water, weighed and homogenised. Chemical spe- cies were separated as described elsewhere by high
performance liquid chromatography HPLC on an Alltech MF-Plus Metal-Free HEMA-SEC
BIO 1000 size-exclusion column with
95m
TcO
4 −
as internal standard to correct for possible artefacts.
A 8.3 × 10
− 3
mol l
− 1
N-2-hydroxylethyl- piperazine-N-ethanesulfonic acid Hepes buffer
pH 7.0 at a flow rate of 1 ml min
− 1
was used. In this way, two species were detected: TcO
4 −
and reduced Tc-compounds. A further separation of
the reduced Tc-compound as described in Krijger et al. 1999a was not carried out. For more
details and retention times of the different Tc-spe- cies, see Harms et al. 1996a,b, 1999.
3
.
6
. Competition experiments Both in excess 10
− 3
mol l
− 1
of nitrate, phos- phate, sulphate, and chloride as well as in their
absence, duckweed was incubated in a solution of 2.6 × 10
− 11
mol l
− 1
Tc and of 8.3 × 10
− 3
mol l
− 1
calcium acetate solution pH 7.0 for 1 h. Calcium acetate was chosen to maintain equal
ionic strengths in all solutions. Nitrate was sup- plied in the form of CaNO
3 2
, KNO
3
, or MgNO
3 2
; phosphate as KH
2
PO
4
or NaH
2
PO
4
; sulphate as K
2
SO
4
, MgSO
4
, or Na
2
SO
4
; and chlo- ride as CaCl
2
, MgCl
2
, or NaCl. Samples were spin dried for 10 min, weighed and placed in counting
vials for g ray measurements.
3
.
7
. Radionuclides and detection
99
Tc b-emitter, E
max
= 292 keV, half-life 2.1 ×
10
5
years was obtained from Amersham Buck- inghamshire, UK as KTcO
4
in 1 M NH
4
OH;
95m
Tc g-emitter of mainly 204 keV 66 and 835 keV 28 half-life: 60 days was obtained from
Los Alamos National Laboratory Los Alamos, NM as NH
4
TcO
4
in 1 M NH
4
OH;
99m
Tc g-emit- ter of 141 keV, half-life 6.0 h was obtained from
a
99
Mo
99m
Tc generator Malinckrodt, Petten, The Netherlands.
99m
Tc and
95m
Tc were measured with a Wallac Wallay Oy, Turku, Finland 1480
automatic 3¦ g counter, using a well type NaTlI scintillator. Energy windows
99m
Tc 104 – 162 keV,
95m
Tc 163 – 240 keV were chosen for optimal detection and possible dual label counting, data
were corrected for spill over, background and Compton radiation automatically WALLAC,
1995.
99
Tc in the nutrient solution was measured with a Packard liquid scintillation counter LSC
in Ultima Gold™ Packard Instruments, Gronin- gen, The Netherlands, using appropriate correc-
tion for quenching. Energy windows were set on 5 – 290 keV, and the counting efficiency under
these conditions was 95.
3
.
8
. Data analysis The decrease of Tc concentration in the
medium as a result of Tc uptake was negligible B 0.1. Linear regression was performed in
Quattro Pro for Windows version 1.0 Borland International using the build in linear regression
function, extended with an estimation of the S.E. for the intercept. The reduction rate was obtained
directly from the fitted slope from Eq. 8 to the data sampled from 1 h on, the influx from Eq. 6,
using the data sampled within the first 30 min. Flux constants were calculated using Eqs. 6 and
8, or Eqs. 7 and 8 if the TcO
4 −
concentration in duckweed was measured. The efflux rate
V
efflux
, was calculated by multiplying the efflux rate constant by the calculated or measured equi-
librium level of TcO
4 −
, Eq.7: V
efflux
k
1
k
2
+ k
3
[TcO
4 −
]
solution
10 S.E. were calculated using the Gaussian error
propagation rules.
4. Results
4
.
1
. Test of the model Fig. 2 shows the uptake of TcO
4 −
by duckweed over 5 h; the solid curve presents the results of the
two-compartmental model, which is fitted to the experimental data of accumulation of total Tc
solid squares. Clearly, two compartments can be distinguished: a fast compartment representing
the TcO
4 −
, and a ‘sink’ compartment representing reduced Tc-compounds. The TcO
4 −
concentration pdf text line
Fig. 2. Bioaccumulation of Tc at 2.6 × 10
− 11
mol l
− 1
TcO
4 −
during a 5-h uptake period at a light intensity of 120 mmol photons s
− 1
m
− 2
. Solid squares
are measured total Tc concentration in duckweed n = 1, open circles are the
measured TcO
4 −
concentration in duckweed n = 2 per point; A or reduced Tc-compounds B, open triangles are the
residual Tc after efflux B, error bars represent S.D. Lines indicate fitted two-compartmental bioaccumulation model
Eq. 3, with k
1
= 0.189 9 0.001 l kg
− 1
h
− 1
, k
2
= 1.812 9
0.025 h
− 1
, and k
3
= 0.602 9 0.008 h
− 1
. R
2
= 0.964 for the fit
on total Tc concentration factors.
mental values. Additional measurements of the TcO
4 −
concentration open circles and concentra- tion of reduced Tc-forms concentration in duck-
weed are in good agreement with the model. These points were not used to fit the model.
4
.
2
. Kinetics of TcO
4
−
accumulation Fig. 3 shows Van’t Hoff plots for all fluxes at
10
− 14
– 10
− 5
mol l
− 1
TcO
4 −
concentrations in the nutrient solution; additional measurements of
influx only were carried out till 10
− 2.6
mol l
− 1
TcO
4 −
. The slope of the influx graph is 1.01 9 0.02, indicating a first-order process, with a rate constant
k
1
of 0.151 9 0.004 l kg
− 1
h
− 1
. Calculated values for the pseudo first-order efflux rate constant k
2
and the reduction rate constant k
3
are 1.58 9 0.09 and 0.65 9 0.06 h
− 1
, respectively. Data for efflux and reduction above 10
− 5
mol l
− 1
TcO
4 −
were not collected.
4
.
3
. Temperature dependence Fig. 4 shows the temperature dependency of the
rate constants between 5 and 35°C. Both influx and efflux rate constants show a linear relationship with
temperature and with Q
10
-values of about 1.5 and 1.3, respectively Fig. 4A,B. The reduction rate
constant shows a typical parabolic dependency, characteristic for enzymatic processes. Fig. 5 gives
the Arrhenius plot for the TcO
4 −
equilibrium con- centration in duckweed.
4
.
4
. Light dependence Fig. 6 shows the influence of light on the fluxes.
Fig. 6A – C show influx, efflux and reduction rates, respectively. The influx is independent of light
intensity. Both efflux and reduction rates show a correlation with light intensity. With low light
intensities the efflux increases, while the reduction rate constant shows a strong positive dependency
on light intensity. An empirical saturation model could be fitted to the reduction rates with a
maximum transformation rate of 2.1 9 0.2 × 10
− 12
mol kg
− 1
fresh wt h
− 1
, and ‘K
i
’ of 40.7 9 4.3 mmol photons s
− 1
m
− 2
light intensity when half of the maximum transformation rate is reached.
pdf text line in duckweed reaches a steady state as a result of
efflux and reduction. Hereafter, the formation rate of reduced Tc-compounds will become constant.
Fig. 2B focuses on the formation of reduced compounds. The open symbols represent experi-
4
.
5
. Competition studies Table 1 presents the results of the competition
study. The accumulation of Tc was not inhibited by 10
− 3
mol l
− 1
nitrate, chloride, phosphate, or sulphate. Higher concentrations of nitrate or chlo-
ride up to 35 × 10
− 3
mol l
− 1
also could not inhibit the Tc accumulation data not shown.
Fig. 3. Van’t Hoff plots for all fluxes. Squares
are experiments carried out between 10
− 11
and 10
− 5
mol TcO
4 −
l
− 1
. Additional measurements of influx are carried out till 10
− 2.6
mol TcO
4 −
l
− 1
open circles []. Error bars represent propagated S.E. The TcO
4 −
concentration in duckweed was calculated on a fresh weight basis by k
1
k
2
+ k
3
× [TcO
4 −
]
nutrient solution
where k
1
, k
2,
and k
3
are influx, efflux, and reduction rate constants, respectively. Lines are drawn through the points with slope equal to one.
pdf text line
Fig. 4. Fluxes as a function of temperature at 2.6 × 10
− 11
mol l
− 1
TcO
4 −
and a light intensity of 120 mmol photons s
− 1
m
− 2
. Data are expressed on a fresh weight basis. Error bars represent propagated S.E.
High calcium concentrations were applied to avoid electrostatic effects which might mask the
competitive effect. Electrostatic effects were ob- served in studies without calcium acetate, and will
be elaborated on in a forthcoming article.
5. Discussion
