Acute and Sublethal Effects of Boron on Daphnia magna: Assessment Using Behavioral, Physiological and Oxidative Stress Endpoints

Abstract

Boron serves as a necessary micronutrient, but elevated concentrations may exert toxic effects, which has raised concern over its increasing presence in the environment owing to anthropogenic activities. This study assessed the sublethal effects of boron on Daphnia magna, which is a commonly employed model species in freshwater ecotoxicology. D. magna neonates were subjected to boron concentrations ranging from 0.5 to 350 mg B/L over 48 h, and acute toxicity (EC₅₀), along with swimming velocity, heart rate, and oxidative stress responses, were evaluated as toxicological endpoints. Swimming velocity increased significantly at 80 mg B/L before declining, while heart rate significantly decreased at 250 mg B/L (p < 0.05). In addition, significant increases in oxidative stress responses were observed at sublethal concentrations of 40 and 80 mg B/L (p < 0.05), highlighting the sensitivity of oxidative stress responses to boron exposure. These findings demonstrate the previously underexplored sublethal effects of boron on D. magna, including alterations in swimming velocity, heart rate, and antioxidant defenses, emphasizing the need for integrated endpoints in ecotoxicological assessments.

1. Introduction

Boron (B), a metalloid, is widely distributed in minerals throughout the Earth’s crust. B is present in water, air, and soil with average environmental concentrations of 0.1 mg/L, 5 × 10⁻⁵ mg/m³, and 26 mg/kg, respectively [1]. In freshwater systems, it is mainly present as boric acid, with smaller amounts as borate ions. Environmental B originates from natural processes such as weathering of boron-rich sedimentary rocks, the release of sea salt aerosols from seawater, and the emission of vapors and fine particles from volcanic activity [2,3]. Moreover, anthropogenic activities contribute significantly to environmental B through its extensive use in glass, ceramics, fiberglass, pharmaceuticals, cosmetics, fertilizers, and biocides [2,4,5]. At low concentrations, B functions as a vital trace element required for plants and animals [6,7] and is known to be important for reproduction, bone growth, regulation of enzymes and hormonal processes [6,8,9]. At high concentrations, however, B has been noted to have toxic effects which has led to its use as a pesticide against terrestrial insects [10,11,12].

In recent decades, the presence of B in aquatic environments and its potential toxicity has drawn increasing attention [8]. Borates are highly stable in water and are difficult to remove using conventional water treatment processes [1,5]. Although many countries, including Japan, regulate drinking water B concentrations below 1 mg B/L, elevated levels have been reported due to geological sources and wastewater discharge [13]. For instance, natural waters in Canada and the USA have shown B concentrations ranging from 0.02 to 360 mg B/L, while drinking water in Germany, the USA, Chile, and the United Kingdom has been reported to contain up to 15 mg B/L [5]. In Japan, hot spring water from the Jozankei area in Sapporo, containing approximately 40 mg B/L, mixes with the Toyohira River, a major source of municipal drinking water [14]. The Environmental Protection Agency of the United States has included B on its Contaminant Candidate List, indicating that it could be designated as a drinking-water contaminant in the near future [10,15].

Studying the effects of sublethal B concentrations is crucial because of the potential risk to aquatic organisms. Major ecological phenomena such as the bioaccumulation and biomagnification of B remain uncertain and understudied [16]. Although most studies suggest low bioaccumulation in aquatic organisms, it has been observed in certain plants and algae, with evidence indicating species-specific variation, such as differing bioconcentration factors in duckweed species [17]. However, the extent of B biomagnification in aquatic food webs remains uncertain. Hence, further studies are needed because increasing the quantity and variety of available B toxicity data for different species is crucial to assessing the relative sensitivity of organisms to B exposure.

The effects of B toxicity have been well documented for terrestrial species [12,18,19] and for aquatic organisms such as fish [4,8,20], and cladocerans to a certain extent. Microcrustaceans such as cladocerans play a critical role in aquatic ecosystems. Daphnia magna, known widely as the water flea, is a fundamental species in freshwater habitats, occupies the role of a primary consumer in aquatic food chains [21]. Even slight biological changes in Daphnia sp. can affect the entire trophic structure, making them valuable indicators of freshwater ecosystem health [22]. Moreover, D. magna is one of the most widely used freshwater invertebrates in ecotoxicological research. Its short life cycle, parthenogenetic reproduction, sensitivity to contaminants, ease of laboratory handling and well-established standardized test protocols make it a valuable model organism for assessing the effect of various contaminants [23]. The comprehensive toxicological database available for D. magna facilitates comparisons across studies and enhances the interpretation of contaminant-induced responses [23,24]. Furthermore, the transparent body of D. magna enables non-invasive physiological measurements, such as heart rate monitoring, while its swimming velocity can be easily quantified, making both endpoints sensitive indicators of sublethal stress [25]. The majority of research investigating the toxic effects of various chemicals, including B, on Daphnia sp. have focused on mortality (LC₅₀) and immobilization (EC₅₀) [26,27]. To illustrate the acute toxicity of B, Gersich [28] reported an LC₅₀ value of 133 mg B/L depending on the outcomes of a 48 h static acute test. Maier and Knight [29] reported an LC₅₀ value of 141 mg B/L after 48 h exposure, while Lewis and Valentine [30] found an LC₅₀ of 226 mg B/L following a 48 h static acute exposure. According to Strigul et al. [31] D. magna exposed to B nanoparticles exhibits a 48 h LD₅₀ of 6.7 mg B/L. In addition, Soucek et al. [20] documented an LC₅₀ of 102 mg B/L for Ceriodaphnia dubia, a related daphniid species belonging to the same family (Daphniidae) as D. magna. Nevertheless, data on highly sensitive and earlier indicators, such as boron-induced behavioral, physiological, and oxidative stress responses, are limited.

Behavioral endpoints, particularly swimming velocity, are recognized as highly sensitive indicators of sublethal stress in Daphnia sp. [21] and have been widely used to assess the effects of pharmaceuticals [32,33], metals [34,35], and pesticides [36,37]. Similarly, heart rate is an important physiological biomarker for evaluating toxic stress [25]. Although numerous studies have explored Daphnia’s heart rate responses to diverse xenobiotics [21,25], information on the potential effects of B on heart rate remains scarce.

Oxidative stress responses are typically regarded as more sensitive than whole-organism responses to toxicants because alterations at the subcellular level typically precede organism-level effects [38]. Enzymatic antioxidants, including catalase (CAT), glutathione peroxidase (GPx), and detoxification enzymes such as glutathione S-transferase (GST), are vital for counteracting oxidative stress in cells by neutralizing reactive oxygen species (ROS), thus preventing genetic damage and protecting cellular lipids from oxidative degradation [39]. Boron-induced ROS production and oxidative stress have been studied in rats, ostrich chicks, wax moth (Galleria mellonella), clams (Ruditapes decussatus), and algae (Chodatodesmus mucronulatus) [40,41,42,43,44], but no data are available for the boron-induced oxidative stress in D. magna.

The present study focused on addressing the knowledge gap on more sensitive indicators that can facilitate the assessment of aquatic B toxicity on D. magna. Although sublethal effects are typically associated with chronic toxicity studies, incorporating sublethal endpoints in acute exposure scenarios offers valuable prompt insights into the physiological stress responses that may occur prior to severe toxic effects. Therefore, the aim of this study was not only to characterize acute B toxicity, but also to evaluate and compare the sensitivity of behavioral, physiological, and oxidative stress endpoints in D. magna. By integrating responses across multiple levels of biological organization, this study aimed to improve understanding of B toxicity mechanisms and identify sensitive endpoints that may complement conventional toxicity assessments and contribute to ecological risk evaluation.

2. Results and Discussion

2.1. Acute Toxicity: Effective Concentration Based on Immobilization

The actual B concentrations obtained via inductively coupled plasma optical emission spectrometer (ICP-OES; Agilent 5800, Santa Clara, CA, USA) closely corresponded with the nominal values (

Table 1. Nominal and measured B concentrations (obtained via ICP-OES) in test media prepared during the acute test.

Nominal B Concentration (mg B/L)	Actual B Concentration (mg B/L)
0.5 (control)	0.54 ± 0.1
2	1.9 ± 0.1
10	9.8 ± 0.9
20	20.3 ± 0.9
40	39 ± 1.7
80	78 ± 3.5
175	177 ± 3.2
250	250 ± 1.2
350	348 ± 3.5

). Hence, nominal B concentrations were applied for data presentation and analysis.

As shown in Figure 1, exposure to B, induced an increase in immobilization in D. magna, which intensified with both concentration and exposure duration. Individuals in the control group did not show any immobility throughout the test period. Exposure to B levels of below or equal to 20 mg B/L did not result in any detectable immobilization. Individuals exposed to 350 mg B/L showed 100% immobilization after 48 h. Based on the immobility data, the EC₅₀ values for 24 and 48 h were calculated as 238 ± 10 and 138 ± 21 mg B/L, respectively. Control immobilization was below 10%, satisfying the OECD validity criteria.

2.2. Boron-Induced Alterations in Behavioral and Physiological Responses

2.2.1. Effects on Swimming Velocity

Upon completion of 48 h of exposure, swimming velocity in D. magna was not significantly affected at B concentrations up to 40 mg B/L, although a slight increase was observed (Figure 2). However, a significant increase was observed at 80 mg B/L compared to the control. Beyond this concentration, a concentration-dependent decline in swimming velocity relative to the control was observed at higher B levels, with reductions of 60% and 75% recorded at 175 and 250 mg B/L, respectively (p < 0.05; one-way ANOVA). The swimming velocity was not assessed at 350 mg B/L because no individuals were alive after 48 h.

Behavioral assays such as swimming velocity serve as important indicators of sublethal toxicity, capable of identifying detrimental effects at far lower concentrations than conventional toxicological measures [45]. Swimming behavior is considered a sensitive biomarker of sublethal stress in D. magna as it reflects alterations in neuromuscular activity and energy metabolism. In this study, swimming velocity remained stable up to 40 mg B/L, indicating that low-to-moderate concentrations of B did not considerably impair swimming performance. However, the elevated swimming velocity at the higher sublethal concentration of 80 mg B/L, may reflect a stress-induced behavioral response, characterized by increased locomotor activity under sublethal exposure conditions. Alternatively, the increased swimming velocity may indicate stress-induced hyperactivity, which has been observed in organisms exposed to sublethal levels of toxicants. Alterations in neuromuscular regulation may also contribute to this response, as toxicants have been reported to affect swimming behavior through disturbances in sensitive systems involved in locomotor control [46]. Marked reductions observed after 175 mg B/L, suggesting that high levels of B interfere with swimming activity. Because the locomotion of D. magna requires continuous muscular activity with substantial energy consumption [47], the observed reduction in swimming velocity may be associated with increased energy demands for maintaining essential physiological processes under boron-induced stress. Moreover, the reduction in swimming velocity at higher B concentrations may be related to oxidative stress responses, as suggested by the increased activities of antioxidant enzymes observed in the present study. This suggests that swimming response in D. magna appears to be a sensitive marker of higher sublethal B exposure, while significant behavioral impairment becomes evident at concentrations near or above the EC₅₀. Similar reductions in swimming performance under chemical stress have been reported for D. magna exposed to different toxicants, and such impairments are ecologically important because swimming behavior is essential for feeding and predator avoidance. For instance, Cano et al. [48] reported a reduction in swimming velocity in D. magna exposed to carbon-based nanomaterials. A decline in swimming velocity may adversely impact filter feeding activity and in turn reduce food intake, which can reduce growth and reproduction rates [47]. Uttieri et al. [49] suggested that alterations in swimming performance may influence prey–predator dynamics and alter trophic interactions between zooplankton and fish as well as between zooplankton and phytoplankton. For instance, O’Keefe et al. [50] reported that Daphnia sp. with a higher swimming velocity were more likely to be predated upon by the fish Lepomis macrochirus.

2.2.2. Effects on Heart Rate

As shown in Figure 3, B exhibited a mild stimulatory effect up to 175 mg B/L (p > 0.05; one-way ANOVA), followed by a strong inhibitory effect at the highest concentration (p < 0.05; one-way ANOVA). The initial increase in heart rate may represent a compensatory physiological response to boron-induced stress aimed at maintaining homeostasis under increasing exposure conditions. In contrast, the significant reduction in heart rate observed at 250 mg B/L suggests physiological impairment at higher exposure levels. Significant increases in CAT, GPx, and GST activities even at higher sublethal B concentrations indicate activation of antioxidant defense systems and suggest the involvement of oxidative stress responses. The co-occurrence of these biochemical and physiological responses implies that oxidative stress may contribute to the observed alterations in cardiac activity. Similar biphasic heart-rate responses have been reported in Daphnia sp. exposed to other toxicants supporting the interpretation that physiological stress responses vary according to exposure intensity. Szabelak and Bownik [25] examined the effects of salicylic acid on the heart rate of D. magna and reported an increase in heart rate at low concentrations and a decrease at higher concentrations. The authors suggested that these responses may be associated with physiological and neuroregulatory disturbances induced by toxicant exposure. The biphasic pattern observed in the present study is consistent with these findings. Furthermore, the elevated activities of CAT, GPx, and GST observed under B exposure suggest the involvement of oxidative stress responses, which may contribute to the observed heart-rate alterations.

2.3. Biochemical Activity and Oxidative Stress

Figure 4 shows that the CAT activity of D. magna increased with B concentration. The highest CAT activities were observed at 40 and 80 mg B/L and exhibited a statistically significant elevation above the CAT activity of the control group (p < 0.05; Welch’s ANOVA). Nearly 100% increase in CAT activity was observed at 40 and 80 mg B/L compared to the control.

As shown in Figure 5, the GPx activity also showed a concentration-dependent increase. At the highest exposure concentration, the GPx activity showed a 150% increase compared with the control.

The observed increase in CAT and GPx activity with increasing B concentrations may reflect the activation of antioxidant defense mechanisms that help maintain cellular integrity and function under boron-induced stress conditions. The elevated activities of antioxidant enzymes observed in the present study suggest the involvement of oxidative stress responses in D. magna following B exposure. Notably, the most pronounced CAT responses occurred at 40 and 80 mg B/L, highlighting the sensitivity of CAT as a biomarker of oxidative stress at higher sublethal levels. In D. magna, CAT and GPx activities have been reported to increase following exposure to redox-cycling compounds (paraquat) and Cu, whereas CAT activity decreased under chronic Cr exposure [51,52]. Studies on other species have reported elevated CAT activity following B exposure. For example, increased CAT activity was observed in the midgut of wax moth larvae and in the gills and digestive gland of Palourde clam [42,43]. Moreover, rats exposed to higher B concentrations (40 mg/kg) exhibited increased CAT activity in both the kidney and liver, indicating an upregulated antioxidant response [53]. B toxicity may also cause cellular damage by interfering with cellular structures. A study on Saprolegnia sp. suggested that excessive B exposure (175 mg B/L) potentially contributes to oxidative stress responses by disrupting mitochondrial functions [54].

As shown in Figure 6, the GST activity in D. magna showed a clear concentration-dependent response to B exposure. At lower concentrations (2, 10 and 20 mg B/L), the GST activity did not differ significantly from that of the control group. However, exposure to 40 and 80 mg B/L resulted in a significant increase in the GST activity (p < 0.05; one-way ANOVA), reaching nearly 230% higher than the control. This pattern suggests that GST activity remains relatively stable at lower exposures but increases when oxidative stress reaches high levels.

GST is classified as a phase II enzyme and contributes significantly to cellular detoxification mechanisms. It catalyzes the conjugation of reduced glutathione to reactive electrophilic compounds, thereby protecting cells from oxidative damage [55]. Enhanced GST activity suggests the involvement of phase II detoxification process as a response to B exposure [43]. Moreover, pronounced increase in GST activity (230%) compared to CAT (100%) at 40 mg B/L suggests that GST exhibits higher sensitivity to boron-induced stress at higher sublethal concentrations. Similar results have been documented in both aquatic and terrestrial organisms following exposure to B. For example, exposure to B resulted in a significant enhancement in GST activity in snails, as reported by Radwan and Gad [19]. Wax moth larvae exposed to B also demonstrated an increase in GST activity in the midgut as well as in the fat body [42]. Upregulation of GST activity following B exposure have also been reported for clams (Ruditapes decussatus) and German cockroaches (Blattella germanica) [12,43]. Together, these findings support the role of GST as a sensitive biomarker of boron-induced oxidative stress in D. magna.

In addition to oxidative stress, B toxicity may involve more specific biochemical mechanisms. B has been reported to form reversible complexes with cis-diol–containing biomolecules such as ATP, NAD⁺, and RNA, potentially interfering with cellular metabolism and energy production. In biological systems, B exists in equilibrium between boric acid and borate, and the relative abundance of these species is pH dependent, which may influence the formation of such complexes. However, studies in animal systems have shown that B can influence enzyme activity, membrane transport, and cell signaling pathways through interactions with ribose-containing cofactors [56,57,58]. Such metabolic disruptions may lead to impaired physiological processes and secondary oxidative stress responses. These mechanisms have been reported in animal models including fish and vertebrate systems [59], suggesting that boron-induced toxicity may involve metabolic and signaling disturbances in addition to generic oxidative stress. These mechanisms may collectively contribute to the physiological and biochemical alterations observed in D. magna, including changes in heart rate, swimming behavior, and antioxidant enzyme activities following exposure to elevated B concentrations. Moreover, in addition to direct oxidative stress responses, interactions between B species and ribose-containing cofactors such as NAD⁺/NADH [60] and potentially other ribose-containing cofactors (NADP⁺/NADPH) could be proposed as a potential mechanism affecting cellular redox regulation. However, as cofactor availability and glutathione metabolism were not assessed in the present study, the contribution of this mechanism to the observed enzymatic responses remains uncertain.

2.4. Behavioral, Physiological, and Oxidative Stress Responses as Indicators for Higher Sublethal B Concentrations

In this study, the responses of D. magna to B exposure varied across the oxidative stress, behavioral, and physiological endpoints evaluated. Antioxidant enzyme activities were among the most sensitive responses to higher sublethal B exposure. Significant increases in CAT and GST activities (as shown in Figure 4 and Figure 6) were observed at 40 mg B/L, even though immobility remained low at only 7% (Figure 1). Similarly, GPx activity increased at 80 mg B/L (Figure 5), where immobility was still limited to 11% (Figure 1). These results indicate activation of antioxidant and detoxification pathways, at concentrations below those causing substantial immobilization. The pronounced increase in enzymatic activity at these concentrations implies the possible onset of oxidative stress at higher concentrations in D. magna. The gradual increase in CAT activity with the B concentration indicates a consistent elevation of oxidative stress. In contrast, the GST activity remained unchanged at lower sublethal concentrations (≤20 mg B/L) and then increased significantly at the higher sublethal concentrations of 40 and 80 mg B/L. These enzymatic responses reflect different patterns of antioxidant enzyme activity, where CAT exhibited a gradual increase with rising B concentrations and GST showed a pronounced response at higher sublethal concentrations, particularly at 40 and 80 mg B/L in the present study.

At higher sublethal concentrations, behavioral responses became evident. Swimming velocity showed a slight increase up to 80 mg B/L, with a significant elevation compared to the control, indicating a potential stress-induced hyperactivity response below the acute EC₅₀ (138 mg B/L). However, at higher concentrations, swimming velocity decreased, indicating behavioral impairment under elevated B levels. These findings suggest that swimming velocity may serve as a sensitive indicator of higher sublethal B exposure, while pronounced behavioral impairment became evident at higher concentrations near or above the EC₅₀.

Physiological responses, such as heart rate were primarily affected only at higher exposure levels (>175 mg B/L). A slight increase in heart rate was observed up to 175 mg B/L, and a significant decrease at 250 mg B/L, indicating physiological impairment at higher exposure levels. Considering that this decrease occurred at concentrations exceeding the EC₅₀ (138 mg B/L), these findings suggest that physiological endpoints reflect more advanced stages of stress and toxicity. Overall, the results demonstrate differing sensitivities among the measured endpoints. Antioxidant enzyme activities responded at lower B concentrations than behavioral and physiological endpoints, highlighting their value as sensitive indicators of B exposure. This integrated interpretation emphasizes the importance of combining multiple endpoints to better understand B toxicity mechanisms and to identify sensitive indicators for aquatic ecotoxicological assessments.

In addition to the observed behavioral, physiological, and biochemical responses related to oxidative stress, B speciation in the exposure medium should be considered when interpreting the results. Under the pH conditions of the M4 Elendt medium (7.84 ± 0.14), B was expected to exist mainly as boric acid, the neutral species present below the pKa of boric acid (~9.24). Because boric acid is uncharged, it exhibits relatively high membrane permeability and may be transported through passive diffusion and aquaporin-mediated pathways [61,62,63]. Therefore, the predominance of boric acid in the exposure medium likely enhanced B bioavailability to D. magna and may have contributed to the observed responses.

From an environmental toxicology perspective, the effect concentrations identified in the present study should be interpreted in the context of environmental B occurrence. Although the concentrations associated with significant biological responses are higher than those commonly reported in most freshwater ecosystems, elevated B levels have been documented in certain natural and anthropogenically impacted waters. Consequently, the present findings are most relevant to elevated exposure scenarios and provide insight into the progression of boron-induced toxicity across multiple levels of biological organization. In addition to encompassing environmentally relevant exposure levels, the inclusion of higher B concentrations enabled the evaluation of concentration-dependent sublethal responses and facilitated the identification of toxicity thresholds across biochemical, behavioral, and physiological endpoints. Furthermore, the results demonstrate differences in the sensitivity of these endpoints, with oxidative stress responses occurring at lower concentrations than organism-level effects. Such information contributes to a more comprehensive understanding of B toxicity and may assist in the selection of appropriate endpoints for ecological risk assessment.

One constraint of the present investigation is the relatively short exposure duration (48 h), compared to a chronic test, may not adequately capture the chronic effects of B toxicity in D. magna. Future studies on potential risk should focus on chronic exposure and underlying mechanisms to better understand the long-term effects of sublethal B concentrations in freshwater environments. Although this study focused on enzymatic and physiological endpoints, future research employing transcriptomic and metabolomic approaches would be valuable for identifying gene expression changes and metabolic alterations, thereby offering a more comprehensive understanding of the mechanisms through which D. magna responds to boron-induced stress. In addition, future studies should examine whether interactions between B and ribose-containing cofactors such as NAD⁺/NADH and NADP⁺/NADPH contribute to alterations in cellular redox balance and antioxidant enzyme responses. While four replicates were used in this study, consistent with standard ecotoxicological practices, the relatively small sample size may limit statistical power, especially for detecting subtle effects, and should be considered when interpreting the results.

3. Materials and Methods

Vital behavioral (swimming velocity) and physiological (heart rate) parameters, as well as biomarkers of the oxidative stress response (CAT, GPx, and GST activities), were assessed along with the conventional toxicological endpoint (i.e., immobility) to enhance the sensitivity and specificity of the B toxicity assessment.

3.1. Laboratory Cultures of D. magna

Adult D. magna, sourced from a commercial supplier, were cultured over multiple generations in 1 L glass beakers. M4 Elendt solution was used as the culture medium, and organisms were fed daily with Chlorella vulgaris at a ration equivalent to 0.1–0.2 mg C/individual/day. All cultures were maintained under gentle aeration conditions with a dissolved oxygen measured at 7.7 ± 0.3 mg/L, temperature of 22 ± 0.9 °C, conductivity of 684 ± 9 µS/cm, and pH 7.9 ± 0.1 (arithmetic mean ± standard deviation). Cultures were maintained by renewing the medium thrice per week, and the conditions were monitored regularly to avoid stressing D. magna. Prior to experiments, clonal cultures were acquired from healthy and actively swimming adult female D. magna individuals cultured in 1 L glass beakers. The holding conditions were the same as for the main culture, and the stocking density was 50–75 individuals per liter.

3.2. Acute Toxicity Test

Boric acid acquired from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, (H₃BO₃; 99.5% m/m, Cat No. 021-02195) was dissolved to prepare a 2 g/L stock solution and eight test concentrations (2, 10, 20, 40, 80, 175, 250 and 350 mg B/L). Concentrations were identified following the results of a preliminary assessment and were designed to cover environmentally relevant levels as well as elevated concentrations approaching sublethal toxicity. This broad concentration series allowed accurate estimation of acute toxicity (EC₅₀) while also characterizing behavioral and physiological responses across a gradient of B exposure. M4 Elendt medium was used as the dilution medium for the test and control solutions. Acute toxicity was evaluated based on the procedure outlined in Organization for Economic Co-operation and Development (OECD) guideline 202 [64]. Female D. magna neonates under 24 h of age, were obtained from parthenogenetic cultures that were not the progeny of the first brood. Twenty five D. magna individuals were introduced into each vessel filled with 100 mL of the experimental solution, resulting in 100 individuals per concentration. Each concentration was tested in four replicates. The test was conducted with a 16:8 light/dark photoperiod, without aeration and food under semi-static renewal conditions with the medium refreshed at 24 h intervals. Throughout the exposure, the test medium maintained stable conditions, with dissolved oxygen at 7.86 ± 0.03 mg/L, temperature at 21.3 ± 0.2 °C, conductivity at 687 ± 5 µS/cm, pH at 7.84 ± 0.14, and hardness at 267 ± 3 mg/L as CaCO₃. All animals were acclimated and exposed under identical conditions, maintaining valid treatment comparisons. Individuals in each replicate were checked for immobility at both 24 h and 48 h of exposure. The acute EC₅₀ value indicated that sublethal exposure corresponded to B concentrations below 80 mg B/L. Throughout the manuscript, exposure concentrations are expressed as mg B/L (elemental B) rather than boric acid equivalents. The actual B concentrations in the test media were measured via ICP-OES (Agilent 5800, Santa Clara, CA, USA).

3.3. Determination of Behavioral and Physiological Responses

3.3.1. Swimming Velocity

The swimming velocity was measured by following the procedure of Samarakoon and Fujino [65] with minor modifications. Five individuals were randomly selected from each replicate, corresponding to twenty individuals per concentration. Sufficient numbers of mobilized individuals were available in each replicate at lower concentrations, while the number of mobilized individuals decreased at higher concentrations. Therefore, five representatives were selected from the total individuals available at each concentration and transferred to 24-well plates (i.e., one individual per well), and 300 µL of the test solution was added to each well. After 48 h of exposure, the swimming activity of each individual was observed by using a stereomicroscope (SZX10, Olympus Corporation, Tokyo, Japan) equipped with a 0.8× objective lens and recorded for 30 s at 29 frames per second (1600 × 1200 pixels; calibration 11.7614 µm/pixel). The vertical movements of the individuals were minimal due to the limited depth of the 300 µL solution. Thus, the movement of each individual was represented by X and Y coordinates. The videos were then analyzed using Fiji (ImageJ 1.54p) an open-source software for precise analysis of biological images [66]. The swimming velocity and swimming trajectories were determined by a manual tracking plugin.

3.3.2. Heart Rate

The heart rate was determined by using a microscopic method [21]. After 48 h of acute exposure, five individuals from each test vessel (i.e., twenty individuals per test concentration) were placed on a microscope slide with approximately 50 µL of exposure medium. A small amount of excess medium was gently removed to limit swimming activity and maintain the organisms within the field of view during observation, while animals remained submerged throughout the measurement. The heart rate was observed by using a BZ-X810 microscope (Keyence, Osaka, Japan) equipped with a high-resolution 4× objective lens, operated under transmitted-light (brightfield) illumination and recorded for 30 s. The videos were reviewed frame by frame via Windows Media Player to quantify the heart rate of the D. magna.

3.4. Determination of Biochemical Biomarkers and Oxidative Stress Responses

Utilizing EC₅₀ values obtained from the acute toxicity assessment, five exposure levels were selected to represent concentrations below EC₅₀, including considerably low doses ≤ EC₁ (lower sublethal—2, 10 and 20 mg B/L), as well as concentrations corresponding to EC₇ and EC₂₅ (higher sublethal—40 and 80 mg B/L, respectively). This experimental design incorporated lower and higher sublethal concentrations, while allowing assessment of responses occurring at sublethal levels before occurrence of visible immobilization effects. The enzyme activity induced by the B concentration was then tested to evaluate the potential risks of B in aquatic environments. For the oxidative stress enzyme assays, each B concentration and the control were tested using four replicate vessels, each containing 60 D. magna neonates in 250 mL of test medium. Following the exposure period, 30–40 surviving individuals were collected from each replicate separately and used for enzyme analyses. All other conditions were similar to those of the acute toxicity test.

3.4.1. Sample Preparation

After exposure to the sublethal concentration (or control solution), 30–40 surviving individuals from each experimental vessel were gently rinsed twice with potassium phosphate buffer (100 mM) at pH 7.4 before being transferred into 1.5 mL microcentrifuge tubes containing 1 mL of ice-cold buffer of same concentration and pH. The samples were then homogenized by using an ultrasonic processor (Astrason XL 2020, Misonix Inc., New York, NY, USA), and the tissue homogenates were subjected to centrifugation at 10,000× g for 10 min at 4 °C [67]. The resulting supernatant was collected for the determination of enzyme activities and protein concentration.

3.4.2. CAT Activity

The CAT activity was assessed through spectrophotometry following the protocol outlined by Claiborne [68] and detailed by Samarakoon et al. [67]. First, 50 µL of D. magna homogenate was combined with hydrogen peroxide (H₂O₂) solution (950 µL, 19 mM) in 50 mM potassium phosphate buffer at pH 7. The reduction in absorbance (at 240 nm over a 2 min) was measured by the spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan). The CAT activity was quantified as the rate of H₂O₂ decomposition (µmol min/mg/protein) applying the H₂O₂ molar extinction coefficient of 0.0436 mM⁻¹ cm⁻¹.

3.4.3. GPx Activity

GPx activity was measured following Regoli and Principato [69]. The assay was based on the oxidation of NADPH to NADP⁺ during the reduction of oxidized glutathione to reduced glutathione by glutathione reductase. The decrease in absorbance at 340 nm was monitored after initiating the reaction with H₂O₂. GPx activity was calculated using the molar extinction coefficient of NADPH (ε = 6.22 mM⁻¹cm⁻¹).

3.4.4. GST Activity

The enzymatic activity of GST was evaluated based on the assay developed by Habig et al. [70] utilizing 1-chloro 2,4-dinitrobenzene (CDNB). First, 3 mM solution of CDNB and reduced glutathione solution (0.01 M) were freshly prepared in potassium phosphate buffer (100 mM, pH 6.5). The reaction mixture contained 50 µL of enzyme supernatant, 1.65 mL of potassium phosphate buffer (100 mM), 1 mL of CDNB (3 mM) and 300 µL of reduced glutathione (0.01 M). The increase in absorbance at 340 nm was measured via the spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan) for 5 min. The GST activity was expressed as nanomoles of the CDNB–GHS conjugate produced per minute per mg of protein. The molar extinction coefficient of the conjugate is 9600 M⁻¹cm⁻¹ [67].

3.4.5. Protein Content

Protein content for each homogenate was quantified with the Pierce™ BCA Protein Assay Kit (Cat. no. 23227, Thermo Scientific, Rockford, IL, USA) following the manufacturer’s standard test-tube protocol, applying a sample-to-working reagent ratio of 1:20.

3.5. Statistical Analysis

Data were statistically analyzed with IBM SPSS software (version 25, IBM Corp., Armonk, NY, USA). The EC₅₀ values (mean ± SD, based on four independent replicates) were obtained by Probit analysis of the concentration–response data (immobilization after 24 and 48 h of B exposure). Prior to statistical analysis, the assumption of normal distribution was tested using the Shapiro–Wilk normality test (p > 0.05), and equality of variances among groups was examined by Levene’s test (p > 0.05). One-way analysis of variance (ANOVA) followed by Dunnett post hoc test was used to compare the means and to determine significant differences (p < 0.05) among the control and treatment groups in terms of swimming velocity, heart rate and oxidative stress responses. For datasets meeting the normality assumption but violating the homogeneity of variances (Levene’s test, p < 0.05), a Welch’s ANOVA followed by Games–Howell post hoc tests was applied.

4. Conclusions

This study investigated both the acute and sublethal impacts of B on D. magna using both conventional (EC₅₀) and sublethal toxicological metrics. In addition to the immobility-based EC₅₀, sublethal endpoints such as the swimming velocity, heart rate, and oxidative stress responses provided a comprehensive evaluation of boron-induced toxicity. An initial stimulation in swimming velocity and heart rate was observed at lower B concentrations, followed by concentration-dependent declines, with significant reductions occurring at 175 and 250 mg B/L, respectively. Elevated activities of CAT, GST, and GPx at higher sublethal concentrations of 40 and 80 mg B/L indicated the occurrence of oxidative stress-related responses following B exposure. These findings indicate that B exposure is associated with oxidative stress-related biochemical responses as well as behavioral and physiological alterations in D. magna. Among the measured endpoints, oxidative stress biomarkers responded at lower concentrations than behavioral and physiological parameters, suggesting their usefulness as sensitive indicators of sublethal B exposure. These results highlight GST activity may be a sensitive biomarker for assessing B toxicity at higher sublethal concentrations. In this study, B exposure adversely affected various aspects of oxidative stress, behavior and physiology in D. magna, with oxidative stress responses proving particularly valuable as sensitive indicators of boron-induced stress at higher sublethal levels.