Chromium Removal by Dunaliella salina in High-Salinity Environments: An Investigation Based on Microalgal Cytotoxic Responses and Adsorption Capacity

Abstract

Chromium (Cr) is a widespread heavy metal contaminant in aquatic environments, posing serious risks to phytoplankton due to its persistence, biotoxicity, and mutagenic potential. Microalgae have emerged as promising biological agents for Cr remediation. In this study, the Cr removal potential of living Dunaliella salina (D. salina) was evaluated by examining the toxic effects and adsorption behavior of trivalent Cr(III) and hexavalent Cr(VI) through short-term exposure experiments. This study elucidated the mechanisms by which Cr disrupts key photosynthetic metabolic pathways, quantified the short-term toxicity thresholds of Cr(III) and Cr(VI) to D. salina, and characterized the saturation adsorption capacity and adsorption kinetics of Cr on algal cells. The results showed that Cr(VI) at concentrations of 5–20 mg/L inhibited the growth of D. salina in a dose-dependent manner throughout the culture period, with inhibition rates ranging from 22.8% to 70.9%. After 72 h of exposure, the maximum growth inhibition rates caused by Cr(III) and Cr(VI) reached 42.5% and 52%, respectively. Interestingly, low concentrations of Cr(VI) (0.1–1 mg/L) slightly enhanced the growth of D. salina. However, Cr(VI) exhibited stronger biotoxicity than Cr(III). Exposure to both Cr species significantly reduced the levels of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car), resulting in damage to the photosynthetic reaction centers and suppression of the photosynthetic electron transport system. The adsorption of Cr(VI) by D. salina followed a pseudo-second-order kinetic model, with a maximum adsorption capacity of 38.09 mg/g. The process was primarily governed by monolayer chemisorption. These findings elucidate the toxic mechanisms of Cr in D. salina and highlight its potential application as an effective bioremediation agent for heavy metal pollution, particularly Cr(VI), in marine environments.

1. Introduction

Currently, heavy metal pollution resulting from industrial wastewater discharge, mining activities, and landfill leachate has become widely introduced into natural water bodies, posing serious risks to human health and the aquatic ecosystem [1,2]. Among these pollutants, chromium (Cr) primarily exists in two oxidation states: trivalent Cr(III) and hexavalent Cr(VI). Cr(III) can be oxidized to Cr(VI) under certain environmental conditions, and both forms exhibit considerable toxicity to plants and animals. Excessive exposure to chromium in humans can lead to various adverse health effects, including cephalalgia, nausea, diarrhea, and emesis [3]. Chromium primarily exerts its toxic effects by disrupting essential biochemical processes, such as hydrolysis, protein denaturation, and precipitation of nucleic acids and nucleoproteins, while simultaneously interfering with enzymatic systems.

Dunaliella salina is a halotolerant, unicellular, eukaryotic green alga that thrives in high-salinity environments and interacts with heavy metal ions through distinct cellular structures and metabolic mechanisms [4]. Recent studies have demonstrated the remarkable potential of microalgae in heavy metal removal [5,6]. Compared to other biosorbent materials, including bacteria, fungi, and activated carbon, microalgae possess distinct advantages such as biodegradability, abundant availability, and environmental friendliness. The surface of microalgal cells contains various functional groups, such as carboxyl, hydroxyl, and sulfate groups, which confer strong metal-binding capabilities. Moreover, microalgae exhibit rapid growth even under low-nutrient conditions and can be directly cultivated in contaminated wastewater. Furthermore, both viable and inactivated algal biomass can be employed for metal remediation, providing greater operational flexibility. Living microalgae metabolically transform metal ions mainly through transport systems such as facilitated diffusion and active transport, leading to the internalization of heavy metals and a consequent reduction in their environmental toxicity and mobility. Non-viable algal biomass acts as a passive biosorbent, effectively removing metal ions through ion exchange, complexation, and chelation mechanisms. These biosorption processes can also occur in living cells [7,8,9]. In a comprehensive review, Khushbu S. Parmar systematically evaluated the potential of microalgae and macroalgae in biosorption and bioremediation processes. The study particularly emphasized the successful applications of algal-based systems in removing metallic pollutants such as lead, cadmium, arsenic, and chromium [10]. These distinctive characteristics position algal-based technologies as a promising approach for heavy metal remediation, allowing the use of either living algal systems or immobilized algal biomass to achieve efficient heavy metal removal.

The unique advantage of living algal cells lies in their integrated biological functions of adsorption, transformation, and regeneration. Studies by Chen [11] and Thabet [12] have shown that microalgae possess outstanding metal accumulation capabilities, rendering them both reliable biomarkers for heavy metal contamination and highly efficient candidates for metallic pollutant removal [13]. A study by Jihen Elleuch demonstrated that D. salina could survive for at least 15 days in systems containing copper or chromium, with significant biosorption efficiency. At a chromium concentration of 29.4 mg/L, the biological removal rate reached approximately 95% [14]. Similarly, in a study on the adsorption of Cr, Cu, Fe, and Ni using microalgae, Baba Imoro Musah reported a maximum removal efficiency of 99.7%, with corresponding biosorption capacities of 14.1, 15.8, 21.6, and 13.5 mg/g, respectively [15]. Elleuch explored Dunaliella species as efficient biosorbents and applied the Box–Behnken experimental design to optimize zinc removal conditions, confirming the strong potential of Dunaliella in zinc remediation [16]. Moreover, several studies have examined the metabolic responses of Dunaliella cells under heavy metal exposure, revealing intrinsic links between their adaptive metabolic mechanisms and exceptional biosorption capabilities [2,17].

Heavy metals exert pleiotropic effects on algal cells through highly complex toxicological processes [18]. Chromium in aquatic systems readily accumulates in algal biomass. Excessive intracellular accumulation of chromium leads to interactions with macromolecules such as nucleic acids and proteins, causing structural and conformational alterations that impair cellular growth and metabolic functions. Furthermore, chromium interferes with electron transport and photophosphorylation, suppresses photosynthetic enzyme activity, and ultimately inhibits both photosynthesis and respiration [19]. Chlorophyll content, a key indicator of algal physiological status, was comprehensively assessed in this study. Balaji reported that increasing chromium concentrations progressively inhibited the activity of the electron transport system, leading to the accumulation of NADH and H⁺, while simultaneously causing a gradual decline in the total protein content of Oscillatoria acuminate [20]. Previous studies have revealed that chromium exposure disrupts the normal function of photosystem II by diminishing electron transport activity and reducing the density of reaction centers, which in turn significantly compromises overall photosynthetic efficiency [21]. However, the effects of chromium on the photosynthetic electron transport chain in microalgal cells remain insufficiently elucidated. To further determine the short-term toxicity thresholds of chromium in D. salina and to specifically investigate its effects on the photosynthetic electron transport system, this study analyzes the chromium-induced damage mechanisms through a comprehensive examination of the photosynthetic reaction centers, as well as the donor and acceptor sides of photosystem II (PSII), to elucidate its disruptive effects on key metabolic pathways in algal photosynthesis.

The primary objective of this study is to elucidate the physiological and photosynthetic response mechanisms of D. salina to chromium stress and to systematically evaluate its potential for chromium bioremediation. To achieve this, the study is designed to accomplish the following specific technical tasks: (1) assessing the growth status, pigment composition, and chlorophyll fluorescence parameters of algae under varying chromium concentration stress. (2) determining the tolerance of D. salina in chromium-containing solutions. (3) quantitatively analyzing the adsorption kinetic characteristics of Cr(VI) under different exposure durations. The findings provide valuable insights into the ecotoxicity of chromium pollutants and demonstrate the promising and environmentally sustainable potential of D. salina for marine chromium remediation.

2. Materials and Methods

2.1. Cultivation and Treatment of Microalgal Materials

Dunaliella salina strain IOCAS 879ss was obtained from the Institute of Oceanology, Chinese Academy of Sciences. The alga were pre-cultured in a modified Johnson medium (The medium contains the following components per liter: KNO₃ 0.2 g, KH₂PO₄ 27 mg, NaHCO₃ 1.0 g, Na₂EDTA 1.89 mg, FeCl₃·6H₂O 2.44 mg, H₃BO₃ 0.61 mg, ZnCl₂ 414 µg, (NH₄)₆Mo₇O₂₄·4H₂O 0.38 mg, CuSO₄·5H₂O 60 µg, CoCl₂·6H₂O 51 µg, MnCl₂·4H₂O 41 µg.) Simultaneously, 30 g/L of sea salt (Qingdao Haizhiyan Aquarium Technology Co., Ltd., Qingdao, China) was added to the medium, and the pH was adjusted to 8.5. Under controlled conditions: room temperature of 20 ± 2 °C, light intensity of 100 μmol/m²/s, and a photoperiod of 14 h:10 h light-dark cycle. The cultures were pre-cultured until the exponential growth phase was reached [22], with subculturing performed every 20 days and algal growth monitored throughout the process. The other reagents in the culture medium were purchased from China National Pharmaceutical Group Co., Ltd. (Beijing, China).

2.2. Chromium Ion Contamination Exposure Experiment

An aliquot of exponentially growing algal culture was aseptically transferred into sterilized fresh medium and acclimatized under ambient temperature with natural illumination. Upon re-attainment of the logarithmic growth phase, the culture was utilized for exposure assays. Standard aqueous solutions of Cr(III) and Cr(VI) were prepared from oven-dried (105 °C, 2 h) CrCl₃·6H₂O and K₂Cr₂O₇, respectively. D. salina suspensions (150 mL; initial density: 8.6 × 10⁵ cells/mL) were established, with experimental treatments supplemented with Cr(III) (1.0, 10, 20, 50, 80, and 100 mg/L, respectively) or Cr(VI) (0.1, 0.5, 1.0, 5.0, 10, and 20 mg/L, respectively), while controls received no chromium amendment. All cultures were subjected to fluorescent illumination (100 μmol·m⁻²·s⁻¹ PAR) under a 14:10 h photoperiod with triplicate biological replicates per concentration. During incubation, flasks were manually agitated daily to maintain suspension homogeneity and systematically repositioned following agitation to mitigate positional light-distribution artifacts.

2.3. Measurement of Algal Cell Density and Quantification of Half-Maximal Effective Concentration (EC₅₀) of Toxicant

Throughout the experimental duration, 2 mL aliquots were collected daily from control and treatment groups. Absorbance measurements at 750 nm (OD₇₅₀) were obtained using UV-Vis spectrophotometry (756 S, Shanghai Lengguang Technology Co., Ltd., Shanghai, China). Algal cell density (Y) across treatment concentrations was determined via a pre-established calibration curve correlating OD₇₅₀ (X) with cell counts, facilitating Dunaliella salina growth curve construction.

In compliance with the U.S. EPA Ecological Effects Test Guidelines (OCSPP 850.4500: Algal Toxicity²³), growth curves were generated for 72 h and 96 h exposures at varying chromium concentrations. Growth inhibition rates (I%) were quantified at both intervals. Concentration-response relationships were established by plotting probit-transformed inhibition values against log₁₀-transformed concentration data. Median effective concentrations (EC₅₀) at 72 h and 96 h were derived from the resulting regression equation.

2.4. Measurement of Pigment Content

Pigment content was measured at 24 h, 48 h, 72 h, and 96 h after the initiation of Cr exposure. Chlorophyll and carotenoids were extracted using 95% ethanol. The extraction and quantification of chlorophyll a (Chl a), chlorophyll b (Chl b), and total carotenoids (Car) followed the methodology described by Ritchie [23]. This method is applicable to Synechococcus, Ostreobium, Phaeodactylum, Rhodomonas, and Acaryochloris. Briefly, 5 mL of culture medium was centrifuged at 10,000× g for 10 min. After discarding the supernatant, the cell pellet was resuspended in 5 mL of 95% ethanol and all samples were subjected to extraction at a 1:1 ratio, then subjected to dark extraction for 24 h. The extract was then centrifuged at 10,000× g and 4 °C for 10 min. The resulting supernatant was used to measure absorbance at specific wavelengths. Pigment quantification was performed as follows:

Chl a = 0.0604 × A632_nm − 4.5224 × A649_nm + 13.2969 × A665_nm − 1.7453 × A696_nm

(1)

Chl b = − 4.1982 × A632_nm + 25.7205 × A649_nm − 7.4096 × A665_nm − 2.7418 × A696_nm

(2)

Car = (1000 × A470_nm − 2.05 × Chl a)/245

(3)

where A represents the absorbance at the indicated wavelength. Pigment content is expressed in mg/L.

2.5. Measurement of Chl a Fluorescence Transient

Following the methodology established by Che et al. [24], cuvettes containing algal suspensions treated with varying concentrations of Cr(III) and Cr(VI) were initially dark-adapted for 20 min. Chlorophyll a fluorescence transients were subsequently measured using a FluorPen system (AquaPen Ap110-C, FluorPen1.1, Dràsov, Czech Republic). Saturation red light (peak 650 nm) at an intensity of 3000 μmol/m²/s was generated by a light-emitting diode (LED) array. The Chl a fluorescence transients were captured during a 2 s exposure to saturating light, and OJIP transient analysis was performed according to the protocol proposed by Strasser and Strasser [25].

Table 1. Calculation Methods and Biological Significance of Fast Chlorophyll Fluorescence Parameters.

	Calculation Formula	Biological Significance
RC/ABS	=1/[MO·(1/VJ)·(1/φPo)]	RC/ABS reflects the efficiency of the reaction center.
φPO/(1 − φPO)	=[1 − (Fo/FM)]/(Fo/FM)	It indicates the light energy absorption efficiency of the antenna system.
ψO/(1 − ψO)	=(1 − VJ)/VJ	It represents the acceptance efficiency of electron acceptors.
Pi_Abs	=(RC/ABS)·[φPo/(1 − φPo)]·[ψo/(1 − ψo)]	The performance index, based on absorbed light energy, consists of three components: (RC/ABS), [φPo/(1 − φPo)], and [ψo/(1 − ψo)].
Fv/Fm	=(Fm − Fo)/Fm	Fv/Fm represents the maximum photochemical efficiency of PSII under dark-adapted conditions.
VJ	=(FJ − Fo)/(Fm − Fo)	On the fluorescence induction curve, the variable fluorescence at the J-point reflects the extent of QA− accumulation.
Mo	=4(FK − Fo)/(FM − Fo)	It describes the maximum rate of QA reduction, specifically the rate at which QA is reduced during the O-J phase.
Wk	=(FK − Fo)/(FJ − Fo)	It indicates changes in the PSII oxygen-evolving complex (OEC).
ETo/RC	=MO·(1/VJ)	It represents the energy captured per reaction center that is utilized for electron transport.

showed that Calculation Methods and Biological Significance of Fast Chlorophyll Fluorescence Parameters.

2.6. Liquid-Solid Adsorption Equilibrium of Chromium on Algal Cell Surface

150 mL aliquot of algal suspension (biomass concentration: 0.15 g/L) was amended with potassium dichromate (K₂Cr₂O₇) stock solution to establish target Cr(VI) concentrations of 0.5, 1, 5, 10, and 20 mg/L. The experimental systems were incubated at ambient temperature (20 ± 1 °C) under continuous illumination (100 μmol photons/m²/s) throughout the 6 h adsorption phase. Post-incubation, the supernatant was harvested via centrifugation for subsequent analytical procedures.

Equilibrium Adsorption Capacity:

$$q_e={\displaystyle \frac{V(C_0-C_e)}{m}}$$

(4)

Langmuir Adsorption Isotherm:

$${\displaystyle \frac{1}{q_e}}={\displaystyle \frac{1}{q_{max}{K}_L{C}_{\mathrm{e}}}}+{\displaystyle \frac{1}{q_{max}}}$$

(5)

Freundlich Adsorption Isotherm:

$$\lg q_e={\displaystyle \frac{1}{n}}\lg c_e+\lg K_F$$

(6)

In the equation, C_e represents the Cr(VI) concentration at adsorption equilibrium (mg/L), and q_e denotes the equilibrium adsorption capacity (mg/g). q_max represents the maximum adsorption capacity (mg/g); K_L denotes the Langmuir adsorption constant (mg/L); while n (reflecting adsorption intensity) and K_F are the Freundlich constants.

2.7. Adsorption Kinetics Studies

Cr(VI) concentration was determined spectrophotometrically at 540 nm based on the formation of a violet-red complex through its reaction with diphenylcarbazide in acidic solution. For the determination of Cr(III) concentration, potassium permanganate (KMnO₄) is first used to oxidize Cr(III) to Cr(VI). The total chromium concentration is then measured by the diphenylcarbazide spectrophotometric method at 540 nm. The Cr(III) concentration is calculated as the difference between the total chromium concentration and the Cr(VI) concentration, where the latter is measured directly before the oxidation step. The chromium removal efficiency and equilibrium adsorption capacity are presented below.

Chromium Removal Efficiency:

$$\eta ={\displaystyle \frac{C_0-C_e}{C_0}}\times 100\%$$

(7)

For adsorption kinetic investigations, cultivation conditions remained identical to those specified in Section 2.6. Aliquots were collected at adsorption time intervals of 15, 60, 120, 180, 240, and 300 min. Supernatants from each sampling point underwent dilution with deionized water to achieve concentrations amenable to Cr(VI) quantification. The adsorption kinetics of Cr(VI) by Dunaliella salina were subsequently modeled using pseudo-first-order and pseudo-second-order kinetic frameworks.

Pseudo-first-order kinetic model:

$$\lg (q_e-q_t)=\lg q_e-{\displaystyle \frac{k_{1}t}{2.303}}$$

(8)

$$q_t={\displaystyle \frac{V(C_0-C_t)}{m}}$$

(9)

where q_e and C₀ maintain the same definitions as previously described; k₁ represents the pseudo-first-order adsorption rate constant (min⁻¹); t denotes adsorption time (min); q_t indicates the adsorption capacity at time t (mg/g); C_t refers to the Cr(VI) concentration at time t (mg/L); V represents the volume of the Cr(VI) containing solution (L); and m corresponds to the dry weight of algal cells in the suspension (g). If a linear relationship is obtained by plotting lg(q_e − q_t) versus t, it indicates that the adsorption mechanism conforms to the pseudo-first-order kinetic model.

Pseudo-Second-order kinetic model:

$${\displaystyle \frac{\mathrm{t}}{q_t}}={\displaystyle \frac{1}{k_2{q}_e^2}}+{\displaystyle \frac{t}{q_e}}$$

(10)

In the equation, k₂ represents the pseudo-second-order adsorption rate constant (g/(mg·min)), while the other parameters retain the same definitions as previously described. If the adsorption process follows pseudo-second-order kinetics, plotting t/q_t against t will yield a linear relationship. Compared to the pseudo-first-order model, the pseudo-second-order adsorption model provides a more comprehensive description of the overall adsorption behavior.

The suitability of the models was evaluated using the correlation coefficient (R²) of the linear regression and the chi-square test statistic (χ²).

$${\chi }^2={\displaystyle \sum \frac{{(q_1-q_0)}^2}{q_0}}$$

(11)

In the equation, q₀ represents the calculated value (mg/g) derived from the fitted slope and intercept, while q₁ denotes the experimentally determined value (mg/g). A smaller χ² value indicates a closer agreement between the experimental results and the model prediction.

2.8. Statistical Analysis

Data presented in the figure (expressed as mean ± standard error (SE) of replicates) were analyzed by one-way ANOVA using SPSS Statistics 26.0 software. All datasets underwent tests for normality and homogeneity of variance. Statistical significance was defined as p < 0.05. The figure was prepared using Origin software (Origin 2025b).

3. Results

3.1. The Growth Effects of Varying Concentrations of Cr(III) and Cr(VI) on Algal Cells

Algal cell proliferation was significantly inhibited at Cr(III) concentrations above 1 mg/L, with the inhibitory effect becoming more pronounced at higher levels. As illustrated in Figure 1, algal growth at Cr(III) levels above 1 mg/L was significantly lower than in the control group. In the 10 mg/L and 20 mg/L treatment groups, cell density reached only 5 × 10⁶ cells/mL, while the 50 mg/L treatment group exhibited almost no growth. Low concentrations of Cr(VI) (below 1 mg/L) slightly promoted algal growth, whereas higher concentrations inhibited proliferation in a clear dose-dependent manner. Exposure to both Cr(III) and Cr(VI) caused D. salina to enter the stationary phase earlier than normal. These results suggest that chromium concentrations above 1 mg/L, regardless of oxidation state, suppress algal growth, induce premature onset of the stationary phase, and may ultimately lead to cell death.

3.2. EC₅₀ of Cr on D. salina

Based on curve fitting, the 72 h-EC₅₀ and 96 h-EC₅₀ values for D. salina exposed to Cr(III) were 57.54 mg/L and 37.15 mg/L, respectively, while for Cr(VI) they were 13.8 mg/L and 22.9 mg/L, respectively (

Table 2. Probit regression of log₁₀[Cr] versus response under Cr treatment at 72 h and 96 h (y, Probit; x, log₁₀[Cr]).

	Time (h)	Standard Curve	R2	EC50 (mg/L)
Cr(III)	72	y = 0.25271x + 4.5552	0.9795	57.54
	96	y = 0.58351x + 4.08244	0.9919	37.15
Cr(VI)	72	y = 1.09266x + 3.74654	0.9386	13.8
	96	y = 1.00122x + 3.63591	0.9891	22.9

).

3.3. Effects of Chromium on Pigment Contents

As shown in Figure 2, under Cr(III) treatments ranging from 0 to 20 mg/L, the concentrations of Chl a, Chl b, and Car in the algal cultures generally increased. Pigment levels declined in cultures exposed to 50–100 mg/L Cr(III). Similarly, D. salina exposed to 0–20 mg/L Cr(VI) demonstrated an overall increase in Chl a, Chl b, and Car levels. Analysis of pigment content under varying Cr(VI) concentrations at the same exposure duration showed that Cr(VI) levels above 5 mg/L significantly reduced Chl a content, while concentrations exceeding 1 mg/L caused significant decreases in both Chl b and Car.

3.4. Effects of Chromium on Primary Photochemical Reactions in D. salina

3.4.1. Photochemical Efficiency

Figure 3 demonstrates that Fv/Fm values remained largely unchanged in experimental groups exposed to 1–20 mg/L Cr(III) over four days. However, at Cr(III) concentrations of 50–100 mg/L, Fv/Fm values declined sharply, with maximum photosynthetic efficiency dropping to 17.8%, 17.2%, and 16.5% of the control group values. The RC/ABS parameter decreased by 26.1%, 48.7%, and 25.9% at Cr(III) concentrations of 1–20 mg/L, while φP_O/(1 − φP_O) remained largely unchanged. For Cr(VI) exposure, Fv/Fm values showed no significant change in the 0–1 mg/L treatment group, whereas concentrations of 5–20 mg/L resulted in reductions to 95.1%, 95.7%, and 88% of the control values, respectively. At elevated Cr(VI) concentrations (5–20 mg/L), RC/ABS decreased by 22.1%, 7.7%, and 34.1%, respectively, while φP_O/(1 − φP_O) showed no significant change. These results indicate that high chromium levels impair the photochemical efficiency of D. salina. The Fv/Fm parameter provides clear evidence of Cr-induced PSII inhibition, and the reduced Pi_Abs under high chromium stress reflects a diminished capacity for utilizing absorbed light energy.

3.4.2. PSII Reaction Centers

As shown in Figure 4A,B, ETo/ABS values remained largely unchanged at low concentrations of Cr(III) (1–20 mg/L) and Cr(VI) (0.1–1 mg/L). However, exposure to Cr(III) at 50–100 mg/L led to substantial reductions of 66.4%, 73.8%, and 81.5% compared to the control, indicating severe disruption of the photosynthetic electron transport chain. Similarly, Cr(VI) at concentrations of 5–20 mg/L caused reductions of 8.6%, 3.1%, and 12.4% in ETo/ABS values. These results indicate that high chromium levels inhibit the functionality of PSII reaction centers, as reflected by the decreased energy per reaction center available for electron transport.

3.4.3. Donor and Acceptor Sides of PSII

The W_k parameter reflects the status of the donor side, or water-splitting system, of PSII reaction centers, providing insight into the condition of the oxygen-evolving complex (OEC). An increase in W_k values suggests damage to the donor side, whereas a decrease may suggest the activation of alternative regulatory mechanisms. Figure 4C,D illustrates the changes in W_k values under varying concentrations of Cr(III) and Cr(VI). No significant changes in W_k values were observed at Cr(III) concentrations of 0–20 mg/L. However, Cr(VI) exposure at 5–20 mg/L resulted in significant reductions of 6.1%, 10.5%, and 7.3% compared to the control group.

Figure 4 illustrates changes in V_j (Figure 4E,F) and Mo (Figure 4G,H) under varying concentrations of Cr(III) and Cr(VI), showing that both parameters increased significantly in the Cr(III)-treated groups compared to the control. At Cr(III) concentrations of 1, 10, and 20 mg/L, Mo values increased by 11.1%, 32.3%, and 15.3%, respectively, while V_j values rose by 11.6%, 30.0%, and 11.7%. These changes indicate that Cr(III) exposure disrupts electron transport on the acceptor side in D. salina, causing QA⁻ accumulation and/or reduced electron transfer rate to QB. Under Cr(VI) concentrations of 0–20 mg/L, Mo decreased by 10.7% at 10 mg/L, while V_j increased by 8.9% at 5 mg/L compared to the control.

Analysis of key chlorophyll fluorescence parameters (Fv/Fm, Pi_Abs, ETo/ABS, Wk, Mo, and Vj) indicates that chromium primarily impairs photosynthetic efficiency by targeting the acceptor side of PSII reaction centers. These findings confirm that Cr exposure disrupts electron transport on the acceptor side, effectively elucidating its toxic mechanism in D. salina.

3.5. Analysis of Adsorption Equilibrium and Kinetics

Table 3. Kinetic parameters of the Cr(VI) adsorption process.

	C0 (mg/L)	k1 (min−1)	k2 (g/(mg·min))	R2	qe (mg/g)	χ2
Pseudo-first-order reaction	20	0.0083	-	0.9505	10.25	1.05
Pseudo-second-order kinetics	0.5	-	0.0481	0.9862	2.1436	0.3701
	1	-	0.3405	0.9955	3.0512	0.0471
	5	-	0.0083	0.9949	11.9617	1.1935
	10	-	0.0073	0.9928	20.3915	1.1104
	20	-	0.0029	0.9969	37.4672	2.3515

presents the linear fitting results of the adsorption kinetic models.

Table 4. Adsorption Constants and Correlation Coefficients for Two Adsorption Isotherms of Cr(VI).

		Langmuir Isotherm			Freundlich Isotherm
Parameter	qmax (mg/g)	KL (mg/L)	R2	KF	n	R2
	38.09	0.1649	0.9958	5.7364	1.5230	0.9838

presents the linear fittings of the Langmuir and the Freundlich adsorption isotherms. Both models exhibited high correlation coefficients (R² > 0.95), with the Langmuir model (R² = 0.9958) demonstrating a better fit than the Freundlich model (R² = 0.9838). These results indicate that D. salina has strong adsorption potential for Cr(VI), especially in low-concentration contaminated environments.

At an initial Cr(IV) concentration of 20 mg/L, the adsorption process followed the pseudo-first-order kinetic model (R² = 0.95054), with a rate constant (k₁) of 0.00833 min⁻¹ and a calculated equilibrium adsorption capacity (q_e) of 10.25 mg/g. Across initial Cr(VI) concentrations of 0.5, 1, 5, 10, and 20 mg/L, the adsorption data were highly consistent with the pseudo-second-order kinetic model, with R² values ranging from 0.9862 to 0.9969.

As shown in Figure 5, at initial Cr(VI) concentrations of 0.5, 1, 5, 10, and 20 mg/L, D. salina rapidly removed 51.93%, 41.73%, 28.1%, 27.19%, and 22.15% of Cr(VI) within the first 60 min. The removal rates gradually stabilized after 180 min, reaching equilibrium values of 63.59%, 46.09%, 32.83%, 27.88%, and 26.42%, respectively.

4. Discussion

4.1. The Effects of Cr(III) and Cr(VI) on the Growth of D. salina

In this study, Cr(III) and Cr(VI) exerted different impacts on the growth of D. salina. Algal proliferation was significantly inhibited at concentrations above 1 mg/L for Cr(III) and 5 mg/L for Cr(VI), resulting in visible culture lightening and premature transition into the stationary or decline phase. Elevated chromium levels disrupted the physiological structure and metabolic functions of D. salina, hindering normal cell division and growth. Low Cr(VI) concentrations (<1 mg/L) slightly promoted algal growth, likely through hormesis-induced activation of stress adaptation mechanisms. This observation, consistent with Zhang [26], illustrates the typical low-concentration stimulation and high-concentration inhibition pattern, reflecting the complex adaptive responses of algae to environmental stress.

The 72 h-EC₅₀ values of Cr(III) and Cr(VI) for D. salina were 57.54 mg/L and 13.8 mg/L, respectively, while the 96 h-EC₅₀ values were 37.15 mg/L and 22.9 mg/L, respectively. According to the European Commission [27], pollutants are classified based on EC₅₀ values as follows: very toxic (EC₅₀ < 1 mg/L), toxic (EC₅₀ = 1–10 mg/L), and harmful to aquatic organisms (EC₅₀ < 100 mg/L). The results of this study indicate that Cr(VI) is more toxic to D. salina than Cr(III), and both should be regarded as toxic to aquatic environments.

4.2. Cr(III) and Cr(VI) Induced Damage to Photosynthesis in D. salina

The study revealed that both Cr(III) and Cr(VI) adversely affected pigment accumulation and photosynthetic electron transport in D. salina (Figure 6). Cr(III) concentrations above 1 mg/L and Cr(VI) concentrations above 5 mg/L caused significant reductions in Chl a, Chl b, and Car, with Chl a showing the greatest decline per cell, indicating that chromium preferentially damages the core pigments of photosystem II. High concentrations of Cr(III) (50–100 mg/L) and Cr(VI) (5–20 mg/L) significantly decreased Fv/Fm and Pi_Abs values, indicating PSII damage and disruption of the photosynthetic electron transport chain. Both Cr(III) and Cr(VI) affected the donor side, acceptor side, and reaction center of PSII to varying degrees. At Cr(III) concentrations of 1–20 mg/L, significant increases in Mo and Vj indicate impaired electron transfer on the acceptor side, resulting in QA⁻ accumulation or a reduced electron transfer rate to QB. High concentrations of Cr(III) (50–100 mg/L) and Cr(VI) (5–20 mg/L) caused a significant decrease in ETo/ABS, indicating structural damage to the PSII acceptor side. At Cr(III) concentrations of 1–20 mg/L and Cr(VI) concentrations of 5–20 mg/L, ETo/RC decreased, suggesting that chromium impairs the electron capture and transfer capacity of PSII reaction centers, blocking electron flow from QA to QB and enhancing energy dissipation per reaction center. The RC/ABS ratio showed the largest changes across varying Cr(III) and Cr(VI) concentrations, indicating that chromium preferentially damages PSII reaction centers. These findings demonstrate that chromium disrupts photosynthetic function in D. salina, leading to increased energy absorption by PSII, reduced electron transport capacity, decreased photosynthetic efficiency, and impaired energy supply and biomolecule synthesis in algal cells.

4.3. Analysis of Cr(VI) Adsorption Isotherms and Adsorption Kinetics by D. salina

The two most common and stable forms of chromium are trivalent Cr(III) and hexavalent Cr(VI). Cr(VI) is more toxic than Cr(III) due to its greater ability to penetrate cell membranes, disrupt membrane integrity, and pose serious health risks. In aqueous solutions, Cr(III) exists primarily as a cationic species, while Cr(VI) occurs as an anionic species. Under strongly acidic conditions (pH < 1), H₂CrO₄ (chromic acid) is the predominant species. In the weakly acidic to neutral range (pH 1–6), HCrO₄⁻ (bihydrogen chromate ion) dominates, while in alkaline conditions (pH > 7), CrO₄²⁻ (chromate ion) becomes the main species. Numerous studies have explored adsorption-based strategies for chromium removal [17,28,29,30,31,32,33,34,35] (

Table 5. Adsorption Performance of Different Adsorbent Materials for Cr(VI).

Microalgae Strain	Chemical Species	Temp (°C)	Optimal pH	Salinity g/L	Initial Metal Conc. (mg/L)	Time (min)	Max. Sorption (mg/g)	Removal Efficiency (%)	References
Spirulina platensis	CrO42−	25	1.5	0.5	250	600	148.64	59.5	[28]
Immobilized Chlorella minutissima	Cr2O72−	30	2	0.85	100	2160	57.33	99.7	[29]
Scenedesmus quadricauda	Cr2O72−	25	6	2.5	100	120	-	98.3	[30]
Chitosan/bioactive glass	Cr2O72−	-	4		50	300	188.71	-	[31]
Bacillus laterosporus	Cr2O72−	25	7		-	120		-	[32]
Hortaea werneckii	-	20	7		-	180	-	75	[33]
D. salina	Cr2O72−	20	8.5	30	150	480	38.9	-	This study
Rhizoclonium hookeri	Cr2O72−	-	2	-	1000	45	67.3	-	[34]
Spirulina platensis	Cr2O72−	60	1	0.6	500	90	59.6	-	[35]
D. salina	-	20	8.6	6	-	120 h	-	66.4	[17]

). A review by Keyvan Aeini reported that Saccharomyces cerevisiae can efficiently remove heavy metals from aqueous solutions within a pH range of 5.0–9.0. Under optimal conditions of pH 6.0 and 55 °C, the yeast achieved a maximum arsenic removal efficiency of 90.46% [36]. As presented in the table, the adsorption capacities of various materials are summarized. Hortaea werneckii NIOT129A8 demonstrated the following heavy metal removal efficiencies: Cu²⁺ (79%), Pb²⁺ (76%), Cr(VI) (75%), Cd²⁺ (74%), Ni²⁺ (73%), and Co²⁺ (69%) [33]. S. Revathi utilized a binary mixture of chitosan (CS) and bioactive glass (BAG) to optimize the removal of hexavalent Cr(VI) at pH 4 and Cu(II) at pH 5. With a contact time of 300 min, an adsorbent dosage of 0.5 g, and an initial metal concentration of 50 ppm, the maximum adsorption capacities were 188.71 mg/g for Cr(VI) and 181.84 mg/g for Cu(II). Isothermal modeling indicated that adsorption of both Cr(VI) (R² = 0.9529) and Cu(II) (R² = 0.9615) followed the Freundlich isotherm, while kinetic analysis showed that the pseudo-second-order model best described the adsorption process for Cr(VI) (R² = 0.9836) and Cu(II) (R² = 0.9893) [31]. A.I. Zouboulis applied inactivated Bacillus licheniformis and B. laterosporus to remove Cd(II) and Cr(VI), achieving maximum adsorption capacities of 142.7 mg/g and 159.5 mg/g for Cd(II), and 62 mg/g and 72.6 mg/g for Cr(VI), respectively [32]. Further, numerous studies have demonstrated that green microalgae possess remarkable metal accumulation capabilities, making them highly suitable for both the removal of metallic pollutants and the assessment of environmental risks [12,18,37]. Numerous studies have highlighted the significant potential of the genus Dunaliella as an effective biosorbent for heavy metals [16,38], demonstrating its adaptive metabolic responses to metal exposure [2,17].

The primary mechanism for chromium adsorption by microalgae is ion exchange, where metal ions are captured through two pathways: direct adsorption onto oppositely charged sites on the biomass surface, or indirect reduction followed by adsorption [39]. In particular, hexavalent chromium (Cr(VI)) can be effectively removed via an adsorption-coupled reduction process [40]. Studies have confirmed that functional groups, including aldehydes and alkyl chains, play a key role in chromium biosorption. Under acidic conditions, the biomass surface is enriched with hydronium ions, enhancing the interaction between Cr(VI) and microalgal binding sites. As summarized in Table 3, many studies focus on acidic conditions to achieve maximum adsorption capacity. Trivalent chromium occurs in forms such as CrOH²⁺ and shows stronger binding affinity for negatively charged functional groups, typically reaching maximum adsorption at pH 6. Hexavalent chromium exists as anionic species, including HCrO₄⁻ and CrO₄²⁻, with the highest removal efficiency observed at pH 1–2 [34]. However, seawater’s high pH often limits the achievement of saturated adsorption capacity under optimal conditions. Given the significantly higher toxicity of hexavalent chromium compared to trivalent chromium, its adsorption is of particular importance. Kaisar Raza isolated and identified 15 strains of D. salina from the high-pH environment of Sambhar Salt Lake, demonstrating their strong potential for Cr(VI) removal from industrial wastewater under extreme conditions of high pH and salinity [41]. These findings were further confirmed through spectroscopic, crystallographic, and microscopic analyses, including scanning electron microscopy (SEM) and X-ray diffraction (XRD), validating D. salina as an effective biosorbent for Cr(VI) [17]. To address chromium adsorption in high-salinity environments, clarify the relationship between chromium concentration and adsorption behavior, and elucidate microalgal adsorption mechanisms, this study examined the adsorption characteristics of D. salina for Cr(VI). The results demonstrated that as the initial Cr(VI) concentration increased, the removal rate decreased, while the adsorption capacity increased.

This phenomenon is attributed to the limited number of active sites on algal cell surfaces. At low Cr(VI) concentrations, ions readily occupy these sites, resulting in a high removal rate. At higher concentrations, Cr(VI) ions increasingly occupy the available active sites, raising the adsorption capacity, but as the sites approach saturation, further increases in ion concentration no longer enhance adsorption, leading to a decline in the removal rate. Adsorption kinetics (Table 3) indicated that for the high-concentration Cr(VI) group (20 mg/L), the process followed the pseudo-first-order model (R² = 0.95054), indicating a dominance of physical adsorption. However, across a broader concentration range (0.5–20 mg/L), the experimental data closely followed the pseudo-second-order kinetic model (R² = 0.9862–0.9969), suggesting that chemical adsorption predominates in these cases.

In the isothermal adsorption experiments (Table 4), both the Langmuir (R² = 0.9958) and the Freundlich (R² = 0.9838) models effectively described the adsorption equilibrium, with the Langmuir model showing a slightly better fit, indicating that Cr(VI) adsorption predominantly follows a monolayer mechanism involving cooperative interactions among multiple active sites on the algal surface. The Freundlich parameters also demonstrated strong performance, suggesting surface heterogeneity or partial multilayer adsorption. The adsorption process of hexavalent chromium (Cr(VI)) by D. salina aligns more closely with the pseudo-second-order kinetic model, indicating a predominance of chemical adsorption. Moreover, both the Langmuir and Freundlich isotherm models adequately describe the adsorption equilibrium. D. salina demonstrated significant Cr(VI) adsorption potential, particularly under low-concentration contamination conditions. The D. salina strain used in this study achieved a high Cr(VI) adsorption capacity of 38.9 mg/g under high-pH conditions, providing a theoretical basis for its application in remediating chromium-contaminated seawater, particularly at low chromium concentrations.

Living algal cells possess unique biological characteristics of adsorption, transformation, and regeneration, coupled with metabolism-dependent mechanisms such as intracellular sequestration and extracellular precipitation or accumulation. However, cell surface adsorption and complexation are independent of cellular viability and can occur in both living and non-living cells. Studies have shown that the biosorption of hexavalent chromium onto extracellular polymeric substances is the primary mechanism for chromium removal by the diatoms Phaeodactylum tricornutum and Navicula pelliculosa [42]. Within microalgal cells, organelles, granular structures, and heat-stable cytosolic peptides/proteins are responsible for accumulating the majority of hexavalent chromium [43]. In addition to extracellular adsorption and intracellular accumulation, chromate reductase also plays a key role in heavy metal ion removal [44]. Functional groups on the cell walls of living microalgae, including carboxyl, ester, and hydroxyl groups, bind metal ions and facilitate adsorption. Damage to the microalgal cell wall reduces the efficiency of heavy metal removal [45]. Living D. salina is especially effective for treating low-concentration, multi-metal pollution systems. Although its operation can be relatively complex, its sustainability and cost-effectiveness make it a highly promising candidate for environmental remediation.

5. Conclusions

Exposure experiments were conducted on the more toxic form of chromium, Cr(VI). The results showed that analysing the growth of microalgae and their photosynthetic responses under varying concentrations of Cr(VI) revealed that low concentrations (0.1–1 mg/L) stimulated the growth of D. salina. Cr(VI) concentrations above 1 mg/L significantly inhibited algal cell proliferation. However, the cells maintained growth throughout the 20-day experimental period, demonstrating substantial tolerance and stress resistance to high chromium levels. High chromium concentrations disrupted both the donor and acceptor sides of PSII, inhibiting photosynthetic electron transport, impairing reaction center function, and lowering photochemical efficiency. Notably, the adsorption kinetics followed a pseudo-first-order model at a Cr(VI) concentration of 20 mg/L, a salinity of 30 g/L and a pH of 8.5 (Table 5), indicating a dominant physisorption process. The algal strain exhibited excellent chromium removal capability, with D. salina attaining a maximum adsorption capacity of 38.9 mg/g under high-salinity conditions. This high adsorption capacity, achieved without any external osmotic adjusters, underscores its innate suitability for applications in high-salinity environments like marine or industrial wastewater. This study demonstrates the toxic effects of chromium on D. salina and characterizes its adsorption behavior, highlighting the microalga’s significant potential in high-salinity marine environments. The maintained adsorption rate and capacity despite PSII damage suggest that biosorption, potentially via extracellular polymeric substances or cell wall functional groups (e.g., carboxyl, hydroxyl), plays a crucial role alongside metabolic uptake. Its ability to efficiently remove heavy metals while maintaining regenerative capacity under metal stress positions D. salina as a promising agent for the remediation of heavy metal pollution in marine ecosystems. Further investigation into the specific binding mechanisms and synthesis of the link between physiological stress and adsorption kinetics is warranted to fully optimize its application.