Lead and cadmium toxicity effects on the Pinus roxburghii seed germination and early seedling growth in different environments

Experimental setup

The experiments were conducted in two distinct environments: a heterogeneous (nursery) and a homogeneous (laboratory) setting. The open-air heterogeneous experiment occurred at the Bandagai Nursery in the Dir Lower district of Khyber Pakhtunkhwa, Pakistan, at 71.8195001°E longitude and 34.7460993°N latitude. Meanwhile, the homogeneous experiment was conducted under standard laboratory conditions at the Department of Botany, University of Malakand, Khyber Pakhtunkhwa, Pakistan. The experimental trials were commenced in April 2021. Pb and Cd were applied four times, at 12-day intervals, with equivalent water supplementation provided to control seedlings. After one and a half months of growth, the seedlings’ photosynthetic and physiological characteristics were measured. The seedlings were harvested at the end of June 2021, with roots, shoots, and leaves harvested separately for morphological analysis.

Germination assay

The same germination method was employed for nursery and laboratory experiments. P. roxburghii seeds were obtained from a local nursery. Seeds of uniform size were surface-sterilized for one minute using a 0.01% mercuric chloride (HgCl₂) solution before germination. Any residues of the hazardous solution were then thoroughly removed by washing with distilled water. Stock solutions of heavy metals—lead nitrate (Pb(NO₃)₂) and cadmium chloride (CdCl₂) were prepared in distilled water at concentrations of 0, 30, 60, 90, 120, 150, and 180 mg/L, respectively, based on a prior study by Pavel et al. (2013). For the germination of P. roxburghii seeds, plastic pots (16 cm length and 8 cm diameter) filled with natural soil were utilized. Each treatment included three duplicates of nine seeds, which were watered with 100 mL of appropriate heavy metal solution (Pb (NO₃)₂ and CdCl₂) per pot, while controls received the same amount of distilled water. Germination was recorded over 12 days, and the germination percentage (GP) was calculated using the following formula. ${\displaystyle \text{GP}=\frac{GerminatedSeed}{TotalSeed}\times 100.}$

Determination of morphological indices

The effects of Pb and Cd concentrations on the growth of seedlings were assessed under heterogeneous (nursery) and homogeneous (laboratory) conditions. After a four-week germination period, seven seedlings were randomly selected from each replicate to measure root, shoot, and leaf lengths, as well as to calculate the mean number of leaves, fresh and dry weights of the seedlings.

Root and shoot lengths were measured using a ruler. In the laboratory, the seedlings were initially placed in an oven at 100 °C for 30 min, followed by drying at 70 °C until a constant weight was achieved before being weighed.

Stress Tolerance Index

This step aimed to determine the tolerance threshold of (P. roxburghii) to varying levels of Pb and Cd. We measured and weighed both metal-treated seedlings and control seedlings, which were grown in a nursery and under laboratory conditions. Plant materials were initially dried in an oven at 100 °C for 30 min to assess their dry biomass. Subsequently, they were further dried at 70 °C until they reached a constant weight and then weighed. The stress tolerance index (STI) (%) for the seedling was computed using the following formula based on the dry biomass:

${\displaystyle \text{STI}=\frac{DryweightofseedlingdevelopedonmediawithPb{\left(N{O}_3\right)}_{2}orCdC{l}_2}{DryweightofseedlingdevelopedonmediadevoidofPb{\left(N{O}_3\right)}_{2}orCdC{l}_2}\times 100.}$

Determination of the biochemical indices

From the three replicates of each treatment, three mature fresh leaves were indiscriminately selected from both heterogeneous and homogeneous grown seedlings and placed separately into an ice box to measure each biochemical index.

Proline

Fresh needles (0.2 g) were homogenized in 10 mL of 3% sulfosalicylic acid solution in test tubes and then centrifuged at 10,000 rpm for 6 min to adjust the supernatant. Equal volumes (2 mL each) of the supernatant, ninhydrin reagent, and glacial acetic acid were mixed in the test tube. The solution was then shaken and heated in the water bath at 100 °C for 1 hour. After heating, the solution was cooled, and 4 mL of toluene was added to each test tube (Bates, Waldren & Teare, 1973), with toluene serving as a blank sample. The absorbance of the supernatant was measured using a UV spectrophotometer at 520 nm. The proline concentration was determined using a proline standard curve.

Sugar

The sugar content in the needles of P. roxburghii (Sarg.) was determined using the phenol sulphuric acid method described by Dubois et al. (1956). To each test tube, 10 mL of 80% methanol and 0.2 g of needle samples were added. The solution was heated in a water bath at 95 °C for 10 min and then cooled to room temperature. Subsequently, 0.5 mL of the sample was mixed with 1 mL of 18% phenol and incubated at room temperature for 1 hr. Then, 2.5 mL of H₂SO₄ was supplemented, and the mixture was vortexed for 1 min. The absorbance of the sugar content was measured at 490 nm using a spectrophotometer in one mM cuvettes against the blank.

Photosynthetic pigments

One gram of fresh pine needles was cut into small fragments and ground with a mortar and pestle to determine chlorophyll content. Then, 20 ml of 80% acetone and 0.5 g of MgCO₃ powder were added, and the mixture was ground further according to the method of Kamble et al. (2015). The mixture was incubated at 4 °C for 3 hrs. After incubation, the mixture was centrifuged at 1,000 rpm for 10 min. The supernatant solution was transferred to a 100 mL volumetric flask and diluted to 100 ml with 80% acetone. The absorbance of the supernatant was measured for chlorophyll a/b at 663 nm and 645 nm using a spectrophotometer, with 80% acetone used as a blank sample. Chlorophyll content was determined using the appropriate formula given below:

Chlorophyll a$=\frac{12.7\left(A663\right)-2.69\left(A645\right)\times V}{1000\times W}$

Chlorophyll b $=\frac{22.9\left(A645\right)-4.68\left(A663\right)\times V}{1000\times W}$

Total chlorophyll $=\frac{20.2\left(A645\right)+8,02\left(A663\right)\times V}{1000\times W}$.

Carotenoid

The carotenoid content was estimated following the method described by Lichtenthaler (1987). Needles of P. roxburghii (Sarg.) were cut and extracted with 100% acetone using a pistil and mortar. The pigment extracts were centrifuged in glass tubes for 3 to 5 min. The resulting extracts were immediately analyzed using spectrophotometry.

Data analysis

The study employed a completely randomized design (CRD) for data collection to ensure unbiased distribution of treatments across experimental units. The collected data were analyzed using one-way analysis of variance (ANOVA), a robust statistical method that assesses significant differences among group means. To further explore pairwise comparisons between treatment groups, a post-hoc test was conducted, which helps identify specific groups that differ significantly from each other. Pearson correlation coefficients were calculated to determine the strength and direction of relationships between variables, specifically assessing the effects of Pb and Cd treatments. The statistical analyses, including ANOVA with post-hoc tests and correlation assessments, were performed using OriginLab version 24.0b (OriginLab, Northampton, MA, USA), a powerful software for data visualization and statistical analysis. The significance level was set at P < 0.05, ensuring that only results with a probability of occurrence of less than 5% were considered statistically significant.

Germination assessment in the heterogeneous and homogeneous environment

The Pb and Cd treatments did not significantly affect the final seed germination rate, as all seeds germinated, regardless of the environmental conditions or the concentration of Pb and Cd. The only noticeable impact of these metal treatments was a negligible delay in seed germination compared to the control (Table 1). At higher concentrations (180 mg/L), germination began 5–6 days after seeding, whereas in the control, control group, germination started 3–4 days after seeding.

Growth assessment in a heterogeneous environment

Pb and Cd considerably inhibited the morphological traits of P. roxburghii seedlings at various concentrations (30–180 mg/L). At 180 mg/L of Pb, root, shoot, and leaf lengths decreased by 55.40%, 72.24%, and 42.71%, respectively. At 30 mg/L of Pb, these traits were reduced by 17.82%, 18.94%, and 3.64%, respectively. Pb treatment had no significant effect on the number of leaves at concentrations ranging from 30 to 90 mg/L, though a slight reduction in leaf number was observed at concentrations ranging from 90 to 180 mg/L. Pb also substantially decreased the seedling’s fresh and dry weights (Table 1). Likewise, Cd exposure significantly (P < 0.05) reduced morphological traits. At 180 mg/L, root, shoot, and leaf lengths were decreased by 44.93%, 70.04%, and 41.42%, respectively, while total fresh biomass decreased by 57.81% and dry biomass by 70.18%. A similar decreasing tendency in morphological traits was also observed at 30 mg/L of Cd (Table 1). Cd concentrations did not significantly affect the number of leaves, with effects comparable to the control.

Biochemical assessment in heterogeneous conditions

Proline content initially increased and then decreased with the application of Pb and Cd compared to the control. The increase in proline ranged from 28.57% to 51.42% with Pb 30 to 180 mg/L, and 48.57% to 65.71% with Cd 30 to 180 mg/L (Table 2). Regarding sugar content, seedlings treated with low levels of Pb and Cd had higher sugar concentrations than those treated with high levels (Table 2). The content of photosynthetic pigments was also affected by Pd and Cd. Chlorophyll content in the treated seedling decreased progressively with increasing levels of Pb and Cd (Table 2). Total chlorophyll content decreased by 15.55 to 78.51%, and carotenoid dropped to 30.56 to 83.41% with Pb (30–180 mg/L). With Cd (30–180 mg/L), total chlorophyll content dropped from 25.37 to 77.41%, and carotenoid content decreased by 16.06 to 68.91% (Table 2).

Growth assessment in homogeneous conditions

The impact of various Pb and Cd concentrations on the growth of P. roxburghii seedlings is detailed in Table 3. Both Pb and Cd positively enhanced morphological traits at a concentration of 30 mg/L (Table 3). However, at concentrations above 30 mg/L, the morphological traits showed only slight variations compared to the control (Table 3). The Pb and Cd most pronounced phytotoxic effects were observed at levels (180 mg/L), compared to lower levels (60 mg/L). Specifically, Pb at 180 mg/L caused a 28.26% decline in root length, a 55.47% decline in shoot length, a 32.03% decline in leaf length, a 38.80% decline in total fresh biomass, and a 55.76% decline in total dry biomass. Similarly, Cd at 180 mg/L resulted in a 31.25% decrease in root length, a 39.67% decrease in shoot, a 35.03% decrease in leaf, a 22.38% decrease in fresh biomass, and a 53.20% decrease in dry biomass. At a concentration of 60 mg/L, Pb caused a 3.26% reduction in root length, 11.76% in shoot length, 10.82% in leaf length, 17.91% in fresh biomass, and 8.01% in dry biomass. Cd at 60 mg/L induced a 7.03% decrease in root length, 8.15% in shoot length, 6.01% in leaf length, 2.98% in fresh biomass, and 10.57% in dry biomass. Notably, the number of leaves was not significantly inhibited by Pb and Cd concentrations and remained comparable to the control.

Biochemical assessment in homogeneous conditions

The results presented in Table 4 show that exposure to 30–180 mg/L of Pb resulted in a 13.04–39.13% increase in proline content, while Cd led to a 43.47–26.08% increase (Table 4). Additionally, the sugar content in treated seedlings increases by 22.85% to 34.28% with Pb (30–120 mg/L) and by 17.14% to 28.57% with Cd (30–120 mg/L). At higher concentrations of Pb and Cd (150 and 180 mg/L), sugar levels returned to values similar to the control (Table 4). The treated seedling also showed a decreasing tendency in photosynthetic pigments across all levels of Pb and CD. Total chlorophyll decreased by 17.56–81.53% with Pb (30–180 mg/L), and by 14.41–80.63% with Cd (30–180 mg/L), as shown in Table 4. Carotenoid levels in P. roxburghii seedlings decreased by 33.99–71.14% and 32.01–69.96% under Pb and Cd at 30–120 mg/L, respectively. The respective values of carotenoids decreased by 77.86–85.37% and 77.07–83.39% with Pb and Cd at 150–180 mg/L, respectively Table 4).

Stress tolerance index

In experiments with P. roxburghii seedlings in a heterogeneous environment, the STI exhibited a negative correlation with increasing concentrations of Pb and Cd. Specifically, STI values for Pb ranged from 83.77% to 36.30%, while for Cd, they ranged from 86.81% to 29.84% at concentrations of 30–180 mg/L (Table 5). Conversely, in a homogeneous environment, at a low concentration of 30 mg/L, the STI for Pb was 109%, whereas, for Cd, it was 112%, showing a positive correlation between STI and the concentrations of Pb and Cd at this low level. However, at higher concentrations (above 30 mg/L), the seedlings showed the lowest STI values, reaffirming a negative correlation between STI and the concentrations of Pb and Cd (Table 5).

Correlation analysis

The correlation between various morphological traits and organic solutes in P. roxburghii seedlings exposed to Pb and Cd under both field and laboratory conditions is shown in Figs. 1 and 2, respectively. Our findings reveal that while not all morphological characteristics were significantly correlated, the majority exhibited strong correlations in both heterogeneous and homogeneous conditions. For example, stem length (SL) under Pb exposure in laboratory conditions was strongly correlated with root length (RL), leaf length (r = 0.695, P < 0.001), and leaf number (r = 0.714, P < 0.001). However, under homogeneous conditions, only root length (r = 0.60, P < 0.01) and leaf length (r = 0.576, P < 0.01) showed a strong correlation with root length, while leaf number and the fresh and dry weight of seedlings did not show significant correlations, except for leaf length (r = 0.466, P < 0.05) and leaf number (r = 0.446, P < 0.05), respectively.

Similarly, the intercorrelation among morphological traits with Cd treatment was generally stronger than with lead (Fig. 2). However, like in the Pb-treated seedlings, leaf number, fresh weight, and dry weight were not correlated with any other morphological variables. In terms of organic solutes, those extracted from P. roxburghii seedlings in both field and laboratory conditions showed some significant relationships (Fig. 2). For instance, chlorophyll content (a and b) and carotenoid levels were significantly correlated with stem and leaf length in Pb-treated seedlings. In contrast, these relationships were even stronger in seedlings treated with Cd (Fig. 2).