Subscriber access provided by Kaohsiung Medical University
Environmental Processes
Pharmacokinetics in Plants: Carbamazepine and Its Interactions with Lamotrigine Myah Goldstein, Tomer Malchi, Moshe Shenker, and Benny Chefetz Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01682 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 32
Environmental Science & Technology
ACS Paragon Plus Environment
Environmental Science & Technology
Page 2 of 32
Pharmacokinetics in Plants:
1
Carbamazepine and its Interactions with Lamotrigine
2 3
Myah Goldstein,†,‡ Tomer Malchi,†,‡ Moshe Shenker,† and Benny Chefetz†,‡,*
4 5
†
Department of Soil and Water Sciences, The Robert H. Smith Faculty of Agriculture,
6
Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot
7
7610001, Israel
8
‡
9
The Hebrew University Center of Excellence in Agriculture and Environmental
Health
10 11
*
12
Benny Chefetz
13
Tel.: 972-8-9489384; email:
[email protected] 14
Corresponding Author:
1 ACS Paragon Plus Environment
Page 3 of 32
Environmental Science & Technology
ABSTRACT
15
Carbamazepine and lamotrigine prescribed antiepileptic drugs are highly
16
persistent in the environment, and were detected in crops irrigated with reclaimed
17
wastewater. This study reports pharmacokinetics of the two drugs and their
18
metabolites in cucumber plants under hydroponic culture, testing their uptake,
19
translocation and transformation over 96 h in single and bi-solute systems at varying
20
pH. Ruling out root adsorption and transformations in the nutrient solution, we
21
demonstrate that carbamazepine root uptake is largely affected by the concentration
22
gradient across the membrane. Unlike carbamazepine, lamotrigine is adsorbed to the
23
root and undergoes ion trapping in root cells thus its translocation to the shoots is
24
limited. Based on that, carbamazepine uptake was not affected by the presence of
25
lamotrigine, while lamotrigine uptake was enhanced in the presence of
26
carbamazepine. Transformation of carbamazepine in the roots was slightly reduced in
27
the presence of lamotrigine. Carbamazepine metabolism was far more pronounced in
28
the shoots than in the roots, indicating that most of the metabolism occurs in the
29
leaves, probably due to higher concentration and longer residence time. This study
30
indicates that the uptake of small non-ionic pharmaceuticals is passive and governed
31
by diffusion across the root membrane.
32
2 ACS Paragon Plus Environment
Environmental Science & Technology
INTRODUCTION
Page 4 of 32
33
Carbamazepine (CBZ) is the most frequently prescribed antiepileptic drug
34
worldwide, used by patients of all ages.1–4 It acts as a voltage-dependent sodium-
35
channel blocker.5–7 Once administered, CBZ is metabolized, mainly in the liver, into
36
various different metabolites8,9 which are subsequently excreted from the body. Of the
37
total administrated dose, 13.8% was reported to be excreted as the parent compound
38
(mostly in the feces); 32% of the total as the metabolite 10,11-dihydro-10,11-
39
dihydroxy-carbamazepine (DiOH-CBZ), 5.1% as 3-hydroxy-carbamazepine (3-OH-
40
CBZ), 4.3% as 2-hydroxy-carbamazepine (2-OH-CBZ), and 1.4% as 10,11-dihydro-
41
10,11-epoxy-carbamazepine (EP-CBZ), all excreted in the urine, with several other
42
metabolites excreted in both feces and urine.10 Low removal efficiency and minimal
43
degradability of CBZ and its metabolites have been reported in municipal wastewater-
44
treatment plants.11–13 Thus CBZ and several of its metabolites are frequently detected
45
in reclaimed-wastewater used for irrigation and in freshwater bodies receiving
46
effluents.10,13–16 Once CBZ is introduced into the soil via irrigation with reclaimed-
47
wastewater or biosolids application, it has been shown to undergo only limited
48
biodegradation17, attributed to binding to soil organic matter and/or clays18, and to be
49
highly resistant to microbial decomposition.17,19 The shortest half-life reported for
50
CBZ in soils is approximately 42 days, whereas in most cases it is more than 200
51
days.17,20 CBZ is a neutral compound within the range of environmental pH.15
52
Lamotrigine (LTG), also a sodium-channel blocker,5–7 is a frequently
53
prescribed antiepileptic drug which may be co-administered with other antiepileptic
54
drug such as CBZ.1,3,4 LTG is excreted in urine mainly in its glucuronide form while
55
only 10% is excreted as the parent compound 21,22. LTG is of low removal efficiency
56
in municipal wastewater-treatment plants and is found at concentrations above 1 µg L-
57
3 ACS Paragon Plus Environment
Page 5 of 32
Environmental Science & Technology
1
in water bodies and in reclaimed-wastewater.13,23 Lamotrigine-N2-glucuronide is the
58
main LTG metabolite found in reclaimed-wastewater.13,23 Following reclaimed-
59
wastewater irrigation LTG was found to accumulate in the top soil exhibiting little to
60
no biodegradation.17,24 LTG is a weak base with a pKa of 5.7 and a Log D of 2.12 (at
61
pH of 7.5).25,26
62
Being a relatively restrictive barrier to organic compounds, the casparian strip
63
in plant roots exhibits traits similar to those of tight junctions which make up the
64
blood–brain barrier in mammals,27,28 thus implying that compounds known to cross
65
the blood–brain barrier through diffusion, such as CBZ29 and LTG,22 may also be
66
taken up and translocated by plant roots. We hypothesize that CBZ and LTG are taken
67
up as non-ionic species by diffusion through root cell membranes and therefore are
68
not expected to directly affect each other. However, based on well documented
69
pharmacokinetic interactions reported in humans30, we suggest that co-introduction of
70
CBZ and LTG will affect the kinetics of CBZ metabolism within the plant. The main
71
objective of this study was to understand the kinetics governing CBZ and LTG
72
uptake, translocation and metabolism in plants and to reveal whether these processes
73
are affected by co-introduction with other drugs (i.e., the environmental scenario). We
74
also aimed to reveal if the CBZ metabolites found in plant materials are a result of in-
75
plant metabolism or due to direct uptake of the metabolites from the irrigation water
76
and/or soil solution.
77 78
EXPERIMENTAL SECTION
79
Chemicals. CBZ (>97% purity) was purchased from Sigma-Aldrich Israel Ltd.
80
(Rehovot, Israel), and LTG (>99%) from EnzoBiochem Inc. (New York, NY). The
81
following CBZ metabolites were purchased from Toronto Research Chemicals Inc.
82
4 ACS Paragon Plus Environment
Environmental Science & Technology
Page 6 of 32
(Toronto, Canada): EP-CBZ, DiOH-CBZ, and 2-OH-CBZ. Selected properties of the
83
studied compounds are presented in Table 1. The following labeled compounds were
84
purchased from Toronto Research Chemicals: CBZ-13C-D2, LTG-13C3, DiOH-D3,
85
EP-CBZ-D8.
86
Hydroponic Culture Setup. Cucumber seeds (Cucumis sativus, Patriot, Hazera
87
Genetics Ltd., Berurim, Israel) were germinated in vermiculite moistened with
88
CaSO4·H2O-saturated solution. Seven-day-old seedlings were transferred and each
89
single plant was cultivated in 1-L darkened glass jars in a continuously aerated
90
nutrient solution, at an initial volume of 800 ± 10 mL, with the following composition
91
of macronutrients (mM): K2SO4, 0.7; KCl, 0.1; Ca(NO3)2·4H2O, 2.0; MgSO4, 0.5;
92
KH2PO4, 0.1, and micronutrients (µM): Fe-EDTA, 10; MnSO4·H2O, 0.5;
93
ZnSO4·7H2O, 0.5; CuSO4, 0.2; (NH4)6Mo7O24·4H2O, 0.01; H3BO3, 10. The nutrient
94
solution was prepared in deionized water, with a final pH of 5.7. The plants were
95
grown in a temperature-controlled chamber with a 16/8-h day/night cycle at 25 ± 1 °C
96
and 20 ± 1 °C day and night, respectively31 and relative humidity of 60-70% during
97
the day and 80-100% during the night. The nutrient solution was replaced every 3–4
98
days. Following 29 days of cultivation in the nutrient solution, the plants were
99
introduced into a fresh nutrient solution containing: (i) CBZ, 1.82 ± 0.05 µM (430 µg
100
L-1); or (ii) LTG, 1.80 ± 0.03 µM (461 µg L-1); or (iii) EP-CBZ, 1.69 ± 0.00 µM (428
101
µg L-1); or (iv) DiOH-CBZ, 1.78 ± 0.00 µM (480 µg L-1); or (v) a mixture of CBZ and
102
LTG at concentrations of ~2 µM each.
103
Experimental Design. Plants were exposed to either CBZ, LTG, EP-CBZ, or DiOH-
104
CBZ (single-solute experiments), or to CBZ and LTG (bi-solute experiments) for up
105
to 96 h, maintaining a pH of 6-7 in all experiments by adding 4-(2-hydroxyethyl)-1-
106
piperazineethanesulfonic acid (HEPES) to the nutrient solution at a concentration of 5
107
5 ACS Paragon Plus Environment
Page 7 of 32
Environmental Science & Technology
mM. During this time, the nutrient solution was not replenished and continuously
108
aerated. The plants exhibited a healthy root system throughout the entire cultivation
109
and experimental period. The nutrient solution was sampled at 1, 3, 6, 8, 24, 48, 72
110
and 96 h for the CBZ and LTG single-solute experiments and CBZ and LTG bi-solute
111
experiments, and once after 72 h for the EP-CBZ and DiOH-CBZ single-solute
112
experiments. The plants exposed to CBZ in the presence or absence of LTG were
113
sampled at 8, 24, 48, 72 and 96 h; the plants exposed to the metabolites were sampled
114
after 72 h. The experiments were conducted in 5 replicates for each sampling time. At
115
each sampling time, a whole plant was sacrificed for sap sampling and plant analyses.
116
After plants were detopped, xylem sap was collected over a period of approximately
117
15 min after detopping, collecting 2-3 mL of sap from each plant, while roots were
118
still in the nutrient solution, first drops being discarded. Then roots and shoots were
119
washed, weighed and frozen at -20 °C until extraction.
120
CBZ and LTG uptake and translocation in a bi-solute experiment was also tested
121
comparatively at pH of 4.5 and 7.5. In this experiment, the exposure time was 48 h.
122
To maintain pH 7.5, HEPES was added to the nutrient solution at a concentration of 5
123
mM. To maintain pH 4.5, 2(N-morpholino)ethane sulfonic acid (MES) was added at a
124
concentration of 5 mM. Since the MES buffer alone was not capable of maintaining a
125
steady, low pH, the Ca(NO3)2·4H2O (2 mM) in the nutrient solution was substituted
126
with 0.8 mM NH4NO3, 1.2 mM Ca(NO3)2·4H2O and 0.8 mM CaCl2 in the low pH
127
treatment, so that the N was composed of 20% NH4+ and 80% NO3-.
128
Analysis. Analyte concentrations in all solutions (sap and nutrient solutions) were
129
quantified using a Waters Alliance HPLC system equipped with a LiChrospher 100
130
RP-18 column (25 cm × 4.6 mm, 5 µm particle size; Merck, Darmstadt, Germany)
131
and photodiode array detector. The analytes were eluted from the column at a constant
132
6 ACS Paragon Plus Environment
Environmental Science & Technology
Page 8 of 32
flow rate of 1 mL min-1 and constant temperature of 45 °C; sample injection volume
133
was 10 µL. The initial mobile phase consisted of a mixture of methanol (14%),
134
acetonitrile (20%) and water (66%) (v/v). A gradient program was applied by raising
135
the level of acetonitrile from 20 to 50% over 12 min. CBZ, EP-CBZ and DiOH-CBZ
136
were quantified based on absorption at 210 nm, and LTG was quantified at 307 nm.
137
Limit of detection (LOD) and limit of quantification (LOQ) values, determined using
138
a signal to noise ratio of 3:1 and 10:1 respectively, were 25 and 50 µg L-1,
139
respectively, for CBZ and its metabolites and 50 and 100 µg L-1, respectively, for
140
LTG.
141
Analyte concentration in the plant material (roots and shoots) was analyzed as
142
detailed in Goldstein et al.15 In brief, plant material was ground to a fine powder and
143
extracted with methanol using accelerated solvent extractor (ASE 350, Dionex,
144
Sunnyvale, CA) in two static 5-min cycles with 100% methanol at 80°C under a
145
constant pressure of 10.34 MPa. Extracts were evaporated to dryness and
146
reconstituted in 1 mL acetonitrile:water:acetic acid (20:80:0.01) spiked with 10 µL of
147
a mixture of isotopically labeled internal standards, centrifuged at 13500 rpm for 20
148
minutes and filtered (0.22 µm PTFE) before LC-MS analysis. Extracts were analyzed
149
by Agilent 1200 Rapid Resolution LC system (Agilent Technologies Inc., Santa
150
Clara, CA) equipped with a Gemini C-18 column (150 × 2 mm, 3-µm particle size;
151
Phenomenex, Torrance, CA) coupled to an Agilent 6410 triple quadruple mass
152
spectrometer with ESI ion source, in multiple reaction monitoring (MRM) mode, with
153
positive or negative ionization. LOD and LOQ, determined using a signal to noise
154
ratio of 3:1 and 10:1 respectively, were 1and 2 µg kg-1, respectively, for LTG; 0.05
155
and 0.1 µg kg-1, respectively, for CBZ; 0.1 and 0.2 µg kg-1, respectively for DiOH-
156
7 ACS Paragon Plus Environment
Page 9 of 32
Environmental Science & Technology
CBZ; 0.05 and 0.1 µg kg-1, respectively, for EP-CBZ; 0.2 and 0.5 µg kg-1,
157
respectively for 2-OH-CBZ and 0.05 and 0.1 µg kg-1, respectively for 3-OH-CBZ.15
158
Data Analysis. Statistical analysis (non-parametric Wilcoxon/Kruskal–Wallis test and
159
non-parametric multiple comparisons using Dunn method for joint ranking, p < 0.05)
160
was performed using JMP Pro 10 software (JMP®, Version 10. SAS Institute Inc.,
161
Cary, NC, 1989-2007). Mass balance was calculated for CBZ and its metabolites as
162
the ratio between the sum of CBZ and its metabolites at each sampling time (the
163
amount of CBZ in the nutrient solution and CBZ and its metabolites in the plant
164
material) and the initial amount of CBZ in the nutrient solution, and found to be 95.36
165
± 0.76% in the single-solute system and 94.36 ± 0.77% in the bi-solute system. Mass
166
balance calculation for LTG in the bi-solute system revealed a growing deficit over
167
time, starting with 92.08 ± 2.18% at 8 h and declined to 57.33 ± 3.49% at 96 h.
168 169 170
8 ACS Paragon Plus Environment
Environmental Science & Technology
Page 10 of 32
Table 1. Selected physicochemical properties of the studied compounds Compound
Structure
Carbamazepine (CBZ)
log Kow (ref)
2.77
(24)
171
pKa (ref) pKa1 = -0.5 (24)
pKa2 = 14.4 pKa1 = -0.9 (24)
10,11-dihydro-10,11-epoxy1.97
(24)
carbamazepine (EP-CBZ)
pKa2 = 14.8
pKa1 = -1.5 (24) 10,11-dihydro-10,11dihydroxy-carbamazepine
0.81
(24)
pKa2 = 11.7 pKa3 = 12.3
(DiOH-CBZ)
pKa4 = 14.0 2-hydroxy-carbamazepine 2.66 (32)
pKa = 9.30 (32)
2.66 (32)
pKa = 9.46 (32)
1.93 (24)
pKa = 5. 7 (25)
(2-OH-CBZ) 3-hydroxy-carbamazepine (3-OH-CBZ)
Lamotrigine (LTG)
172
9 ACS Paragon Plus Environment
Page 11 of 32
Environmental Science & Technology
RESULTS AND DISCUSSION
173
CBZ and LTG Uptake. The concentrations of CBZ in the nutrient solution,
174
as well as in the sap, were similar for the single- and bi-solute systems (i.e., plants
175
exposed only to CBZ, and plants exposed to both CBZ and LTG as a mixture,
176
respectively) during the exposure time (Figure 1a). Therefore, the following
177
discussion about CBZ uptake by the roots describes both treatments as a single
178
scenario. CBZ concentration in the nutrient solution was constant (~1.76 µM) for the
179
first 48 h, after which a minor, but statistically significant rise in concentration was
180
observed (1.98 µM at 72 h and 2.3 µM at 96 h; Figure 1a). LTG concentration in the
181
single-solute system (Figure 1b) was constant both in the nutrient solution and in the
182
sap during the exposure time. However, for the bi-solute system LTG concentration in
183
the nutrient solution decreased significantly with time. Unlike the steady increase in
184
CBZ concentration in the nutrient solution, CBZ concentration in the plant sap
185
remained constant from 24 h to the end of the experiment (Figure 1a). It is important
186
to note that the CBZ concentration in the sap was always lower than in the nutrient
187
solution and this difference increased with time from 24 h to the end of the
188
experiment. CBZ, being neutral with an intermediate lipophilicity (Table 1), is
189
relatively easily translocated from root to shoot via the sap.33,34 It is important to note
190
that metabolites of CBZ were not detected in the sap. As for LTG concentration in the
191
sap, it was consistently much lower than in the nutrient solutions in both the single-
192
and bi-solute systems (Figure 1b). The sap LTG in the bi-solute system exhibited a
193
significant decrease in concentration following 48 h of exposure.
194
10 ACS Paragon Plus Environment
Environmental Science & Technology
Page 12 of 32
195
Figure 1. Concentrations of carbamazepine (CBZ; left) and lamotrigine (LTG; right) in the plant sap and in the nutrient solution throughout the exposure period in the absence (single-solute system) and presence (bi-solute system) of the companion compound. Averages and standard errors are shown (n = 5).
196 197 198 199 200
The kinetics analysis of CBZ and LTG influx into the cucumber roots,
201
calculated from the decreased amount of each compound in the nutrient solution, and
202
water uptake are presented in Figure S1. The initial CBZ uptake rate was high (11.98
203
± 2.42 and 13.52 ± 1.48 µmol-1 kg root-1 h-1 in the single- and bi-solute systems
204
respectively; average ± standard error) in the first 8 h, and decreased thereafter to 5.45
205
± 0.33 and 6.30 ± 0.36 µmol-1 kg root-1 h-1, in the single- and bi-solute, respectively.
206
The water-uptake rate largely affected the CBZ-uptake rate, but this does not mean
207
that CBZ was actually taken up with the water-influx stream. Alternatively, we
208
suggest different routes for these two components. Water transport across the roots
209
occurs via three pathways: (i) through the cell walls (apoplastic path), (ii) from cell to
210
cell through plasmodesmata (symplastic path), and (iii) across membranes
211
(transcellular path). In order to pass the casparian band and reach the sap, water and
212
solutes must enter the symplast and pass through root cell membranes in the
213
endodermis.35,36 Water uptake is largely driven by water potential gradients, however
214
11 ACS Paragon Plus Environment
Page 13 of 32
Environmental Science & Technology
studies have demonstrated the importance of water channels known as aquaporins in
215
the active regulation of water uptake and for elevated water influx rates.35,37 The non-
216
ionic CBZ and LTG molecules are mainly translocated by a passive diffusion
217
mechanism across root-cell membranes, and is thus largely affected by the
218
concentration gradient across the membrane according to Fick’s law, thus their initial
219
influx rate is rapid and slows down as the concentration gradient decrease (Figure S1).
220
Later on, the influx rate of CBZ depends on a steady concentration gradient that is
221
maintained by the water influx. For LTG, the translocation rate in the sap is much
222
slower, as is evident from its lower concentration in the sap (Figure 1b) and thus its
223
influx rate diminishes with the time of exposure. It is hypothesized that CBZ will be
224
transported with water in both apoplastic and symplastic pathways, and as water
225
influx and transpiration increase, a larger CBZ gradient across the membrane will be
226
maintained and larger rates of CBZ uptake will prevail.
227
The apparent LTG uptake rate, as calculated from LTG disappearance from
228
the nutrient solution, was higher than that of CBZ throughout most of the exposure
229
period (Figure S1). The apparent LTG uptake rate decreased gradually over time. The
230
high initial apparent uptake rate calculated for LTG may be attributed to two separate
231
mechanisms, the first being the high root sorption affinity as demonstrated in the root
232
adsorption experiments where LTG adsorption was shown to be substantial and rapid
233
(Figure S2). The second mechanism is ion trapping within the root vacuole. The root
234
vacuole has a pH of ~5.5 under which ~50% of LTG molecules are positively charged
235
and thus vacuole serve as a sink for LTG,38 and the concentration gradient between
236
the external concentration (nutrient solution) and the internal cytoplasm concentration
237
is preserved, resulting in a greater driving force for diffusion across the root cell
238
membrane.
239
12 ACS Paragon Plus Environment
Environmental Science & Technology
Page 14 of 32
To demonstrate the uptake mechanism for both drugs, we calculated the
240
apparent influx concentration (µM) as the ratio between CBZ or LTG uptake rate
241
(µmol kg root-1 h-1) and water-uptake rate (L kg root-1 h-1). This is shown in Figure 2.
242
For CBZ the initial (8 h) apparent influx concentration was about 60% higher than the
243
CBZ concentration in the nutrient solution, similar to it at 24 and 48 h and diminished
244
later to be lower than the nutrient solution concentration (Figure 2a). Since CBZ
245
adsorption to the external root surfaces was shown to be negligible (see SI; Figure
246
S2a) and CBZ transformation products were not detected in the nutrient solution, we
247
conclude that the calculated apparent CBZ-influx concentration truly represents CBZ
248
influx across the root membrane (i.e., uptake).
249
250
Figure 2. Concentrations of carbamazepine (CBZ; left side) and lamotrigine (LTG; right side) in the nutrient solution and the apparent CBZ and LTG influx concentrations in the absence (single-solute system) and presence (bi-solute system) of the companion compound throughout the duration of the exposure period. Average data are shown (n = 5); bars represent standard errors.
251 252 253 254 255 256
Based on the greater initial influx rate of CBZ molecules through the root cell
257
membrane, followed by a slower and steady influx rate we suggest that water and
258
CBZ are taken up through separate pathways, while water uptake occurs mainly
259
13 ACS Paragon Plus Environment
Page 15 of 32
Environmental Science & Technology
through regulated water channels or aquaporins, and is affected by the plant's demand
260
for water, the non-ionic CBZ molecule is mainly translocated by a diffusion
261
mechanism across root-cell membranes.
The CBZ influx is driven by the
262
concentration gradient between the outer and inner sides of the root cell membrane in
263
accordance with Fick’s law: the initial gradient is steep facilitating the high apparent
264
CBZ-influx concentration; this is followed by a decreasing gradient across the
265
membrane and results in decreased apparent CBZ-influx concentration (Figure 2).
266
This also explains the increase in CBZ concentration in the nutrient solution with time
267
(Figure 1a). The apparent LTG-influx concentration was higher than the LTG
268
concentration in the nutrient solution and significantly higher than the apparent CBZ-
269
influx concentration throughout the entire exposure period, although declining over
270
time (Figure 2b). The large initial difference between the two compounds is probably
271
reflecting not only real uptake but also a rapid adsorption of LTG to the roots, as
272
shown in Figure S2. After this initial step, it reflects a higher concentration gradient
273
that is maintained across the root cell membranes due to the trapping mechanisms
274
described above.
275
The transpiration stream concentration factor (TSCF; Figure S3) is the ratio
276
between the concentration in the xylem sap and the concentration in the nutrient
277
solution. The TSCF indicates the efficiency of the uptake of a chemical from the
278
nutrient solution and efficiency of its translocation from roots to shoot. TSCF values
279
equal to 1 are usually interpreted as indicating that the compound is taken up and
280
translocated with the water transportation stream, values >1 indicate active transport,
281
and values