Phytoremediation of cadmium-trichlorfon co-contaminated water by Indian mustard (Brassica juncea): growth and physiological responses

Abstract In this study, the morphological and physiological responses of Brassica juncea to the stresses of Cadmium (Cd) and trichlorfon (TCF), and the phytoremediation potential of B. juncea to Cd and TCF were investigated under hydroponics. Results showed that Cd exhibited strong inhibition on biomass and root morphology of B. juncea as Cd concentration increased. The chlorophyll a fluorescence intensity and chlorophyll content of B. juncea decreased with the increased Cd concentration, whereas the malondialdehyde and soluble protein contents and superoxide dismutase activity increased. TCF with different concentrations showed no significant influence on these morphological and physiological features of B. juncea. The biomass and physiological status of B. juncea were predominantly regulated by Cd level under the co-exposure of Cd and TCF. B. juncea could accumulate Cd in different plant parts, as well as showed efficient TCF degradation performance. A mutual inhibitory removal of Cd and TCF was observed under their co-system. The present study clearly signified the physiological responses and phytoremediation potential of B. juncea toward Cd and TCF, and these results suggest that B. juncea can be used as an effective phytoremediation agent for the Cd-TCF co-contamination in water. NOVELTY STATEMENT Combined pollution of heavy metals and pesticides in agricultural water systems is a common phenomenon. In previous phytoremediation studies, limited information is available on the co-contamination of heavy metals and pesticides. In this study, we aimed to investigate the concentration-dependent morphological and physiological characteristics of B. juncea under single and co-stress of Cd and trichlorfon (TCF), and the phytoremediation ability of B. juncea to remove Cd and TCF through hydroponic experiment. B. juncea exhibited efficient removal performance of Cd and TCF alone and simultaneous exposure of both pollutants, indicating that B. juncea is an effective phytoremediation agent for the Cd-TCF co-contaminated water.


Introduction
A large amount of heavy metals and pesticides enter the environment with the rapid development of industry and agriculture, thus forming combined pollution in various environmental media (Chen et al. 2014;Qie et al. 2023).Compared to single pollution, combined pollution is more likely to pose a greater threat to the environment (Zhang et al. 2012).Industrial wastewater discharge, sewage irrigation, and the application of agrochemicals including plant nutrients, fertilizers, and pesticides, can result in a sharp increase in heavy metal concentration in the agricultural environment (Rattan et al. 2005;Dmuchowski et al. 2011;Lopes et al. 2011).In aquatic ecosystems, the bioaccumulation, toxicity, and persistence of heavy metals can cause serious pressure on water self-purification.Pesticides are chemical or biological agents that aim to destroy, prevent, or mitigate pests.Although many pesticides can be degraded naturally (Zhang et al. 2010, Herrero-Hernandez et al. 2020), the original pesticides and their degradation products can lead to the contamination of water, soil, and living organisms and thus may become a public health problem (Escoto et al. 2019).
Cadmium (Cd) has attracted increasing attention due to its strong toxicity at even low concentrations (Valderrama et al. 2012).Cd stress can affect the growth and physiological characteristics of plants, such as causing leaf chlorosis and withering, inhibiting biomass and root elongation, and altering metabolic processes and nutrient uptake and distribution (Wang et al. 2008;Gomes et al. 2012).Moreover, Cd also causes severe threats to human health through the food chain (Adhikari et al. 2018).Trichlorfon (TCF) is a widely used organophosphorus insecticide and used as an agricultural pesticide to control pests on crops, a human medicine to combat internal parasites, and an ectoparasiticide in livestock and aquaculture (Li et al. 2011;Chang et al. 2013).However, the long-term widespread use and overuse of trichlorfon pose risks to public and environmental health, for example, causing histopathological injury (Mataqueiro et al. 2009), the toxic effect on hepatocytes (Woo et al. 2018) and immune system (Baldissera et al. 2018), and other biochemical effects (Woo et al. 2018) of animals and even humans.Therefore, conducting remediation research on Cd and TCF combined pollution is of great significance for protecting the agricultural ecological environment.
Phytoremediation, which uses plants for the degradation, stabilization, and uptake of pollutants, is an effective technology to control pollution because of its cheap, effective, and environment-friendly properties, as well as no secondary pollution (Bharti and Kumar Banerjee 2012;Lin and Li 2016).As an important technology of bioremediation, phytoremediation has shown great potential in aquatic ecological remediation and wastewater treatment for combined polluted water of heavy metals and organics.Indian mustard (Brassica juncea) is a well-acknowledged Cd hyperaccumulator because of its fast growth and high biomass production and has been widely used in the field of heavy metal pollution remediation (Bauddh and Singh 2012;Mohamed et al. 2012).In addition to its advantages in heavy metal pollution remediation, B. juncea can also remove organic pollutants from the environment through uptake, rhizodegradation, and other pathways (Bartha et al. 2010;Ramamurthy and Memarian 2012).Up to now, there are few reports on the application of B. juncea in the remediation of the co-contamination of heavy metals and pesticides, especially in the treatment of contaminated water.Therefore, the present study aimed to investigate the concentration-dependent morphological and physiological characteristics of B. juncea under single and co-stress of Cd and TCF, and the phytoremediation ability of B. juncea to remove Cd and TCF through hydroponic experiment.This study can provide a theoretical basis for the phytoremediation of co-contaminated water of heavy metals and organic compounds by B. juncea.

Materials
Standard TCF (99% purity) was obtained from Aladdin Reagent Co., Ltd.(Shanghai, China).Except for chromatography-grade methanol, all other reagents used in this study were of analytical grade.The Cd used in this study was Cd(NO 3 ) 2 Á4H 2 O.

Plant growth conditions and treatments
B. juncea seeds were surface sterilized with 0.5% sodium hypochlorite solution for 20 min, and then sprouted in a humid condition for 48 h at 28 C in the dark.The germinated seeds were evenly sown in sterilized quartz sands and then cultivated with the order of quarter-, half-, and full-strength Hoagland solution every 2 days, respectively, in a greenhouse at the schedule of 122 mmolÁm À2 Ás À1 light/dark 14/10 h and 25/15 C. One week later, healthy and consistently tall B. juncea seedlings were selected and transplanted into 1.5-L plastic basins, and cultivated using the fullstrength Hoagland solution for ten days.During this period, the cultivation solution was renewed every three days.Afterward, the cultivation solution was replaced with the fresh full-strength Hoagland solution containing Cd and TCF with different concentrations, and the concentrations (mgÁL À1 ) of pollutants in different treatments were shown in Table 1.All treatments were performed in three repetitions.The cultivation solution was replenished one time per day with the full-strength Hoagland solution (without pollutants) throughout the experiment period.The plant tissues and cultivation solutions of different treatments were collected after two-week exposure.And all the plant samples needed to be washed to remove residual pollutants adhered to the surface of the plants when harvesting.
Taking into account the natural dissipation of TCF, the cultivation solution containing TCF (without B. juncea plantation) was set as the treatment of natural remediation to measure the natural dissipation rate of TCF during the entire experiment period.

Morphological measurements
Dry weights of shoots and roots were measured through drying at 105 C for 24 h.Root morphology was analyzed as follows: the roots were washed thoroughly with distilled water and then placed in a rectangular transparent tray filled with distilled water.The rootlets were softly separated from each other.Then, the roots of each plant were scanned by Imagery Scan Screen (EPSON Expression 1680).The measured root indicators including the total root length (cm), surface area of root (cm 2 ), the average diameter of root (mm), root length per volume (cm/cm 3 ), total volume of root (cm 3 ), number of root tips, number of root forks and number of root crossings.These indicators were calculated by the WinRHIZO image analysis software (2005b, Regent Instruments Inc., Canada).

Determination of physiological parameters
Chlorophyll a fluorescence determination Chlorophyll a (Chl a) fluorescence was tested by a portable plant efficiency analyzer (PEA, Hanstech, UK).The leaves of the plants were darkly adapted for 20 min before the measurement (Schansker et al. 2005).In the testing process, the excitation light intensity was 1500 mE m À2 s À1 , and the recording time was 2 s.The obtained fluorescence data by PEA show a four-step Chl a fluorescence transients (Zhang et al. 2018).In this pot, the fluorescence signals at 50 ms (Ostep), 2 ms (J-step), and 30 ms (I-step) were denoted as Fo (original fluorescence), F J , and F I , respectively, and the peak signal (P-step) was recorded as Fm (maximal fluorescence).
The variable fluorescence-to-maximal fluorescence (Fv/Fm) ratio calculated by (Fm-Fo)/Fm, which is considered as not only an indicator of maximal photochemical quantum yield but also an indicator demonstrating the maximal light energy conversion efficiency of photosystem II (PSII).In addition, RC/CSo is the density of photosystem II (PSII) reaction centers in each excited cross-section.ABS/RC is the light energy absorbed by the unit PSII reaction center.
ETo/TRo is the efficiency in that a trapped excitation can move an electron into the electron transport chain beyond Q A -(primary plastoquinone electron acceptor of PSII).ETo/ABS is the probability that an absorbed photon moves an electron into the electron transport chain beyond Q A -.And TRo/RC is the energy captured by the unit PSII reaction center for the reduction of Q A -.All these parameters are calculated as stated by the O-J-I-P test (Strasserf et al. 1995).
Determination of chlorophyll, malondialdehyde and soluble protein contents and superoxide dismutase activity Chlorophyll was measured by the method of Lichtenthaler and Wellburn (1983).0.2 g of leaf was extracted with 80% (v/v) acetone overnight.Then the absorbance of the extract was measured at the wavelength of 663 and 646 nm with a UV-visible spectrophotometer (UV-2600, SHIMADUZ, Japan), respectively.
Malondialdehyde (MDA) was determined according to the method of Smeets et al. (2005).Briefly, 1.0 g of leaf was ground into homogenate with 10% trichloroacetic acid (TCA).The homogenate was centrifuged for 10 min at 5000 rpm. 2 mL of 0.6% TBA was added into an equal amount of supernatant, heated at 100 C for 15 min, and then rapidly cooled and centrifuged.The absorbance of the mixture was measured at 532, 600, and 450 nm, respectively.
Soluble protein was measured via Coomassie Brilliant Blue G-250 staining and using bovine serum albumin as a standard, as described by Bradford (1976).Briefly, 0.2 g of the leaf was ground into homogenate with phosphate buffer (pH ¼ 7) and centrifuged at 3000 rpm for 10 min.To 1 mL aliquot of the supernatant, 5 mL of Coomassie Brilliant Blue reagent was added.Shaking well and holding for 2 min, then the absorbance of the mixture was measured at 595 nm.
Superoxide dismutase (SOD) activity was determined according to the method of Dhindsa et al. (1981).Briefly, 0.50 g of the leaf was ground into homogenate with icy 10 mL of 50 mM phosphate buffer (pH ¼ 7.8) and centrifuged at 4000 rpm for 15 min.Then, 0.1 mL of obtained supernatant was mixed with the following reagent: 1.5 mL of 50 mM phosphate buffer (pH ¼ 7.8), 0.3 mL of 130 mM methionine, 0.3 mL of 750 mM nitroblue tetrazolium, 0.3 mL of 100 mM EDTA-Na 2 , 0.5 mL of distilled water, and 0.3 mL of 20 mM riboflavin.After reacting for 20 min under the white light, the absorbance was measured at 560 nm.
Cd content analysis 0.1 g of dry plant samples were digested with HNO 3 -HClO 4 (3:1, v/v) at 200 C in a graphite digestion furnace until the digestion solution was transparent.The cultivation solutions containing Cd were digested at 80 C plus with HNO 3 .The Cd contents in plants and cultivation solutions were measured using an atomic absorption spectrophotometer (TAS-990, Persee, Beijing, China).The Cd translocation factor (TF) is calculated as: Cd concentration in the shoots (mgÁkg À1 )/Cd concentration in the roots (mgÁkg À1 ).The bioaccumulation factor (BAF) of Cd was calculated as: Cd content in plant tissues (mgÁkg À1 )/initial Cd content in the cultivation solution (mgÁg À1 ).

TCF residue analysis
The residual contents of TCF and DDVP in the cultivation solution were measured with high-performance liquid chromatography (HPLC) system (UltiMate TM 3000, Thermo Scientific, USA).Before entering the HPLC system analysis, the cultivation solution was passed through a 0.22-mm polytetrafluoroethylene membrane filter.The mobile phase was methanol/distilled water (50:50, v/v) injected at a flow rate of 1.0 mLÁmin À1 .The detection wavelength was 206 nm.The column temperature was 30 C and the injection volume was 20 mL.The peak times of TCF and DDVP were 3.9 and 8.8 min, respectively.

Statistics analysis
All the data are expressed in mean ± standard deviation.A one-way analysis of variance (ANOVA, p < 0.05) on ranks with Duncan's multiple comparisons through SPSS 21.0 software was used to analyze the significant differences among treatments.

Results
Effects of Cd and TCF on the growth and root morphology of B. juncea B. juncea showed obvious morphological changes induced by increased Cd stress (Figure S1).The B. juncea biomass in different treatments is presented in Figure 1.Compared to the control, Cd alone exposure showed a significant inhibitory effect on the B. juncea biomass.A gradual decrease in the shoot and root biomass was observed with increasing Cd levels in the solution, and the higher Cd concentration (10 and 50 mgÁL À1 ) significantly decreased the biomass of shoots and roots.TCF alone exposure slightly increased the biomass with the increase of TCF level, indicating that TCF may promote the growth of B. juncea.In Cd-TCF treatments, the shoot and root biomass decreased with increased Cd concentration and showed a similar trend to Cd alone treatments.Comparatively, Cd influenced dominant stress on B. juncea growth than that of TCF.
Figure 2 shows the root morphological characteristics of B. juncea in different treatments.High Cd concentration (50 mgÁL À1 ) exhibited a significant inhibition on total length, surface area, total volume, number of tips, number of forks, and number of crossings, and a significant enhancement in root length per volume.There was no significant difference in the average diameter among treatments (Figure 2c), implying that Cd stress had little effect on root diameter.TCF alone exposure with different concentrations showed no significant effect on root morphology.In Cd-TCF treatments, Cd dominantly affected root morphology.

Effects of Cd and TCF on Chl a fluorescence transients of B. juncea
Chl a fluorescence transients are used for assessing the effects of pollutants on the PSII electron transport of plants.As shown in Figure 3, it is obvious that all treatments showed a polyphasic rise in fluorescence induction (O-J-I-P) (Figure 3).The fluorescence yield at phases J, I, and P obviously decreased under Cd alone exposure with increased Cd concentration (Figure 3a), while no significant difference was observed under TCF alone exposure with different concentrations (Figure 3b).In Cd-TCF treatments, the fluorescence yield decreased with increased Cd concentration, irrespective of the TCF levels (Figure 3c-e).Moreover, a slightly enhanced fluorescence yield was observed with the increase of TCF concentration under the same Cd level (Figure 3c-e).
The selected photosynthetic parameters of B. juncea in different treatments are shown in Table 2. Cd inhibited the Fv/Fm, RC/CSo, ETo/TRo, and ETo/ABS of B. juncea in Cd alone and Cd-TCF treatments, and this inhibition increased with the increased Cd concentration.Conversely, the ABS/RC and TRo/RC increased under the Cd stress.TCF alone exposure with different concentrations showed no significant influence on these photosynthetic parameters.
Effects of Cd and TCF on the chlorophyll, MDA, and soluble protein contents and SOD activity of B. juncea Low Cd concentration (2 mgÁL À1 ) showed no significant impact on the chlorophyll content of B. juncea in Cd alone and Cd-TCF treatments, but the higher Cd concentration (10 and 50 mgÁL À1 ) significantly decreased chlorophyll content compared to the control Figure 4a).The presence of Cd and TCF in different treatments increased the MDA content to varying degrees Figure 4b).In Cd alone and Cd-TCF treatments, the MDA and soluble protein contents, and SOD activity increased as the Cd concentration increased (Figure 4b-d).The high Cd concentration (50 mgÁL À1 ) significantly increased the MDA and soluble protein contents and SOD activity compared to the control.TCF alone exposure with different concentrations showed no significant influence on the chlorophyll and soluble protein contents, and SOD activity (p < 0.05).
Uptake and bioaccumulation of Cd in B. juncea tissues B. juncea showed an excellent ability to uptake and accumulate Cd, especially in the roots (Figure 5).In the treatments of Cd alone exposure, the Cd contents in the roots were 2535, 5687, and 11951 mgÁkg À1 at the Cd concentration of 2, 10, and 50 mgÁL À1 in cultivation solutions, while the Cd contents in the shoots were 220, 639, and 3071 mgÁkg À1 , respectively.In Cd-TCF treatments, TCF with different concentrations showed no significant impact on the uptake of the lower Cd concentration (2 and 10 mgÁL À1 ) by B. juncea.However, in the presence of 50 mgÁL À1 Cd, the Cd accumulation significantly decreased in the shoots and increased in the roots with the increase of TCF concentration (from 10 mgÁL À1 to 200 mgÁL À1 ).
The Cd translocation factor (TF) and bioaccumulation factor (BAF) of B. juncea, and Cd removal rate from water were calculated to evaluate the phytoremediation ability of B. juncea to Cd in water (Table 3).The TF value significantly increased with the increased Cd concentration in the treatments of Cd exposure alone and Cd with the lower concentration of TCF (10 and 50 mgÁL À1 ), while the TF value increased firstly and then decreased with the increased Cd level at the presence of 200 mgÁL À1 TCF.The BAF value was higher in the roots than in the shoots, and the BAF value of shoots and roots all decreased with the increased Cd concentration in different treatments.The highest Cd removal rate was obtained in the treatment of 2 mgÁL À1 Cd exposure alone.The Cd removal rate significantly decreased with the increased Cd concentration in the treatments of Cd exposure alone and Cd with 10 mgÁL À1 TCF, but there was no significant difference in the treatments of Cd with the higher concentration of TCF (50 and 200 mgÁL À1 ).Moreover, a higher Cd removal rate was observed in Cd-alone treatments than that in Cd-TCF treatments at the same Cd level.

Degradation of TCF by B. juncea
TCF can naturally dissipate, especially in neutral or alkaline conditions.As shown in Figure 6a, it was clear that B. juncea significantly promoted the degradation of TCF in all treatments, and the maximum TCF removal efficiency (84.60%) was obtained under 10 mgÁL À1 TCF alone exposure.In Cd-TCF treatments, Cd inhibited the degradation of TCF, and this inhibitory effect increased as the Cd concentration increased.DDVP is the primary degradation product of TCF.As shown in Figure 6b, the DDVP production was higher in the presence of B. juncea than that of natural degradation in all treatments, which proved that B. juncea could accelerate the degradation of TCF to DDVP. Figure 6c illustrated the total residues of TCF and DDVP in different treatments.Obviously, B. juncea showed a higher TCF degradation efficiency than that of natural degradation.

Discussion
The response characteristics of growth and physiology in plants under the stress of contaminants are an important aspect to evaluate the feasibility of phytoremediation.In this study, growth inhibition of B. juncea is the primary sign under Cd stress, Pietrini et al. (2015) and Ahmad et al. (2016) also found consistent results of heavy metals inhibiting plant growth under hydroponics.TCF alone exposure with different concentrations (10, 50, and 200 mgÁL À1 ) slightly increased B. juncea biomass compared with control, which could be attributed that TCF might promote plant growth as an exogenous stimulator or be transformed into nutrients for plant growth under plant metabolism, thereby increasing plant biomass.In the treatments of Cd alone and Cd-TCF co-exposure, B. juncea biomass decreased with the increased Cd concentration (from 2 to 50 mgÁL À1 ) whether TCF existed or not.And no significant difference in plant biomass was observed between Cd-TCF co-exposure and Cd alone exposure at the equal Cd level, indicating that the growth of B. juncea predominantly depended on the Cd concentration under the stress of Cd-TCF coexistence.
The chlorophyll content of B. juncea decreased with the increased Cd concentration regardless of the presence of TCF (Figure 4a).Previous studies also found that heavy metal stress decreased chlorophyll content in various plant species (Bilal Shakoor et al. 2014;Ahmad et al. 2016).MDA level is an indirect indicator of cell membrane damage, which is positively correlated with ROS concentration (Chan et al. 2016).The excess ROS are produced beyond the elimination capacity of the antioxidative system in plants under abiotic stress, thus causing membrane lipid peroxidation (Habiba et al. 2015).MDA is the final decomposition product of membrane lipid peroxidation, and its content can reflect the damage degree of plants under environmental stress (Zhang et al. 2020).Cd and TCF with different concentrations increased the MDA content of B. juncea, and the highest MDA content was obtained at 50 mgÁL À1 Cd  (Figure 4b), implying that ROS production at this stress level was possibly beyond the antioxidative capacity of B. juncea, thereby might affect the normal growth or even survival of B. juncea.As important components of plant cells, proteins are easily damaged under adverse environments (Hu et al. 2015).Soluble protein is important osmoregulation substance in plants, and its increase can enhance the water retention ability of plant cells and play a protective role in cell biofilms (Khalilzadeh et al. 2020).In this work, the soluble protein content increased as the Cd concentration increased (Figure 4c), which might be due to the adaptation of B. juncea to Cd stress.A similar result was found by Yılmaz and Parlak (2011) with Groenlandia densa under Cd stress.Plants tend to produce more ROS under high Cd load, thus, more oxidative stress induces the increased protein content.The antioxidant enzymes in plants can scavenge cytotoxic ROS.The SOD activity of B. juncea increased under Cd stress, and a remarkable SOD rising was observed under 50 mgÁL À1 Cd exposure (Figure 4d).The overexpression of antioxidant enzymes in plants under heavy metal stress may be an effective means for the survival of plants with high metal accumulation ability (Habiba et al. 2015;Tauqeer et al. 2016).SOD is the first defense line against oxidative stress as it can convert superoxide (O 2 .-) to H 2 O 2 and O 2 , and H 2 O 2 could be efficiently eliminated by SOD (Waszczak et al. 2018).The overproduction of SOD has been shown to enhance plants' tolerance to oxidative stress (Gupta et al. 1993).As a result, the increased SOD activity may be one of the possible mechanisms of Cd tolerance in B. juncea.Previous studies have demonstrated a positive correlation between metal stress and SOD activity in plants within the affordable range of metal concentration (Feigl et al. 2013;Riaz et al. 2017).
B. juncea is widely acknowledged as a potential Cd hyperaccumulator.In this work, B. juncea showed a strong ability to accumulate Cd, particularly in the roots.The metal bioaccumulation capacity of various plant species is an important trait of phytoremediation under hydroponics, given the adequate supply of nutrients, water, and bioavailability of heavy metals (Pietrini et al. 2015).The Cd accumulation in B. juncea increased with the increased Cd level (Figure 5   TCF could naturally dissipate in water, and B. juncea exhibited excellent degradation ability to TCF (Figure 6a).The TCF removal efficiency decreased with the increased Cd level under both natural degradation and phytoremediation, indicating that Cd inhibited the removal of TCF.Linking with the results of Cd removal in the presence of TCF, we concluded that TCF and Cd mutually inhibit each other's removal by B. juncea.DDVP is the primary degradation intermediate of TCF.The DDVP concentrations in water were higher under phytoremediation than those under natural degradation in different treatments (Figure 6b), which demonstrated that B. juncea can degrade TCF to DDVP.The results of the total residues of TCF and DDVP were also lower under phytoremediation than that under natural degradation (Figure 6c), indicating that B. juncea can not only degrade TCF to DDVP, but also degrade DDVP to more simple compounds.Previous study found that TCF can be phytodegraded to DDVP, dimethyl hydrogen phosphate and methyl dihydrogen phosphate (Talebpour et al. 2006).

Conclusions
In this study, B. juncea performed well in treating Cd-TCF co-contaminated water and showed a high tolerance of up to 50 mgÁL À1 Cd, but the biomass and physiological status of B. juncea deteriorated rapidly at such a level.TCF showed little influence on the growth and physiology of B. juncea even at a high concentration (200 mgÁL À1 ).Cd accumulation in different B. juncea tissues increased with the increased Cd concentration.B. juncea exhibited efficient removal performance of Cd and TCF alone and simultaneous exposure of these pollutants.Moreover, a mutual inhibition of Cd and TCF was observed in the process of their removal, especially in both high concentrations.Overall, B. juncea shows good potential as a phytoremediation tool for the Cd-TCF co-contamination in water.

Figure 1 .
Figure 1.Shoot and root biomass of B. juncea under different treatments.Values are means ± standard deviation (n ¼ 6).Means with different letters indicate that values are significant different at p < 0.05.

Figure 2 .
Figure 2. Total root length (a), surface area of root (b), average diameter of root (c), root length per volume (d), total volume of root (e), number of root tips (f), number of root forks (g), and number of root crossings (h) of B. juncea under different treatments.Values are means ± standard deviation (n ¼ 6).

Figure 3 .
Figure 3. Variance of Chl a fluorescence transients of B. juncea under different treatments.Values are means of three replicates.

Figure 4 .
Figure 4. Variance of chlorophyll content (a), MDA content (b), soluble protein content (c) and SOD activity (d) of B. juncea under different treatments.Values are means ± standard deviation (n ¼ 3).Means with different letters indicate that values are significant different at p < 0.05.

Figure 5 .
Figure 5. Contents of Cd in B. juncea tissues under different treatments.Values are means ± standard deviation (n ¼ 3).Means with different letters indicate that values are significant different at p < 0.05.
), which showed the high Cd tolerance of B. juncea.The TF value of Cd in B. juncea increased with the increased Cd concentration in the treatments of Cd alone and Cd with the lower concentration of TCF (10 and 50 mgÁL À1 ), indicating that B. juncea can hyper-accumulate Cd from water, and the low TCF concentration showed no significant effect on Cd uptake by B. juncea.However, a decreased TF value was observed with the increased Cd concentration in the treatments of Cd with a high concentration of TCF (200 mgÁL À1 ), which suggested that a competition existed between TCF and Cd with both high concentrations in water, and TCF thus inhibited the Cd uptake by B. juncea.The Cd removal rate in water significantly decreased with the increased Cd concentration under Cd alone exposure, suggesting that B. juncea showed good remediation potential for treating low-concentration Cd wastewater.The Cd removal rate in Cd-TCF treatments was lower than in Cd alone treatments at the same Cd level, which further confirmed that TCF inhibited the removal of Cd.

Figure 6 .
Figure 6.Residues of TCF and DDVP in water under different treatments.Values are means ± standard deviation (n ¼ 3).

Table 1 .
Single and combined treatments of Cd and TCF in this study.

Table 2 .
Variance of selected photosynthetic parameters of B. juncea under different treatments.Values are means ± standard deviation (n ¼ 3).Means with different letters indicate that values are significant different at p < 0.05.

Table 3 .
Cd translocation factor (TF), bioaccumulation factor (BAF) of B. juncea tissues, and Cd removal rate in water under different treatments.Values are means ± standard deviation (n ¼ 3).Means with different letters indicate that values are significant different at p < 0.05.