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Uptake of nicotine from discarded cigarette butts – A so far unconsidered path of contamination of plant-derived commodities
Environmental Pollution xxx (2018) 1e5
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities* Dirk Selmar a, *, Alzahraa Radwan a, b, Neama Abdalla c, Hussein Taha c, Carina Wittke a, €chter a, Ahmed El-Henawy d, Tarek Alshaal d, Megahed Amer e, Maik Kleinwa a d Melanie Nowak , Hassan El-Ramady a
Institute for Plant Biology, TU Braunschweig, Braunschweig, Germany Agriculture Genetic Engineering Research Institute (AGERI), Egypt Plant Biotechnology Department, National Research Center, Egypt d Soil & Water Sciences Department, Faculty of Agriculture, Kafrelsheikh University, Egypt e Soil, Water & Environment Research Institute, Sakha Agricultural Research Station, ARC, Egypt b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2017 Received in revised form 19 January 2018 Accepted 31 January 2018 Available online xxx
This study aimed to elucidate the origin of the widespread nicotine contamination of plant-derived commodities, by conducting field experiments with various herbs and spice plants. By scattering tobacco and cigarette butts on the field and subsequent nicotine analyses of the acceptor plants, we verified that the alkaloid is leached out into the soil and is taken up by the crop plants. This path of contamination pertains even when there is only one cigarette butt per square meter. Even such minor pollution results - at least in the case of basil and peppermint - in considerable high nicotine contaminations, which exceed the maximum residue level by more than 20-fold. The data reported here clearly outline the large practical relevance of this soil-borne contamination path and imply that unthoughtful disposal of cigarette butts in the field by farm workers may be the reason for the widespread occurrence of nicotine contamination in plant-derived commodities. Therefore, such misbehavior needs to be prevented using education and sensitization, and by including this issue into the guidelines of good agricultural practice. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Nicotine Contamination Horizontal natural product transfer Plant-derived commodities Field studies
1. Introduction In 2009, the EU banned all nicotine-containing insecticides (Commission decision, No. 396/2005, 2009) and a maximum residue level (MRL) of 0.01 mg nicotine per kg d.w. was set. As a consequence, routine controls have been conducted. Surprisingly, a tremendously high number of the tested plant-derived commodities contain nicotine levels far above the MRL. In 2011, a comprehensive survey by the EFSA (2011) revealed a high prevalence of nicotine contaminations in fruits, teas, spices, and medicinal plants. Moreover, this study revealed that many samples contained massively enhanced nicotine levels, which exceeded the prescribed MRL by more than 100-fold. In order to prevent the withdrawal of major shares of these commodities from the market, the EFSA
*
This paper has been recommended for acceptance by Dr. Jorg Rinklebe. * Corresponding author. E-mail address: [email protected] (D. Selmar).
(2011) temporarily increased the MRL for nicotine to 0.05 mg per kg d.w. Initially, it was argued that the observed nicotine contaminations might be due to prolonged illegal usage of nicotinecontaining insecticides. However, high concentrations of nicotine were detected even in plant-derived commodities produced in well-audited farms, wherein the possibility of illegal use of nicotine-containing insecticides could be excluded. Accordingly, the question of the origin of such contamination remained unanswered. In the case of the widespread contamination of plant-derived commodities by pyrrolizidine alkaloids, it is well established that the main source of contaminations is an accidental co-harvest of alkaloid-containing weeds (Stegelmeier et al., 1999; Van Wyk et al., 2017). In contrast, due to the rare and very restricted occurrence of nicotine-containing weeds, this classical path of contamination could also be excluded as a source of the nicotine contamination observed. Consequently, an optional path of contamination must be responsible for the widespread occurrence of nicotine in plant
https://doi.org/10.1016/j.envpol.2018.01.113 0269-7491/© 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Selmar, D., et al., Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities, Environmental Pollution (2018), https://doi.org/10.1016/j.envpol.2018.01.113
2
D. Selmar et al. / Environmental Pollution xxx (2018) 1e5
products. Recently, it was demonstrated that, in principle, there are two further possibilities: either the nicotine in the acceptor plants may result from an alkaloid transfer by cigarette smoke, or it is due to an uptake of nicotine from the soil (Selmar et al., 2015a). In the latter case, the alkaloid would have to leach out from tobacco products, such as cigarettes butts, which had been discarded in the field (Selmar et al., 2015a). Yet, these studies had been conducted in green house and pot experiments. Accordingly, the question remained whether these coherences are also valid under field conditions. Whereas a direct nicotine transfer by cigarette smoke seems likely in closed factory buildings, e.g., processing and drying (Selmar et al., 2015a), it could be excluded in the fields owing to the considerable dilution of smoke outdoors. Thus, nicotine contamination in the field should only be due to the uptake of nicotine from the soil, as shown by Selmar et al. (2015a). In this paper, we report that - even under field conditions - nicotine is leached out from discarded cigarette butts and effectively taken up by crop plants. As a result, this paper unveils the major path for the widespread nicotine contamination of plant-derived commodities. 2. Material and methods
as internal standard. After intensive stirring (30 s), further extraction was performed in an ultrasonification bath (30 min, room temperature). After centrifugation (15 min at 5000 g), the supernatant was filtrated using a syringe with cotton wool in the tip. The filtrate was transferred into a round-bottom flask. To ensure complete extraction, the pellet was resuspended in 10 mL sulfuric acid (0.005 M) and stirred for 30 s, followed by 30 min of ultrasonification and a centrifugation step (15 min at 5000 g). The supernatant was filtered and transferred into the round-bottom flask. After weighing, the combined acidic extracts were frozen and lyophilized. Due to the acidic conditions, no nicotine was lost during freeze-drying. The dried residue was resuspended in 1M NaOH (1 mL) and intensively stirred. The liquid was transferred into sealable glass centrifugation tubes. The flask was rinsed with 0.5 mL NaOH (0.5 M), which was added to the first extract. After addition of 1 mL dichloromethane, the suspension was stirred intensively for 1 min to ensure that the nicotine, which is present in the alkaline solution as free base, is quantitatively transferred into the organic solvent. For effective phase separation, the liquid was centrifuged (5000 g at 17 C for 60 min). The dichloromethanephase was filtered through a PTFE syringe filter and transferred directly into the sample vials for the GC-MS analyses.
2.1. Field experiments 2.4. Quantification of nicotine Plants were cultivated in the experimental fields of the Agricultural Research Centre in Sakha, in common Egyptian clay soils (clay: ~53%; sand: ~22%; silt: ~25%; pH: 8.6; electrical conductivity: ~4.5 dS m1). In the winter season, the typical “winter crops” such as parsley and coriander were cultivated, whereas basil and peppermint were grown in the summer. Different amounts of cigarette tobacco and quantities of cigarette butts were applied randomly in distinct plots of 10 m2 each, resulting in six different treatments: 0 g tobacco/m2, 0.25 g/m2, 2.5 g/m2, 25 g/m2, 1 cigarette butt/m2, and 5 cigarette butts/m2. On an average, about 9 plants (in case of peppermint and basil) and about 25 plants (in case of parsley and coriander) were cultivated per square meter. All treatments were carried out in triplicate. Using a garden rake, the tobacco material was mixed with the soil. To ensure appropriate leaching of the nicotine, directly after mulching, the plots were watered by spraying. In the course of further cultivation, watering was performed according to the plant needs, i.e., 2e3 times a month. Sampling was performed 7 and 14 days after mulching in the case of basil, and 7 and 12 days for the peppermint plants. Parsley and coriander plants were harvested at days 3, 7, 14, and 21 after the treatment. In addition, for all trials - as a negative control samples were taken just before the mulching was performed. 2.2. Sample preparation Freshly harvested plant material was dried at ambient temperatures in aerated rooms on benches and tables until the relative water content decreased below 10%. The dry samples were wrapped in aluminum foil and sent by airfreight from Cairo to Braunschweig for nicotine quantification. 2.3. Extraction of nicotine The dried samples were pulverized using a two-step procedure: first, they were shredded in a blender, and then the resulting material was powdered using a grinder. A two-step extraction was carried out to ensure comprehensive quantification of nicotine, and to avoid any loss of the volatile alkaloid: 2 g of dried plant material were weighed into plastic sample tubes (50 mL) and extracted with 20 mL sulfuric acid (0.005 M). For reliable quantification, 100 mL deuterated nicotine (2,4,5,6-d4 pyridine-d4, 10 mg/mL) was added
Quantification of nicotine was carried out on an Agilent GC/ MSD-System 5977B. Separation was achieved using a capillary column (DB-5MS; 30 m; diameter: 0.25 mm; film thickness 0.25 mm). The oven temperature was increased from 45 C to 300 C, following a temperature gradient of 40 C/min. The selected ion monitoring (SIM) mode was used in mass analysis. As selected ion, m:z values of 162 and 166 were used as quantifiers for nicotine and d4-nicotine, respectively, and 133 and 137 as corresponding qualifiers. 3. Results and discussion In order to elucidate whether the origin of the observed nicotine contaminations of plant-derived commodities could be due to an uptake of alkaloids from the soil, field experiments using various herbs and spice plants were conducted. Various amounts of tobacco or cigarette butts were applied onto the soil, in where the plants were then cultivated. 3.1. Nicotine uptake in crop plants Apart from the controls, in all trials, nicotine was detectable in the acceptor plants (Figs. 1e4); this accounts for all mulching approaches with tobacco as well as for those with cigarette butts. Although the data reveal a tremendous variation in the nicotine contents, in all cases the highest nicotine levels in the acceptor plants were detected within the first week after the mulching. The highest nicotine contaminations, e.g., nearly 17 mg/g d.w. in coriander leaves and about 4 mg/g d.w. in parsley leaves correspond to nicotine concentrations, which exceed even the temporarily enhanced - MRL of 0.05 mg/kg d.w. (mg/g d.w.) by more than 300fold and 80-fold, respectively. Independent of the applied dose, in the plants from all tobacco treatments, the MRL for nicotine was exceeded during the first week after treatment. The most astonishing finding, which has undoubtedly the highest relevance for practical considerations, corresponds to the contamination resulting from only a single cigarette butt applied to one square meter soil (¼ 10 cigarette butts per 10 m2). Such - at first glance - minor pollution of the soil resulted, at least in the case of basil and peppermint, in considerable high nicotine contamination, which
Please cite this article in press as: Selmar, D., et al., Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities, Environmental Pollution (2018), https://doi.org/10.1016/j.envpol.2018.01.113
D. Selmar et al. / Environmental Pollution xxx (2018) 1e5
Fig. 1. Nicotine content in peppermint plants. Different amounts of cigarette tobacco and cigarette butts were applied in distinct plots of 10 m2. The control corresponds to plants, that were harvested directly before the mulching. The dotted line denoted by *, corresponds to the temporarily enhanced MRL (EFSA, 2011). Each column represents the mean value of three independent replicates. Standard deviations are represented as error bars In the case of the lower graph (12 days after application), only two samples were analyzed. Accordingly, the standard deviation was not calculated.
exceeded the MRL by more than 20-fold. In contrast, in coriander, the contamination caused by the application of one cigarette butt per m2 was just twice the MRL, and in the case of parsley, the MRL was barely reached.
3.2. Differences in the pattern and extent of the nicotine uptake The second conjuncture that needs to be highlighted is the presence of extremely large variations in the nicotine levels in different acceptor plants and - at least in parts - the high standard deviations. Whereas the highest nicotine content in coriander (Fig. 4) and parsley (Fig. 3) was seen in plants grown on soils tainted by tobacco, the application of cigarette butts just caused minor contaminations. The opposite effect was observed in peppermint plants (Fig. 1), i.e., the application of cigarette butts resulted in high nicotine content, whereas the mulching with tobacco caused only relatively low nicotine pollution of the acceptor plants. The results for basil are in between; both treatments show similar, relatively low and medium nicotine content (Fig. 2). When discussing the putative reasons for these massive differences and the high standard deviations, we have to consider several facts and putative coherences: * * * *
inhomogeneous distribution of mulch material differences in the extent of leaching differences in the uptake of alkaloids differences in the ability to metabolize the imported alkaloids.
3
Fig. 2. Nicotine content in basil plants. Different amounts of cigarette tobacco and cigarette butts were applied in distinct plots of 10 m2. The control corresponds to plants that were harvested directly before the mulching. The dotted line denoted by * corresponds to the temporarily enhanced MRL (EFSA, 2011). Each column represents the mean value of three independent replicates. Standard deviations are represented as error bars.
3.2.1. Inhomogeneous distribution of mulch material In contrast to the very small range of variations in corresponding pot experiments (Selmar et al., 2015a), the variation in the nicotine levels of the acceptor plants derived from the field experiments is tremendous. In this context, it needs to be considered that a homogeneous distribution of the mulch material in the field could hardly be achieved. This especially accounts for the application of cigarette butts, i.e., it is not possible to uniformly distribute a single cigarette butt in one square meter. Accordingly, the plants directly neighboring the butt would be exposed to a much higher nicotine concentration than those further afield. 3.2.2. Differences in the extent of leaching The leaching of nicotine requires the presence of water, e.g., by rain or watering. Indeed, watering directly after the mulching (see material and methods) should ensure that the leaching was initiated, but the efficiency and extent of the elution depend on the timespan exhibiting an appropriate moisture content. This, however, depends on the specific climatic conditions, e.g., wind and rain intensity, humidity, and temperature. As the field trials were performed at different times, and thus - at least in parts - also in different seasons, these parameters are slightly different. Moreover, it has to be taken into consideration that - depending on the climatic conditions - parts of nicotine might be lost by evaporation. Indeed, in the plants, the nicotine is trapped as a non-volatile salt within the acidic vacuoles (Matile, 1976), but when present in the soil, it might be volatilized. In contrast to these minor effects, for cigarette butts, massive differences in the nicotine evaporation are possible, as the alkaloid is present mainly as a volatile free base.
Please cite this article in press as: Selmar, D., et al., Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities, Environmental Pollution (2018), https://doi.org/10.1016/j.envpol.2018.01.113
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D. Selmar et al. / Environmental Pollution xxx (2018) 1e5
Fig. 3. Nicotine content in parsley plants. Different amounts of cigarette tobacco and cigarette butts were applied in distinct plots of 10 m2. The control corresponds to plants that were harvested directly before the mulching. The dotted line denoted by * corresponds to the temporarily enhanced MRL (EFSA, 2011). Each column represents the mean value of three independent replicates. Standard deviations are represented as error bars.
Fig. 4. Nicotine content in coriander plants. Different amounts of cigarette tobacco and cigarette butts were applied in distinct plots of 10 m2. The control corresponds to plants that were harvested directly before the mulching. The dotted line denoted by * corresponds to the temporarily enhanced MRL (EFSA, 2011). Each column represents the mean value of three independent replicates. Standard deviations are mentioned as error bars.
Accordingly, the evaporation rate could be much higher depending on the climatic conditions. Consequently, these factors would strongly affect the actual nicotine content of the cigarette butts, and thereby, the amount of the alkaloid that could putatively leach out. These coherences might explain, why in the plants cultivated in winter, i.e., coriander and parsley, only minor amounts of nicotine were present in the acceptor plants, whereas in the plants grown in the summer season (peppermint and basil), high nicotine concentrations resulted from the application of cigarette butts.
strongly reduced (Nowak and Selmar, 2016). Indeed, all plants were grown in the same soil, and thus, basically, in an environment experiencing the same pH. However, we have to consider that plant roots create their own rhizosphere, e.g., by exuding organic acids (Hinsinger et al., 2003). As a consequence, the extent of the acidification might depend on the plant species (e.g. Zhou et al., 2009). Accordingly, the pH in the rhizosphere, and thus, the diffusion velocity of the alkaloids and their uptake into the plants, could vary for different plant species. Another factor that needs to be considered is the rate of transpiration. The alkaloids taken up by the roots of the acceptor plant are translocated via the xylem into the leaves (Nowak and Selmar, 2016). The extent of this transport depends strongly on the rate of transpiration, which varies, depending on the plant species as well as on the environmental conditions. Furthermore, the velocity of translocation strongly influences the concentration of alkaloids remaining in the roots, and thereby, the gradient between the soil and the root. As a consequence, the diffusion-driven import into the roots depends indirectly on the rate of transpiration.
3.2.3. Differences in the uptake of alkaloids As the uptake of alkaloids is due to their simple diffusion through the plasmalemma of the root cells, and no active import by transporters is required (Yahyazadeh et al., 2017), in principle, all plants should reveal the same import rate. Nonetheless, some environmental factors do influence the extent of the alkaloids present in the acceptor plants, i.e., the pH of the soil and the rate of transpiration of the plant leaves. Due to the higher degree of protonation in acidic compartments, less free alkaloids are present as free bases, and thus, the rate of diffusion across biomembranes is
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3.2.4. Differences in the ability to metabolize the imported alkaloids In general, the content of nicotine strongly declines with time (Figs. 1e4). The same effect has been observed for nicotine uptake in the pot experiments (Selmar et al., 2015a). As outlined above, a simple evaporation of nicotine could only contribute to a minor extent to this phenomenon, since the alkaloids accumulated within acidic vacuoles are trapped as non-volatile salts. Accordingly, there must be another explanation for the time-dependent decline of the nicotine concentration in the acceptor plants. A similar concentration decrease was determined, when analyzing the concentrations of the non-volatile pyrrolizidine alkaloids in acceptor plants (Nowak et al., 2016). Preliminary results indicate that the imported alkaloids are modified (Selmar et al., unpublished data), analogous to the metabolization of imported xenobiotics. It is well established that xenobiotics - after their uptake into the acceptor plants - are modified by oxidation and hydroxylation, and by the attachment of glucose or glutathione (Sandermann, 1994). As these processes may differ depending on the plant species, different shares of the imported nicotine should be modified and metabolized, respectively. Indeed, it is not currently known that which of the four abovementioned causes represent the most relevant factor for the observed high variation in nicotine uptake. Nevertheless, it needs to be clearly emphasized that, in all the acceptor plants tested, considerably high nicotine contamination was detected. 4. Conclusion As already outlined, the most important finding with respect to practical considerations corresponds to the contamination resulting from just a single cigarette butt applied to one square meter of soil. These data clearly reveal that the unthoughtful disposal of cigarette butts in the field by farm workers indeed could be the reason for the widespread occurrence of nicotine contaminations of plant-derived commodities. It is recommended that, in future, such misbehavior will be prevented, by education and sensitization, and by including this issue in the guidelines of good agricultural and collection practice (GACP). Apart from the nicotine uptake from the soil, we have to keep in mind that, in closed buildings, there might be a second path of contamination, i.e., the transfer of nicotine via the atmosphere by cigarette smoke (Selmar et al., 2015a). Therefore, smoking in closed compartments that contain plant materials, i.e., buildings for processing and drying, or transportation vessels, has to be prevented as well. A further nicotine source might be related to the compost applied as fertilizer to the fields. Unfortunately, no information on the occurrence of nicotine in composting materials and its biological half-life is available. In addition to applied aspects outlined above, the data on the uptake of nicotine under field conditions verifies the concept of the
5
horizontal transfer of natural products (Selmar et al., 2015b), which, up to date, was based on corresponding pot experiments. This investigation unequivocally proves that these processes are also relevant in the field, and thus, in nature. Disclosure All authors declare no competing financial interest. Acknowledgements This research project was supported by the German Egyptian Research Fund Programme (GERF), which is co-financed by the Federal Ministry of Education and Research (BMBF) of the Federal Republic of Germany and the Science and Technology Development Fund (STDF) of the Arab Republic of Egypt under project number 01DH14019 (BMBF) and GERF II-ID 5310 (STDF), respectively. References Commission Decision, 2009. Concerning the non-inclusion of nicotine in Annex I to Council Directive 91/414/EEC and the withdrawal of authorisations for plant protection products containing that substance. Offic. J. Eur. Union. L 5/ 9e9.1.2009. EFSA - European Food Safety Authority, 2011. Setting of temporary MRLs for nicotine in tea, herbal infusions, spices, rose hips and fresh herbs. EFSA J. 9 (3), 2098. Hinsinger, P., Plassard, C., Tang, C., Jaillard, B., 2003. Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: a review. Plant Soil 248, 43e59. Matile, P., 1976. Localization of alkaloids and mechanism of their accumulation in vacuoles of Chelidonium majus laticifers. Nova Acta Leopold. 7, 139e156. Nowak, M., Selmar, D., 2016. Cellular distribution of alkaloids and their translocation via phloem and xylem: the importance of compartment pH. Plant Biol. 18, 879e882. €chter, M., Selmar, D., 2016. Nowak, M., Wittke, C., Lederer, I., Klier, B., Kleinwa Interspecific transfer of pyrrolizidine alkaloids: an unconsidered source of contaminations of phytopharmaceuticals and plant-derived commodities. Food Chem. 213, 163e168. Sandermann, H., 1994. Higher plant metabolism of xenobiotics: the ‘green liver’ concept. Pharmacogenetics 4, 225e241. €nsel, S., Thra €ne, C., Nowak, M., Kleinwa €chter, M., Selmar, D., Engelhard, U.H., Ha 2015a. Nicotine contamination sources: nicotine uptake by peppermint plants (Mentha piperita). Agron. Sustain. Dev. 35, 1185e1190. Selmar, D., Radwan, A., Nowak, M., 2015b. Horizontal natural product transfer: a so far unconsidered source of contamination of plant-derived commodities. J. Anal. Toxicol. 5, 4. Stegelmeier, B.L., Edgar, J.A., Colegate, S.M., Gardner, D.R., Schoch, T.K., Coulombe, R.A., Molyneux, R.J., 1999. Pyrrolizidine alkaloid plants, metabolism and toxicity. J. Nat. Toxins 8, 95e116. Van Wyk, B.-E., Stander, M.A., Long, H.S., 2017. Senecio angustifolius as the major source of pyrrolizidine alkaloid contamination of rooibos tea (Aspalathus linearis). S. Afr. J. Bot. 110, 124e131. Yahyazadeh, M., Nowak, M., Kima, H., Selmar, D., 2017. Horizontal natural product transfer: a potential source of alkaloidal contaminants in phytopharmaceuticals. Phytomedicine 34, 21e25. Zhou, L.L., Cao, J., Zhang, F.S., Li, L., 2009. Rhizosphere acidification of faba bean, soybean and maize. Sci. Total Environ. 407, 4356e4362.
Please cite this article in press as: Selmar, D., et al., Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities, Environmental Pollution (2018), https://doi.org/10.1016/j.envpol.2018.01.113
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities* Dirk Selmar a, *, Alzahraa Radwan a, b, Neama Abdalla c, Hussein Taha c, Carina Wittke a, €chter a, Ahmed El-Henawy d, Tarek Alshaal d, Megahed Amer e, Maik Kleinwa a d Melanie Nowak , Hassan El-Ramady a
Institute for Plant Biology, TU Braunschweig, Braunschweig, Germany Agriculture Genetic Engineering Research Institute (AGERI), Egypt Plant Biotechnology Department, National Research Center, Egypt d Soil & Water Sciences Department, Faculty of Agriculture, Kafrelsheikh University, Egypt e Soil, Water & Environment Research Institute, Sakha Agricultural Research Station, ARC, Egypt b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2017 Received in revised form 19 January 2018 Accepted 31 January 2018 Available online xxx
This study aimed to elucidate the origin of the widespread nicotine contamination of plant-derived commodities, by conducting field experiments with various herbs and spice plants. By scattering tobacco and cigarette butts on the field and subsequent nicotine analyses of the acceptor plants, we verified that the alkaloid is leached out into the soil and is taken up by the crop plants. This path of contamination pertains even when there is only one cigarette butt per square meter. Even such minor pollution results - at least in the case of basil and peppermint - in considerable high nicotine contaminations, which exceed the maximum residue level by more than 20-fold. The data reported here clearly outline the large practical relevance of this soil-borne contamination path and imply that unthoughtful disposal of cigarette butts in the field by farm workers may be the reason for the widespread occurrence of nicotine contamination in plant-derived commodities. Therefore, such misbehavior needs to be prevented using education and sensitization, and by including this issue into the guidelines of good agricultural practice. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Nicotine Contamination Horizontal natural product transfer Plant-derived commodities Field studies
1. Introduction In 2009, the EU banned all nicotine-containing insecticides (Commission decision, No. 396/2005, 2009) and a maximum residue level (MRL) of 0.01 mg nicotine per kg d.w. was set. As a consequence, routine controls have been conducted. Surprisingly, a tremendously high number of the tested plant-derived commodities contain nicotine levels far above the MRL. In 2011, a comprehensive survey by the EFSA (2011) revealed a high prevalence of nicotine contaminations in fruits, teas, spices, and medicinal plants. Moreover, this study revealed that many samples contained massively enhanced nicotine levels, which exceeded the prescribed MRL by more than 100-fold. In order to prevent the withdrawal of major shares of these commodities from the market, the EFSA
*
This paper has been recommended for acceptance by Dr. Jorg Rinklebe. * Corresponding author. E-mail address: [email protected] (D. Selmar).
(2011) temporarily increased the MRL for nicotine to 0.05 mg per kg d.w. Initially, it was argued that the observed nicotine contaminations might be due to prolonged illegal usage of nicotinecontaining insecticides. However, high concentrations of nicotine were detected even in plant-derived commodities produced in well-audited farms, wherein the possibility of illegal use of nicotine-containing insecticides could be excluded. Accordingly, the question of the origin of such contamination remained unanswered. In the case of the widespread contamination of plant-derived commodities by pyrrolizidine alkaloids, it is well established that the main source of contaminations is an accidental co-harvest of alkaloid-containing weeds (Stegelmeier et al., 1999; Van Wyk et al., 2017). In contrast, due to the rare and very restricted occurrence of nicotine-containing weeds, this classical path of contamination could also be excluded as a source of the nicotine contamination observed. Consequently, an optional path of contamination must be responsible for the widespread occurrence of nicotine in plant
https://doi.org/10.1016/j.envpol.2018.01.113 0269-7491/© 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Selmar, D., et al., Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities, Environmental Pollution (2018), https://doi.org/10.1016/j.envpol.2018.01.113
2
D. Selmar et al. / Environmental Pollution xxx (2018) 1e5
products. Recently, it was demonstrated that, in principle, there are two further possibilities: either the nicotine in the acceptor plants may result from an alkaloid transfer by cigarette smoke, or it is due to an uptake of nicotine from the soil (Selmar et al., 2015a). In the latter case, the alkaloid would have to leach out from tobacco products, such as cigarettes butts, which had been discarded in the field (Selmar et al., 2015a). Yet, these studies had been conducted in green house and pot experiments. Accordingly, the question remained whether these coherences are also valid under field conditions. Whereas a direct nicotine transfer by cigarette smoke seems likely in closed factory buildings, e.g., processing and drying (Selmar et al., 2015a), it could be excluded in the fields owing to the considerable dilution of smoke outdoors. Thus, nicotine contamination in the field should only be due to the uptake of nicotine from the soil, as shown by Selmar et al. (2015a). In this paper, we report that - even under field conditions - nicotine is leached out from discarded cigarette butts and effectively taken up by crop plants. As a result, this paper unveils the major path for the widespread nicotine contamination of plant-derived commodities. 2. Material and methods
as internal standard. After intensive stirring (30 s), further extraction was performed in an ultrasonification bath (30 min, room temperature). After centrifugation (15 min at 5000 g), the supernatant was filtrated using a syringe with cotton wool in the tip. The filtrate was transferred into a round-bottom flask. To ensure complete extraction, the pellet was resuspended in 10 mL sulfuric acid (0.005 M) and stirred for 30 s, followed by 30 min of ultrasonification and a centrifugation step (15 min at 5000 g). The supernatant was filtered and transferred into the round-bottom flask. After weighing, the combined acidic extracts were frozen and lyophilized. Due to the acidic conditions, no nicotine was lost during freeze-drying. The dried residue was resuspended in 1M NaOH (1 mL) and intensively stirred. The liquid was transferred into sealable glass centrifugation tubes. The flask was rinsed with 0.5 mL NaOH (0.5 M), which was added to the first extract. After addition of 1 mL dichloromethane, the suspension was stirred intensively for 1 min to ensure that the nicotine, which is present in the alkaline solution as free base, is quantitatively transferred into the organic solvent. For effective phase separation, the liquid was centrifuged (5000 g at 17 C for 60 min). The dichloromethanephase was filtered through a PTFE syringe filter and transferred directly into the sample vials for the GC-MS analyses.
2.1. Field experiments 2.4. Quantification of nicotine Plants were cultivated in the experimental fields of the Agricultural Research Centre in Sakha, in common Egyptian clay soils (clay: ~53%; sand: ~22%; silt: ~25%; pH: 8.6; electrical conductivity: ~4.5 dS m1). In the winter season, the typical “winter crops” such as parsley and coriander were cultivated, whereas basil and peppermint were grown in the summer. Different amounts of cigarette tobacco and quantities of cigarette butts were applied randomly in distinct plots of 10 m2 each, resulting in six different treatments: 0 g tobacco/m2, 0.25 g/m2, 2.5 g/m2, 25 g/m2, 1 cigarette butt/m2, and 5 cigarette butts/m2. On an average, about 9 plants (in case of peppermint and basil) and about 25 plants (in case of parsley and coriander) were cultivated per square meter. All treatments were carried out in triplicate. Using a garden rake, the tobacco material was mixed with the soil. To ensure appropriate leaching of the nicotine, directly after mulching, the plots were watered by spraying. In the course of further cultivation, watering was performed according to the plant needs, i.e., 2e3 times a month. Sampling was performed 7 and 14 days after mulching in the case of basil, and 7 and 12 days for the peppermint plants. Parsley and coriander plants were harvested at days 3, 7, 14, and 21 after the treatment. In addition, for all trials - as a negative control samples were taken just before the mulching was performed. 2.2. Sample preparation Freshly harvested plant material was dried at ambient temperatures in aerated rooms on benches and tables until the relative water content decreased below 10%. The dry samples were wrapped in aluminum foil and sent by airfreight from Cairo to Braunschweig for nicotine quantification. 2.3. Extraction of nicotine The dried samples were pulverized using a two-step procedure: first, they were shredded in a blender, and then the resulting material was powdered using a grinder. A two-step extraction was carried out to ensure comprehensive quantification of nicotine, and to avoid any loss of the volatile alkaloid: 2 g of dried plant material were weighed into plastic sample tubes (50 mL) and extracted with 20 mL sulfuric acid (0.005 M). For reliable quantification, 100 mL deuterated nicotine (2,4,5,6-d4 pyridine-d4, 10 mg/mL) was added
Quantification of nicotine was carried out on an Agilent GC/ MSD-System 5977B. Separation was achieved using a capillary column (DB-5MS; 30 m; diameter: 0.25 mm; film thickness 0.25 mm). The oven temperature was increased from 45 C to 300 C, following a temperature gradient of 40 C/min. The selected ion monitoring (SIM) mode was used in mass analysis. As selected ion, m:z values of 162 and 166 were used as quantifiers for nicotine and d4-nicotine, respectively, and 133 and 137 as corresponding qualifiers. 3. Results and discussion In order to elucidate whether the origin of the observed nicotine contaminations of plant-derived commodities could be due to an uptake of alkaloids from the soil, field experiments using various herbs and spice plants were conducted. Various amounts of tobacco or cigarette butts were applied onto the soil, in where the plants were then cultivated. 3.1. Nicotine uptake in crop plants Apart from the controls, in all trials, nicotine was detectable in the acceptor plants (Figs. 1e4); this accounts for all mulching approaches with tobacco as well as for those with cigarette butts. Although the data reveal a tremendous variation in the nicotine contents, in all cases the highest nicotine levels in the acceptor plants were detected within the first week after the mulching. The highest nicotine contaminations, e.g., nearly 17 mg/g d.w. in coriander leaves and about 4 mg/g d.w. in parsley leaves correspond to nicotine concentrations, which exceed even the temporarily enhanced - MRL of 0.05 mg/kg d.w. (mg/g d.w.) by more than 300fold and 80-fold, respectively. Independent of the applied dose, in the plants from all tobacco treatments, the MRL for nicotine was exceeded during the first week after treatment. The most astonishing finding, which has undoubtedly the highest relevance for practical considerations, corresponds to the contamination resulting from only a single cigarette butt applied to one square meter soil (¼ 10 cigarette butts per 10 m2). Such - at first glance - minor pollution of the soil resulted, at least in the case of basil and peppermint, in considerable high nicotine contamination, which
Please cite this article in press as: Selmar, D., et al., Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities, Environmental Pollution (2018), https://doi.org/10.1016/j.envpol.2018.01.113
D. Selmar et al. / Environmental Pollution xxx (2018) 1e5
Fig. 1. Nicotine content in peppermint plants. Different amounts of cigarette tobacco and cigarette butts were applied in distinct plots of 10 m2. The control corresponds to plants, that were harvested directly before the mulching. The dotted line denoted by *, corresponds to the temporarily enhanced MRL (EFSA, 2011). Each column represents the mean value of three independent replicates. Standard deviations are represented as error bars In the case of the lower graph (12 days after application), only two samples were analyzed. Accordingly, the standard deviation was not calculated.
exceeded the MRL by more than 20-fold. In contrast, in coriander, the contamination caused by the application of one cigarette butt per m2 was just twice the MRL, and in the case of parsley, the MRL was barely reached.
3.2. Differences in the pattern and extent of the nicotine uptake The second conjuncture that needs to be highlighted is the presence of extremely large variations in the nicotine levels in different acceptor plants and - at least in parts - the high standard deviations. Whereas the highest nicotine content in coriander (Fig. 4) and parsley (Fig. 3) was seen in plants grown on soils tainted by tobacco, the application of cigarette butts just caused minor contaminations. The opposite effect was observed in peppermint plants (Fig. 1), i.e., the application of cigarette butts resulted in high nicotine content, whereas the mulching with tobacco caused only relatively low nicotine pollution of the acceptor plants. The results for basil are in between; both treatments show similar, relatively low and medium nicotine content (Fig. 2). When discussing the putative reasons for these massive differences and the high standard deviations, we have to consider several facts and putative coherences: * * * *
inhomogeneous distribution of mulch material differences in the extent of leaching differences in the uptake of alkaloids differences in the ability to metabolize the imported alkaloids.
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Fig. 2. Nicotine content in basil plants. Different amounts of cigarette tobacco and cigarette butts were applied in distinct plots of 10 m2. The control corresponds to plants that were harvested directly before the mulching. The dotted line denoted by * corresponds to the temporarily enhanced MRL (EFSA, 2011). Each column represents the mean value of three independent replicates. Standard deviations are represented as error bars.
3.2.1. Inhomogeneous distribution of mulch material In contrast to the very small range of variations in corresponding pot experiments (Selmar et al., 2015a), the variation in the nicotine levels of the acceptor plants derived from the field experiments is tremendous. In this context, it needs to be considered that a homogeneous distribution of the mulch material in the field could hardly be achieved. This especially accounts for the application of cigarette butts, i.e., it is not possible to uniformly distribute a single cigarette butt in one square meter. Accordingly, the plants directly neighboring the butt would be exposed to a much higher nicotine concentration than those further afield. 3.2.2. Differences in the extent of leaching The leaching of nicotine requires the presence of water, e.g., by rain or watering. Indeed, watering directly after the mulching (see material and methods) should ensure that the leaching was initiated, but the efficiency and extent of the elution depend on the timespan exhibiting an appropriate moisture content. This, however, depends on the specific climatic conditions, e.g., wind and rain intensity, humidity, and temperature. As the field trials were performed at different times, and thus - at least in parts - also in different seasons, these parameters are slightly different. Moreover, it has to be taken into consideration that - depending on the climatic conditions - parts of nicotine might be lost by evaporation. Indeed, in the plants, the nicotine is trapped as a non-volatile salt within the acidic vacuoles (Matile, 1976), but when present in the soil, it might be volatilized. In contrast to these minor effects, for cigarette butts, massive differences in the nicotine evaporation are possible, as the alkaloid is present mainly as a volatile free base.
Please cite this article in press as: Selmar, D., et al., Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities, Environmental Pollution (2018), https://doi.org/10.1016/j.envpol.2018.01.113
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D. Selmar et al. / Environmental Pollution xxx (2018) 1e5
Fig. 3. Nicotine content in parsley plants. Different amounts of cigarette tobacco and cigarette butts were applied in distinct plots of 10 m2. The control corresponds to plants that were harvested directly before the mulching. The dotted line denoted by * corresponds to the temporarily enhanced MRL (EFSA, 2011). Each column represents the mean value of three independent replicates. Standard deviations are represented as error bars.
Fig. 4. Nicotine content in coriander plants. Different amounts of cigarette tobacco and cigarette butts were applied in distinct plots of 10 m2. The control corresponds to plants that were harvested directly before the mulching. The dotted line denoted by * corresponds to the temporarily enhanced MRL (EFSA, 2011). Each column represents the mean value of three independent replicates. Standard deviations are mentioned as error bars.
Accordingly, the evaporation rate could be much higher depending on the climatic conditions. Consequently, these factors would strongly affect the actual nicotine content of the cigarette butts, and thereby, the amount of the alkaloid that could putatively leach out. These coherences might explain, why in the plants cultivated in winter, i.e., coriander and parsley, only minor amounts of nicotine were present in the acceptor plants, whereas in the plants grown in the summer season (peppermint and basil), high nicotine concentrations resulted from the application of cigarette butts.
strongly reduced (Nowak and Selmar, 2016). Indeed, all plants were grown in the same soil, and thus, basically, in an environment experiencing the same pH. However, we have to consider that plant roots create their own rhizosphere, e.g., by exuding organic acids (Hinsinger et al., 2003). As a consequence, the extent of the acidification might depend on the plant species (e.g. Zhou et al., 2009). Accordingly, the pH in the rhizosphere, and thus, the diffusion velocity of the alkaloids and their uptake into the plants, could vary for different plant species. Another factor that needs to be considered is the rate of transpiration. The alkaloids taken up by the roots of the acceptor plant are translocated via the xylem into the leaves (Nowak and Selmar, 2016). The extent of this transport depends strongly on the rate of transpiration, which varies, depending on the plant species as well as on the environmental conditions. Furthermore, the velocity of translocation strongly influences the concentration of alkaloids remaining in the roots, and thereby, the gradient between the soil and the root. As a consequence, the diffusion-driven import into the roots depends indirectly on the rate of transpiration.
3.2.3. Differences in the uptake of alkaloids As the uptake of alkaloids is due to their simple diffusion through the plasmalemma of the root cells, and no active import by transporters is required (Yahyazadeh et al., 2017), in principle, all plants should reveal the same import rate. Nonetheless, some environmental factors do influence the extent of the alkaloids present in the acceptor plants, i.e., the pH of the soil and the rate of transpiration of the plant leaves. Due to the higher degree of protonation in acidic compartments, less free alkaloids are present as free bases, and thus, the rate of diffusion across biomembranes is
Please cite this article in press as: Selmar, D., et al., Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities, Environmental Pollution (2018), https://doi.org/10.1016/j.envpol.2018.01.113
D. Selmar et al. / Environmental Pollution xxx (2018) 1e5
3.2.4. Differences in the ability to metabolize the imported alkaloids In general, the content of nicotine strongly declines with time (Figs. 1e4). The same effect has been observed for nicotine uptake in the pot experiments (Selmar et al., 2015a). As outlined above, a simple evaporation of nicotine could only contribute to a minor extent to this phenomenon, since the alkaloids accumulated within acidic vacuoles are trapped as non-volatile salts. Accordingly, there must be another explanation for the time-dependent decline of the nicotine concentration in the acceptor plants. A similar concentration decrease was determined, when analyzing the concentrations of the non-volatile pyrrolizidine alkaloids in acceptor plants (Nowak et al., 2016). Preliminary results indicate that the imported alkaloids are modified (Selmar et al., unpublished data), analogous to the metabolization of imported xenobiotics. It is well established that xenobiotics - after their uptake into the acceptor plants - are modified by oxidation and hydroxylation, and by the attachment of glucose or glutathione (Sandermann, 1994). As these processes may differ depending on the plant species, different shares of the imported nicotine should be modified and metabolized, respectively. Indeed, it is not currently known that which of the four abovementioned causes represent the most relevant factor for the observed high variation in nicotine uptake. Nevertheless, it needs to be clearly emphasized that, in all the acceptor plants tested, considerably high nicotine contamination was detected. 4. Conclusion As already outlined, the most important finding with respect to practical considerations corresponds to the contamination resulting from just a single cigarette butt applied to one square meter of soil. These data clearly reveal that the unthoughtful disposal of cigarette butts in the field by farm workers indeed could be the reason for the widespread occurrence of nicotine contaminations of plant-derived commodities. It is recommended that, in future, such misbehavior will be prevented, by education and sensitization, and by including this issue in the guidelines of good agricultural and collection practice (GACP). Apart from the nicotine uptake from the soil, we have to keep in mind that, in closed buildings, there might be a second path of contamination, i.e., the transfer of nicotine via the atmosphere by cigarette smoke (Selmar et al., 2015a). Therefore, smoking in closed compartments that contain plant materials, i.e., buildings for processing and drying, or transportation vessels, has to be prevented as well. A further nicotine source might be related to the compost applied as fertilizer to the fields. Unfortunately, no information on the occurrence of nicotine in composting materials and its biological half-life is available. In addition to applied aspects outlined above, the data on the uptake of nicotine under field conditions verifies the concept of the
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horizontal transfer of natural products (Selmar et al., 2015b), which, up to date, was based on corresponding pot experiments. This investigation unequivocally proves that these processes are also relevant in the field, and thus, in nature. Disclosure All authors declare no competing financial interest. Acknowledgements This research project was supported by the German Egyptian Research Fund Programme (GERF), which is co-financed by the Federal Ministry of Education and Research (BMBF) of the Federal Republic of Germany and the Science and Technology Development Fund (STDF) of the Arab Republic of Egypt under project number 01DH14019 (BMBF) and GERF II-ID 5310 (STDF), respectively. References Commission Decision, 2009. Concerning the non-inclusion of nicotine in Annex I to Council Directive 91/414/EEC and the withdrawal of authorisations for plant protection products containing that substance. Offic. J. Eur. Union. L 5/ 9e9.1.2009. EFSA - European Food Safety Authority, 2011. Setting of temporary MRLs for nicotine in tea, herbal infusions, spices, rose hips and fresh herbs. EFSA J. 9 (3), 2098. Hinsinger, P., Plassard, C., Tang, C., Jaillard, B., 2003. Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: a review. Plant Soil 248, 43e59. Matile, P., 1976. Localization of alkaloids and mechanism of their accumulation in vacuoles of Chelidonium majus laticifers. Nova Acta Leopold. 7, 139e156. Nowak, M., Selmar, D., 2016. Cellular distribution of alkaloids and their translocation via phloem and xylem: the importance of compartment pH. Plant Biol. 18, 879e882. €chter, M., Selmar, D., 2016. Nowak, M., Wittke, C., Lederer, I., Klier, B., Kleinwa Interspecific transfer of pyrrolizidine alkaloids: an unconsidered source of contaminations of phytopharmaceuticals and plant-derived commodities. Food Chem. 213, 163e168. Sandermann, H., 1994. Higher plant metabolism of xenobiotics: the ‘green liver’ concept. Pharmacogenetics 4, 225e241. €nsel, S., Thra €ne, C., Nowak, M., Kleinwa €chter, M., Selmar, D., Engelhard, U.H., Ha 2015a. Nicotine contamination sources: nicotine uptake by peppermint plants (Mentha piperita). Agron. Sustain. Dev. 35, 1185e1190. Selmar, D., Radwan, A., Nowak, M., 2015b. Horizontal natural product transfer: a so far unconsidered source of contamination of plant-derived commodities. J. Anal. Toxicol. 5, 4. Stegelmeier, B.L., Edgar, J.A., Colegate, S.M., Gardner, D.R., Schoch, T.K., Coulombe, R.A., Molyneux, R.J., 1999. Pyrrolizidine alkaloid plants, metabolism and toxicity. J. Nat. Toxins 8, 95e116. Van Wyk, B.-E., Stander, M.A., Long, H.S., 2017. Senecio angustifolius as the major source of pyrrolizidine alkaloid contamination of rooibos tea (Aspalathus linearis). S. Afr. J. Bot. 110, 124e131. Yahyazadeh, M., Nowak, M., Kima, H., Selmar, D., 2017. Horizontal natural product transfer: a potential source of alkaloidal contaminants in phytopharmaceuticals. Phytomedicine 34, 21e25. Zhou, L.L., Cao, J., Zhang, F.S., Li, L., 2009. Rhizosphere acidification of faba bean, soybean and maize. Sci. Total Environ. 407, 4356e4362.
Please cite this article in press as: Selmar, D., et al., Uptake of nicotine from discarded cigarette butts e A so far unconsidered path of contamination of plant-derived commodities, Environmental Pollution (2018), https://doi.org/10.1016/j.envpol.2018.01.113