Ecotoxicological effects of tungsten on celery (Apium graveolens L) and pepper (Capsicum spp.)

Plants incubation

Celery and pepper seeds were purchased from the Shouhe Seeds Industry Co., Ltd. (Shandong province, China). Before the experiment, the seeds were kept in a dark and dry environment. For all vegetables, 150 uniform size seeds were selected. The seeds were sterilized with 10% H₂O₂ for 20 min, then washed with ultra-pure water for three times. The seeds were then placed in a Petri dish (d = 90 mm) with a double layer of filter paper (150 seeds per dish) and wetted with 10 mL of ultra-pure water. The seeds were germinated at a greenhouse chamber for 7 days. The chamber temperature was maintained at 25 °C, with 16 h/per daylighting and the intensity set at 120 µmol/ (m²⋅ s).

Seedlings with the same growth conditions were selected and transplanted into hydroponic solution with different W concentrations. Hydroponic solutions were prepared by sodium W and the W concentration were 0, 10, 20, 40, and 60 mg/L. And the seedings growth condition was consistency with the seeds germination. Nine plants were placed in each treatment. The culture solution was renewed every three days to prevent changes in the form and concentration of substances in the hydroponic solution. Therefore, a total of 10 treatments (two plants at each of the 5 W concentrations) were harvested after14 days of hydroponics cultivation, washed, and left to be measured. Plants were divided into three tissues for the enzyme activities and W concentration measurement: leaves, stems, and roots.

Growth of plants determination

Each plant’s shoot height (SH) and root length (RL) were measured using a ruler. The fresh weight (FW) is obtained by weighing after removing water from the surface with absorbent paper.

Biochemical indicator determination

To evaluate the physiological changes of vegetables exposed to W, we measured a group of biomarkers commonly used in toxicological research. About 1.000 g of plant material was homogenated to measure them with 4.0 mL normal saline (0.9% sodium chloride). Then, the homogenate was centrifuged for 10 min at 3,500 rpm, and the supernatant was used for analysis. All the above indicators were measured using the test kits purchased from Nanjing Jiancheng Bioengineering Institute.

Specifically, membrane damage was detected in terms of “lipid peroxidation” using the biomarker of malondialdehyde (MDA) content measured using the test kit (A003-1, Nanjing Jiancheng Bioengineering Institute). Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and total antioxidant capacity (TAOC) were measured using the test kits of A001-1, A084-3-1, A007-1-1, and A015-1 (Nanjing Jiancheng Bioengineering Institute), respectively.

Pigments (chlorophyll ‘a’, ‘b’ and total chlorophyll)

The concentration of chlorophyll ‘a’ (C_a) and chlorophyll ‘b’ (C_b) was measured by following the standard acetone-ethanol mixed extraction method. The 0.5 g fresh sample (different tissue) was crushed and the contents were mixed with 10 ml of 80% acetone and preserved in the refrigerator for 5 h to allow complete digestion of chlorophyll pigments in the dark. The filtrate was prepared for analysis with a spectrophotometer at 663 and 645 nm wavelength for determination of chlorophyll a, b, (mg/g fresh weight), the sum of C_a and C_b represented the total chlorophyll as follows. (1)${\displaystyle C\text{\_}a=\left(12.21\times A_{663}\right)-\left(2.81\times A_{645}\right)}$ (2)${\displaystyle C\text{\_}b=\left(20.13\times A_{645}\right)-\left(5.03\times A_{663}\right)}$ (3)${\displaystyle \text{Total chlorophyll}=C\text{\_}a+C\text{\_}b}$ where A₆₄₆ andA₆₆₃ are the absorbance of the sample at 645 and 663 nm wavelengths. Reference materials (ASB-00003459-00A and SHAM_105119; Weiye Metrological Reference Materials Research Center, China) were used for quality control with a less than 5% deviation.

Integrated biomarker response (IBRv2) index

To comprehensively assess the physiological and biochemical parameters in response to tungsten exposure, the Integrated Biological Response version 2 (IBRv2) index was employed. This approach aids in the identification and screening of sensitive biomarkers for W contamination. The calculation of the IBRv2 index follows the methodology outlined in existing literature (Li et al., 2023a; Li et al., 2023b). The calculation of IBRv2 is as follows. (4)${\displaystyle Y_i=log\left(X_i/X_0\right)}$ (5)${\displaystyle Z_i=\left(Y_i-X_0\right)/\sigma }$ (6)${\displaystyle A_i=Z_i-Z_0}$

where X_i is the mean of the individual biomarker data, X₀ is the mean of the reference data, Y_i is the standardized biomarker response, Z_i is the mean of standardized biomarker response, μ represents the general mean of Y_i, σ is the deviation of Y_i, A_i is the biomarker deviation index; Z₀ is the mean of the reference biomarker data. The sum of the absolute values of the A_i calculated for each biomarker in each treatment studied: (7)${\displaystyle IBRv2=\sum _{i=1}^n\left|Ai\right|}$ where the n is the concentration gradient of W (n = 5).

Statistical analysis

All measurements were performed with three parallel samples in all case, the results are expressed in terms of mean and standard deviation (sd). One-way analysis of variance (ANOVA, p < 0.05) was used to determine the significant differences among treatments, and then LSD test was performed. Pearson correlation analysis were performed to analyze the correlation among morphophysiological indicators with Origin 2021 and the “ggplot2” package in R (version 4.1.1).

Effect of W of celery and pepper growth

The evaluation of plant root or shoot elongation is a widely employed method in environmental biomonitoring (Wang & Williams, 1990). Celery and pepper, due to their sensitivity and vitality, are commonly utilized to assess the toxicity of hazardous compounds (Altaf et al., 2022; Long et al., 2003; Mumtaz et al., 2022; Scoccianti et al., 2006). In this study, significant (p ≤ 0.05) linear relationships were observed between the inhibitory effects (%) on root and shoot elongation and the concentration of W. Table 1 outlines the effects of different W concentrations on the growth of celery and pepper. As the W concentration increased, the RL and SH of celery were inhibited. Notably, a stimulating effect on celery RL was observed at a 10 mg/L W concentration. In contrast, no such effect was observed for either RL and SH in pepper at any W concentration. When the W concentration reached 40 mg/L, a noticeable inhibition rate on RL was observed, with 27.6% and 43.7% for celery and pepper, respectively. Pepper’s RL was more sensitive to W concentration, with the inhibition rate slightly increasing as the concentration increased. Across the range of 0 to 40 mg/L, the SH of both plants was significantly inhibited (28.6% for celery and 25.1% for pepper). While the inhibition rate for celery’s SH remained relatively stable beyond 40 mg/L, the inhibition rate for pepper increased with higher concentrations, indicating greater sensitivity of pepper’s SH to W. W exhibited a low-promotion-high-inhibition effect on the FW of both plants. The inflection point for celery occurred at a concentration of 10 mg/L, beyond which inhibitory effects were observed, whereas the inflection point for pepper was at 20 mg/L. These results suggest that pepper may exhibit greater tolerance to W compared to celery. Furthermore, based on the SH indicator, both plants can serve as biomonitor organisms for W pollution, with pepper demonstrating a broader applicability range.

The dissolution of W is related to the decrease in oxygen availability and hydrogen ion concentration in water and/or soil, which may be the reason for delayed seedling growth caused by tungstate at plant toxicity levels (Strigul et al., 2005). Like most heavy metals, Adamakis, Panteris & Eleftheriou (2010) pointed out that W regulates cell formation and elongation by disrupting cortical microtubules, thereby inhibiting root growth. Celery and Pepper have not been reported as accumulators of W. Therefore, W may oxidize proteins in these plants, denature some important enzymes, and affect reactions related to substituting essential metal ions from biological components (Ghori et al., 2019). These changes trigger the loss of membrane integrity by altering basic plant metabolic pathways and stimulating the production of reactive oxygen species (Sallam et al., 2019).

Effect of W on pigments

Figure 1 illustrates the impact of W concentration on the chlorophyll content of both celery and pepper, including C_a and C_b. At a W concentration of 0 mg/L, the C_a and C_b content for celery were 0.45 and 0.29 mg/g, respectively, while for pepper, these values were 0.82 and 1.98 mg/g, respectively. For celery, W concentrations ranging from 0 to 40 mg/L had minimal effects on C_a and C_b. However, at a W concentration of 60 mg/L, both C_a and C_b were suppressed, exhibiting reductions of 48.9% and 72.4%, respectively, compared to the 0 mg/L condition.

In the case of pepper, the impact of W concentration on C_a was negligible, and C_a increased with increasing W concentration (with no significant difference) from 0 to 20 mg/L. For C_b in pepper, W exhibited a promoting effect at concentrations up to 20 mg/L; however, further increases in concentration led to the inhibitory impacts, following a trend similar to the changes in pepper’s FW. Thus, the influence of W concentration on C_b may play a determining role in pepper FW. The inhibitory effect of W on pigments in celery plants was stronger than that in pepper, influenced by plant characteristics, where the larger FW of pepper may have a significant role. In conclusion, W exerts a negative or negligible impact on the pigments’ content in plants, consistent with previous studies.

Effect of W on active substances (MDA)

Figure 2 depicts the changes in MDA content in various plant organs (root, stem, and leaf) under W stress. Compared to the control group (0 mg/L), cultivation in 60 mg/L W resulted in an increase of 67.3%, 225.8%, and 292.9% in MDA content for celery roots, stems, and leaves. For pepper, the MDA content in roots stems, and leaves increased by 229.1%, 120.5%, and 40.1%, respectively. MDA content reflects the degree of lipid peroxidation within tissues, indirectly indicating the extent of cellular damage (He et al., 2024). It can be inferred that W stress on celery roots is relatively low, while stress on leaves is significant. Conversely, W stress on pepper roots is more substantial, while stress on leaves is less pronounced. The changes in chlorophyll content in Fig. 1 also support the notion that celery leaves are more susceptible to W stress.

Oxidative damage is considered an important cellular effect and has become a commonly used technique for evaluating and comparing the toxicity of various pollutants. The W may induce oxidative stress and accumulate reactive oxygen species in the plant. In this study, the reactive oxygen species of plants were not measured, but the content of MDA can reflect changes in reactive oxygen species. When exposed to environmental stressors, plants secrete active substances such as proline, proteins, soluble sugars, MDA, etc., to increase cell osmotic pressure and enhance tolerance to stressors (Fang, Hou & Liang, 2021). In this study, the MDA content in plant organs increased with the rise in W concentration, but the magnitude of the increase varied between different plants and organs of the same plant. Both plants exhibited the lowest stem MDA content, attributed to their classification as vascular plants with a tough outer layer (bark) and a woody interior (xylem). Research indicates that plant stem tissues primarily transport nutrients and water, making them less susceptible to W stress.

Effect of W on the antioxidative defense system (CAT, SOD, POD and TAOC)

In this study, the antioxidant system of two plants under different W concentrations was investigated by analyzing SOD, CAT, POD, and TAOC in various plant organs (Fig. 3). Compared to the control group, W stress increased the SOD content in celery roots, stems, and leaves by 360.6–521.1%, 96.8–186.6%, and 152.9–248.7%, respectively. For pepper, the respective increments in SOD content in different tissues were 1.8–51.8%, 23.1–57.7%, and 5.9–20.4%. The POD content in celery tissues showed an increment ranging from 5.9% to 202.8%, while in pepper, the increment ranged from 14.8% to 157.6%. The increment of CAT content in celery tissues ranged from 61.2% to 2645.6%, while in pepper, the increment ranged from 9.1% to 890.0%.

Plants subjected to heavy metals or organic pollutants may generate reactive oxygen species such as hydrogen peroxide (H₂O₂), superoxide anion radical (⋅ O₂⁻), and hydroxyl radical (⋅OH) (Guo et al., 2022; Gao et al., 2022). To counteract the cellular damage caused by reactive oxygen species and maintain the homeostatic balance of plant cells, the defense system of the plant organism can eliminate reactive oxygen species through the production of enzymes (including CAT, POD, and SOD, etc.) or non-enzymatic antioxidants (such as glutathione and hydroxybenzene, etc.) (Jan et al., 2023). The study indicates that plants counteract heavy metal stress by increasing SOD, POD, and CAT to eliminate excess reactive oxygen species (Zhou, Wang & Inyang, 2021). Additionally, TAOC reflects the sum of the antioxidant capacity of enzymes and non-enzymatic components (Huang et al., 2023). As shown in Fig. 3D, the TAOC content in plant tissues is positively correlated with W concentration. Table 2 presents the correlation analysis between the antioxidative defense system in plant organs and W stress. The results indicate that the antioxidant capacity of enzymes in celery roots is not significantly correlated with W concentration, which may be attributed to the promoting effect of low concentration W on celery (Table 1). While, antioxidant capacity of enzymes in pepper roots has a significant correlation ship with W concentration. The antioxidant capacity of enzymes in pepper roots may be a primary contributor to pepper’s tolerance to W stress.

Evaluation of comprehensive phytotoxicity of W

The IBRv2 index combines the biological effects of multiple biomarkers and is used to screen for sensitive biomarkers. In this study, data from five biomarkers (MDA, SOD, POD, CAT, and TAOC) were standardized to create a radar chart (Fig. 4). It is important to note that in this section, our primary focus is on the roots and leaves of the plants. The baseline (depicted in red) represents the control group, and when the index is greater than 0, it indicates biomarker activation. All five biomarkers in the roots are activated for celery except for POD at a W concentration of 20, suggesting a deactivation. In the case of pepper, W at 10 mg/L does not significantly activate MDA, SOD, CAT, and POD in the roots, possibly due to pepper’s tolerance to low concentrations of W. Figs. 4E, 4F present the IBRv2 values for the five indicators. Generally, the indicator with the highest IBRv2 value is selected as the sensitive biomarker. The maximum IBRv2 values for celery roots and leaves are observed in SOD, while for pepper roots and leaves, they are observed in TAOC and POD, respectively. The levels of these antioxidant enzymes (SOD and POD) are crucial indicators in plant toxicity tests, closely related to the degree of oxidative damage. They are important markers for assessing the impact of environmental pollutants on plant growth (Zhang et al., 2018). TAOC represents the activity of the non-enzymatic antioxidant defense system. Our results reflect the potentially toxic effects of W on the celery and pepper, which complements research on the celery and pepper as bio-indicator.