Impact of a snail pellet on the phytoavailability of different metals to cucumber plants (Cucumis sativus L.).

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Downloaded by Universiteit Utrecht on 11/04/2013 22:31:22. Published on 21 December 2012 on http://pubs.rsc.org | doi:10.1039/C2EM30806A

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Impact of a snail pellet on the phytoavailability of different metals to cucumber plants (Cucumis sativus L.) ¨ rg Feldmann Sabine Freitag,* Eva M. Krupp, Andrea Raab and Jo FePO4 based molluscicides (snail pellets) also contain a chelating agent. The influence of the chelating agent, which is intrinsically present in the molluscicide, on the phytoavailability of other metals present in the growth medium was investigated in the present study. Cucumber plants (Cucumis sativus) were grown in a hydroponic nutrient solution and exposed for one week to different metals in combination with a chelating agent containing molluscicide. Oven dried roots and shoots of plants were HNO3/H2O2 microwave digested and analysed regarding total Fe, stable isotopic

Received 2nd October 2012 Accepted 4th December 2012

54

Fe, Cd, Pb, and Bi concentrations

using ICP-MS. The results showed that the addition of a chelating agent enhances the Fe phytoavailability to the plant, whether as an intrinsic part of the molluscicide or added individually.

DOI: 10.1039/c2em30806a

Additionally, the chelating agent present in the pesticide mobilises externally added metals and thus increases their phytoavailability. In particular the significantly higher Cd concentration in shoots from

rsc.li/process-impacts

plants exposed to chelating agents indicates a potentially detrimental environmental effect.

Environmental impact Iron(III) phosphate based molluscicides contain in addition a chelating agent. FePO4 alone is a stable compound with a very low solubility. While the latter minimizes its dispersal beyond where it is applied, the addition of a chelating agent leads to an increased solubilisation and thus mobilisation linked to increased bioavailability of iron but potentially also other heavy metals present in the growth medium. With our study we could show that the addition of a chelating agent either externally added or intrinsically present in the molluscicide leads to an increased bioavailability of externally present heavy metals in Cucumis sativus grown in a hydroponic nutrient solution. These results indicate that the chelating agent present in the molluscicide has a detrimental effect.

Introduction Currently approved active molluscicidal substances listed in the EU Pesticides Database comprise amongst others metaldehyde, methiocarb and ferric phosphate.1 While the rst two are synthetic substances, the latter is an inorganic, naturally occurring compound. Ferric phosphate has a very low toxicity to mammals2 and occurs naturally in the minerals strengite and metastrengite and also as a component of several other minerals.3,4 The biodegradation products of ferric phosphate are iron and phosphate, which are released into the soil for plant uptake. Ferric phosphate and its breakdown products thus will have no adverse effects in the environment.5 It is a stable, non-volatile compound with a very low solubility in water, which minimizes ferric phosphates dispersal beyond where it is applied. FePO4 based molluscicidal pellets also contain chelating agents such as ethylenediamine-N,N0 -disuccinic acid (EDDS) or ethylenediamine tetraacetic acid (EDTA), depending on the commercial pesticide.6,7 Chelating agents are organic TESLA-Trace Element Speciation Laboratory, College of Physical Sciences – Chemistry, University of Aberdeen, Meston Walk, Aberdeen AB24 3UE, Scotland, UK. E-mail: [email protected]; Fax: +44 (0)1224-272921; Tel: +44 (0)1224 272911

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compounds, which complex metal ions by removing these from their insoluble salt form and therefore keeping them in solution. While EDTA is persistent in nature, (S,S0 )-EDDS, which is a structural isomer of EDTA, is biodegradable.8 Unlike ferric phosphate, which is found in nature, the additional presence of a chelating agent in the molluscicide at the same or even higher amounts than FePO4 might have an impact on the mode of action in snails and slugs but importantly also have environmental consequences. The exact mode of EDTA/EDDS based molluscicide pellets is not fully elucidated, but iron appears to be deposited in the digestive gland and body wall which leads to reduced levels of feeding and potentially slug death.9 It was suggested that ferric phosphate alone interferes with the calcium metabolism within the slug and eventually causes cellular pathological changes in the slug's crop and hepatopancreas. The process from feeding to dying usually takes about three to six days.5 Another study on the other hand showed that Fe(III) levels in tissues from snails fed with FeEDTA pellets were 10–100 times higher than the levels in snails fed solely with FePO4 pellets.7 Although the exact mechanism of iron toxicity in the snails is not known, it was suggested that high concentrations of FeEDTA present in the tissues may be due to the fact that under physiological conditions, where the stability of the FeEDTA complex could change,

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Environmental Science: Processes & Impacts essential metals such as copper could become complexed by EDTA. Copper in turn is used by snails to carry oxygen in their haemolymph rather than iron.7 If this was the case, however, EDTA alone should behave similarly and lead to similar results, but the US patent 19956 clearly states that individually neither the simple iron compound nor the second component (EDTA) is toxic to terrestrial molluscs. The environmental fate of chelating agents has received considerable attention for more than 50 years as chelating agents have the potential to perturb the natural speciation of metals10 and consequently inuence their phytoavailability.11 Chelating agents mobilize metals from soils and this knowledge has been used for soil washing and phytoremediation procedures to clean metal contaminated soils.12 The aim of this study was to investigate the phytoavailability of Fe from the FePO4 salt but also other externally added metals without and with the chelating agents ethylene diamine tetraacetic acid (EDTA) and ethylene diamine disuccinic acid (EDDS), either present as individual substances or intrinsically present in the commercially available molluscicide Ferramol. It was of importance to analyse the inuence of the intrinsically present EDDS in the molluscicide on externally present metals in a hydroponic nutrient solution to investigate the potential environmental impact of the molluscicide on the mobilisation and thus phytoavailability of other metals present in the growth medium. The approach of this study was divided into three experiments, where the plants were initially exposed to pure FePO4 alone or in combination with pure chelating agents or the molluscicide containing both FePO4 and EDDS. Following,

Paper a 54Fe tracer study was performed in order to investigate the inuence of the intrinsically present EDDS in the molluscicide on the phytoavailability of externally present iron. This then lead to the third experiment, where plants were exposed to three metals including bismuth, cadmium and lead alone or in combination with the molluscicide or pure EDDS. These metals were chosen due to their high complexation constants but also their environmental importance. For the experiments a hydroponic nutrient system was chosen as the initial system, as it provides a system with known composition and concentration of micro- and macronutrients and pH and rules out soil physico-chemical and ecological factors interfering with metal uptake in the plants. Cucumber plants were selected as they produce relatively high biomass in a short time period where the experiments were performed. In addition they are typical arable crop plants which oen suffer from snail/slug pests and have been used in previous nutritional experiments in a hydroponic system.13,26

Results and discussion Phytoavailability of iron in the presence of chelating agents Prior to the treatment with the recommended application rates of FePO4 as well as the commercially available molluscicide Ferramol, the plants were grown in Hoagland's solution without the addition of the usually applied FeEDTA concentration (Table 1) for one week. The aim was to iron deprive the plants in order to increase their desire to take up iron, when applied, in any form. Aer four days, these plants

Table 1 Overview of performed plant cultivation experiments indicating growth conditions and the theoretical concentrations (mg L1) of metals added to the hydroponic nutrient solution. HS ¼ Hoagland's solution; EDDS ¼ ethylendiaminedisuccinic acid

Treatment

Full strength HS followed by 54 Fe (mg L1) Bi Vermiculite treatment (when treatment, Fe Cd Pb EDDS nal (mg L1) (mg L1) (mg L1) (mg L1) (duration) Half strength HS no FeEDTA in HS) (mg L1) Tracer

1. Experiment: natural abundant iron with and without chelating agents FePO4 10 days 7 days with 14 days with, 7 days FeEDTA w/o FeEDTA Ferramol 10 days 7 days with 14 days with, 7 days FeEDTA w/o FeEDTA FePO4 + 10 days 7 days with 14 days with, 7 days EDDS FeEDTA w/o FeEDTA 2. Experiment: stable iron isotope tracer with Tracer only 10 days 7 days w/o FeEDTA Ferramol + 10 days 7 days w/o tracer FeEDTA Fe + tracer + 10 days 7 days w/o EDDS FeEDTA

200

N/A

N/A

N/A

N/A

N/A

200

N/A

N/A

N/A

N/A

0.98

200

N/A

N/A

N/A

N/A

1.4

and without chelating agents 7 days w/o FeEDTA in HS

N/A

200

N/A

N/A

N/A

N/A

7 days w/o FeEDTA in HS

200

200

N/A

N/A

N/A

0.98

7 days w/o FeEDTA in HS

200

200

N/A

N/A

N/A

1.4

N/A

N/A

200

50

200

N/A

200

N/A

200

50

200

0.98

N/A

N/A

200

50

200

1.4

3. Experiment: heavy metal mix with and without chelating agents Metal mix 10 days 7 days with 7 days with FeEDTA FeEDTA Metal mix + 10 days 7 days with 7 days with FeEDTA Ferramol FeEDTA Metal mix + 10 days 7 days with 7 days with FeEDTA EDDS FeEDTA

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Paper did not show any obvious differences, such as chlorotic leaves for example, compared to the control plants, which were grown in Hoagland's solution with FeEDTA (data not shown). Aer exposing the plants to FePO4 and the slug pellets, chlorosis on mostly upper leaves was observed on all plants except for control plants. This is likely to be due to the fact that the application rate according to the recommendation for the application of the snail pellets was well below the normal iron concentration in Hoagland's solution. Traditional Hoagland's solution has an iron concentration of 1.4 mg L1, while plants treated with Ferramol in Fe-EDTA free Hoagland's solution were exposed to a nal iron concentration of 0.2 mg L1 (Table 1). These iron concentrations are seven times lower compared to the concentration in the traditional Hoagland's solution and might thus not be sufficient for chlorophyll synthesis in the plants. The plants were at the treatment time already six weeks old (Table 1), and thus chlorosis was apparent. However, minimal concentrations of iron required for normal growth of plants range from 109 to 105 M (0.56 mg L1 to 56 mg L1), depending on other nutritional factors and also the type of plant and its developmental stage.14,15 The mean tissue iron content of roots and shoots was investigated for the cucumber plants (Cucumis sativus) exposed for one week to the different treatments (Table 1). Iron is an essential micronutrient for plants, required for respiration, photosynthesis, and many other cellular functions such as DNA synthesis and hormone production.16 The general trend between all treatments is that the iron content in roots is higher compared to leaves. This trend is comparable to other plants such as Arabidopsis.17 The results clearly showed that the addition of a chelating agent is a necessary requirement to make the iron in the FePO4 phytoavailable for the cucumber plants (Cucumis sativus) (Fig. 1). This can be seen in the signicantly higher shoot iron (90–130 mg kg1; p < 0.05 for EDTA and Ferramol, p < 0.001 for EDDS; Fig. 1a) and signicantly lower root iron concentrations (167–197 mg kg1 p < 0.05 for EDDS, EDTA and Ferramol, Fig. 1b) in comparison to plants that were not exposed to an additional chelating agent (54 mg kg1 in shoots, 618 mg kg1 roots). As the sum of the adsorptive rhizosphere and the uptake of iron cannot be unravelled, the translocation from the roots to the shoots was investigated in order to get some information about the phytoavailability of the pure FePO4, FePO4 pellets with chelating agents and the snail pellets. Signicantly higher translocation rates of iron from the roots to the shoots were observed in chelate treated plants (p < 0.01 for EDTA and Ferramol, p < 0.001 for EDDS, Fig. 1c) in comparison to plants solely exposed to pure FePO4. The results suggest that pure FePO4 is not phytoavailable for the plants, as FePO4 has a very low solubility in water (pH 7: 1.86 1012 g L1 at 25 C) and the addition of the chelating agents is necessary to make the iron soluble in water and consequently phytoavailable. For hydroponic experiments this fact has been known for a long time18 and it is a common procedure to use Fe(III) EDTA as iron fertilizer in hydroponic nutrient solutions (see experimental, plant cultivation).


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Fig. 1 Mean (n ¼ 4) tissue iron concentration (per dry weight) with 95% confidence interval in (a) shoots and (b) roots of Cucumis sativus exposed for one week to FePO4 (1 wt%), FePO4 (1 wt%) with EDTA, EDDS and the molluscicide Ferramol (1 wt% FePO4) in its recommended application rate, resulting in iron concentrations of 200 mg L1. Additionally the translocation rate of iron from roots to shoots is shown (c). The significance level is shown in comparison to FePO4 treated plants: *p < 0.05, **p < 0.01, ***p < 0.001.

Phytoavailability of molluscicide

54

Fe tracer iron in the presence of a

The question arose as to whether chelating agents, present either as intrinsic compounds in the molluscicide or additionally added in a 1 : 1 molar ratio to the nutrient solution would


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inuence the uptake behaviour of additionally externally added stable iron isotopes. Interestingly, the obtained results clearly showed that 54Fe tracer concentration is signicantly higher in shoots exposed to Ferramol pellets (p < 0.001) as well as EDDS solution (p < 0.001) in comparison to control plants (control plants were only exposed to 54Fe tracer, Fig. 2a). Results regarding root 54Fe concentrations were not as straightforward to interpret as expected. While roots of Ferramol treated plants had signicantly lower tissue 54Fe concentrations (p < 0.01), roots of EDDS treated plants on the other hand had signicantly higher 54Fe tissue concentrations (p < 0.05, Fig. 2b).

Fig. 2 Mean (n ¼ 4) tissue 54Fe tracer concentration (per dry weight) in (a) shoots and (b) roots shown with 95% confidence interval. Furthermore, the translocation rate of 54Fe from roots to shoots is illustrated (c). The significance level is shown in comparison to control plants (tracer treated only): *p < 0.05, **p < 0.01, ***p < 0.001.


Paper The washing procedure of the roots was carefully performed with deionised water, yet existing iron plaques on the outside of the roots cannot be ruled out. However, translocation rates of isotopic 54Fe were signicantly higher in chelate treated plants (Fig. 2c; p < 0.01 for Ferramol, p < 0.05 for EDDS). These ndings indicate that the addition of the chelating agent EDDS which is either intrinsically present in the molluscicide Ferramol or externally added as a pure substance enhances the uptake of externally added iron in the plant.

Phytoavailability of the three metals cadmium, bismuth and lead in the presence of a molluscicide Whether other metals, in particular toxic metals are also mobilised by the addition of Ferramol to the Hoagland solution was further investigated. The possibility that the addition of a chelating agent in the form of a molluscicide might enhance the mobilisation of other metals than iron exists, leading to an increased phytoavailability and thus accumulation of those metals in crop plants. As shown in Table 1 in experiment 3, plants grown in the Hoagland solution were exposed to the commercial FePO4/ EDDS based snail pellets in combination with a metal mix (MM). Bismuth, lead and cadmium were the metals of choice. While bismuth, cadmium and lead occur naturally in pristine areas at concentrations between 0.1 and 0.2 mg kg1 at the earth's crust,19,20 these metals are at high concentrations toxic to plants, as they disrupt enzyme functions, replacing essential metals in pigments or producing reactive oxygen species.21,22 High concentrations of these three selected metals illustrate a problem in regions of general anthropogenic activity. Lead and bismuth pollution in particular occur in shooting ranges. Lead has been used in the past for bullets, but has now been replaced by the new bismuth-based bullets.23 This is of concern since bismuth has been shown to be bioactive.24 During the time of exposure, plants subjected to the metal mix alone or in combination with EDDS showed rst signs of chlorosis due to iron deciency in contrast to Ferramol treated plants (data not shown). This might be due to the fact, that Ferramol provides an additional iron source in the form of FePO4 (active compound) plus the chelating agent EDDS, which is sufficient for the cucumber plant at that particular developmental stage (Table 1). The phytoavailability of the selected metals, here indicated in the shoot/root concentrations and translocation rates, differed depending on the treatment. Ferramol treated plants had signicantly higher Bi shoot concentrations (p < 0.01) in comparison to plants exposed to the pure metal mix (Fig. 3a). Roots on the other hand had signicantly lower Bi tissue concentrations for both, Ferramol and EDDS treated plants (p < 0.001, Fig. 3b). Thus, the application of chelating agents either externally (EDDS) or intrinsically present in the molluscicide (Ferramol) signicantly enhances bismuth translocation rates from the roots to the shoots (p < 0.01 and p < 0.05, Fig. 3c). In comparison to the other metals used in the present experiment, bismuth has a very high complexation constant of log K ¼ 27.8 with EDTA.


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For cadmium, signicantly higher concentrations were found in shoots of plants treated with Ferramol (p < 0.01) and EDDS (p < 0.01) in comparison to plants which were exposed to the metal mix without any chelating agent (Fig. 3a). In contrast, signicantly lower Cd concentration were only observed in roots of Ferramol treated plants (Fig. 3b). However, translocation rates from roots to shoots were signicantly higher for both Ferramol (p < 0.05) and EDDS (p < 0.001) treated plants (Fig. 3c). This would indicate that the chelates helped not necessarily the uptake but the translocation of cadmium into the shoots. These results would imply that molluscicides containing chelating agents may have a detrimental impact on plants when grown on soils with elevated toxic metal concentrations. CD-EDDS is almost as strong as EDTA and can help to extract Cd from soil.25

Experimental Chemicals

Fig. 3 Mean (n ¼ 4) metal tissue content (per dry weight) in (a) shoots and (b) roots shown with 95% confidence interval. Furthermore, the translocation rate of the respective metal from roots to shoots is illustrated (c). The significance level is shown in comparison to control plants (only): (1 symbol) p < 0.05, (2 symbols) p < 0.01, (3 symbols) p < 0.001 of * – bismuth, † – cadmium and ‡ – lead. MM ¼ Metal Mix containing bismuth, cadmium and lead.

Lead in contrast showed a different phytoavailability behaviour. Strikingly, Ferramol treated plants had signicantly lower Pb concentrations in shoots (p < 0.05, Fig. 3a) and roots (p < 0.001, Fig. 3b) of 1 and 33 mg kg1, respectively, in comparison to non-treated or EDDS treated plants. The exact composition of the Ferramol molluscicide is not known, but the possibility of it containing sulphates which can form PbSO4 exists, which itself has a very low solubility and thus a much lower phytoavailability. The translocation rates of Pb2+ from the roots to the shoots did not differ signicantly between the chelating agent treated and non-treated plants (Fig. 3c). In comparison to the other metals, Pb has a lower complexation constant with EDTA of log K ¼ 18. This journal is ª The Royal Society of Chemistry 2013

Stock solutions for Hoagland's solution were made up in ultra pure water (Elga, UK). All chemicals including KNO3, Ca(NO3)2$4H2O, MgSO4, NH4NO3, KH2PO4, FeSO4$7H2O, MnCl2$4H2O, Na2B4O7$10H2O, ZnSO4$7H2O, CuSO4$3H2O, Na2MoO4$2H2O were purchased from Sigma-Aldrich (Dorset, UK) and were at least of analytical grade. Samples for iron treatment were obtained from an unopened package of the commercially available molluscicide Ferramol (W. Neudorff GmbH, KG, Germany). Ferramol pellets contain 1% FePO4 and 1.63% EDDS. Additionally, 1% containing FePO4 pellets (217) were obtained from Lonza Ltd (Basel, Switzerland), which do not contain any chelating agents. Analytical data for concentration of iron and chelating agents in both products were provided by the company Catalyse – Etudes & recherch´ e sur les polymers. Chelating agents EDDS (35% in H2O) and EDTA (Sigma-Aldrich, Dorset, UK) were added in a 1 : 1 molar ratio to the pure FePO4 pellets (217, Lonza Ltd), respectively. Isotopically enriched Fe was purchased as metal from CK Gas Products (Hampshire, UK). The certied isotopic abundances were given by the manufacturer as 54Fe 99.88%, 56Fe 0.11% 57Fe 0.005% and 58Fe 0.005 (CK Gas Products, Certicate of Analysis lot no. 2129, 03/2007). Solutions of isotopically enriched Fe were made up by dissolving the metal in concentrated acidic acid and diluting it to a working solution of 973 mg L1. 100 mg Bi L1 and 100 mg Pb L1 solutions were prepared from the metal salts Bi(NO3)3$5H2O and Pb(CH3COO)2 in bidistilled water. Additionally, a 31 mg Cd L1 solution was prepared from CdS in HCl (Sigma Aldrich, Lab grade, Dorset, UK). A metal mix stock solution containing all three metals was then prepared using the three stock solutions to result in nal concentrations of 25 mg Bi L1, 25 mg Pb L1 and 6 mg Cd L1. This stock solution was added to the 2 L Hoagland solution to result in nal concentrations of 0.2 mg Bi L1, 0.2 mg Pb L1 and 0.05 mg Cd L1. Plant cultivation Cucumber seeds (Cucumis sativus, “Italian Kitchen”, Cetriolo Marketer) were sown on Vermiculite soil damped with tap


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Table 2 Reference materials used for quantitative quality control of different elements (n ¼ 3). Values are given for the measured, targeted concentrations and recovery rates for the three metals with 95% confidence interval and relative standard deviation in brackets. No certified values for bismuth are given for the used CRM

CRM/element Fe

Pb


Cd

Measured Targeted Recovery Measured Targeted Recovery Measured Targeted Recovery

IAEA-140 TM (Fucus sp.)

SPS-WW2 (wastewater)

989 13 mg kg1 (1.3%) 1256 35 mg kg1 (2.8%) 78.8 0.8% (1.0%) 2.0 0.03 mg kg1 (1.6%) 2.2 0.3 mg kg1 (12.8%) 90.1 1.5% (1.7%) 0.43 0.014 mg kg1 (3.2%) 0.54 0.037 mg kg1 (6.9%) 81.1 2.6% (3.2%)

4.8 0.3 mg L1 (5.6%) 5.0 mg L1 95.8 3.9% (4.1%) 0.49 0.0053 mg L1 (1.1%) 0.5 mg L1 98.2 1.1 mg L1(1.1%) 0.10 0.0023 mg L1 (2.3%) 0.1 mg L1 102.3 2.3% (2.3%)

water in a seedling tray. The tray was placed in a greenhouse with a temperature of ca. 24 2 C. Temperature control was achieved using a heating system. Humidity was not adjusted. The seeds were le for approximately 10 days to germinate until they reached the two leaves stage. Following, young seedlings were removed from the Vermiculite soil, their roots cleaned and transferred into plastic containers, holding 2 L of hydroponic Hoagland's solution. (Hoagland full strength solution: 1.4 mg Fe L1 (added as Fe(III)EDTA), 2.5 mM KNO3, 2.5 mM Ca(NO3)2, 1.0 mM MgSO4, 0.5 mM NH4NO3, 0.5 mM KH2PO4 (adjusted to pH 6.3), 9.1 mM MnCl2, 50 mM Na2B4O7, 0.76 mM ZnSO4, 0.36 mM CuSO4, 0.49 mM Na2MoO4.) Four plants were placed in one container per treatment. These are the standard conditions which have shown good statistical values.26 The biological variability is larger than the differences in the diffusion relative uptake. For the rst week, the plants were grown in half strength Hoagland's solution. The following week, the plants were grown in full strength Hoagland's solution. Duration of plant growth and Hoagland particulars regarding FeEDTA are listed in Table 1. Experimental design and treatment In total three separate experiments were performed (Table 1): (1) plants exposed to natural iron pellets with and without chelating agents but also the FePO4/EDDS containing molluscicide Ferramol. (2) Plants exposed to stable iron tracer with and without chelating agents and the molluscicide Ferramol. (3) Plants exposed to a mixture of metals including Bi, Pb and Cd with and without chelating agents and the molluscicide Ferramol. All pellets were added as a whole to the hydroponic solution and their application rate was adjusted according to the recommendation on the packaging (5 g m2). As the application rate refers to an area, the area of the plant containers was calculated (0.0368 m2) and the recommended application rate adjusted. This resulted in 0.12 g Ferramol per 2 L, i.e. nal iron concentrations of 200 mg L1 for Ferramol but also FePO4 treated plants (Table 1). Regarding experiment 2, a stable iron isotope tracer was applied to result in a nal starting concentration of 200 mg L1. In order to control the real starting concentration of the added stable isotopic iron, water samples of the three different treatments were measured prior to exposure, resulting in an initial added 54Fe concentration in the hydroponic nutrient


solution of 178 11 mg L1. Bismuth and lead were applied to result in nal concentrations of 200 mg L1 (see Table 1), while cadmium was applied to result in a nal concentration of 50 mg L1. Initial average concentrations of Bi, Pb and Cd prior to plant exposure were determined and resulted to be 206 13 mg L1, 182 9 mg L1 and 53 2 mg L1. Following the addition of the stable iron isotope tracer but also the metal mix solution to the Hoagland solution, the pH was adjusted with 1.5 M NaOH (Sigma Aldrich, Dorset, UK) to 6.5. The pH of the Hoagland solution was controlled with a portable pH meter (Hanna Instruments, Bedfordshire, UK), which was two points calibrated prior to each measurement. The metal concentrations of the respective treatments were measured aer pH adjustment. Sample preparation and tissue digestion Following treatment, as indicated in Table 1, plants were harvested. The roots and rhizomes themselves were white, showing no macroscopically discernible plaque. Aer separating the roots from the upper plant, they were repeatedly rinsed by shaking them by hand in demineralised water, which has been shown to be more important than the choice of rinsing agent.17 The plant material was then dried at 35 C for 3 days. Approximately 0.1 g dry weight of nely ground shoot and 0.02 g dry weight root material were placed in 50 mL polypropylene tubes (Greiner Bio-One, Kremsmuenster, Austria) and 1 mL of 69% HNO3 (67%, trace select grade, BDH Merck Ltd, UK) was added and le overnight. The following day 2 mL of H2O2 (30%; w/w, Aldrich, Buchs, Switzerland) were added to the samples and digested using the open vessel microwave procedure (MARS microwave, CEM). The settings were as follows: 5 minutes at 50 C (2 min temperature ramp), 5 minutes at 75 C (2 minute temp. ramp) and 15 minutes at 95 C (2 min temperature ramp). For quality control, the certied reference material IAEA-140/ TM (Fucus sp.) was digested and analysed in the same way as the samples. Furthermore, procedural blanks were included for quality control. Chemical analysis and quality control Prior to analysis, all digests were diluted with deionised water (>18 MU cm) so that concentrations of targeted elements would range between 0 and 100 mg L1 and the HNO3 concentration This journal is ª The Royal Society of Chemistry 2013

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Paper would not exceed 2%. Total iron concentrations of diluted digests from shoots and roots were determined by magnetic sector eld ICP-MS (measured on m/z 54, 56, and 57 for iron) in medium resolution (m/D, >4000) (Element 2, Thermo Fisher Scientic, Waltham, Massachusetts, USA). Respective calibration was performed for the natural iron standard (High Purity Standards, Charleston, USA) and the stable iron isotope tracer (54Fe) at the beginning of the sample sequence and then repeated aer 30 samples, respectively. Scandium was added as continuous internal standard via a tee piece before the nebulizer and its signal was measured on m/z 45. Total element concentrations of Bi, Pb and Cd in shoots and roots were determined by low resolution ICP-MS (Agilent 7500c, Santa Clara, USA). The elements were measured on m/z 209, m/z 206–208 and m/z 111 respectively. Calibration was performed using a 1000 mg L1 bismuth (Fluka, Dorset, UK), lead (Alfa Aesar, Massachusetts, USA) and cadmium (High Purity Standards, Charleston, USA) standard. Thallium (1000 mg L1, Alfa Aesar, Massachusetts, USA) was added as continuous internal standard at a concentration of 20 mg L1 before the nebulizer and its signal was measured on m/z 205. Total elemental analysis regarding tracer 54Fe, Bi, Cd and Pb of water samples was performed prior to exposure of the plants. For quality control, the certied reference material SPS-WW2 (wastewater) was used. Measured, targeted concentration and recovery rates for IAEA-140/TM and SPS-WW2 are shown in Table 2.

Conclusion This study clearly showed that the addition of the chelating agent EDDS, either present in the snail pellet (molluscicide) or externally added, led to an increased phytoavailability of the metals cadmium and bismuth. It appears that the molluscicide Ferramol enhances metal uptake in crop plants. These results, of course, apply to hydroponic nutrient solutions and it is of importance to test the effect of those EDDS containing molluscicides on the mobilisation and related phytoavailability of metals present in a soil pot but also a eld experiment. This, in turn will depend on a number of factors: (1) the exposure time, which is dependent on the number of snails; (2) the weather conditions, where high precipitation would cause the release of EDDS; (3) the type, concentration and mobility of metals present in the root zone and soil; (4) the type of soil (chemical properties); and (5) the crop species (e.g. if leaf or root vegetable or grain crop).

Acknowledgements We thank Lonza Ltd for their nancial support and in particular Dr Markus Bieri and Ms Susanne Apppoloni for valuable discussions. A great thanks is extended to Prof. Detlef G¨ unther


Environmental Science: Processes & Impacts for establishing the collaboration between Lonza Ltd and the Trace Element Speciation Laboratory (TESLA)/University of Aberdeen.

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Transformation of cucumber (Cucumis sativus L.) plants with Agrobacterium rhizogenes.

Phytotoxic substances in root exudates of cucumber (Cucumis sativus L.).

Transformation of cucumber (Cucumis sativus L.) plants using Agrobacterium tumefaciens and regeneration from hypocotyl explants.

Evidence of translocation and physiological impacts of foliar applied CeO2 nanoparticles on cucumber (Cucumis sativus) plants.

Synthesis of a dicyclohexylcarbodiimide-binding proteolipid by cucumber (Cucumis sativus L.) mitochondria.

Loss-of-Function Mutations in CsMLO1 Confer Durable Powdery Mildew Resistance in Cucumber (Cucumis sativus L.).

A New Glabrous Gene (csgl3) Identified in Trichome Development in Cucumber (Cucumis sativus L.).

Fine Mapping of Virescent Leaf Gene v-1 in Cucumber (Cucumis sativus L.).

Inheritance and linkage relationships of isozyme loci in cucumber (Cucumis sativus L.).

Exogenous Melatonin Improves Antioxidant Defense in Cucumber Seeds (Cucumis sativus L.) Germinated under Chilling Stress.

Effects of Heat Shock on Photosynthetic Properties, Antioxidant Enzyme Activity, and Downy Mildew of Cucumber (Cucumis sativus L.).

Trichome-Related Mutants Provide a New Perspective on Multicellular Trichome Initiation and Development in Cucumber (Cucumis sativus L).

Integrative Analyses of Nontargeted Volatile Profiling and Transcriptome Data Provide Molecular Insight into VOC Diversity in Cucumber Plants (Cucumis sativus).

A High-Density Genetic Linkage Map for Cucumber (Cucumis sativus L.): Based on Specific Length Amplified Fragment (SLAF) Sequencing and QTL Analysis of Fruit Traits in Cucumber.

Ethylene is Involved in Brassinosteroids Induced Alternative Respiratory Pathway in Cucumber (Cucumis sativus L.) Seedlings Response to Abiotic Stress.

Rapid identification of fruit length loci in cucumber (Cucumis sativus L.) using next-generation sequencing (NGS)-based QTL analysis.

Glutathione-dependent induction of local and systemic defense against oxidative stress by exogenous melatonin in cucumber (Cucumis sativus L.).

Ultrahigh-density linkage map for cultivated cucumber (Cucumis sativus L.) using a single-nucleotide polymorphism genotyping array.

CeO₂ and ZnO nanoparticles change the nutritional qualities of cucumber (Cucumis sativus).

Transcriptome profiling reveals roles of meristem regulators and polarity genes during fruit trichome development in cucumber (Cucumis sativus L.).

High-Throughput Sequencing Identifies Novel and Conserved Cucumber (Cucumis sativus L.) microRNAs in Response to Cucumber Green Mottle Mosaic Virus Infection.

[Virus particles in phloem exudate from Cucumis sativus L. after infection by Cucumis Virus 2 A].

Transcriptomic Analysis Implies That GA Regulates Sex Expression via Ethylene-Dependent and Ethylene-Independent Pathways in Cucumber (Cucumis sativus L.).

Inheritance of seed weight in Cucumis sativus (L.) var. sativus and var. hardwickii (Royle) Kitamura.