of which can serve as useful metrics in the initial phase of preclinical toxicity studies of inorganic pharmaceutical substances.

CONCLUSION

The method for determining the toxicity of water-soluble drug solutions on the protozoan Spirostomum ambiguum as an alternative to animal testing is an efficient method with ethical value. This method meets the requirements of pharmaceutical research and development under the 3Rs (Refine, Reduce, Replace) strategy, which is oriented towards sustainable ecosystems. The results of the study demonstrate that the zinc sulphate monohydrate solution exhibits the highest toxicity among the three hydrators with observed activity energy (68±7 kJ/mol). The present study demonstrated that the biological activity of the Spirotox test is dependent on the pH of solutions comprising different hydrated forms of zinc sulphate. The Spirotox test can be employed to monitor the toxicity of pharmaceutical substances and to prescribe the less toxic form of zinc sulphate at equivalent dosages in medical practice.

Conflicts of Interest: The authors declare that they have no conflicts of interest.

Contributors: All authors have read and approved the final manuscript.

Acknowledgment: The authors would like to express their deepest gratitude to the staff of the Department of Pharmaceutical and Toxicological Chemistry RUDN University.

Source of Support: This paper has been supported by the RUDN University Strategic Academic Leadership program.

Funding: This publication has been supported by the RUDN University Scientific Projects Grant System, project № 033323-2-000

Data Availability Statement: The data presented in this study are available on request from the corresponding author.

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