Migration Behaviour of the Combined Pollutants of Cadmium and 2,2 ′ ,4,4 ′ ,5,5 ′ -Hexabrominated Diphenyl Ether (BDE-153) in Amaranthus mangostanus L. and Their Toxicity to A. mangostanus

: The effects of different concentrations of cadmium and 2,2 ′ ,4,4 ′ ,5,5 ′ -hexabrominated diphenyl ether (BDE-153) on the growth and related physiological and biochemical indexes of Amaranthus mangostanus L. (amaranth) were studied. The results showed that the presence of BDE-153 promoted the absorption of Cd by the amaranth and inhibited the migration of Cd from the roots to the shoots. At the same time, 0.1 mg/L of Cd had a synergistic effect on the migration of BDE-153, but 5 mg/L Cd inhibited the accumulation of BDE-153 in the aboveground part of the amaranth. In addition, the kinetics of the uptake of pollutants by the amaranth showed that both Cd and BDE-153 could be transported by amaranth, but Cd and BDE-153 were mainly enriched in the roots, and the presence of Cd may cause a lag in the uptake of BDE-153 in the shoots. Compared with the control group, the biomass of the amaranth affected by BDE-153 and a high concentration of Cd (5 mg/L) decreased by 30.2–49.5%, the chlorophyll content decreased by 43.0–60.3%, the Evans blue increased, and the MDA content was higher. The activities of superoxide dismutase (SOD) and catalase (CAT) also decreased with an increase in the BDE-153 concentration. This indicates that the interaction between BDE-153 and a high concentration of Cd (5 mg/L) is more toxic to amaranth than single Cd pollution. This paper provides the necessary data support for phytoremediation of heavy metal and organic compound pollution.


Introduction
Inappropriate methods of dismantling and recycling e-waste cause a large number of heavy metals and polybrominated diphenyl ethers (PBDEs) from the e-waste to enter the soil environment, causing the soil to be contaminated with heavy metal-polybrominated diphenyl ethers, which seriously threatens the soil's ecological environment and the health of its residents.Cd and 2,2 ′ ,4,4 ′ ,5,5 ′ -hexabrominated diphenyl ether (BDE-153) are frequently detected in e-waste and can be ingested by plants [1][2][3][4].Therefore, it is necessary to evaluate the effects of Cd and BDE-153 on the growth and related physiological and biochemical indexes of terrestrial plants in order to provide a basis for control of their combined pollution.
The heavy metals and organic pollutants produced by different pollution sources can enter the soil in different ways, such as through industrial wastewater discharge, waste stacking, atmospheric deposition, and sewage irrigation.As such, there are often refractory organic pollutants and heavy metals in the soil.Cd is a heavy metal that usually exists in e-waste and is easily introduced into the soil through human activities and industrial discharge.The Cd content in the soil of China's irrigation area ranged from 0.01 to 54.05 mg/kg, and the average Cd concentration in the soil of its mining and smelting areas was four times that of the irrigation area.The Cd content in urban farmland was relatively low, ranging from 0.05 to 3.15 mg/kg.The average content of Cd in the cultivated soil in China is about 0.27 mg/kg, which is higher than the background value (0.097 mg/kg) measured by China National Environmental Monitoring Centre in 1990 [5].This indicates that most of the agricultural soil suffer from Cd pollution, and Cd has the characteristics of stability, accumulation, and being difficult to eliminate after entering the soil.Therefore, after the soil is polluted with Cd, it is easy for Cd enrichment to be caused in crops [6].The long-term consumption of Cd-contaminated vegetables by humans will lead to a gradual increase in the Cd accumulated in the body [7][8][9].Long-term Cd exposure will cause symptoms such as bone atrophy, osteomalacia, and bone curvature, which will lead to natural fractures [10].The chemical properties of polybrominated diphenyl ethers are very stable and they are easily disassembled into the soil due to unreasonable e-waste discharge, resulting in polybrominated diphenyl ethers being adsorbed into the soil.For example, the average concentration of PBDEs in the soil of Shanghai, China, was 735 ng/g [11], and the average concentration of PBDEs in the soil of an e-waste treatment site in Taizhou, Zhejiang, China, reached 625 ng/g (dry weight, dw), which was higher than the concentration in the surrounding area (mean: 209 ng/g) [12].They experience persistent, long-distance migration and bioaccumulation in the environment and can enter the human body through the food chain and endanger human health [13], mainly manifested in liver damage, thyroid hormone interference, and neurodevelopment and reproductive system damage.For example, studies have found that exposure of fish embryos to PBDEs could lead to the increased malformation and mortality of the juvenile fish [14].2,2 ′ ,4,4 ′ ,5,5 ′ -hexabrominated diphenyl ether (BDE-153) is one of the most widely studied hydrophobic (log Kow = 7.9) PBDE congeners and has been detected in the air, soil, surface water, vegetables, and fruits, and even in human blood and serum [15][16][17][18][19][20].
There are complex interactions between heavy metals and organic pollutants in the environment, including in the adsorption behaviour, chemical processes (complexation and dissociation, redox, acid base reaction, precipitation and dissolution, etc.), and microbial processes [21].These interactions make heavy metals and organic compounds form more complicated combined pollution.Combined pollution not only has the toxicity of heavy metals or organic compounds but also can produce additive, synergistic, or antagonistic effects, thus changing the nature of the pollutants, biological toxicity, and environmental behaviour.Electronic waste and its dismantling sites contain large amounts of heavy metals and PBDEs and plants are vulnerable to compound pollution during growth and development.It was found that under the combined pollution of copper and BDE-209, the presence of copper could induce damage in the cell membranes in maize roots, resulting in an increase in the BDE-209 content in the aboveground parts of the plants [22].Similarly, the growth of pumpkin and the absorption and metabolism of BDE-209 were also affected by copper stress to varying degrees.Higher concentrations of copper inhibited the absorption and metabolism of BDE-209, while the hydroxylation of polybrominated diphenyl ethers was enhanced at the 50 mg/kg copper level [23].In addition, heavy metal pollution usually reduces the soil's enzyme activity and weakens soil respiration, thereby indirectly extending the half-life of organic pollutant degradation to a certain extent [24].The combined effects of combined pollution also affect plant growth, rhizosphere biological populations, and microecological processes, including the soil's physical and chemical properties and biological changes, thereby further affecting plants' physiological and biochemical properties and the uptake and transformation of pollutants [25].However, the toxicity of co-pollutants in Amaranthus mangostanus L. has still scarcely been reported.Hence, the environmental risk of co-pollutants is clearly elucidated to provide a basis for establishing a reasonable control technology to prevent the migration of co-pollutants into Amaranthus mangostanus L.
In this study, we exposed amaranth to Cd, BDE-153, and both Cd and BDE-153 under simulated hydroponic conditions to better analyse the effects of Cd and BDE-153 on the growth and related physiological and biochemical indexes of the amaranth under single and interactive stress.The photosynthetic parameters, antioxidant mechanisms, cell integrity, and time accumulation characteristics of the Cd and BDE-153 in the plants under Cd/BDE-153 combined pollution stress were studied, which provided data support for the study of the phytoremediation of heavy metal-polybrominated diphenyl ether combined pollution.As far as we know, this is the first time the physiological response of amaranth under the combined pollution of Cd and BDE-153 has been studied, and it is the first time that different concentrations of a heavy metal environment have been to found to have different effects on the absorption and transport of polybrominated diphenyl ethers in the same plant.

Hydroponic Design
In this study, the hydroponic approach of Jia et al. was followed [26].In short, the germinated amaranth was cultivated to have 3 or 4 fully developed leaves, and then amaranth seedlings of a uniform size were selected for exposure.Two amaranth plants were planted in each bottle and 180 mL of Hoagland nutrient solution at full concentration was added.Different concentrations of Cd (0-5 mg/L) and BDE-153 (0-20 µg/L) were added to each exposure bottle.The specific concentration settings are shown in Table S1.In addition, in order to study the time accumulation characteristics of Cd and BDE-153 in the plants under Cd/BDE-153 combined pollution stress, treatments with Cd and BDE-153 concentrations of 1 mg/L and 20 µg/L were set up, and samples were taken and analysed after a certain time.When all the plants had been harvested, the amaranth roots were rinsed with deionised water.We measured the fresh biomass in all treatments and stored all plants at −80 • C (DW-86L388A freezer, Qingdao Haier Electric Co., Ltd., Qingdao, China) until further analysis.

Cd Measurement
We weighed 0.1 g of the plant samples in a microwave digestion tank, added 6 mL of HNO 3 and 2 mL of H 2 O 2 to the mix, and let it stand for 10 min to end the initial reaction.It was then mixed and sealed and we placed the digestion tank in a microwave rapid digestion instrument (MDS-8G, Shanghai Xinyi Microwave Chemical Technology Co., Ltd., Shanghai, China).The program was set as shown in Table S2.The digestion solution was diluted to 35 mL with deionised water and filtered through a 0.45 µm water filter membrane.Finally, the Cd content was determined using liquid chromatography-inductively coupled plasma mass spectrometry (ELAN DRC-e, PerkinElmer, Shelton, CT, USA).

GC/MS Analysis
Wang et al. modified the extraction procedure of BDE-153 and debrominated diphenyl ethers (de-BDEs) from plant tissues [3].In short, 0.1 g of the plant samples were placed in an 8 mL sample bottle, BDE-154 was used as the internal standard, and dichloromethane was used as the extractant for ultrasonic extraction.Each sample was ultrasonically extracted three times with 2 mL of extractant for 30 min.All extracts were combined, concentrated to nearly dry using nitrogen blowing, and adjusted in volume to 1-2 mL with n-hexane.Improved commercial silica gel columns (CNW Technology, Shanghai, China) were used for cleaning.The eluent was concentrated again using nitrogen blowing, diluted to 0.4 mL with n-hexane, transferred into a gas chromatography injection bottle, and stored at 4 • C for GC-MS analysis.
The concentration of BDE-153 was analysed using an Agilent 7890A gas chromatography-mass spectrometry (GC/MS) negative ion chemical source (NCI).A DB-5HT (15 m × 0.25 mm × 0.10 µm, Agilent, Santa Clara, CA, USA) capillary column was used, and high purity helium (1 mL/min) was used as the carrier gas.Detailed information on the sample cleaning and measurement is provided in S2.
We defined the ratio of Cd/BDE-153 content in the aboveground part of the plants to the content in the underground part of the plants as the translocation factor (TF) of amaranth to Cd/BDE-153.

Determination of the Chlorophyll Content
The chlorophyll content was measured according to the Arnon formula [27].We weighed 0.2 g of the cut and mixed plant leaves into a 40 mL brown bottle, added 25 mL of 80% acetone, and extracted it in the dark for 24 h at room temperature until the leaves were all white.The absorbance of the extract at 645 nm and 633 nm was measured using a spectrophotometer with 80% acetone as the control and the chlorophyll concentration was calculated.The Evans blue absorption method can be used to determine the integrity of plants' root tip cell membrane [28].The approach of Yamamoto et al. was followed: the accumulation of superoxide anions (•O 2 − ) in the stained tissue was observed using confocal laser-scanning microscopy (excitation wavelength: 450-490 nm, radiation wavelength: 500-530 nm).The root fluorescence intensity was quantified using the Image J (1.8.0) software.Detailed information is provided in S3.

Antioxidant Enzymes
The activity of SOD was determined using the NBT photoreduction method and the number of enzymes with a relative percentage of 50% of SOD inhibiting the NBT photoreduction was used as an enzyme activity unit (U).
The CAT activity was determined using ultraviolet spectrophotometry.H 2 O 2 has a characteristic absorption peak at a 240 nm wavelength.CAT can decompose H 2 O 2 , so the absorbance of the reaction solution at 240 nm decreases with the reaction time.The enzyme activity unit (U) is the amount of D 240 reduced by 0.1 per minute.

Data Analysis
All the experimental data were the average of three parallel samples.The Excel 2013 and SPSS 18.0 statistical software were used to record, collate, and analyse the data.Significant differences between the data in the chart were tested using Duncan's test in one-way ANOVA.

Accumulation and Transfer of the Combined Pollutants in Amaranth
In the present study, BDE-153 and Cd were not detected in the roots, stems, or leaves of the plants in the control group (Cd 0 B 0 ).After eight days of the hydroponic exposure experiment, the content of pollutants in the plants after harvest was analysed to understand the uptake and transport of various pollutants into the plants.Amaranth is divided into three parts-root, stem, and leaf-to determine the content of pollutants in order to more accurately grasp the absorption and transfer of Cd and BDE-153 in amaranth.
According to the report [29], there are three main ways for plants to uptake pollutants after absorbing pollutants from soil or solution: the plant roots migrate them to the tissues and organs such as the stems and leaves through the tension mechanism of transpiration through the xylem.Via the leaf tissue of plants, pollutants enter from the atmospheric environment by being absorbed; contaminated dust settles on the surface of the plant and diffuses into the plant tissues and organs through the plant cuticle.Therefore, the effects of leaf uptake and atmospheric dust deposition on the experimental results can be ignored, that is, the accumulation of Cd and BDE-153 in plants from soil all occurs through the plant roots.As shown in Table S3 and Figure 1, after 8 days of hydroponics, amaranth root systems under different pollution treatments accumulated Cd from the nutrient solution.Regardless of Cd alone, the Cd content in the amaranth roots, stems, and leaves increased significantly with an increasing Cd concentration in the nutrient solution.For example, when treated with Cd alone, the Cd content in the roots, stems, and leaves of the 5 mg/L Cd treatment group was 6.4 times, 20.9 times, and 5.3 times that of the 0.1 mg/L Cd treatment group, respectively.The translocation factor decreased with an increase in the Cd concentration in the nutrient solution, indicating that the toxicity of Cd in amaranth increased with the increase in Cd concentration, which inhibited the accumulation of Cd in amaranth (Figure 1).

BDE-153 Content in the Amaranth as Affected by Cd
When the concentration of BDE-153 in the nutrient solution was 5 µg/L, with the increase in Cd from 0 to 5 mg/L, the accumulation of BDE-153 in the leaves gradually decreased from 62.5 ng/g to 39.0 ng/g, and the accumulation of BDE-153 in the stems gradually decreased from 69.8 ng/g to 25.3 ng/g.At the same time, the accumulation in the roots increased first and then decreased, and the transfer coefficient also gradually decreased from 0.09 to 0.05.The same pattern was observed when the concentration of BDE-153 in the nutrient solution was 20 µg/L.The accumulation of BDE-153 in the root system also decreased.This indicated that the migration of BDE-153 at a low concentration of Cd was influenced by a synergistic effect, but the presence of a high concentration of Cd inhibited the accumulation of BDE-153 in the aboveground parts of the amaranth.This may be because low concentrations of Cd stimulated the growth of amaranth and promoted the absorption of BDE-153 by the amaranth roots, but high concentrations inhibited the growth and metabolism of the plants, thereby reducing the absorption of BDE-153 by the plants.
The accumulation of BDE-153 in the roots, stems, and leaves of the amaranth increased significantly with an increase in the BDE-153 concentration in the nutrient solution when Cd was held at the same pollution level as used under the single treatment of BDE-153 and the interaction of Cd/BDE-153.
In addition, the transfer coefficient of BDE-153 in all treatment groups was very low (0.05-0.10), and the transfer coefficients gradually decreased from 0.09 and 0.1 to 0.05 with an increase in the BDE-153 concentration, indicating that the BDE-153 taken up by the roots of the amaranth mostly accumulated in them, and the transfer ability to the shoots was relatively low (Figure 2).The transport ability of PBDEs in plants may be related to Under the composite treatment of Cd and BDE-153, with an increase in the BDE-153 concentration in the nutrient solution, the Cd content in the roots, stems, and leaves of the amaranth was higher than that of the single Cd contamination treatment group, indicating that BDE-153 promoted the uptake of Cd by the amaranth to a certain extent, which may be the result of the cotransporter of Cd and BDE-153 pollutants at a certain concentration.However, the translocation factor of Cd decreased with an increase in the BDE-153 concentration, which indicated that the interaction or competition between BDE-153 and Cd had a certain inhibitory effect on the transfer of Cd from the amaranth roots to the aboveground parts, and the higher the concentration of BDE-153 was, the more obvious the inhibitory effect was.In addition, the translocation factors of all treatment groups were between 0.17 and 0.44, which were less than 1, indicating that the absorption and enrichment ability of Cd in the underground parts of the amaranth was much greater than that in the aboveground parts.
Combined with Table S3 and Figure 1, it can be concluded that the addition of BDE-153 will promote the uptake of Cd from nutrient solution by amaranth but will inhibit the transfer of Cd from the roots to the shoots in amaranth to a certain extent.This may be due to the addition of low-concentration pollutants, which promotes the secretion of carbohydrates and organic acids in the roots and changes the composition of the root exudates, thus improving the uptake of Cd from the solution by the plants [3].At the same time, studies have shown that BDE-153 can cause damage to plants' cell walls, resulting in an increased uptake of pollutants by the plant roots [30].Under the interaction of pollutants, the toxic effect of the pollutants on the roots of amaranth is greater, which leads to the failure of the roots to transport the heavy metal Cd to the shoots.

BDE-153 Content in the Amaranth as Affected by Cd
When the concentration of BDE-153 in the nutrient solution was 5 µg/L, with the increase in Cd from 0 to 5 mg/L, the accumulation of BDE-153 in the leaves gradually decreased from 62.5 ng/g to 39.0 ng/g, and the accumulation of BDE-153 in the stems gradually decreased from 69.8 ng/g to 25.3 ng/g.At the same time, the accumulation in the roots increased first and then decreased, and the transfer coefficient also gradually decreased from 0.09 to 0.05.The same pattern was observed when the concentration of BDE-153 in the nutrient solution was 20 µg/L.The accumulation of BDE-153 in the root system also decreased.This indicated that the migration of BDE-153 at a low concentration of Cd was influenced by a synergistic effect, but the presence of a high concentration of Cd inhibited the accumulation of BDE-153 in the aboveground parts of the amaranth.This may be because low concentrations of Cd stimulated the growth of amaranth and promoted the absorption of BDE-153 by the amaranth roots, but high concentrations inhibited the growth and metabolism of the plants, thereby reducing the absorption of BDE-153 by the plants.
The accumulation of BDE-153 in the roots, stems, and leaves of the amaranth increased significantly with an increase in the BDE-153 concentration in the nutrient solution when Cd was held at the same pollution level as used under the single treatment of BDE-153 and the interaction of Cd/BDE-153.
In addition, the transfer coefficient of BDE-153 in all treatment groups was very low (0.05-0.10), and the transfer coefficients gradually decreased from 0.09 and 0.1 to 0.05 with an increase in the BDE-153 concentration, indicating that the BDE-153 taken up by the roots of the amaranth mostly accumulated in them, and the transfer ability to the shoots was relatively low (Figure 2).The transport ability of PBDEs in plants may be related to phloem transporters because there are fewer phloem transporters in amaranth, which affects the transfer of BDE-153 from the ground [31].

Variation in Kinetics of Pollutant Uptake by Amaranth
Under the combined pollution of Cd (1 mg/L) and BDE-153 (20 µg/L), the concentration of Cd in the amaranth roots increased with time, and the accumulation amount tended to be stable after 96 h, reaching 1615.2 mg/kg after 144 h.The logarithmic growth rate of Cd in the amaranth roots increased at first and reached the maximum at 24 h, then gradually decreased, indicating that the amaranth roots rapidly absorbed and accumulated Cd within 24 h and then the absorption rate gradually decreased.After 72 h, the logarithmic growth rate was basically stable and tended to 0, indicating that the absorption and accumulation of Cd in the roots basically stopped.The absorption of Cd into the stems and leaves of the amaranth also showed the same pattern.Similarly, the content of

Variation in Kinetics of Pollutant Uptake by Amaranth
Under the combined pollution of Cd (1 mg/L) and BDE-153 (20 µg/L), the concentration of Cd in the amaranth roots increased with time, and the accumulation amount tended to be stable after 96 h, reaching 1615.2 mg/kg after 144 h.The logarithmic growth rate of Cd in the amaranth roots increased at first and reached the maximum at 24 h, then gradually decreased, indicating that the amaranth roots rapidly absorbed and accumulated Cd within 24 h and then the absorption rate gradually decreased.After 72 h, the logarithmic growth rate was basically stable and tended to 0, indicating that the absorption and accumulation of Cd in the roots basically stopped.The absorption of Cd into the stems and leaves of the amaranth also showed the same pattern.Similarly, the content of BDE-153 in the amaranth also showed the same pattern, but in contrast, the maximum absorption rate in the amaranth stems and leaves occurred at 72 h, later than the maximum absorption rate of the amaranth roots at 24 h, indicating that the absorption of BDE-153 into the amaranth shoots has a certain lag.
In addition, according to Figure 3, the concentration ratios of Cd and BDE-153 in the amaranth leaves and stems and the roots were 1.0:2.3:6.9 and 1.0:1.2:25.4,respectively, after 144 h of exposure, indicating that Cd and BDE-153 were mainly accumulated in the roots of amaranth and that BDE-153 had a weaker transport capacity to the stems and leaves and was mostly enriched in the roots.After 72 h of the experiment, the absorption of Cd by the amaranth gradually stabilised, and the shoots of the amaranth reached the maximum absorption rate of BDE-153, indicating that the presence of Cd may have been the reason for the lag in BDE-153 absorption in the aboveground parts of amaranth.

Amaranth Biomass Affected by the Combined Pollutants of Cd and BDE-153
The changes in the fresh weight of amaranth under the combined effect of C BDE-153 are shown in Figure S1.It can be seen from the figure that the fresh wei amaranth under the condition of single Cd pollution, with an increase in the Cd co tration, increased first and then decreased.The variation in the amaranth's fresh w ranged from 8.82 to 13.96 g.For BDE-153 pollution alone, an increase in the BDE-15 centration had no significant effect on the fresh weight of amaranth.Studies have s that the proliferation activity of stem cells will be enhanced when stimulated by oxi stress [32], DNA damage [33], and heat stress [34], so our previous studies suggeste this may be due to the anti-oxidative stress of plants at low concentrations of BDE-1 In addition, BDE-153 is difficult for amaranth to absorb due to its hydrophobicity, may also be one of the reasons why BDE-153 has less effect on the fresh weight o ranth [35].
Under the composite pollutant condition, when the Cd concentration reach highest concentration of 5 mg/L (Cd3), the biomass of the amaranth decreased signifi with the increase in BDE-153 concentration (Figure S1).This may be due to the fa

Amaranth Biomass Affected by the Combined Pollutants of Cd and BDE-153
The changes in the fresh weight of amaranth under the combined effect of Cd and BDE-153 are shown in Figure S1.It can be seen from the figure that the fresh weight of amaranth under the condition of single Cd pollution, with an increase in the Cd concentration, increased first and then decreased.The variation in the amaranth's fresh weight ranged from 8.82 to 13.96 g.For BDE-153 pollution alone, an increase in the BDE-153 concentration had no significant effect on the fresh weight of amaranth.Studies have shown that the proliferation activity of stem cells will be enhanced when stimulated by oxidative stress [32], DNA damage [33], and heat stress [34], so our previous studies suggested that this may be due to the anti-oxidative stress of plants at low concentrations of BDE-153 [3].In addition, BDE-153 is difficult for amaranth to absorb due to its hydrophobicity, which may also be one of the reasons why BDE-153 has less effect on the fresh weight of amaranth [35].
Under the composite pollutant condition, when the Cd concentration reached the highest concentration of 5 mg/L (Cd 3 ), the biomass of the amaranth decreased significantly with the increase in BDE-153 concentration (Figure S1).This may be due to the fact that the high concentration of Cd disrupted the plasma membrane structure of the amaranth root system, making it easier for BDE-153 to enter the cells and increasing the toxicity of the composite pollutant in the amaranth.In addition, under the condition of combined pollution, the fresh weight of the amaranth increased first and then decreased, which was the same as the results under single Cd pollution, indicating that a low concentration of Cd (0.1 mg/L) could stimulate the growth of the amaranth.When the concentration of Cd increased to 1 mg/L and 5 mg/L, the toxic effect of the Cd on the amaranth increased, resulting in a significant decrease in the biomass of the amaranth.
The synergistic and antagonistic effects of organic matter and heavy metals on plants have been reported in some studies.For example, it has been reported that combined pollution of low concentrations of PAH (50 + 50 mg/kg with phenanthrene + pyrene in 1:1 proportion) and Cd (10 or 50 mg/kg) can reduce the toxicity of Cd in Juncus subsecundus and promote its growth compared with Cd pollution alone [36].In addition, studies have found that the germination rate of wheat under the combined pollution of lead (100 mg/L) and low amounts of fluoranthene (0.2 mg/L) is higher than that of low levels of fluoranthene (0.2 mg/L) pollution alone [37].
However, from our present study, it was found that the synergistic and antagonistic effects of organic matter and heavy metals on the plants depend on the concentration of these pollutants.When the concentration of Cd was low, the addition of BDE-153 had little effect on the fresh weight of plants, while at high concentrations of Cd, BDE-153 showed a synergistic effect in its toxicity in the plants.

Chlorophyll Content Affected by the Combined Pollutants of Cd and BDE-153
The chlorophyll content of plants reflects the level of photosynthesis to a certain extent.A decrease in the chlorophyll content decreases the photosynthetic capacity of the plant and vice versa.The strength of photosynthesis will directly affect the normal metabolic activities of plants.By observing the changes in chlorophyll content in plants, the growth status of plants can be reflected [38].It can be seen from Figure S1 that when amaranth is stressed by Cd, its chlorophyll content decreases to varying degrees with the increase in Cd concentration, among which the chlorophyll content under 5 mg/L (Cd 3 ) treatment decreases by 43.0% compared with the control group.From the data of the plant-accumulated pollutants, it can also be seen that with an increase in the Cd concentration, the Cd content in the amaranth leaves increases from 58.3 mg/kg (Cd 1 B 0 ) to 307.4 mg/kg (Cd 3 B 0 ), indicating that the accumulation of heavy metal Cd exerts obvious damage on amaranth's mesophyll cells.With an increase in the BDE-153 concentration, the content of BDE-153 in the amaranth leaves also increased from 62.5 ng/g (Cd 0 B 1 ) to 215.7 ng/g (Cd 0 B 2 ), indicating that amaranth was subjected to BDE-153 stress, but there was no significant effect on Amaranthus's chlorophyll content.
Under the interaction of the highest Cd concentration of 5 mg/L and BDE-153, although the chlorophyll content decreased significantly with the increase in the BDE-153 concentration, the content of BDE-153 in the amaranth leaves was lower than that at other Cd concentrations.For example, the content of BDE-153 in the amaranth leaves of the Cd 3 B 2 group was 92.8 ng/g, which was the lowest value under the same BDE-153 concentration and different Cd concentrations.This indicates that the effect of Cd on the chlorophyll content of amaranth is greater than that of BDE-153 on the chlorophyll content.The synergistic effect of BDE-153 and Cd on the reduction in plant chlorophyll under a high Cd concentration may also be due to the fact that the high concentration of Cd causes severe damage to the leaf cells in amaranth, resulting in easier uptake of BDE-153 and BDE-153 stress.Evans blue staining can be used to determine plasma membrane integrity [28].It can be seen from Figure 4 that the root tips of the amaranth had obvious Evans blue absorption under Cd/BDE-153 interaction, indicating that Cd and BDE-153 had certain destructive effects on the plasma membrane of the root tips.Under Cd or single BDE-153 pollution conditions alone, the absorption of Evans blue increased with the increase in the pollutant concentration.Under the Cd/BDE-153 interaction, the uptake of Evans blue by the amaranth root tips gradually increased with the increase in the Cd contaminant concentration, and the degree of damage to the plasma membrane in the amaranth root tips by Cd increased.
Polybrominated diphenyl ethers have been shown to increase the production of ROS in animal cells [39] and plant cells [4], leading to oxidative stress.Therefore, the production of superoxide anions was detected using a laser confocal scanning microscope (Figure 4b).
It can be seen from Figure 4 that under the treatment of Cd and BDE-153, the fluorescence intensity of •O2 − in the root tips of the amaranth gradually increased with the increase in the pollutant concentration, indicating that plants produced a large amount of •O2 − under Cd/BDE-153 stress.When the concentration of the pollutants is low, the •O2 − fluorescence is mainly distributed in the root epidermis, while when the concentration of the pollutants is high, •O2 − fluorescence is distributed in the root tip and meristem.It can be seen from Figure 4d that Cd pollution alone and BDE-153 pollution alone increased the level of hydrogen peroxide in the roots.The hydrogen peroxide levels were further elevated in the amaranth roots under composite pollution conditions compared to single pollution.For example, the fluorescence intensity of hydrogen peroxide in the roots increased by 66.1% under 5 mg/L Cd pollution.This is similar to the results of confocal laser scanning microscopy, indicating that Cd and BDE-153 indeed lead to an increase in ROS production and are synergistic in combined pollution.Under the interaction of Cd/BDE-153, with an increase in BDE-153 concentration, the absorption of Evans blue increased only at a high concentration of Cd (5 mg/L).Hence, the toxicity to the plasma membrane of the amaranth root tips caused by the highest concentration of Cd when interacting with BDE-153 is greater than that of Cd alone, which is consistent with the results of the BDE-153 and Cd accumulation in the amaranth, amaranth biomass, and chlorophyll contents.Polybrominated diphenyl ethers have been shown to increase the production of ROS in animal cells [39] and plant cells [4], leading to oxidative stress.Therefore, the production of superoxide anions was detected using a laser confocal scanning microscope (Figure 4b).
It can be seen from Figure 4  fluorescence is mainly distributed in the root epidermis, while when the concentration of the pollutants is high, •O 2 − fluorescence is distributed in the root tip and meristem.It can be seen from Figure 4d that Cd pollution alone and BDE-153 pollution alone increased the level of hydrogen peroxide in the roots.The hydrogen peroxide levels were further elevated in the amaranth roots under composite pollution conditions compared to single pollution.For example, the fluorescence intensity of hydrogen peroxide in the roots increased by 66.1% under 5 mg/L Cd pollution.This is similar to the results of confocal laser scanning microscopy, indicating that Cd and BDE-153 indeed lead to an increase in ROS production and are synergistic in combined pollution.

Antioxidant Enzyme Activity Affected by the Combined Pollutants
Plants can produce a large number of reactive oxygen species (ROS) under the stress of pollutants, causing damage to the biological macromolecules of the plant cell membranes, which, in turn, causes membrane lipid peroxidation [40,41].In all the treatments, the activities of two major antioxidant enzymes, SOD and CAT, were measured in the amaranth roots.
The changes in the SOD and CAT activities in plants under Cd and BDE-153 pollution stress are shown in Figure 5.Under the condition of Cd alone, the SOD activities and CAT activities achieved the highest values at concentrations of 0.1 and 1 mg/L of Cd, respectively, and then decreased with an increase in the Cd concentration.Under the condition of BDE-153 alone, the SOD and CAT activities increased with an increase in the BDE-153 concentration.Under the interaction of Cd with BDE-153, the SOD and CAT activities decreased with an increase in the BDE-153 and Cd concentration, indicating that the SOD and CAT activities were inhibited under the interaction.In addition, compared with single Cd pollution, Cd combined with BDE-153 made the SOD and CAT activities decrease with an increasing BDE-153 concentration, indicating that the combined pollution enacted greater stress on the root system than with Cd pollution alone.l.Sci.2024, 14, x FOR PEER REVIEW 11 of

Antioxidant Enzyme Activity Affected by the Combined Pollutants
Plants can produce a large number of reactive oxygen species (ROS) under the str of pollutants, causing damage to the biological macromolecules of the plant cell me branes, which, in turn, causes membrane lipid peroxidation [40,41].In all the treatmen the activities of two major antioxidant enzymes, SOD and CAT, were measured in t amaranth roots.
The changes in the SOD and CAT activities in plants under Cd and BDE-153 polluti stress are shown in Figure 5.Under the condition of Cd alone, the SOD activities and C activities achieved the highest values at concentrations of 0.1 and 1 mg/L of Cd, resp tively, and then decreased with an increase in the Cd concentration.Under the conditi of BDE-153 alone, the SOD and CAT activities increased with an increase in the BDE-1 concentration.Under the interaction of Cd with BDE-153, the SOD and CAT activities d creased with an increase in the BDE-153 and Cd concentration, indicating that the SO and CAT activities were inhibited under the interaction.In addition, compared with sin Cd pollution, Cd combined with BDE-153 made the SOD and CAT activities decrease w an increasing BDE-153 concentration, indicating that the combined pollution enact greater stress on the root system than with Cd pollution alone.
The reactive oxygen species scavenging system had limited protective effects on t plant cells under pollutant stress.The elevated SOD and CAT activities under low co centrations of Cd pollution and BDE-153 stress indicate increased phytotoxicity in the a aranth, as the concentration of reactive oxygen species in the body increased.SOD beg to convert reactive oxygen species into hydrogen peroxide, so it was not surprising th the activity of CAT also increased [42].When the concentration of Cd reached 1 mg/L, t SOD activity and CAT activity began to decline, indicating that under the condition o high concentration of Cd pollution, the plant regulation exceeded its own threshold, a the SOD activity and CAT activity were inhibited, which was a precursor of a plant p soning reaction.

Discussion
In the soil of many contaminated sites, different types of pollutants exist at the sam time.We studied the effects of Cd and BDE-153 on the growth and related physiologi and biochemical indexes of amaranth under single and interactive stress.It was found th The reactive oxygen species scavenging system had limited protective effects on the plant cells under pollutant stress.The elevated SOD and CAT activities under low concentrations of Cd pollution and BDE-153 stress indicate increased phytotoxicity in the amaranth, as the concentration of reactive oxygen species in the body increased.SOD began to convert reactive oxygen species into hydrogen peroxide, so it was not surprising that the activity of CAT also increased [42].When the concentration of Cd reached 1 mg/L, the SOD activity and CAT activity began to decline, indicating that under the condition of a high concentration of Cd pollution, the plant regulation exceeded its own threshold, and the SOD activity and CAT activity were inhibited, which was a precursor of a plant poisoning reaction.

Discussion
In the soil of many contaminated sites, different types of pollutants exist at the same time.We studied the effects of Cd and BDE-153 on the growth and related physiological and biochemical indexes of amaranth under single and interactive stress.It was found that the addition of a low concentration of Cd (0.1 mg/L) promoted the uptake of BDE-153 by the amaranth roots, while the addition of a high concentration of Cd (5 mg/L) inhibited the absorption of BDE-153 by the amaranth roots.Similarly, studies have found that adding copper at a concentration greater than 50 mg/kg to the soil will inhibit the absorption and metabolism of BDE-209 by pumpkins [23], indicating that different concentrations of heavy metals in the environment will indeed lead to different uptake and transport of PBDEs by plants.We believe that the difference in this result is due to the fact that low concentrations of Cd promote the growth of amaranth and thus promote the uptake of BDE-153 by the amaranth roots, while high concentrations cause the opposite.Interestingly, copper exposure caused root damage but enhanced the penetration and translocation of BDE-209 and BDE-47 in maize [19].This indicates that not only will the concentration of heavy metals in the environment of plants affect the uptake and transport of pollutants by plants but the difference in plant species will also lead to different situations, because the transport capacity of polybrominated diphenyl ethers in plants may be related to phloem transporters [31].There are fewer phloem transporters in amaranth, which affects the transport of BDE-153.
Previous studies have shown that there is a significant positive correlation between the root lipid content and root polybrominated diphenyl ether concentration, which indicates that different root lipid contents also cause differences in plants' uptake and transport of polybrominated diphenyl ethers.Our subsequent research will make up for this.In addition, Wang et al. found that the concentration of BDE-209 in the aboveground parts using the soil experiment much lower than that using the hydroponic method and BDE-209 was more difficult to absorb and transport in maize than BDE-47, indicating that we also need to use a variety of PBDEs and expand the soil experiment to better explore the migration behaviour of Cd and PBDE combined pollution in amaranth [22].

Conclusions
In summary, the accumulation of BDE-153 in the amaranth roots was significantly affected by the Cd dose.A low concentration of Cd promoted the uptake of BDE-153 by the amaranth roots and a high concentration inhibited the uptake of BDE-153 by the amaranth roots.Interestingly, the interaction of BDE-153 with Cd also inhibited the migration of Cd from the roots to the shoots of the amaranth.According to the kinetic results on the pollutants accumulated by amaranth, the concentrations of Cd and BDE-153 in the roots, stems, and leaves of the amaranth increased with prolongation of the culture time, but the rate of increase decreased with prolongation of the culture time.In addition, the interaction of BDE-153 with a high concentration of Cd (5 mg/L) significantly reduced the biomass and chlorophyll content, enhanced the toxicity to the root tip plasma membrane, and increased the antioxidant enzyme activity compared to with Cd or BDE-153 alone.Overall, this study provides data support showing that reasonable environmental risk assessment and pollution control technology should also consider the concentration range of combined pollution.

3. 1 . 1 .
Cd Content in the Roots, Stems, and Leaves of Amaranth Affected by BDE-153

Figure 3 .
Figure 3. Changes in Cd and BDE-153 concentrations in amaranth roots, stems, and leaves ov under combined pollution conditions.(a,b) represent the concentration changes in Cd and BD in amaranth, respectively.(c,d) represent the logarithmic growth rates of Cd and BDE-153 i ranth, respectively.

Figure 3 .
Figure 3. Changes in Cd and BDE-153 concentrations in amaranth roots, stems, and leaves over time under combined pollution conditions.(a,b) represent the concentration changes in Cd and BDE-153 in amaranth, respectively.(c,d) represent the logarithmic growth rates of Cd and BDE-153 in amaranth, respectively.
that under the treatment of Cd and BDE-153, the fluorescence intensity of •O 2 − in the root tips of the amaranth gradually increased with the increase in the pollutant concentration, indicating that plants produced a large amount of •O 2 − under Cd/BDE-153 stress.When the concentration of the pollutants is low, the •O 2 −