1577
Environmental Toxicology and Chemistry, Vol. 15, No. 9, pp. 1577?1583, 1996
q 1996 SETAC
Printed in the USA
0730-7268/96 $6.00 1 .00
THE INFLUENCE OF pH AND MEDIA COMPOSITION ON THE UPTAKE OF INORGANIC
SELENIUM BY CHLAMYDOMONAS REINHARDTII
GERHARDT F. RIEDEL* and JAMES G. SANDERS
Benedict Estuarine Research Center, The Academy of Natural Sciences, 10545 Mackall Road, St. Leonard, Maryland 20685, USA
(Received 2 January 1996; Accepted 20 March 1996)
Abstract?The uptake of inorganic selenium species, selenate and selenite, by the green alga Chlamydomonas reinhardtii Dang was
examined as a function of pH over the range 5 to 9 and in media with varying concentrations of major ions and nutrients using 75Se
as a radiotracer. Little difference was noted in the uptake of selenate as a function of pH, with the maximum uptake occurring at pH
8; however, selenite uptake increased substantially at the lower pH values. Selenate uptake was significantly decreased by higher
sulfate concentrations and increased significantly by calcium, magnesium, and ammonium. Selenite uptake was significantly increased
when the phosphate concentrations in the media were reduced. The results of these experiments demonstrate that varying water
chemistry may significantly affect the uptake of inorganic selenium by phytoplankton and the subsequent transfer of the selenium to
higher trophic levels.
Keywords?Selenium Phytoplankton uptake pH Sulfate Phosphate
INTRODUCTION
As a result of man?s activities, selenium has been found to
be an environmental problem in a number of terrestrial and
freshwater systems in recent years. Power generation [1,2] and
agricultural subsurface drainage water [3,4] have been impli-
cated in the release of harmful levels of selenium to the envi-
ronment. Sewage sludge and petrochemical refining are also
significant sources of selenium [5,6]. Selenium has even been
added to lakes to ameliorate high concentrations of mercury
[7].
Selenium is found in aquatic systems in a variety of forms.
Two inorganic species are common, selenite, Se(IV), and sel-
enate, Se(VI) [5]. Selenite is the predominant form released by
the power generation industry [5]; in agricultural drainage sel-
enate is the most common form [8]. In addition, there are a
plethora of organic selenium species, including selenium-con-
taining amino acids and peptides, and methylated species, such
as dimethyl selenide, dimethylselanone, and dimethylselenoxide
[9,10].
Regardless of the original source, adverse environmental ef-
fects appear to result largely from transfer of selenium from
lower to higher trophic levels. In the case of power generation,
game fish populations have suffered reproductive failure after
bioaccumulation of selenium from concentrations of about 10
mg/L dissolved selenium [1]. Dietary intake is the major source
for uptake by fish [2,11]. Reproduction of several wetland bird
species has been affected by mortality, gross malformations,
and internal abnormalities of the young [3,12] in areas where
high selenate concentrations (up to 350 mg/L) were found in
ponds receiving agricultural drainage. As in lakes affected by
selenite, dietary intake is believed to be the source of selenium
[12].
Because of the importance of diet in the environmental effects
of selenium, understanding the role of lower trophic levels in
the uptake of selenium from inorganic forms is critical. One of
* To whom correspondence may be addressed.
the main routes from inorganic selenium in water to higher
organisms is via uptake by phytoplankton, followed by further
passage up the food chain [13,14] (other pathways include up-
take of selenium from sediment by benthic organisms [11] and
accumulation from the water by bacteria and microzooplankton
[15]). Phytoplankton and periphyton accumulate both Se(IV)
and Se(VI) [13,14,16], and zooplankton and benthic infauna are
known to assimilate selenium from phytoplankton with unusu-
ally great efficiency [13,14,17,18], facilitating the passage of
selenium to higher trophic levels. Efforts to model selenium
biogeochemistry, bioaccumulation, and effects in contaminated
lacustrine systems are currently being carried out [19].
It is quite common for the uptake of nutrients or trace ele-
ments by phytoplankton to be coupled to the transport of major
ions. The active uptake of many ions is achieved through sym-
port or antiport of H1, Na1, or K1 [20]. In other cases, ions
may be in competition for uptake at a transport site which will
accept either ion. For example, the uptake and toxicity of chro-
mate ion to phytoplankton is controlled by the concentration of
sulfate in the growth medium; higher sulfate concentrations
reduce the uptake and toxicity of chromate to phytoplankton
[21]. Similarly, arsenate uptake and effects are dependent on
phosphate concentration [22]. Therefore, it is possible that the
composition of the medium will lead to significant variations
in the uptake of selenium to phytoplankton and bioaccumulation
in higher trophic levels. Thus, the wide variation in the com-
position of freshwaters worldwide could produce significant dif-
ferences in selenium behavior and effects between different sys-
tems. The purpose of this study was to examine the possible
influences of pH and medium composition on the uptake of
selenium by a representative phytoplankter, the green alga Chal-
mydomonas reinhardtii.
METHODS
Algae and culturing
Chlamydomonas reinhardtii Dang (UTEX 89) was obtained
from the University of Texas culture collection [23]. The culture
1578 Environ. Toxicol. Chem. 15, 1996 G.F. Riedel and J.G. Sanders
Table 1. Composition of the growth medium used to culture and as
the basis for experimental variations (modified from WC media [22])
mg/L mM
Major salts
CaCl2?H2O
MgSO4?7H2O
NaHCO3
K2HPO4
NaNO3
Na2SiO3?9H2O
KBr
NaF
32.24
36.96
25.20
8.71
85.01
28.42
0.12
0.42
250
150
300
50
1,000
100
1
10
Trace elements
FeEDTAa
CuSO4?5H2O
ZnSO4?7H2O
CoCl2?6H2O
MnCl2?4H2O
Na2MoO4?2H2O
H3BO3
0.01
0.022
0.01
0.18
0.006
0.62
11.7
0.04
0.08
0.05
0.90
0.03
0.16
Vitamins and buffer
Thiamin?HCl
Biotin
B12
HEPESb
0.1
0.0005
0.0005
2,500
a EDTA 5 ethylenediaminetetraacetic acid.
b HEPES 5 N-[2-hydroxyethyl]piparazine-N9-(2-ethanesulfonic acid).
was maintained in a variation of the freshwater medium WC
[24], here denoted as HYCO (Table 1). With the exception of
the pH experiments noted below, all cultures were buffered at
pH 7.0 with 2.5 mM N-[2-hydroxyethyl]piperazine-N9-(2-eth-
anesulfonic acid) (HEPES) [25]. The culture was axenic when
obtained and cultured with sterile techniques; however, tests for
subsequent bacterial contamination were not conducted.
Selenium uptake measurements
Selenium uptake measurements were carried out using 75Se
as a radiotracer. The details of isotope preparation and deter-
mination have been previously described [16]. For all the ex-
periments reported here, concentrations of 75Se added were 3
to 10 mg/L. For both selenate and selenite, these concentrations
are within a range where uptake is approximately proportional
to concentration. Selenate uptake versus time is virtually linear
over 24 h; however, selenite uptake is initially very rapid and
reaches a plateau near 4 to 6 h [16]. Therefore, for the study
of pH on selenium uptake and the initial screening study of the
effect of ions on selenium uptake, experiments were carried out
using short term (4 to 6 h) incubations. In short-term experi-
ments, uptake was examined in three 25-ml cultures, two live
and one heat-killed at 708C to check for adsorptive uptake [16].
The cultures were incubated in a 258C incubator and then filtered
onto 0.4-mm polycarbonate filters using three 5-ml rinses with
buffered medium to remove unincorporated 75Se. For the second
study of the effects of medium composition on uptake, cells
were inoculated at a low level into the media and allowed to
grow over several days. Once cell densities had passed 1 3 105
cells/ml, samples from the cultures were filtered and rinsed as
above to determine selenium uptake. Because of the low initial
cell densities, heat-killed controls were not used.
Effects of pH on selenium uptake
The base HYCO medium was prepared at pH 5.0, 6.0, 7.0,
8.0, and 9.0 using 2.5 mM HEPES or 2-[N-morpholi-
no]ethanesulfonic acid (MES) buffer [25] (MES for pH 5.0 and
6.0; HEPES for pH 7.0, 8.0, and 9.0). Chlamydomonas rein-
hardtii was grown in medium buffered at pH 7.0 with 2.5 mM
HEPES to a cell density of 1.9 3 105 cells/ml. For each pH
tested, 80 ml of the C. reinhardtii culture was added to 320 ml
of buffered medium (for a final cell density of 3.8 3 104 cells/
ml), and the pH adjusted with dilute HCl and/or NaOH. From
each different pH, six 50-ml test tubes received 25 ml of the
buffered medium. Of the six, two tubes were heat-killed. One
was used as a control for absorptive selenate uptake, and one
for selenite uptake at each pH. The remaining tubes were used
to determine selenate and selenite uptake by live cells (2 each).
Selenium additions were 10 mg Se/L as selenate and selenite.
Incubations lasted 6 h.
Effects of medium composition on selenium uptake?
screening
Variations of the base medium were made up as follows: (1)
control, (2) 11,000 mM Na2SO4, (3) 11,000 mM CaCl2, (4)
11,000 mM MgCl2, (5) 11,000 mM KCl, (6) 11,000 mM NaCl,
(7) 1500 mM NH4Cl, (8) 11,000 mM NaHCO3, (9) 1500 mM
Na2SiO3, (10) 2KNO3 (no added KNO3), and (11) 2K2HPO4
(no added K2HPO4). Chlamydomas reinhardtii was grown in 2
L of pH 7.0 HEPES-buffered HYCO medium to a cell density
of 2.5 3 105 cells/ml. For each treatment above, 12 50-ml Pyrext
test tubes were prepared containing 20 ml of medium. Six tubes
were used for selenate uptake and six for selenite. For each form
two selenium concentrations, 3 and 10 mg/L, were tested. Treat-
ment tubes were prepared by adding 5 ml of the culture to the
20 ml of prepared medium with isotope (final cell density 5
5.0 3 104 cells/ml). Incubations lasted 4 h.
Both selenate and selenite results were analyzed by two-factor
(treatment 3 Se concentration) analysis of variance (ANOVA)
[26], and all treatments contrasted to the controls using Dunnet?s
test [27] to control the experimentwise error rate at 0.10. Based
on the results of this experiment, a more detailed examination
of the effects of a few constituents was carried out.
Effects of medium composition on selenium uptake?detailed
examination of major effects
Batches of HYCO medium were made up with the following
deletions: none (control), 2CaCl2, 2MgCl2, 2Na2SO4,
2K2HPO4, and 2Na2SiO3. Twenty Pyrex 50-ml culture tubes
were prepared with 30 ml of each medium. Treatment series
were made in quadruplicate by adding substances back to the
media from which deletions had been made, as follows: (1)
control: 1100, 200, 500, 1,000, or 2,000 mM NaCl; (2) 2CaCl2:
150, 100, 200, 500, or 1,000 mM CaCl2; (3) 2Na2SO4: 150,
100, 200, 500, or 1,000 mM Na2SO4; (4) 2K2HPO4: 15, 10,
20, 50, or 100 mM K2HPO4; and (5) 2Na2SiO3: 110, 20, 50,
100, or 200 mM Na2SiO3.
In addition, eight tubes containing the base medium were
prepared to be used as controls. The addition series for CaCl2,
NaCl, and Na2SO4 received duplicate additions of [75Se]selenate
at 3 and 10 mg/L at each treatment concentration, while the
addition series for K2HPO4 and Na2SiO3 received duplicate ad-
ditions of [75Se]selenite at 3 and 10 mg/L at each treatment
concentration. Because of suspicions that a precipitate in the
silicate addition treatment in the previous experiment may have
led to an artifact in selenite uptake, we made the following
changes: all treatment media were made up a week in advance
to allow the dissolution of any precipitates formed during the
sterilization of the media, and the maximum concentration of
Effect of water chemistry on algal uptake of inorganic selenium Environ. Toxicol. Chem. 15, 1996 1579
Fig. 1. The effect of pH on the uptake of inorganic selenium by
Chlamydomonas reinhardtii. Incubation time, 6 h. (a) 10 mg Se/L
selenate. (b) 10 mg Se/L selenite. Results for live cells show mean
6 SD (n 5 2) V 5 live cells, v 5 heat-killed cells.
Fig. 2. Results of the initial screening for effects of media compo-
sition on inorganic selenium uptake. Incubation time, 4 h. (a) Selenate.
(b) Selenite. Results for live cells show mean 6 SD (n 5 2). * 5
treatments statistically different from control by Dunnet?s test (p ,
0.10, experimentwise error), open bars 5 3 mg/L 75Se additions, filled
bars 5 10 mg/L 75Se additions.
silicate used was reduced from 1,000 mM to 200 mM. The eight
control cultures were divided into four pairs, which received 3
and 10 mg/L of [75Se]selenate or selenite.
Chlamydomonas reinhardtii was grown in pH 7.0 HEPES-
buffered medium to a cell density of 5 3 105 cells/ml. Each
treatment tube was spiked with 0.06 ml of culture, for an initial
cell density of 1 3 103 cells/ml. Treatment and control tubes
were incubated in a 258C incubator with a 12 h light : 12 h dark
cycle. Cell growth was monitored daily using in vivo fluores-
cence, resuspending each tube with a vortex mixer, and inserting
the tube directly into a Turner Designs fluorometer. Cell den-
sities in the treatment tubes were calculated from in vivo flu-
orescence from the ratio of cell density determined by cell
counts to in vivo fluorescence of cells in extra control tubes
without added 75Se. Once cell densities reached approx. 1 3
105 cells/ml, 20- to 30-ml samples were filtered for determi-
nation of selenium uptake.
RESULTS
Effects of pH on selenium uptake
The uptake of selenate by Chlamydomonas was not strongly
affected by pH (Fig. 1a). The average uptake rate of living cells
varied from about 7.5 3 10218 g Se/cell per h at pH 6.0 and
7.0 to 12 3 10218 g Se/cell per h at pH 8.0. Sorptive uptake by
heat-killed cells varied from 1.4 3 10218 g Se/cell per h at pH
7 to 3.5 3 10218 g Se/cell per h at pH 6.0. The variations across
pH appeared not to be systematic.
The uptake of selenite was strongly pH-dependent at the lower
pH values (Fig. 1b). At pH 7.0 or above, the average uptake
rate was 17 3 10218 g Se/cell per h. At pH 6.0 uptake doubled
to approx. 37 3 10218 g Se/cell per h, and at pH 5.0 average
uptake had risen to 167 3 10218 g Se/cell per h. Except for pH
5.0, selenite uptake by heat-killed cells was approximately half
that of uptake by live cells.
Effects of medium composition on selenium uptake?
screening
Mean control uptake rate for selenate in the live control treat-
ment was 12 3 10218 g Se/cell per h in 3 mg/L selenate and 16
3 10218 g Se/cell per h at 10 mg/L selenate. Uptake rates of
selenate by heat-killed algae were less than 10% of the rate by
living algae. With selenite, the control uptake rate of living cells
was 8.8 3 10218 g Se/cell per h at 3 mg/L and 14.5 3 10218 g
Se/cell per h at 10 mg/L. Uptake of selenite by heat-killed cells
varied from about 10 to 50% of live uptake, except for the added
silicate treatment, where the rate for heat-killed algae was 88%
of the rate of living algae for both 3 and 10 mg/L.
Only slight effects on selenate uptake by the compounds test-
ed were noted (Fig 2a). CaCl2, MgCl2, and NH4Cl caused sig-
nificant increases in uptake (30, 23, and 13%, respectively),
while added Na2SO4 caused a significant reduction (16%) (two-
factor ANOVA, Dunnett?s test, a 5 0.10). Added KCl, NaCl,
reduced NaNO3, and reduced K2HPO4 all reduced uptake 13, 7,
5, and 5%, respectively, but not significantly. Added NaHCO3
caused little change (13%).
Two treatments caused significant (two-factor ANOVA, Dun-
nett?s test, a 5 0.10) increases in selenite uptake (Fig 2b).
Reduced K2HPO4 caused an average 253% greater uptake, with
uptake by heat-killed cells increasing proportionally. Added
Na2SiO3 caused an average increase of 311%, with virtually all
the added uptake also being present in the heat-killed treatment.
1580 Environ. Toxicol. Chem. 15, 1996 G.F. Riedel and J.G. Sanders
Fig. 3. Growth of cells during the long-term effect of ions on inorganic selenium uptake experiment. (Top) Control cultures and K2 HPO4 and
Na2SiO3 treatment cultures. (Bottom) CaCl2, NaCl, and Na2SO4 cultures. Mean 6 SD (n 5 4).
The remainder of the treatments ranged from 102 to 134% of
the control treatments.
Effects of medium composition on selenium uptake?detailed
examination of major effects
In general, the variations in media composition did not sub-
stantially affect the growth of C. reinhardtii. However, there
was evidence for somewhat enhanced growth in the three highest
concentrations of the KH2PO4 series, and that added NaCl en-
hanced the growth rate slightly (Fig. 3).
In the Na2SO4 addition series, selenate uptake was lowest at
the highest concentration (1,000 mM) and increased slowly as
sulfate decreased (Fig. 4). At the lowest concentration (50 mM),
selenate uptake increased substantially in both the 3- and
Effect of water chemistry on algal uptake of inorganic selenium Environ. Toxicol. Chem. 15, 1996 1581
Fig. 4. The effect of media composition on inorganic selenium uptake in the long-term experiment. (Left) Effect of Na2SO4 (top), NaCl (middle),
and CaCl2 (bottom) on selenate uptake. (Right) Effect of K2HPO4 (top) and Na2SiO3 (bottom) on selenite uptake. V 5 3 mg/L 75Se additions,
v 5 10 mg/L 75Se additions.
10-mg/L treatments. As a test of whether the Na1 or ionic
strength increase with the added sulfate might be responsible
for the observed effects of Na2SO4, a treatment with the same
molarity of NaCl was made. In this treatment, there was also a
slight decrease in selenate uptake with NaCl added (Fig 4),
noticeably at the lowest concentrations. CaCl2 demonstrated lit-
tle effect on selenate uptake (Fig. 4).
Low K2HPO4 concentrations (below 20 mM) had strong stim-
ulating effects on selenite uptake. At 5 mM K2HPO4, selenite
uptake was two to four times greater than observed at 20 mM
and above (Fig 4). Na2SiO3, however, showed little effect on
selenite uptake (Fig 4), possibly because silicate was added long
before the cell additions, allowing any precipitates formed dur-
ing addition to redissolve and because of the lower, more re-
alistic, concentration range.
DISCUSSION
Effect of pH on selenium uptake
The pH of the medium can influence the uptake of ions in
several ways. First, the extent of protonation of ions in solution
varies as a function of pH. Formation of a more biologically
active form can increase uptake, while formation of a less active
species can decrease uptake. Selenic acid is a strong acid (pK2
5 2.05) and is essentially completely dissociated across the
range of pHs examined here (and found in virtually all natural
waters); thus, we would not expect the uptake of selenate to
vary for this reason. The pH of the medium, over a range of 5
to 9, had little effect on selenate uptake. This suggests that over
the pH range of many freshwater environments, selenate uptake
can be effectively considered constant. Certainly, pH values
outside this range on either side occur, and the generality of
this relationship (or lack thereof) should be determined with
other acid- and base-tolerant algae.
Selenous acid is a weak acid (pK1 5 2.57, pK2 5 7.31) [28],
and thus important changes in its chemical form occur across
the normal environmental range of pH. At pH values above 7.3,
the selenite ion SeO is predominant; at pH values between223
2.5 and 8, HSeO is predominant; while below pH 2.5 un-23
charged selenous acid (H2SeO3) predominates. The sharp rise
in selenite uptake by cells at low pH values well below 7 sug-
gests that the selenous acid molecule may readily enter the cell,
while the charged species are largely excluded (or transported
by a separate ion specific transport system). This increase in
selenite uptake at low pH raises the question of whether the
selenium concentrations in phytoplankton from relatively acid
environments are enhanced relative to more neutral environ-
ments with similar selenite concentrations. No systematic stud-
ies of dissolved selenium occurrence, speciation, and bioac-
cumulation in lakes, across a gradient of pH, are available to
determine whether there is environmental evidence for this hy-
pothesis.
The other major effect of pH on the uptake of ions would be
the effect of pH on uptake transport proteins. This would nor-
mally result in an optimum pH for uptake. For other enzymes
1582 Environ. Toxicol. Chem. 15, 1996 G.F. Riedel and J.G. Sanders
the shape of the pH activity profile is known to be quite variable,
with optima ranging from pH 2 to 10 and from very narrow to
quite broad [29]. For selenate, there is weak evidence for an
optimum uptake near pH 8. For selenite there is no sign of an
optimum, unless it lies somewhere below pH 5, a result better
explained by changes in the inorganic speciation of selenite.
Effects of medium composition on selenium uptake
Of the ions tested in both ion experiments, only sulfate con-
sistently demonstrated significant effects on selenate uptake.
Mean sulfate concentrations of freshwater in the United States
are about 100 mM [30], although concentrations can range from
less than 10 to greater than 1,000 mM. For concentrations of
sulfate near average or above, the effect on selenate uptake was
rather small, with a decrease of about a factor of two for a
10-fold increase in sulfate concentration to 1,000 mM. Below
100 mM, the effect appears to be stronger, with a nearly twofold
increase in cellular selenate concentration with a decrease to 50
mM. Sulfate ion is, therefore, an ion that should be considered
in efforts to model selenate uptake of natural systems, partic-
ularly in soft-water systems with low sulfate ion concentrations.
We had, however, anticipated a proportional reduction of sel-
enate uptake with elevated sulfate concentrations. Previous work
has suggested that for several species of algae, selenate toxicity
was approximately inversely proportional to sulfate concentra-
tion of the medium [31]. Analogous situations are found with
sulfate and chromate [21] and arsenate and phosphate [22].
Given the similarity of selenate and sulfate, it was somewhat
surprising that sulfate and selenate did not show a more strongly
competitive pattern for uptake by C. reinhardtii. Given the en-
vironmental variability of sulfate concentrations, and the non-
linear response of selenate uptake by C. reinhardtii to sulfate
concentration, this relationship should be examined in a wider
variety of algae.
The two most notable effects observed in this study were the
effects of silicate and phosphate on selenite uptake. The con-
current increase of selenite uptake in the heat-killed silicate
treatments of the screening experiment suggested that the in-
creased uptake was due to a silica precipitate in the medium at
the rather high concentrations of silicate added. The second
experiment, with a more reasonable range of silicate concen-
trations added in a manner to minimize precipitate formation,
found no effect of silicate on selenite uptake by Chlamydomonas
and lends credence to this conclusion.
Phosphate appears to exert strong control over the uptake of
selenite and should be included in a biogeochemical model of
selenium transport in lacustrine systems. Phosphate ion con-
centrations in lacustrine systems vary from undetectable levels
in strongly phosphate-limited systems to very high concentra-
tions in lakes with phosphatic geology or extreme nutrient ad-
ditions [30], with normal concentrations of approx. 0.3 to 1.5
mM. However, because the effect of phosphate on selenite up-
take occurs at low phosphate levels, systems in which phosphate
is potentially limited are more likely to be influenced by changes
in phosphate concentration. Many freshwater systems are at
least potentially phosphate-limited [32]; hence, understanding
and correctly modeling the relationship between phosphate and
selenite uptake by algae is crucial to understanding the biogeo-
chemistry of selenium. If phosphate-limited algae take up sub-
stantially more selenite than nitrogen- or light-limited algae,
seasonal changes in selenite uptake, food web transfer, and trans-
formations could be considerably different than predicted by
selenium concentrations and biomass changes alone. From a
practical point of view, this interaction may be extremely im-
portant to modeling the fate of selenite in lacustrine or riverine
systems.
Previous experiments with three different species of phyto-
plankton, C. reinhardtii, the diatom Cyclotella meneghiania,
and the cyanophyte Anabaena flos-aquae, suggest that selenate,
selenite, and selenomethionine uptake among different taxa of
algae have similar rates of uptake, adsorption, and substrate
saturation [16] and thus likely share similar mechanisms of
uptake. Further research should be carried out to determine
whether the effects of sulfate and phosphate on selenium uptake
are common features of algal selenium uptake. If so, models of
selenium bioaccumulation in natural systems will need to in-
corporate this finding.
Acknowledgement?The laboratory assistance of Laura Cole, Douglas
Talaber, and Dorothea Ferrier are gratefully acknowledged. This work
was supported by the Electric Power Research Institute under RP2020/
11.
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