Response of St John’s wort (Hypericum empetrifolium) plants to cadmium (Cd) treatment in relation to substrate acidity/alkalinity

The effect of cadmium (Cd) on growth and Cd accumulation in shoots and roots St John’s wort (Hypericum empetrifolium) was studied over three months in a greenhouse. Plants were cultivated in pots containing a uniform mixture of either acid or alkaline substrate consisting of peat and perlite (1:1 v/v). The pots were arranged in a completely randomized block design within two groups (acid substrate and alkaline substrate) with four Cd treatments (0-control, 1, 2, and 5 mg Cd L) and six replicates per treatment. Cadmium was applied as CdSO4*8/3H2O. The total amount of Cd applied per pot was 260 ml, corresponding to 0.26, 0.52, and 1.3 mg Cd per pot for doses 1, 2, and 5 mg L, respectively. No visual symptoms of toxicity or nutrient deficiency, as well as no differences in plant height were observed in response to Cd application, irrespective of the growth stage or substrate. There were also no differences in height development rate between the plants grown in an acidic or alkaline substrate. Cd accumulation in shoots and roots increased with increasing concentrations of applied Cd and was higher in the acidic substrate. Thus, St John’s wort plant is a Cd accumulator, especially in an acidic environment, and this in combination with its high tolerance to Cd, makes it a suitable species to remove Cd from cadmium-contaminated sites. However, for its use in the preparation of medical products, St John’s wort must be grown in a Cd-free soil so as not to pose a risk to human health. Cd extraction by (DTPA-TEA) can be employed to predict Cd accumulation in this plant.


Introduction
The cadmium concentration in soils depends on geogenic and anthropogenic factors. Generally, Cd concentrations are higher in sedimentary rocks (0.01-2.6 mg kg -1 ) than in igneous and metamorphic rocks (0.11 to 1.0 mg kg -1 ). Anthropogenic Cd sources include mining, atmospheric deposition of combustion emissions, and the use of Cd-containing fertilizers (Kubier et al., 2019). The cadmium content of P mineral fertilizers

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Notulae Botanicae Horti Cluj-Napoca Agrobotanici 2 depends on its concentration in the rock phosphate used for their manufacture. Because of its high mobility in the soil-plant system and its low retention by soil colloids, Cd can easily enter the food chain (Alloway, 1995). Ingested cadmium is excreted slowly and so may accumulate in the human body (biological half-life 10-35 years), particularly the kidney, ultimately resulting in kidney and bone disease (Genghi et al., 2020). Hence, Cd is one of the metals for which the European Food Safety Authority (EFSA, 2012) has set limits -the maximum tolerable monthly intake of Cd being 25 μg kg -1 body weight. Reducing the amount of cadmium in the environment would help reduce Cd exposure and concomitant health risks. St John's wort (Hypericum empetrifolium) is small evergreen shrub that is native to the Mediterranean region and commonly found at low altitudes in the southern Greek Aegean and western Turkey (Davis, 1988). St John's wort is a traditional medicinal plant with high antioxidant and antibacterial activity. Decoctions of the flowers are drunk for their diuretic activity and are applied externally for the treatment of wounds and herpes (Vokou et al., 1993).
Naphthodianthrones and flavonoids have been identified in crude extracts of the flowers (Kitanov, 2001) and the composition of essential oil has been determined (Petrakis et al., 2005). Since Cd may enter the human food chain through medicinal plants, it is important to determine their potential toxicity and health risk. The aim of this study was to examine the effects of Cd on the growth, dry weight and Cd accumulation of St John's wort grown in acid and alkaline substrates.

Experimental conduction and design
The experiments were conducted in a greenhouse of the Agricultural University of Athens Greece, over a period of approximately five months. St John's wort plants were grown in pots (15 cm in diameter) filled 2.0 kg of a dry, uniform mixture of peat and perlite (1:1 v/v). Two types of peat were employed: one acidic and one alkaline. The organic matter and moisture contents were 90% and 50-65% by weight, respectively and the electrical conductivity of both forms of peat was 10 mS cm -1 . The perlite particles were 1-5 mm in diameter (Perloflor™; ISOCON S.A., Athens, Greece). The pH of the acidic substrate was 5.6 (moderately acid) and that of the alkaline substrate was 7.4 (slightly alkaline). Pots were arranged in a completely randomized block design within two groups: acid substrate and alkaline substrate with four Cd treatments (0-control, 1, 2, and 5 mg Cd L -1 ) and six replications per treatment. All pots were lined with clear polyethylene bags. Cadmium was applied as CdSO4*8/3H2O. The experiment for both substrates started on 1 st March and ended on May 29 th , 2019. Two months' seedlings of St John's wort were transplanted to each pot on March 1 st , 2019. Cd application began with the addition of 20 mL of each treatment to each pot at the concentrations indicated above on the following dates: March 10 th , March 16 th , March 21 st , March 24 th , March 28 th , March 31 st , April 4 th , April 7 th , April 11 th , April 14 th , April 20 th , April 24 th . The total amount applied per pot throughout the cultivation was 260 mL, corresponding to 0.26 mg Cd for dose 1 mg L -1 , 0.52 mg Cd for the dose 2 mg L -1 , and 1.3 mg Cd for the dose 5 mg L -1 . The experiment was terminated on 29 May 2019. The cultivation techniques were the same in both substrates. Pots were irrigated in such a way as to maintain the moisture at a soil matrix potential of -100 cm. This matrix potential ensured the maintenance of moisture in the substrate without the occurrence of leaching. Nutrileaf-60 fertilizer (2 mg each of N, P2O5, and K2O) was applied to each pot on April 3 rd and on May 5 th ; the Cd content of the fertilizer was negligible.

Plant analysis
Plant height was measured on 3 and 18 of April and 3 and 18 May in six plant per treatment. On 29 May, the plants were carefully removed from the pots and separated into the aerial organs (shoots) and underground organs (roots). The dry weight of both plant parts from each pot was measured after drying to a constant weight in an oven at 60 °C. Dried samples were ground in a stainless-steel Wiley mill and passed through a 150 μm plastic sieve to ensure uniformity. 0.5 g of dried samples smaller than 150 μm in diameter 3 were ashed at 550 °C for 4 hours. Upon completion of combustion, the residue was dissolved in 5 mL of 6N HCL. The suspension was filtered into 100 ml volumetric flasks and made up to volume with deionized water. Cd was determined by flame atomic absorption spectrophotometry at 213.9 nm, using an air-acetylene flame (Baker and Amacher, 1982). Cadmium uptake by shoots and roots was calculated by multiplying the dry weight of shoots (SDW) and roots (RDW) by the Cd concentration in shoots (Cd-shoots) and Cd concentration in roots (Cd-roots), respectively, and expressed as μg Cd per plant. The sum of the shoot and root accumulation corresponded to the total plant Cd uptake.

Substrate analysis
Samples of the air-dried substrate taken from each pot at the end of each experiment were passed through a 500 μm plastic sieve and analysed for extractable Cd using the diethylene triamine penta acetic acidtriethanol amine (DTPA-TEA) method (Lindsay and Norvell, 1978). A certified reference material (ERM-CC141 European Reference Material) was used to check the analytical procedures.

Statistical analysis
The influence of Cd treatment on plant growth (dry weight and height), Cd accumulation, and the Cd concentration extracted by DTPA-TEA were evaluated by analysis of variance (ANOVA), using STATISTICA (StatSoft, 2008). Where a significant difference was found, Duncan's Multiple Range Test at the 5% level of probability was used to compare individual treatment means. The Cd content of shoots was examined by regression analysis to establish relationships between Cd concentration in the plant parts and applied Cd. The Cd extracted by DTPA-TEA was examined by regression analysis to assess the suitability of this extraction method for predicting tissue Cd concentrations.

Results and Discussion
Cadmium treatments up to 5 mg L -1 for six weeks did not affect plants height irrespective of the growth stage or substrate (Table 1)  Due to heterogeneity in plant height at the beginning of the experiment and in order to study the differences in plant height between the two substrates at different Cd applications, we calculated the height development rate using the following formula: Height Development Rate (HDR) = difference between the height of two consecutive measurements divided by the number of intervening days. No differences in HDR were detected between Cd treatments on the same substrate (Table 2) or between the two substrates at the same Cd concentration (except for one measurement in the control). Moreover, no symptoms of toxicity or nutrient deficiency were observed in any of the plants, indicating good tolerance to Cd irrespective of substrate acidity or alkalinity. No toxicity or nutrient deficiency symptoms were reported in lettuce, cucumber, radish (Moustakas et al., 2001), endive and rocket (Akoumianakis et al., 2008) at Cd application rates of up to 20 mg Cd kg -1 or in purple coneflower (Salta et al., 2019) at Cd application rates of up to 5 mg L -1 . Acidic substrate A significant difference in shoot dry weight (SDW) was observed between the control and the Cd treatments, but not between the different Cd concentrations (1, 2, and 5 mg Cd L -1 ). Root dry weight (RDW) increased at a Cd concentration of 5 mg L -1 . The cadmium content of shoots (Cd-shoots) and roots (Cd-roots) increased with increasing Cd concentration, as did shoot (SUpt), root (RUpt) and total (TUpt) Cd uptake (Table 3). A significant linear correlation existed between Cd applied to the medium and Cd extracted by DTPA-TEA(Cd-soil), expressed by the equation: Cd-soil = 1.91x(Cd-added) -0.19 (r= 0.98, p<0.001). Alkaline substrate No significant differences were detected in SDW with increasing Cd, while RDW increased in the 5 mg L -1 treatment. Cadmium concentrations in shoots and roots increased with increasing Cd application doses. Consequently, shoots, roots, and total Cd uptake by St John's wort plants increased (Table 4). A significant linear correlation existed between Cd applied in the medium and Cd extracted by DTPA-TEA (Cd-soil), expressed by the equation: Cd-soil = 1.66x(Cd-added) -0.22 (r= 0.99, p<0.001). Statistically significant differences were observed between SDW and RDW in all Cd treatments and substrates (Table 5) and could possibly be explained by the higher sensitivity of young roots to Cd stress (Macarovicova et al., 2004). Statistically significantly differences between acidic and alkaline substrates existed in all the data examined, except for RDW and Cd-soil (Table 5). The higher Cd concentration in plants grown in acidic substrate may be due to the higher bioavailability of Cd in an acid environment. Soil pH is the most important factor affecting Cd bioavailability, which increases linearly with decreasing pH (Kirkham, 2006;Kim et al., 2009). Rieuwerts et al. (1998) reported that Cd absorption by plants became significant at pH 5-6.5. These results agree with those reported by Moustakas et al. (2001) for lettuce, radish nad cucumber, and by Akoumianakis et al. (2008) for endive and rocket.  Column means for the same treatment in different substrates followed by a different letter without parenthesis are significantly different according to Duncan's multiple range test at p≤0.05. Means for the SDW and RDW, Cd-shoots and Cd-roots as well as SUpt and RUpt followed by different letter in parenthesis in the same row are significantly different according to Duncan's multiple range test at p≤0.05.
Shoot, root, and total Cd uptake by St John's wort plants grown in acidic and alkaline substrate increased with increasing Cd application (Tables 3, 4). More than 70% of the total Cd taken up by the plants grown in the acid substrate was accumulated in the shoots. In alkaline substrate Cd taken up by the plants was equally distributed in shoots and roots (Figure 1). This finding in combination with the plants' high Cd tolerance (plant height and dry weight unaffected) indicates that St John's wort can be used not only in conventional phytotherapy but also in the rehabilitation and recovery of Cd contaminated sites, as suggested by Macarovicova et al. (2004). In the controls, Cd-shoots and Cd-roots were 6.3 and 15.5 μg g -1 (DW), respectively 6 for plants grown in the acidic substrate were 2.0 and 7.5 μg g -1 (DW), respectively for plants grown in the alkaline substrate (Table 5). These values, which were much higher (especially in the acid substrate) than those for Cd extracted by DTPA-TEA, are particularly noteworthy since they indicate a significant accumulation of Cd by the plant parts independent of the substrate pH. Akoumianakis et al. (2008), Bingham (1983, and Davis (1984) classified rocket, endive, lettuce, spinach, turnip grass, celery, and cabbage as Cd accumulator. Using linear regression analysis, we found a significant relationship between the amount of Cd that was extracted by DTPA-TEA and Cd accumulation in the shoots and roots of St John's wort, independent of the pH of substrate (Table 6, 7). Hence, this extractant could be effectively used to predict Cd content in St John's wort plant parts. Moustakas et al. (2001), Akoumianakis et al. (2008) reported that Cd extraction by DTPA-TEA could be effectively used to predict Cd uptake by lettuce, radish, cucumber, endive and rocket plants. The Cd content in aerial (SUpt) and underground (RUpt) plant parts increased with increasing Cd application (Tables 3, 4). The average ratio of SDW/RDW and Cd-roots/Cd-shoots was 7 and 2.4, respectively for plants grown in an acidic environment, and 3.8 and 3.3 respectively for plants grown in an alkaline environment. The Cd accumulation capacity was greater in roots than in shoots in both substrates. In the acidic substrate Cd accumulation by dry weight was greater in shoots than in roots, resulting in a higher Cd concentration in the shoots. In the alkaline substrate, however, the equal dry matter and Cd accumulation capacities of the shoots and roots resulted an approximately equal Cd concentrations in these plant parts. Thus, Cd accumulation in St John's wort depended on both the Cd accumulation capacity and the biomass of the plant.
Finally, because the Cd concentrations in the shoots and roots were invariably higher than the maximum permissible Cd concentration of 0.3 mg kg -1 (WHO, 1999), it is clear that to be used safely in the preparation of medicinal products, St John's wort should not be grown in Cd contaminated (especially acidic) soils.

Conclusions
Plant growth (dry weight and height) of St John's wort was not affected by Cd application up to 5 mg L -1 no symptoms of toxicity or nutrient deficiency were observed. Our results confirmed that this plant species is a Cd accumulator and supported previous findings that plants of the Hypericaceae family are Cd accumulators. Cd accumulation in St John's-wort plants is much higher in an acidic than in an alkaline environment and occurs mainly in the aerial plant parts (shoots). St John's wort may therefore be used to remove Cd from contaminated soils. However, caution is required when this plant is to be used in the preparation of medicinal products, since it must be grown in a Cd-free soil. Cd extraction by DTPA-TEA could be used effectively to predict Cd concentration in shoots and roots of St John's-wort plants.

Authors' Contributions
Conceptualization, supervision, validation, writing original draft, writing review and editing: AAI, NM, PB; Methodology, data curation analysis: PB and AS, review and editing: NM, PB, AAI. All authors read and approved the final manuscript.