Novel Solid-Phase Bioassay Kit with Immobilized Chlorella vulgaris Spheres for Assessing Heavy Metal and Cyanide Toxicity in Soil

Abstract

Heavy metal and cyanide contamination in soil presents serious environmental and ecological concerns due to their persistence, bioavailability, and toxicity to soil biota. In this study, a novel solid-phase direct contact bioassay kit was developed using immobilized Chlorella vulgaris spheres to evaluate the toxicity of soils contaminated with mercury (Hg²⁺), silver (Ag⁺), copper (Cu²⁺), and cyanide (CN⁻). The assay was designed using 25 mL glass vials in which algal spheres were directly exposed to spiked soils for 72 h without the need for pollutant extraction. Oxygen evolution in the headspace was measured as the primary endpoint, alongside optical density and chlorophyll a fluorescence (OJIP) to assess photosynthetic inhibition. The assay demonstrated high sensitivity and reproducibility, with strong correlations (R² > 0.93) between oxygen evolution and optical density. EC₅₀ values based on oxygen evolution were 4.43, 4.18, 3.10, and 61.3 mg/kg for Hg²⁺, Ag⁺, CN⁻, and Cu²⁺, respectively, and 7.8, 7.4, 2.9, and 29.7 mg/kg based on optical density. The relatively higher EC₅₀ for copper was attributed to its biological role as an essential micronutrient. OJIP transient profiles supported the observed photosynthetic inhibition, particularly under Hg²⁺, Ag⁺, and CN⁻ exposure. The present study overcomes the limitations of conventional chemical analyses by providing a rapid, low-cost, and ecologically relevant tool for direct soil toxicity assessment, with potential applications in environmental monitoring and contaminated site evaluation.

1. Introduction

The assessment of soil toxicity is essential for environmental risk evaluation, as soil is a critical component of the ecosystem and is regularly impacted by a variety of pollutants [1,2,3]. Assessments of soil toxicity predominantly rely on the evaluation of the toxicity of soil elutriates or extracts [4,5,6]. The methodology of liquid-phase bioassays is contingent upon the extraction of toxicants from solid phases utilizing water or a range of organic solvents, which is then followed by the exposure of the aqueous extracts to microorganisms for testing purposes [7]. However, these approaches do not accurately reflect the biologically available or ecologically relevant fractions of contaminants in soil, as metals may bind to organic matter or soil particles, altering their mobility and toxicity. Conventional analytical techniques such as atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma optical emission spectrometry (ICP-OES), and voltammetric methods are widely used for detecting heavy metals in soil. While these methods offer high sensitivity and precision for quantifying total metal content, they do not provide insight into the actual biological impact of metals on living organisms. In contrast, bioassays provide a functional measure of toxicity by assessing biological responses, thereby capturing the bioavailable fraction of contaminants and supporting more realistic assessments of ecological risk. The solid-phase approach is limited in its ability to demonstrate the actual toxicity of contaminants, primarily because of the indirect relationship that exists between these contaminants and the microorganisms involved [7,8,9,10,11]. Solid-phase direct contact bioassays serve as a means of assessing the toxicity linked to solid materials. This approach permits the direct engagement of microorganisms with untreated solids, thereby allowing for the evaluation of actual toxicity. The results obtained reflect the microbial reactions to bioavailable contaminants found within the soil environment. Numerous studies have described protocols utilizing a range of microorganisms, such as marine bacteria, sulfur-oxidizing bacteria, and spore-forming bacteria, for assessing soil toxicity in solid-phase conditions [12,13,14,15].

The utilization of microalgae in the evaluation of toxicity in aquatic environments is prevalent. However, their use in soil toxicity testing has been limited, as soil particles not only hinder accurate microalgae counting but also obstruct photosynthesis, making the process more challenging [16]. Some studies have explored using soil microalgae for direct contact solid-phase toxicity testing by analyzing chlorophyll content and enzymatic activities, but these methods are often time-consuming and complex. Immobilized microalgae, however, offer a promising alternative, streamlining the process and overcoming the challenges associated with free algae in soil toxicity assessments [17,18]. Microalgae such as Chlorella vulgaris are particularly well suited for bioassays due to their well-characterized physiology, high sensitivity to a wide range of pollutants, and capacity for rapid physiological response to environmental stress. Moreover, their ability to remain viable in immobilized form allows for practical application in solid-phase bioassays involving direct exposure to soil. These features make them ideal candidates for developing simple and robust toxicity screening tools for soil systems. The primary objective of this study is to develop a direct solid-phase toxicity assay using immobilized Chlorella vulgaris within algal balls, enabling efficient soil toxicity testing through direct soil contact. The goal is to create a method that is rapid, sensitive, cost-effective, and easy to use for detecting biologically relevant toxicity.

A key feature of this research is its originality, as it employs the measurement of oxygen evolution in the gaseous phase and chlorophyll fluorescence from immobilized microalgae to evaluate endpoints in a bioassay that involves direct interaction with soil. This study utilized a vial-based bioassay to directly expose immobilized microalgae to mercury (Hg²⁺), silver (Ag⁺), copper (Cu²⁺), and cyanide (CN⁻) in contaminated soils for 72 h. Oxygen evolution and chlorophyll a fluorescence were measured throughout the exposure period to assess toxicity. The research focused on assessing the inhibition rates of microalgae in response to diverse types and levels of spiked heavy metals while also determining the half-maximum effective concentration (EC₅₀) for each heavy metal concerning the microalgae. The discussion highlights the potential of immobilized microalgae as a reliable tool for conducting solid-phase bioassays and evaluating the actual toxicity induced via heavy metals and cyanide in soil environments.

2. Materials and Methods

2.1. Algae Culture

An uncontaminated axenic culture of the freshwater microalgae species Chlorella vulgaris was sourced from the National Institute of Environmental Research (NIER) located in Incheon, South Korea. A master culture photobioreactor (MCBPR) was constructed in the laboratory, employing a 2 L transparent glass container. This apparatus operated in a semi-continuous fashion, incorporating the addition of 1 L of fresh media and the removal of 1 L of culture every 10 days to sustain ideal growth parameters. A continuous supply of carbon was ensured by maintaining a gas mixture consisting of 2% CO₂ and 98% air. The culture was subjected to a regulated photoperiod comprising 16 h of illumination followed by 8 h of darkness, with LED lamps providing an irradiance level of 185 μmol m⁻² s⁻¹. To facilitate optimal growth conditions for microalgae, a modified BG11 medium was formulated and employed [19].

2.2. Algae Ball Preparation

Calcium alginate microspheres were prepared to encapsulate Chlorella vulgaris for use in a solid-phase toxicity detection kit. A 4% (w/v) sodium alginate solution was mixed with actively growing algal cultures (5–7 days old) at three concentrations: 5 × 10⁴, 1 × 10⁵, and 2 × 10⁵ cells/mL. To control and maintain the size of the microspheres, the algal–alginate mixture was extruded dropwise into a 4% (w/v) CaCl₂ solution using a 10 mL syringe held at a fixed height (~10 cm) and drop rate. This method ensured consistent bead formation, producing microspheres with an average diameter of approximately 2 mm. The formed beads were gently agitated in the CaCl₂ solution for 2 h to promote uniform crosslinking and structural hardening. After hardening, the beads were collected using a fine mesh strainer, rinsed thoroughly with distilled water, and stored for further testing.

To evaluate the physiological integrity of immobilized algae, both immobilized and free C. vulgaris cells were simultaneously assessed for growth and photosynthetic activity. Growth was monitored via optical density (OD₆₈₀), while photosynthetic performance was measured using chlorophyll fluorescence (F_V/F_m ratio), a reliable indicator of Photosystem II efficiency. The results showed no significant differences between immobilized and free cells, indicating that the immobilization process did not adversely affect algal vitality.

2.3. Experimental Setup

The schematic diagram (Figure 1) depicts a solid-phase soil toxicity test kit that utilizes algal balls. This kit was constructed with a 25 mL flat-bottom glass vial, which includes a plastic cap and a rubber stopper that is lined with Teflon (Figure 1). The present study utilized standard soil Lufa 2.2, which was amended with different concentrations of heavy metals. Individual glass vials, each containing soil (0–4 g) and 15 mL of BG 11 media, received separate groups of five algal balls. These groups were characterized by initial concentrations of 5 × 10⁴, 1 × 10⁵, and 2 × 10⁵ cells/mL. As a negative control, alginate beads lacking algae were employed. The purging of nitrogen gas into the headspace of the kit for 1–2 min successfully displaced the initial oxygen from that environment. The measurement of gaseous phase photosynthetic oxygen evolution was conducted using a needle-type oxygen sensor during a 72 h incubation of the test kit, which received uninterrupted illumination at an irradiance of 185 µmol·m⁻²·s⁻¹, with a controlled temperature of 25 ± 1 °C. All toxicity tests were performed in triplicate with negative controls (alginate balls without algae) to exclude the dissolved phase oxygen in the liquid portion. Following a 72 h incubation period, the algal balls were extracted from the test kit, rinsed with distilled water, and subsequently dissolved in 0.5 N sodium citrate to facilitate the measurement of optical density and chlorophyll content. This kit was employed in three different tests: (1) Control test to evaluate the impact of different amount of soil on oxygen evolution by microalgae; (2) optimization tests focused on discovering the most favorable conditions for the test kits regarding the photosynthetic performance of Chlorella vulgaris, particularly by manipulating initial algal cell densities and mixing speeds; and (3) toxicity tests aimed to assess the harmful effects of soils enriched with heavy metals.

2.4. Chemicals and Analysis

In the current study, algal balls were prepared and dissolved using sodium alginate, calcium chloride dihydrate, and sodium citrate. Moreover, the evaluation of toxicity for mercury, silver, cyanide, and copper was carried out using mercury chloride (HgCl₂), silver nitrate (AgNO₃), potassium cyanide (KCN), and copper sulfate (CuSO₄), respectively. The chemicals employed in this research were sourced from Sigma-Aldrich in St. Louis, MO, USA, and were of analytical grade.

The concentration of oxygen (%) in the headspace of the solid-phase algal ball kits was assessed utilizing a fixed-needle oxygen sensor (OXF500PT, Pyro-science sensor technology, Aachen, Germany). Concurrently, the pH levels in all experiments were determined with a portable pH meter (410A, Orion, Boston, MA, USA). The correlation between algal cell concentration and optical density was established through calibration using algal counting and absorbance measurements. Optical density (OD) was measured at 680 nm using an Aqua-Pen 110 PSI (Photon System Instruments, Drásov, Czech Republic), while corresponding cell densities were determined under a light microscope with a hemocytometer. In subsequent experiments, optical density was measured using an absorbance density curve. The balls containing immobilized algae were dissolved in sodium citrate to release microalgae prior to determine optical density and to allow measuring chlorophyll a fluorescence. Chlorophyll a fluorescence reflects the physiological and biochemical responses of microalgae.

Measurements of rapid fluorescence induction kinetics, specifically through the OJIP test, were conducted utilizing a dual-modulation induction fluorometer (AquaPen-C 110; Photon System Instruments, Drásov, Czech Republic). The transient kinetics of OJIP were measured over a time span of 50 µs to 2 s using a 3 mL cuvette. The rapid phase of fluorescence induction kinetics, lasting approximately 1 s, commences immediately upon the exposure of the dark-adapted microalgae culture to continuous light. The signal ascends swiftly from the origin point (O) to a peak known as maximum fluorescence (F_m), passing through two inflection points designated as J and I. The phenomenon known as the “polyphasic fluorescence rise” is frequently referred to as the OJIP test [20]. Changes in the redox state of the reaction center of Photosystem II (RCII) serve as indicators of the essential mechanisms underlying photosynthesis [21]. At the onset of the fluorescence induction curve, there exists a foundational value referred to as the minimum fluorescence yield (F₀), which is assessed within a time frame of 10 to 50 µs). The signal is generated via the excited chlorophyll a (Chl a) molecules located in the light-harvesting complex II (LHCII) before the excitons have transferred to the reaction center II (RCII), at which point the plastoquinone (PQ) acceptors are entirely oxidized. F₀ and F_m are the most commonly used empirical parameters, with their difference known as variable fluorescence (F_V). The ratio F_V/F_m is widely applied and is closely linked to the maximum quantum yield of PSII photochemistry [22].

2.5. Statistical Analysis

The inhibition rate of Chlorella vulgaris via heavy metals was analyzed using Equation (1) as follows:

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\mathrm{\%}\right)={\displaystyle \frac{\left({\mathrm{O}}_2 \mathrm{\%} \mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)-({\mathrm{O}}_2 \mathrm{\%} \mathrm{i}\mathrm{n} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e})}{{\mathrm{O}}_2 \mathrm{\%} \mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$

(1)

The analysis of statistical data and the plotting of curves were performed using Origin 10.0 and Sigma Plot 10.0. The Hillslope method was utilized to evaluate the concentration at which 50% inhibition of the microalgae population occurred. A one-way ANOVA was conducted to assess the significant differences between the heavy metal treatments and the control condition.

3. Results and Discussion

3.1. Algal Ball Optimization for Solid-Phase Toxicity Test Kit

The present study was optimized by investigating the impact of the amount of soil, mixing intensity, and initial cell concentration. Different amounts of soil (1, 2, 3, and 4 g) were used to determine the most favorable operating conditions. Increasing the amount of soil showed an inverse relation to photosynthetic oxygen evolution (Figure 2A). Up to 1–2 g soil showed no significant difference in oxygen evolution (14.47% and 14.70%), while, as the amount of soil was increased from 3 to 4 g, oxygen production significantly decreased (9.77–9.62%). In terms of mixing intensity, 15 rpm (revolutions per minute) resulted in the maximum oxygen evolution (14.96%) in the headspace of the kit over 72 h incubation period, while 0, 25, and 50 rpm showed 7.95, 14.28, and 9.74% of oxygen evolution, respectively (Figure 2B). In terms of the initial cell density, a concentration of ×10⁵ cells/mL exhibited the greatest oxygen evolution at 14.45% over a 72-h incubation period. This was in contrast to the densities of 5 × 10⁴ and 2 × 10⁵ cells/mL, which resulted in oxygen evolutions of 11.9% and 12.8%, respectively, as measured in the headspace of the bioassay kit (Figure 2C). The amount of soil and mixing intensities are significant factors impacting the activity of microalgae because high amounts of soil and high mixing intensities may increase turbidity and result in inhibiting photosynthetic activity. In the present study, 2 g of soil, a 25 rpm mixing intensity, and 1 × 10⁵ cell/mL cell densities were found to be optimal operating conditions.

3.2. Heavy Metal-Induced Soil Toxicity Using Solid-Phase Algal Ball Toxicity Test Kit

Figure 3 illustrates oxygen evolution in a microalgae bioassay kit spiked with selected heavy metals. After an incubation period of 72 h, the control treatment recorded an oxygen evolution of 14.47%. Conversely, the treatments with mercury at concentrations of 5, 10, 25, and 100 mg/kg yielded oxygen evolution percentages of 8.08%, 6.51%, 3.4%, and 3.2%, respectively, during the 72 h exposure (Figure 3A). The optical density of the microalgae bioassay kit was also measured—the same trend in inhibition of optical density was observed (Figure 4A). Optical density decreased with increasing concentrations of spiked mercury and showed a strong correlation (R² = 0.98) with oxygen evolution inhibition in the headspace of the microalgae bioassay kits (Figure 5A). The EC50 values of 4.43 mg/kg and 7.8 mg/kg of mercury were determined for oxygen evolution and optical density (Figure 6A). In addition to the oxygen evolution and optical density, OJIP transient kinetics of microalgae treated with heavy metals were recorded to evaluate photosynthetic activity inhibition and depicted the same trend in inhibition (Figure 7).

The effects of silver and cyanide in inhibiting oxygen evolution in C. vulgaris displayed similar trends, indicating that higher concentrations of heavy metals correlate with increased soil toxicity and a significant decrease in microalgae performance. The soils that were spiked with 5 mg/kg of cyanide and silver exhibited oxygen evolution rates of 4.3% and 7.51%, respectively (Figure 3B,C). The obtained EC50 values for silver and cyanide for the inhibition of oxygen evolution were 4.18 and 3.10 mg/kg, respectively (Figure 6B,C). The EC50 values for silver and cyanide for the inhibition of optical density were comparable or relatively high and estimated to be 7.4 and 2.9 mg/kg, respectively (Figure 6B,C). Both oxygen evolution and optical density depicted a strongly associated inhibition with R² values of 0.93 and 0.99 for silver and cyanide, respectively (Figure 5B,C). Significant changes in the OJIP transient curve were observed in soil treated with silver and cyanide, signifying inhibition of Photosystem II of photosynthesis (Figure 7B,C). The observed decrease in fluorescence during the J, I, and P phases may stem from the inhibition of electron transport processes in Photosystem II [23]. Among the tested heavy metals, cyanide showed the highest inhibition of oxygen evolution and optical density along with the lowest EC50 value.

Test kits that were supplemented with copper, and they exhibited a higher rate of oxygen evolution compared to those containing other heavy metals. As an illustration, the exposure of algal balls to soil enriched with 25 mg/kg of copper led to an oxygen evolution of 13.2%. Conversely, the oxygen evolution in test kits treated with mercury, silver, and cyanide was recorded at 3.94%, 5.59%, and 2.20%, respectively (Figure 3). The oxygen evolution and optical density inhibition of the algal test kit spiked with individual heavy metals showed a good correlation (Figure 5D). The EC50 based on oxygen evolution for tested heavy metals is similar or less than the EC50 values based on optical density except for copper. The EC50 values of copper were obtained as 61.3 and 29.7 mg/kg for oxygen evolution and optical density, respectively (Figure 6D). Also, the EC50 values of copper using the current bioassay kit are somewhat high but still lower than the permissible limit of copper in the soil (<100 ppm). Unlike mercury, silver, and cyanide, which are non-essential and highly toxic to microalgae even at low concentrations, copper plays a dual role in algal physiology—as both an essential micronutrient and a potential toxicant at elevated levels [24]. This biological relevance may explain the relatively higher EC₅₀ values observed for copper in this study (61.3 mg/kg for oxygen evolution and 29.7 mg/kg for optical density). At lower concentrations, copper is actively taken up and utilized in algal metabolic processes (e.g., as a cofactor for cytochrome-c-oxidase and photosynthetic electron transport proteins); however, excessive accumulation can disrupt photosynthetic electron transport and enzyme activity by binding to chloroplast membranes and generating reactive oxygen species [24,25]. The delayed toxicity response observed in this study is consistent with copper’s conditional toxicity, where sublethal concentrations initially induce antioxidant defenses before overwhelming cellular detoxification mechanisms [24]. The measured OJIP curve also supports the notion that Photosystem II is more inhibited in the test kits spiked with mercury, silver, or cyanide than via copper-spiked treatments (Figure 7).

Overall, the developed algal bioassay kit demonstrated high analytical performance across all tested heavy metals. Sensitivity was confirmed via low EC₅₀ values for mercury, silver, and cyanide, ranging from 3 to 5 mg/kg. Specificity was supported by strong correlations (R² > 0.93) between oxygen evolution and optical density for each metal, indicating the consistent detection of metal-induced photosynthetic inhibition. Reproducibility was validated through triplicate testing, with the coefficient of variation remaining below 7%, confirming the reliability and precision of the assay for solid-phase soil toxicity assessment. These analytical specifications are particularly important in the context of soil toxicity assessment, where pollutant concentrations are often low, matrix interference is high, and reproducibility is essential for risk evaluation. High sensitivity allows the detection of sublethal contamination, specificity ensures the biological relevance of the observed effects, and reproducibility supports reliability across repeated tests and varying soil types. Together, these attributes make the bioassay kit suitable for practical application in soil quality monitoring and ecological risk assessment.

3.3. Suitability of Solid-Phase Microalgae Bioassay Kits for Soil Toxicity Assessment

Microalgae have been infrequently used in solid-phase soil toxicity assessment due to the pronounced interference of soil particles with both the photosynthetic activity and the accurate cell counting of microalgae [16]. Traditionally, the evaluation of metal toxicity in soils using microalgae has relied on classical methods such as chlorophyll extraction and most-probable-number (MPN) techniques [26,27,28]. However, these established approaches present several notable drawbacks. Chlorophyll extraction often requires the use of organic solvents, which can disrupt cellular integrity and result in the degradation of pigments, thereby compromising the accuracy of toxicity measurements [29,30,31]. Furthermore, MPN assays and similar protocols are time-consuming, frequently necessitating prolonged incubation periods of up to 7–14 days, and are limited by their reliance on a narrow set of endpoints, such as cell enumeration or pigment content, which may not fully capture the spectrum of toxic effects [32,33].

Recent reviews underscore that the vast majority of microalgal bioassays—approximately 89%—are still conducted in aqueous systems, with only a small fraction (about 12%) employing direct contact with soil or sediment matrices. Even the latest standardized protocols for soil microalgae tests, such as those utilizing Micractinium inermum in artificial soils, continue to require lengthy 14-day incubations and often yield higher EC₅₀ values for heavy metals (e.g., Hg²⁺ EC₅₀ of 7.4 mg/kg) compared to the results achieved with our immobilized system [28,29]. The methodologies recommended by organizations such as ASTM, ISO, EC, and OECD predominantly focus on the enumeration of cells and measurement of parameters like optical density, dry weight, cell volume, and fluorescence [34,35,36,37]. These methods typically involve the use of soil extracts or suspensions, resulting in indirect toxicity assessment via the liquid phase. This indirect approach can lead to either underestimation or overestimation of actual soil toxicity, as it is susceptible to the incomplete dissolution of pollutants and the potential for synergistic or antagonistic effects between organic solvents and toxicants [38,39,40,41,42]. For example, non-polar contaminants may be underestimated by 30–40% in such liquid-phase assays due to poor solubility, while grinding and extraction steps can result in 25–51% loss of measurable chlorophyll.

In addition to these challenges, several widely used microbial bioassays for solid-phase direct contact toxicity assessment, such as the Toxi-chemo test and the Microtox solid-phase test kit, rely on endpoints like β-galactosidase inhibition in Escherichia coli or bioluminescence in Vibrio fischeri [13,43,44]. However, sample turbidity adversely affects the measurement of parameters because these endpoints are measured using light absorbance [14,44,45]. Immobilized microalgae have also been utilized for toxicity assessment in sediments by employing cell counting as an endpoint measurement, but cell counting is both time-consuming and labor-intensive [46]. Moreover, the dissolution of algal beads to release microalgae for measurement can result in significant cell loss (12–18%), thereby reducing sensitivity and potentially underestimating toxicity [38,47].

In response to these challenges, the present research introduces a novel bioassay kit featuring immobilized microalgae spheres for evaluating soil toxicity via solid-phase direct contact. This kit uniquely focuses on measuring photosynthetic oxygen evolution in the gaseous phase as a non-destructive and highly sensitive endpoint. The kit’s design-using a 25 mL glass vial with a Teflon-lined rubber cap and requiring only a needle-type oxygen sensor enables straightforward, rapid, and reproducible measurements. By employing alginate-immobilized microalgae, the kit allows for direct interaction between untreated soil and microalgae, thus more accurately reflecting the bioavailable fraction of soil contaminants. An essential concept in the field of toxicology is the dose-response relationship, which illustrates how the quantity of a substance that a target population is exposed to correlates with the manifestation of a biological reaction [14,48]. The present study demonstrated a robust and consistent correlation between increasing concentrations of heavy metals and the suppression of photosynthetic oxygen production in microalgae (Figure 3). This strong dose-response relationship validates the use of oxygen evolution as a reliable indicator of algal photosynthetic activity and, by extension, soil toxicity. Furthermore, the study found a significant correlation between oxygen evolution and inhibition of optical density, reinforcing the reliability of this non-destructive endpoint. Unlike optical density and chlorophyll fluorescence measurements, which require the dissolution of the algal balls and can result in cell loss, oxygen evolution is measured directly in the headspace, preserving the integrity of the immobilized microalgae and minimizing sample manipulation.

The promising performance of the developed bioassay kit suggests its suitability for a wide range of future applications in soil toxicity assessment. It can be employed for the routine monitoring of heavy metal contamination in soil surrounding mining areas, industrial discharge zones, or agricultural fields exposed to chemical inputs. Owing to its simple protocol and rapid endpoint measurement, the kit also has the potential for on-site field deployment where real-time toxicity screening is needed. In future developments, this method could be adapted to assess the toxicity of complex contaminant mixtures, aged or weathered soils, or be integrated with other biological assays to enable a more comprehensive evaluation of soil health and ecological risk. Additionally, its adaptability supports potential use in regulatory frameworks and early-warning systems for soil quality management.

4. Conclusions

The developed solid-phase soil toxicity bioassay kit using immobilized Chlorella vulgaris spheres proved to be an effective tool for evaluating the direct contact toxicity of heavy metals and cyanide in standard soil (Lufa 2.2). The primary endpoint—photosynthetic oxygen evolution—enabled the sensitive detection of pollutant-induced stress under controlled exposure conditions. Optimal assay performance was achieved using 2 g of soil, a mixing speed of 25 rpm, and a cell density of 1 × 10⁵ cells/mL. The EC₅₀ values determined for Hg²⁺, Ag⁺, CN⁻, and Cu²⁺ were 4.43, 4.18, 3.10, and 61.3 mg/kg, respectively, indicating that cyanide exerted the highest toxicity, followed by silver, mercury, and copper. The comparatively higher EC₅₀ value for copper is attributed to its biological role as an essential micronutrient. Strong correlations were observed between oxygen evolution and optical density, with R² values of 0.98, 0.93, 0.99, and 0.85 for mercury, silver, cyanide, and copper, respectively. Additionally, significant alterations in the OJIP fluorescence transients confirmed Photosystem II inhibition in microalgae exposed to toxicants. The study highlights the high sensitivity, specificity, and reproducibility of the developed kit, reinforcing its suitability for rapid and ecologically meaningful assessment of soil toxicity caused by heavy metals and cyanide. This method offers a practical alternative to conventional analytical techniques, with potential applications in environmental monitoring and contaminated site evaluation.