Heavy metal and element profiling of Brassica rapa

Ibrahim Bayoumi Abdel-Farid and Adila El-Saady Mohamed

Supplementary information

1 Materials and methods

1.1 Plant materials

Two varieties of Brassica rapa (B. rapa var. rapa native to Egypt and B. rapa var. Raapstelen native to the Netherlands) were submitted to uniform culture conditions. Seeds of both varieties were germinated in soil in the cold room (4°C) for two days. The pots were placed in a box, provided with sufficient water and covered. These were then transferred to the greenhouse and kept at 25°C, 50-60% relative humidity with 16 h of daylight and 8 h of darkness per day. Seven-day old seedlings were transferred to 10 cm-diameter pots with substrate and placed in the same greenhouse and watered daily. Three individual plants were used as replicates from each variety. The basal second leaves and the upper forth leaves of plants at 4, 5 and 6-week-old stages were harvested separately and plunged directly into liquid nitrogen before freeze drying to avoid possible enzyme degradation. The samples were then ground to a fine powder with a pestle and mortar and stored in the cold room until analyzed.

1.2 Methods

1.2.1 Extraction

Extraction was done following Mohamed et al. (2003). Particular pre-weighted powdered samples were wet ashed using 50 mL (1:1) of a concentrated HNO3/HClO4 acid mixture, few drops of hydrofluoric acid (HF) was added, and the mixtures were then heated to a clear solution and continued to near dryness. The cooled residue was dissolved in 5 mL 2N HCl and heated again until complete digestion. After cooling the clear solution was transferred into 50 mL volumetric flask and completed to the mark using bi-distilled water.

1.2.2 Analysis

Certified atomic absorption spectroscopic standards of Mn, Zn, Cu, Fe, Co, Ni, Ca, Mg and Na were purchased from BDH (UK). Perkin–Elmer (Model HGA-3110) atomic absorption spectrophotometer equipped with a Perkin–Elmer HGA-600 carbon furnace and auto sampler As-60 was used.

1.2.3 Statistical analysis

The significance of the difference between heavy metal contents in the Egyptian variety compared to the Dutch variety at different developmental stages and leaves positions was analyzed by one-way analysis of variance (ANOVA). Also the difference between basal leaves and upper leaves in each variety at each developmental stage was assesses by ANOVA using Minitab version 12.21. Three replicates from each sample were analyzed.

Supplementary References

Mohamed AE, Rashed MN, Mofty A (2003) Assessment of essential elements in some kinds of vegetables. Ecotoxicol Environm Safety 55:251-260

Supplementary Table 1 Means and standard deviations of heavy metal contents (µg/g) of different leaves at different developmental stages of two varieties of Brassica rapa

Mineral elements / Heavy metals
Na / Mg / Ca / Ni / Co / Fe / Cu / Zn / Mn / Variety / Leaf position / Developmental stage
29.56 ± 5.05 / 6.04 ± 0.00 / 221.46 ± 16.25*** / 0.09 ± 0.01*** / 0.03 ± 0.01 / 4.28 ± 0.30 / 0.66 ± 0.01*** / 1.16 ± 0.00** / 1.43 ± 0.25 / Egy. / 4th leaves / 4-week-old
32.00 ± 0.84 / 8.02 ± 0.01*** / 62.88 ± 2.67 / 0.00 ± 0.00 / 0.06 ± 0.03 / 3.77 ± 0.10 / 0.45 ± 0.04 / 0.98 ± 0.05 / 1.14 ± 0.03 / Nether.
59.78 ± 0.70 / 15.12 ± 0.00 / 133.29 ± 2.60 / 0.06 ± 0.03 / 0.01 ± 0.02 / 6.42 ± 0.06*** / 0.64 ± 0.03 / 1.29 ± 0.03*** / 2.26 ± 0.03*** / Egy. / 2nd leaves
75.57 ± 19.72 / 16.45 ± 0.00 / 359.65 ± 14.29*** / 0.03 ± 0.03 / 0.05 ± 0.02 / 5.13 ± 0.11 / 0.63 ± 0.06 / 1.13 ± 0.00 / 1.54 ± 0.03 / Nether.
11.89 ± 1.32 / 2.48 ± 0.00 / 63.31 ± 4.03 / 0.08 ± 0.01 / 0.11 ± 0.01** / 0.84 ± 0.01 / 0.24 ± 0.01 / 0.95 ± 0.01 / 1.01 ± 0.01*** / Egy. / 4th leaves / 5-week-old
27.89 ± 0.12*** / 6.28 ± 0.01*** / 135.0 ± 0.30*** / 0.11 ± 0.01* / 0.05 ± 0.02 / 0.91 ± 0.04* / 0.29 ± 0.01** / 0.65 ± 0.00*** / 0.75 ± 0.01 / Nether.
13.31 ± 2.12 / 4.81 ± 0.01 / 127.24 ± 1.79 / 0.14 ± 0.02** / 0.07 ± 0.02 / 1.75 ± 0.03*** / 0.37 ± 0.02 / 0.95 ± 0.01*** / 1.54 ± 0.02*** / Egy. / 2nd leaves
26.86 ± 2.30** / 6.54 ± 0.01*** / 151.76 ± 1.42*** / 0.04 ± 0.02 / 0.06 ± 0.00 / 1.35 ± 0.01 / 0.39 ± 0.01 / 0.86 ± 0.01 / 1.01 ± 0.00 / Nether.
18.57 ± 1.57 / 5.24 ± 0.01 / 111 ± 0.78 / 0.00 ± 0.00 / 0.00 ± 0.00 / 1.98 ± 0.01 / 0.30 ± 0.01 / 0.82 ± 0.04 / 1.15 ± 0.05 / Egy. / 4th leaves / 6-week-old
26.15 ± 7.85 / 5.31 ± 0.01*** / 112.68 ± 0.42* / 0.00 ± 0.00 / 0.09 ± 0.02*** / 1.48 ± 0.44 / 1.66 ± 0.02*** / 0.87 ± 0.01 / 1.14 ± 0.01 / Nether.
19.24 ± 3.24 / 5.44 ± 0.02 / 128.46 ± 2.00 / 0.03 ± 0.01** / 0.04 ± 0.02 / 1.65 ± 0.51 / 0.44 ± 0.02 / 1.15 ± 0.01 / 2.12 ± 0.02 / Egy. / 2nd leaves
28.49 ± 4.14* / 6.11 ± 0.07*** / 154.00 ± 1.90*** / 0.00 ± 0.00 / 0.04 ± 0.02 / 1.89 ± 0.06 / 0.73 ± 0.01*** / 1.14 ± 0.02 / 2.37 ± 0.02*** / Nether.

*** = Very highly significant at p < 0.001, ** = highly significant at p < 0.01, * = significant at p < 0.05

Egy.= the Egyptian variety, Nether. = the Netherlands variety. Data is mean of three replicates

Supplementary Table 2 Statistical differences between heavy metal and element contents of the basal two leaves and the upper four leaves in each developmental stage of Brassica rapa

Na / Mg / Ca / Ni / Co / Fe / Cu / Zn / Mn / Developmental stages / Variety
*** / ns / *** / ns / ns / *** / ns / ** / ** / 4-weeks-old / Egyptian
ns / *** / *** / ** / * / *** / *** / ns / *** / 5-weeks-old
ns / *** / *** / ** / * / ns / *** / *** / *** / 6-week-old
* / *** / *** / *** / ns / *** / * / ** / *** / 4-weeks-old / The Netherlands
ns / *** / *** / ** / ns / *** / *** / *** / *** / 5-weeks-old
ns / *** / *** / nd / * / ns / *** / *** / *** / 6-week-old

*** = Very highly significant at p < 0.001, ** = highly significant at p < 0.01, * = significant at p < 0.05, ns= non-significant and nd= not determined