Effects of BDE3 and the Co-Existence Copper on Photosynthesis and Antioxidative Enzymes in Salvinia natans (L.)

Abstract

Due to the unregulated handling of e-waste, the co-existence of PBDEs and heavy metals in water bodies and soil has been detected with high frequency. However, the combined toxicity for aquatic creatures remains unclear. This study investigated the single and combined stress of BDE3 and copper on the photosynthesis and antioxidant enzyme system of Salvinia natans (L.). The results indicated that there were no negative effects on photosynthetic pigments under single stress of BDE3 or combined stress with copper. However, to deal with oxidative stress, antioxidant defense enzymes, including SOD and CAT, were activated in S. natans. SOD was sensitive in the first stage, while CAT activity was significantly increased until the end of 14 days of incubation. Malondialdehyde content increased significantly, which indicated that excessive reactive oxygen species from pollution of BDE3 or coexistence with copper could not be eliminated. BDE3 concentration in the aqueous phase declined with time, while copper was accumulated over time in S. natans, with BCF increasing to 0.31 ± 0.073 at the end. Our study indicated that the co-existence of copper could exacerbate the damage caused by BDE3 to S. natans in aqueous environment.

1. Introduction

Polybrominated diphenyl ethers (PBDEs) are widely applied as an additive, not reactive, flame retardant in a large number of products such as electronic devices, plastics, textiles, and other fields [1]. Due to unregulated e-waste recycling treatments, the leakage risk of PBDEs to various environmental matrices was high during the last few decades [2,3,4]. High brominated congeners can be degraded via debromination to congeners with fewer bromines in the environment, which could lead to a higher detection frequency of lower brominated diphenyl ethers (LBDEs) in surface water [5]. LBDEs, whose congeners contain 1–4 brome atoms, have drawn more and more attention for higher volatility, water solubility, bioaccumulation, and toxicity to humans [6,7,8].

As one of the most fundamental mono-BDEs in the environment, 4-bromodiphenyl ether (BDE3) is also the most abundant debromination product of PBDEs [9,10]. The concentration of BDE3 was up to 167 ng g⁻¹ soil (dw) with a detection frequency of 87% from an e-waste recycling site in Guiyu, China, as reported in 2014 [11]. Another study showed that both the concentration and the proportion of BDE3 in Guiyu within the study area increased from 2010 to 2017, and the pollution spread to the surrounding area, including water and air [12]. A newly increased reserve of BDE3 in 2020 was estimated to be 0.42 t due to gradual and continuous debromination reaction [12]. Studies have demonstrated that BDE3 induced significant biotoxicity, including cell apoptosis, DNA damage, and reproductive toxicity in vivo; however, the toxicity for aquatic creatures was still unknown [9,13,14].

Heavy metals such as Cu could be released together with PBDEs from electronic waste during the unregulated dismantling process, leading to co-contamination of PBDEs and metals [2,3,15]. For instance, the concentration of PBDEs was reported up to 1561–10,111 mg kg⁻¹ (dw) in sediments from e-waste contaminated rivers alongside Cu concentrations of 7–1236 mg kg⁻¹ (dw). Copper plays an essential role in multiple biochemical processes for plants [16]. On the contrary, Cu has been proven to be toxic to plants and induce abnormalities in metabolic systems, including the production of reactive oxygen species (ROS) at excess levels [17,18]. Even some congeners of PBDEs like BDE47 were reported to be toxic for aquatic and terrestrial plant leaves as well as inhibit the genes related to chlorophyll synthesis, thus affecting the photosynthesis of the plants [19]. The combined toxicity of PBDEs and Cu on aquatic plants remained not well understood.

As important autotrophs in aquatic ecosystems, aquatic plants are distributed widely and commonly used as indicator organisms for pollution in water [20]. As a floating fern plant, although Salvinia natans (L.) has no true roots, the rhizoid-like structure functions similarly to those of real roots, and the plant grows extremely rapidly and is tolerant to pollutants [21,22]. Previous research has indicated that S. natans can remove eutrophication as well as heavy metals such as Cu (II), Ni (II), Hg (II) in water efficiently [23,24,25]. Therefore, this article selects S. natans as an indicator to study the joint effects of BDE-3 and copper Cu. The aims were (1) to investigate the effects of BDE3 and mixture with Cu on the chlorophyll; (2) to illustrate the antioxidant defense system, such as malondialdehyde (MDA) and antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT) after exposure to BDE3 and together with Cu; (3) to evaluate the potential removal efficiency for Cu under the combined pollution of BDE3 of S. natans.

2. Materials and Methods

2.1. Chemicals

BDE-3 and CuCl₂ were both brought commercially with purity > 99% and >99.95%, respectively (J&K Scientific, Shanghai, China). CaCl₂ was purchased with >99% purity (Merck KGaA, Shanghai, China). Solvents for HPLC analysis were chromatographic purity (Merck KGaA, Shanghai, China). Other reagents and organic solvents were analytic grad (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China).

2.2. Cultivation of Duckweed

All the duckweed (S. natans) was collected from Lake Changxing, Shanghai, and washed with tap water to remove impurities and silt attached. Afterward, all the duckweed were cultivated in a crystallization dish with a diameter of 180 mm filled with 1/10 Hoagland nutrient solution (pH 5.5–6.0) in a climate chamber for acclimatization. A total of 7 days later, each duckweed with leaf diameter > 0.5 cm was selected and rinsed with distilled water thoroughly for the experiment. The incubation situation was set to 60% humidity and 28 ± 1 °C with a photoperiod of 16 h light/8 h dark and a photon flux density of 80 ± 20 μmol/m²/s.

The concentration of Cu²⁺ was set to 0.1 mg L⁻¹, and the concentration of BDE3 was set at 0.5 mg L⁻¹ and 5 mg L⁻¹ according to the environmental concentrations [11]. During the experiment, water and nutrient solution were supplemented every day to ensure approximately constant concentrations of nutrients and pollutants. Each treatment was set for 14 days in triplicates containing duckweed of similar size. Dynamic distribution of Cu²⁺ in nutrient solution and duckweed (roots and leaves) was studied by sampling at 0, 1, 2, 4, 7, and 14 days. Physiological indexes, including photosynthetic pigments, activities of SOD, CAT, and MDA content in duckweed were measured at 1, 4, and 14 days. BDE3 in nutrient solution was analyzed by sampling as well as at 1, 4, and 14 days.

2.3. Photosynthetic Pigment Content

The photosynthetic pigments were measured as described in the previous method [26]. Briefly, ~0.05 g of fresh leaves of duckweed was taken and mixed with 10 mL of a mixture of ethanol and 80% acetone/20% deionized water (v:v = 1:1) in 15 mL flasks in the dark at room temperature for 12 h. The photosynthetic pigments were measured with an ultraviolet-visible spectrophotometer (UV-2450; Shimadzu, Kyoto, Japan) at wavelengths of 470 nm, 645 nm, and 663 nm for chlorophyll a, chlorophyll b, and carotenoids, respectively. The calculation was as follows:

C_a = 12.21 × OD₆₆₃ − 2.81 × OD₆₄₅

(1)

C_b = 20.13 × OD₆₄₅ − 5.03 × OD₆₆₃

(2)

C_c = (1000 × OD₄₇₀ − 3.27 × C_a − 104 × C_b)/229

(3)

where C_a—the concentration of chlorophyll a (g·L⁻¹);

C_b—the concentration of chlorophyll b (g·L⁻¹);

C_c—the concentration of carotenoids (g·L⁻¹).

2.4. Lipid Peroxidation

Level of lipid peroxidation was evaluated by malondialdehyde (MDA) measurement according to the previous method with modifications [27]. Weighing 0.1 g, fresh leaves of duckweed were ground in liquid nitrogen and added with 5 mL of 0.1% trichloroacetic acid (TCA) in a 10 mL plastic centrifuge tube. The homogenate was centrifuged at 12,000× g for 10 min. Moreover, 1 mL of the supernatant was taken and mixed with 4 mL of 20% TCA containing 0.5% thiobarbituric acid (TBA). The mixture was heated in a water bath at 95 °C for 30 min and cooled quickly down to room temperature on ice. Then, the mixture was centrifuged at 12,000× g for another 10 min to obtain the supernatant, whose absorbance was measured at 532 nm and 600 nm. The MDA concentration in the leaves was calculated according to the following formula:

MDA concentration C (μmol L⁻¹) = 6.45(OD₅₃₂ − OD₆₀₀)

2.5. Enzyme Activity Measurement

A total of ~0.1 g fresh leaves of duckweed were homogenized with 1 mL of phosphate buffer (PBS, 50 mM, pH 7.8) after cutting into pieces in a pre-cooled centrifuge tube under ice-water bath conditions. Then, the homogenate was centrifuged (C715RE; Hitachi, Tokyo, Japan) at 12,000× g for 20 min at 4 °C. The supernatant was extracted as enzyme solution and stored in a refrigerator at 4 °C for the determination of superoxide dismutase (SOD) and catalase (CAT).

The nitrotetrazolium blue chloride (NBT) photoreduction method was used to determine the activity of SOD. The activity of SOD is defined as the amount of enzyme required to inhibit 50% of the photoreduction of NBT (the measured sample value should be about half of the value of the maximum tube). 30 μL of enzyme solution was mixed with 3 mL reaction mixture (14.5 mM methionine, 0.01 mM EDTA, 0.006 mM riboflavin and 0.075 mM NBT in 50 mM phosphate buffered saline). Then the mixture was placed in a light incubator and reacted under 4000 Lux of light for 20 min. The procedure was repeated with 30 μL phosphate buffered saline (50 mM) instead of enzyme solution a control. The SOD activity was measured at a wavelength of 560 nm. We calculated the SOD activity according to the following formula:

SOD activity (U·g⁻¹) = (OD_control − OD_reaction) × V × [(0.5OD_control × W × Vt)] ⁻¹

(4)

where Vt—the volume drawn for measurement (mL);

V—the total volume of the sample solution (mL);

W—the fresh weight of the sample (g).

The activity of CAT is determined by ultraviolet absorption method. One CAT activity unit is the amount of enzyme that reduces OD₂₄₀ by 0.01 per minute. Zero is adjusted with PBS control, and the activity of CAT is measured by the absorbance at 240 nm. The reaction mixture was composed of PBS (0.15 M, pH 7.0), concentrated H₂O₂, and 0.1 mL of enzyme solution. The calculation of the CAT activity was as follows:

CAT activity (U·g⁻¹) = ΔOD₂₄₀ × V × (0.01 × W × Vs × t)⁻¹

where ΔOD₂₄₀—the change in absorbance within the reaction time;

Vs—the volume drawn for measurement (mL);

V—the total volume of enzyme solution extracted (mL);

W—the fresh weight of the sample (g);

t—the reaction time (min).

2.6. Cu²⁺ Accumulation in Duckweed

Cu²⁺ concentration in duckweed and nutrient solution were both measured at 0, 1, 2, 4, 7, and 14 days. At each sampling point, 0.5 g of fresh leaves and roots of duckweed in the treatment group and the control group were carefully washed three times with deionized water and wiped with filter paper. Then, leaves and roots were cut into pieces after weighing and digested with 9 mL mixed acids (7 mL concentrated HNO₃ + 2 mL concentrated H₂O₂) in a microwave-accelerated digestion system (TANK40; SINEO, Shanghai, China). The digestion method was modified by previous methods with high digestion efficiency [28]. After the digestion, all the liquid was transferred and diluted with pure water to a final volume of 40 mL with 5% HNO₃ (v/v). The concentration of Cu in leaves, roots, and nutrient solution was measured by inductively coupled plasma mass spectrometry (ICP-MS) (Optima 7300 DV; PerkinElmer, Waltham, MA, USA). The concentration of Cu was quantified using calibration curves from standard solutions. The detection limits estimated from the 3-fold deviation of ten replicates at the lowest concentration (0.1 μg L⁻¹) was 0.03 μg L⁻¹.

2.7. BDE3 Quantification in Nutrient Solution

On 1, 4, and 14 days, 1–5 mL nutrient solution was taken and freezing-dried. BDE3 in nutrient solution was extracted by methanol and analyzed by high-performance liquid chromatography (HPLC) after filtration through 0.22 μm. HPLC measurements were performed with Agilent C18 column (250 mm × 5 mm, 4.6 μm) at 30 °C on an Agilent 1220 Infinity II system (Agilent Technologies, Waldbronn, Germany). About 10–50 μL of samples were injected and ran isostatically with a mobile phase containing 10% water and 90% methanol for 14 min. The flow rate was set at 1 mL min⁻¹. The UV absorbance was recorded at 220 nm and 254 nm.

2.8. Bioconcentration Factor (BCF) and Translocation Factor (TLF) of Cu²⁺

The BCF and TF of Cu²⁺ in S. natans (L.) were as shown in Equations (5) and (6) [29].

BCF = C_plant/C_solution

(5)

TLF = C_leaves/C_roots

(6)

C_plant and C_solution are the concentration of Cu²⁺ in duckweed, and the concentration of Cu²⁺ remained in nutrient solution, respectively. C_leaves and C_roots are the concentration of Cu²⁺ in leaves and the concentration of Cu²⁺ in roots of S. natans (L.), respectively.

2.9. Statistical Data Analysis

All values are expressed as the mean ± standard deviation (n = 3). Data analysis was performed by two-way analysis of variance among treatments followed by Duncan’s test. Statistical data analysis was performed via Prism 10.3.1 (464) (Graphpad Software Inc., San Diego, CA, USA). The difference was considered significant when the statistical probability p < 0.05.

3. Results

3.1. Effects on Photosynthetic Pigments

Similar responses from chlorophyll a, chlorophyll b, total chlorophyll and Carotenoid in S. natans to BDE3 exposure in different concentrations and together with Cu exposure were shown compared with the control group (Figure 1A–C). The responses from chlorophyll were different with incubation time. A significant increase in the content of chlorophyll a, chlorophyll b, and total chlorophyll was detected one day after exposure to BDE3, only in low (0.5 mg L⁻¹) (p < 0.05). The increasing chlorophyll content was detected on 1, and 4 in the treatment of BDE3 in high concentrations (5 mg L⁻¹) (p < 0.05). The addition of Cu together with BDE3 in a high dose (5 mg L⁻¹) did not show any significant difference compared with the control group through the whole incubation. A significant increase (p < 0.05) in chlorophyll content caused by BDE3 exposure in low concentration (0.5 mg L⁻¹) together with Cu before 4 days of incubation disappeared at the end compared with the control group.

A significant increase (p < 0.05) in carotenoid content was caused by BDE3 exposure in both concentrations (0.5 mg L⁻¹ and 5 mg L⁻¹) throughout the whole incubation. No significant effect on carotenoids was observed under BDE3 pollution in high concentration (5 mg L⁻¹) combined with Cu compared to the control group. A significant increase in carotenoid content was detected under pollution with a low concentration of BDE3 (0.5 mg L⁻¹) before 4 days incubation (p < 0.05) but fell back to the control level at the end of 14 days incubation (Figure 1D).

3.2. MDA

The MDA content in leaves showed differently with different incubation times. After one day of incubation, no significant difference was found in MDA content under single exposure with BDE3 in both doses (0.5 mg L⁻¹ and 5 mg L⁻¹). However, MDA content was significantly decreased under combined exposure with Cu and BDE3 in both doses (0.5 mg L⁻¹ and 5 mg L⁻¹) compared with the control group (p < 0.05). Four days later, the content of MDA in S. natans polluted with BDE3 in both concentrations was not different from the control group, which means the damage caused by BDE3 was resisted by physiological adjustments made by S. natans. However, MDA content was significantly increased under BDE3 exposure as well as combined stress of Cu and BDE3 in both doses after 14 days of incubation (p < 0.05). As the final product of lipid peroxidation, MDA can be regarded as a sensitive index to diagnose the oxidative injury of plants [30]. These observations indicated that with a longer time, peroxidation damage induced by BDE3 became obvious on the 14th day (Figure 2).

3.3. Antioxidative Enzyme Activities

With exposure to environmental pollutants, ROS, including free radicals and H₂O₂ produced in plants, could cause oxidative damage to the membrane system, such as macromolecule damage and lipids peroxidizing [31]. Antioxidant defense systems play a crucial role in eliminating prooxidants and scavenging free radicals during the long-term evolution of plants [32]. Antioxidant enzymes, including SOD and CAT, are able to combat ROS accumulation and, therefore, can be used as indicators of oxidative stress for plants [33]. After one day of incubation, there was no significant effect on the SOD activity of S. natans under individual BDE3 stress in higher or low doses and combined stress with Cu (Figure 3). On day one, a significant decline was observed in SOD activity after exposure to BDE3 in both concentrations as well as combined exposure of BDE3 (5 mg L⁻¹) and Cu compared with control (p < 0.05). However, SOD activity was not significantly different under exposure of BDE3 and combined with Cu from control on day 4 and day 14 (Figure 3A).

Different from the activity of SOD, the CAT activity of S. natans was not significantly different one day after BDE3 exposure and combined with Cu compared with control. After four days of incubation, individual stress of BDE3 in both concentrations showed a significant decline in CAT activity, while under combined stress, there were no marked effects on the activity of CAT. At the end of 14 days of incubation, no significant differences were observed in CAT activity after exposure to low BDE3 concentrations (0.5 mg L⁻¹). However, a higher BDE3 dose obviously increased CAT activity (p < 0.05). The activity of CAT also increased compared with the control group at BDE3 (0.5 mg L⁻¹) + Cu and BDE3 (5 mg L⁻¹) + Cu mixture (p < 0.05) (Figure 3B).

3.4. Cu Accumulation in S. natans

Under exposure for 14 days, Cu was absorbed by S. natans from the nutrient solution, as shown in Figure 4. The concentration of Cu in the aqueous phase decreased quickly from 92 ± 2.1 µg g⁻¹ to 24 ± 1.5 µg g⁻¹ during the first 7 days of incubation and then declined to 19 ± 0.56 µg g⁻¹ slowly during the second 7 days. At the same time, Cu was detected not only in roots but also in the leaves of S. natans. Cu concentration in roots was significantly higher than that in leaves of S. natans (p < 0.05). Accumulation of Cu in roots was increasing to 5.1 ± 0.68 µg g⁻¹ fw over time. However, the concentration of Cu in leaves reached the high value (1.3 ± 0.058 µg g⁻¹ fw) after 10 days of incubation and then declined to 0.97 ± 0.028 µg g⁻¹ fw slowly at the end of 14 days of incubation. As a result, the BCF for Cu increased to 0.31 ± 0.073 during 14 days of incubation, as shown in Figure 5. The TF for Cu in S. natans was less than 1, increasing to the top value as 0.76 ± 0.10 in 4 days of incubation and then decreasing to 0.19 ± 0.034 with time, as shown in Figure 5.

3.5. Removal of BDE3 by S. natans

The concentration of BDE3 in the aqueous phase was decreased to 3.8 × 10² ± 4 × 10⁴ mg L⁻¹ with an initial concentration of 0.5 mg L⁻¹ and 0.41 ± 0.028 mg L⁻¹ with an initial concentration of 5 mg L⁻¹ over time as shown in Figure 6. The removal rate of BDE3 with an initial S. natans concentration of 0.5 mg L⁻¹ was 72.5% after 4 days and 92.4% after 14 days, respectively. When the concentration of BDE3 pollution was 5 mg L⁻¹, BDE3 removal by S. natans was slowed down to only 50.8% after 4 days, but the final removal rate of BDE3 after 14 days was not significantly different from the BDE3 exposure with low concentration (0.5 mg L⁻¹), as shown in

Table 1. Removal rate of BDE3 in aqueous phase by S. natans during 14 days incubation.

Concentration of BDE3 (mg L−1)	Day 1	Day 4	Day 14
0.5	9.79%	72.5%	92.4%
5	2.49%	50.8%	91.9%

.

4. Discussion

As a typical annual floating freshwater fern plant, S. natans reportedly possesses a strong metal enrichment ability; however, it is sensitive to organic pollutants like pesticides [34]. The chlorophyll content is crucial to the level of plant photosynthesis, so it can be used to monitor the possible damage from pollutants [35]. In this study, we found there were positive effects on Chl content under BDE3 pollution, almost beginning day one, significantly increasing on day four, and returning to the control level after 14 days. There was also no negative effect for Chl content under combined stress of BDE3 in high dose and Cu compared with control. Based on the results from this study, there were no negative effects on photosynthesis from single BDE3 stress or combined stress together with Cu. 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), another PBDE congener with more bromo atoms than BDE3, is proven to reduce Chl content in duckweed fronds at a very low concentration (0.1~100 µg·L⁻¹). BDE47 was also reported with both direct and indirect toxicity to the PSII complex of duckweed only when the concentration of BDE47 accumulated at a certain high level [36]. As persistent organic pollutants (POPs), BDE47 was reported with endocrine disrupting toxicity and neurotoxicity together with much more difficulty to be degraded than BDE3 [37]. The opposite effects between BDE3 and BDE47 on Chl content could be easier removal and fewer residues from BDE3 in aqueous medium, which was proven in Figure 6.

With exposure to single or combined stresses from BDE3 and Cu, the present study showed no different change in MDA before 4 days but significantly increased MDA content of S. natans compared with control on day 14. A similar situation also happened with the activity of CAT, with a significant increase in CAT activity in S. natans on day 14. The possible reason for CAT activity change could be that severe damage S. natans suffered from accumulating ROS overwhelmed the defense system for a longer time. However, the activity of SOD under exposure to BDE3 and combined stress of Cu was opposite, with only a significant decrease on day one, which indicated that SOD was possibly more sensitive to BDE3 stress. CAT was possibly sensitive to the metabolites from BDE3 degradation. Another PBDE congener, BDE47, was also proved to be toxic for maize (Zea mays L.), including elevated MDA and activities of antioxidant enzymes [38]. BDE47 was also reported to be toxic on microalgae including population growth inhibition, oxidative stress, inhibition of photosynthesis [39,40]. The toxicity of PBDEs varies among microalgal species for the number of bromines. For example, the EC₅₀ for population growth of BDE-47 ranged from 13.5 μg L⁻¹ to >1000 μg L⁻¹ for a wide range of microalgae species [41,42,43].

Cu accumulation in duckweed was also reported with a concentration of 0.38 ± 0.02 mg g⁻¹ after 14 days of incubation [44], 63-fold higher than the result (6.07 ± 0.97 μg g⁻¹) in this study. The reason for this could be the original Cu concentration applied was much higher (50 mg/L). Duckweed was previously reported with high capability and efficiency of metal accumulation from polluted water and wetlands [34]. BCF indicates the concentration of contaminants in a floating fern plant against that in a polluted aqueous phase. If the BCF is over one, it means that the amount of contaminants deposited in a plant is more than that in aqueous phase. For a floating fern plant, the TF indicates the ratio of contaminants in roots to that in the aboveground part of the plant [45]. Both BCF and TF of Cu in this study were less than one, which indicates that Cu absorbed in duckweed was less than that in the aqueous phase, and most of Cu was stored in roots instead of being transported to leaves. It was considered a detoxification system and defense mechanism in plants that sequesters contaminants in the roots and prevents them from being translocated to the shoots in order to minimize the negative effects on photosynthesis by leaves [46]. Metal sequestration capacity is also found in large emergent macrophytes, such as Typha species and Phragmites australis [47]. The high removal rate of BDE3 could be the reason for degradation as well as being taken by duckweed. Another congener, BDE47 was reported to be bioaccumulated and degraded by Chlorella sp., mainly oxidatively transformed to hydroxylated products [40]. With a high BDE3 removal rate and tolerance of Cu, duckweed could act as good candidates for phytoremediation for e-waste polluted areas containing PBDEs and heavy metals with more attention to avoid secondary pollution from harvested S. natans.

5. Conclusions

This study investigated the possible toxicity of duckweed from BDE3 exposure in two concentrations and combined stress of BDE3 and Cu. The results showed no obvious toxicity of BDE3 and combined stress together with Cu to duckweed photosystem. However, oxidative damage from excessive ROS happened from increased MDA content and overactivated enzyme of SOD and CAT under BDE3 stress for a longer time. The co-existence of Cu could exacerbate the damage caused by BDE3 to S. natans in aqueous environment. Our results provide a greater understanding of the phytotoxicity of BDE3 as the lowest brominated congener of PBDEs. With the high detection frequency of BDE3 and Cu in water bodies, more attention should be paid to the toxicity to aquatic creatures.