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Dissolvedorganiccarbonreducesuranium
toxicitytotheunicellulareukaryoteEuglena
gracilis
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Ecotoxicology (2012) 21:1013–1023
DOI 10.1007/s10646-012-0855-x
Dissolved organic carbon reduces uranium toxicity
to the unicellular eukaryote Euglena gracilis
Melanie A. Trenfield • Jack C. Ng •
Barry Noller • Scott J. Markich • Rick A. van Dam
Accepted: 9 January 2012 / Published online: 26 January 2012
Ó Springer Science+Business Media, LLC 2012
Abstract The influence of dissolved organic carbon
(DOC), in the form of Suwannee River fulvic acid (SRFA),
on uranium (U) toxicity to the unicellular eukaryote,
Euglena gracilis (Z strain), was investigated at pH 6. In a
background medium without SRFA, exposure of E. gracilis to 57 lg L-1 U resulted in a 50% reduction in growth
(IC50). The addition of 20 mg L-1 DOC (as SRFA),
reduced U toxicity 4 to 5-fold (IC50 increased to
254 lg L-1 U). This reduction in toxicity was also evident
at more sensitive effect levels with a 10% reduction in
growth (IC10) occurring at 5 lg L-1 U in the background
medium and at 17 lg L-1 U in the SRFA medium,
respectively. This amelioration of toxicity with the addition
of SRFA was linked to a decrease in the bioavailability of
U, with geochemical speciation modelling predicting 84%
of U would be complexed by SRFA. The decrease in
bioavailability of U in the presence of SRFA was also
Electronic supplementary material The online version of this
article (doi:10.1007/s10646-012-0855-x) contains supplementary
material, which is available to authorized users.
M. A. Trenfield (&) R. A. van Dam
Ecotoxicology Program, Environmental Research Institute of the
Supervising Scientist, GPO Box 461, Darwin, NT 0801,
Australia
e-mail: [email protected]
M. A. Trenfield J. C. Ng
National Research Centre for Environmental Toxicology, The
University of Queensland, Coopers Plains, QLD 4108, Australia
B. Noller
Centre for Mined Land Rehabilitation, The University
of Queensland, St. Lucia, QLD 4067, Australia
S. J. Markich
Aquatic Solutions International, Dundas, NSW 2117, Australia
evident from the 11–14 fold reduction in the cellular concentration of U compared to that of E. gracilis in the
background medium. Stepwise multiple linear regression
analyses indicated that UO22? alone explained 51% of the
variation in measured U toxicity to E. gracilis. Preliminary
U exposures to E. gracilis in the presence of a reactive
oxygen species probe, suggest exposure to C60 lg L-1 U
may induce oxidative stress, but this endpoint was not
considered to be a sensitive biological indicator.
Keywords Euglena Fulvic acid Uranium Toxicity Freshwater Dissolved organic carbon
Introduction
The growing demand for uranium (U, IAEA 2008) could
potentially lead to an increased risk of U exposure to
humans and the general environment. Anthropogenic
activities such as uranium mining operations have the
potential to release U in the environment at concentrations
higher than natural background levels (Hynes et al. 1987;
Pyle et al. 2002; Antunes et al. 2007). Concentrations of
600–1,200 lg L-1 U have been measured in creekwater
downstream from a U mine, and 5,500 lg L-1 U in
groundwater influenced by a U processing mill (Landa and
Gray 1995).
Uranium toxicity studies conducted with freshwater
organisms at pH 6–7 have shown that U concentrations as
low as 50 lg L-1 can cause 50% effects on species,
including Hydra viridissima and Chlorella sp. (population
growth; Franklin et al. 2000; Trenfield et al. 2011a),
Moinodaphnia macleayi (reproduction; Semaan et al.
2001) and Daphnia magna (growth and reproduction;
Zeman et al. 2008). Although quite a few studies have
123
1014
reported U toxicity data for aquatic biota, there is only
sparse information regarding the mechanisms of U toxicity.
The limited published material indicates that U can inhibit
ATPase and induce oxidative stress in animal tissue
(Nechay et al. 1980; Ribera et al. 1996; Barillet et al. 2007;
Periyakaruppan et al. 2007) and may disrupt gill, hepatopancreas, muscle and gonadal tissue in aquatic organisms
(Barillet et al. 2010; Kaddissi et al. 2011). Induction of
reactive oxygen species (ROS) in lung cells of rats (Periyakaruppan et al. 2007) and in fish exposed to U (Buet
et al. 2005; Barillet et al. 2007), has been linked to the
failure of cellular antioxidant mechanisms which normally
act to suppress a rise in oxidative species. Uranium has also
been found to cause DNA-strand breaks in Escherichia coli
(Yazzie et al. 2003).
The unicellular eukaryote Euglena gracilis is found
commonly in freshwaters worldwide (Lacky 1968) and
grows optimally at 20–30°C (Suzuki, personal communication). E. gracilis is known to be sensitive to metal contaminants (Hg, Cd, Cr, Ni, Gajdosova and Reichrtova
1996) and can be an effective biological model for the
study of metal toxicity in eukaryotic cells (Ohta et al. 2001;
Einicker-Lamas et al. 2002; Watanabe and Suzuki 2002).
E. gracilis has also been used for oxidative stress studies
with the herbicide Paraquat (Overbaugh 1985), Cd
(Watanabe and Suzuki 2002) and Cr (using gene expression, dos Santos Ferreira et al. 2007). With its highly
developed subcellular organelles being equivalent to those
of higher plants (Watanabe and Suzuki 2002), the photosynthetic Z strain of E. gracilis can be used to provide
insight into the mechanisms of U toxicity in higher plants.
However, much of the research with Euglena (including
those studies cited above) has been conducted in nutrientenriched growth media that bear little resemblance to
natural waters. Hence, the associated toxicity data are not
useful for predicting environmental effects in natural
waters. This study focused on the use of a medium that
contains minimal nutrients, and to the authors’ knowledge,
is the first to assess U toxicity to Euglena.
Around 50% of naturally occurring dissolved organic
carbon (DOC) in aquatic ecosystems exists as humic substances, in the form of humic acids and fulvic acids (FA),
with FA being predominant in aquatic systems (Malcolm
1985). DOC can reduce U toxicity to aquatic organisms
through the complexation of bioavailable species such as
UO22? (Markich et al. 2000; Hogan et al. 2005; Trenfield
et al. 2011a). Metal–FA complexation can be predicted by
aqueous geochemical speciation models, such as HARPHRQ (Brown et al. 1991). In addition to reducing the
toxicity of contaminants via their complexation, DOC can
also adsorb to cell membranes and reduce membrane permeability to toxic contaminants (Parent et al. 1996;
Campbell et al. 1997; Matsuo et al. 2004). In order to
123
M. A. Trenfield et al.
accurately assess the toxicity of U in natural systems, the
nature of DOC within a system and its influence on U
toxicity must be considered.
The aims of this study were to (a) investigate the
influence of DOC on the toxicity of U in a soft, acidic, lownutrient medium to the unicellular eukaryote E. gracilis,
(b) estimate the influence of DOC on U speciation through
geochemical speciation modeling, and (c) undertake a
preliminary investigation into the influence of U on the
generation of ROS in E. gracilis.
Materials and methods
General laboratory procedures
All equipment used to hold E. gracilis or associated
culture and test solutions was made of chemically inert
materials (e.g. Teflon, glass or polyethylene). All plastic
and glassware was soaked in 5% nitric acid for 24 h
before undergoing a detergent wash (Gallay Clean A
powder, Gallay Scientific, Burwood, Australia) and rinse
in a laboratory dishwasher using reverse osmosis (RO)
water. All glassware including test tubes (but not volumetric flasks) was silanised with 2% dimethyldichlorosilane in 1,1,1-trichloroethane (Coatasil, AJAX, Seven
Hills, Australia,) to reduce U adsorption to the glass. All
reagents used were analytical grade and stock solutions
were made up in Milli-Q water (18 Xcm-1, Millipore
Ltd., Billerica, MA, USA).
Euglena gracilis (Z strain) culture
Euglena gracilis (Z strain) was provided by the Graduate
School for the Creation of New Photonics Industries,
Hamamatsu, Japan, and cultured in the laboratory for
approximately six months prior to testing. Organisms were
cultured in sterilized Koren Hutner (KH) medium (Koren
and Hutner 1967) at pH 6.0 ± 0.1, 28 ± 1°C and with a
12:12 h day/night cycle (36 W cool white triphosphor
lighting; 100–140 lmol m-2 s-1) based on the method of
Ohta et al. (2001). Cultures (5 mL) were maintained in
10 mL screw-capped, glass test tubes on an orbital shaker.
Each week, 50 lL of a 7-day old E. gracilis culture was
transferred to 5 mL KH medium under aseptic conditions.
Nutrient-rich KH medium was used to sustain the longterm health of the culture and support a growth rate of
E. gracilis that would provide sufficient numbers of test
organisms within a short time frame (1 week). Four days
prior to testing, 100 lL of 7-day old culture in 100% KH
medium was transferred to test tubes containing 5 mL of
dilute KH medium (2.5% strength, diluted with sterilized
Milli-Q water, under aseptic conditions).
Dissolved organic carbon reduces uranium toxicity
1015
Development of a low nutrient test medium
Table 2 Composition of 20 mg L-1 (150 lM) aspartic acid test
medium
KH medium is a high nutrient medium (Koren and Hutner
1967) in which E. gracilis can reach densities of 16 million
cells mL-1 (Koren and Hutner 1967). However, the medium has a high DOC concentration (10 g L-1), containing
high levels of glucose and organic acids, which can
potentially bind metals and reduce their toxicity to test
organisms. Exposure trials were conducted with the aim of
establishing a test medium that was less nutrient-rich and
more relevant to natural freshwater environments (Meybeck 2003), but which still supported a suitable growth rate
of E. gracilis (considered by the authors to be 0.4 ± 0.2
doublings day-1). Media containing minimal concentrations of a single carbon source, such as glucose or citric
acid, in addition to vitamin B1 and B12 (essential for
growth of E. gracilis) were trialed (Table 1), following the
test procedure outlined in the General toxicity test method
section. 2-N-morpholinoethanesulfonic acid sodium salt
(MES) buffer was used to maintain the test media at pH
6.0 ± 0.1, as this buffer is considered to have minimal
binding to metals (Good et al. 1966) and was predicted (by
speciation modeling) to have negligible binding to U
(\0.2% of U). The composition of the test medium selected
for the toxicity testing (150 lM aspartic acid medium) is
provided in Table 2.
Name
L-Aspartic
Concentration (mg L-1)
acid
20
Potassium hydrogen orthophosphate
1
Ammonium chloride
10
Thiamine HCl salt (Vitamin B1)
2.5
Cyanocobalamin (Vitamin B12)
0.005
MES buffer
390
uranium stock solutions were prepared by dissolving
UO2SO43H2O (supplied by AJAX, Seven Hills, Australia)
in Milli-Q water. Test solutions were prepared by adding U
stock solution to the aspartic acid medium, and were pH
adjusted to 6.0 ± 0.15 using 0.05 M NaOH and H2SO4,
and dispensed to test tubes under aseptic conditions.
Solutions were then stored and refrigerated for 48 h to
allow equilibration of U with DOC.
Euglena gracilis were tested on three separate occasions
at a range of U concentrations in the background medium
and then on two separate occasions in background medium
?20 mg L-1 DOC medium (as SRFA). Uranium concentration ranges of up to 6.6 mg L-1 (measured U) were
selected in order to obtain a full toxic response for
E. gracilis.
Preparation of U test solutions
General toxicity test method
Suwannee River fulvic acid (SRFA, 1S101F, International
Humic Substances Society, University of Minnesota) was
the standard DOC source used in this study (IHSS 2008).
Physicochemical characteristics of SRFA have been
described previously (IHSS 2008; Trenfield et al. 2011b).
SRFA (38.14 mg L-1) was dissolved into the aspartic acid
medium (containing background DOC of 10 mg L-1) to
produce a medium containing an additional 20 mg L-1
DOC, a concentration typical of that found naturally in
wetlands (Mladenov et al. 2005). 10 and 50 mg L-1
A standard number of E. gracilis cells (1 9 104
cells mL-1) were exposed to a control (1 lg L-1 U) or one
of six U concentrations at each DOC treatment for 96 h at
28 ± 1°C in 5 mL. Treatments were prepared in duplicate in
a laminar flow using silanized 10 mL screw-capped
glass test tubes. Tests were conducted using exponentially
growing cells from a 4-day old culture (density *3 9
105 cells mL-1). Cells were rinsed twice in aspartic acid
medium (pH 6 ± 0.15 and 28 ± 1°C) and concentrated
Table 1 Various test media trialed for 96 h uranium exposures with Euglena gracilis at pH 6
Medium
DOC (mg L-1)d
IC50 (lg L
-1
U)
e
Control doublings d-1
f
1.5% KHa
0.5% KHa
333 lM Glucoseb
150 lM Aspartic acidc
150
55
60
10
8,900
3,500
[4,000
300
1.2 (1.1–1.3)
0.7 (0.6–0.8)
0.6 (0.45–0.7)
0.55 (0.54–0.56)
a
-1
KH Koren Hutner medium diluted with milli-Q and 2.5 mg L
b
Medium contained 60 mg L-1 glucose, 10 mg L-1 NH4CO3, 10 mg L-1 KH2PO4, 2.5 mg L-1 Vit B1 and 0.005 mg L-1 Vit B12
c
Medium ingredients shown in Table 2
d
DOC Dissolved organic carbon prior to addition of MES buffer
Vit B1 and 0.005 mg L
-1
Vit B12 added
e
IC50 U concentration at which there was 50% reduction in growth over 96 h, representing total U unadjusted for adsorption to glass test tubes
f
Mean (range) 96 h doublings, n = 2
123
1016
using centrifugation (780 g for 1 min) in order to remove
the nutrient-enriched KH culture medium, which may lower
toxicity due to its ability to strongly complex trace metals.
Growth of E. gracilis was measured by counting cells at 48
and 96 h using a compound microscope (9160 mag) and
calculating the cell division rate (as doublings d-1) using
linear regression analysis. Cell morphology was also
observed during cell counts. Treatments were prepared for
counting by taking a 250 lL sub-sample, adding 10 lL
Lugols iodine fixative and transferring the subsample to a
haemocytometer. Growth rates of E. gracilis exposed to U
were expressed as a percentage of the control growth rate. A
test was considered valid if the cell division rate in controls
was 0.4 ± 0.2 doublings day-1, with a coefficient of variation (CV) of less than 20%.
Physico–chemical analyses
The dissolved oxygen (DO), pH and conductivity of test
solutions were measured at 0 and 96 h for each treatment
using WTW Multiline P4 and Inolab multiline level 1
(Weilheim, Germany) instruments. Incubator temperature
readings were taken every 5 min. over the 96 h using a
Tinytag data logger (Gemini Data Loggers UK Ltd.). At
0 h, sub-samples of control solutions were analysed for
total Al, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Na, Ni, Pb, S, Se
and Zn using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES, Thermo Iris Intrepid 2
Radial, Waltham, USA) or Inductively Coupled Plasma
Mass Spectroscopy (ICP-MS, Agilent 7500 ce, Japan). All
treatments were analysed for total and 0.45 lM filtered U
(Sartorious, Minisart RC cellulose nitrate filters, pre-rinsed
with Milli-Q water and test solutions) at 0 h using ICP-MS.
Following one of the tests, 96 h old water from each
treatment was 0.45 lM filtered to remove E. gracilis cells
and analysed for U to assess the proportion of U remaining
in solution. Samples for metal analyses were acidified to
1% HNO3 (AR grade, Crown Scientific). Alkalinity was
determined using method 2320B (APHA et al. 2005) with a
Radiometer Analytical Tim 900 titration manager. Hardness was calculated using method 2340B (APHA et al.
2005) based on measured Ca and Mg concentrations (i.e.
2.497 9 [Ca, mg L-1] ? 4.118 9 [Mg, mg L-1]). DOC
analysis was conducted using the high-temperature combustion method 5310B (APHA et al. 2005; TOC-VCSH,
Shimadzu Scientific Instruments, Oceania Pty. Ltd.,
SD \ 0.1 mg L-1, maximum CV of 2%). Nitrate (NO3-)
and phosphate (PO43-) were measured in controls from
two tests using Flow Injection Analysis (Lachat Quikchem
FIA?, 8000 series). The background medium was analysed
for chloride using Flow Injection Analysis method
4500-Cl-G (APHA et al. 2005).
123
M. A. Trenfield et al.
Investigation of U partitioning in test system
Following the observation of the loss of U from test
solutions and the potential uptake of U by E. gracilis over
96 h, further investigation was conducted to determine the
fraction of bioavailable U in the test system over 96 h.
Both the background medium and medium containing
SRFA were investigated using a control (1 lg L-1) and
three nominal U concentrations (20, 180, 1,000 lg L-1).
These concentrations were chosen in order to characterise
the partitioning of U at background, low, medium and high
concentrations of U in terms of the toxic response of
E. gracilis. Test solutions were inoculated with
1 9 104 cells mL-1 E. gracilis and cell counts performed
at 48 and 96 h to check for acceptable cell growth. Total U,
0.45 lM filtered U, U adsorbed to the inner surface of the
test tubes and U adsorbed to/absorbed by E. gracilis were
measured at 48 and 96 h. Filtered U and U adsorbed to the
test tubes was also measured at 24 h, but there was insufficient E. gracilis tissue mass to measure cellular U for that
period. In total, 16 replicates were prepared for each
treatment. Four ‘sacrificial’ replicates of each U treatment
were pooled to provide 24 and 48 h samples, while eight
replicates were pooled to provide two separate samples of
each treatment at 96 h. After pooling replicates, the test
solution was 5 lm filtered (Millipore, nitrocellulose
47 mm membrane housed in a Swinnex polypropylene
filter holder) to remove E. gracilis cells (which were typically 6 lm wide and 50 lm in length). Filter membranes
were dried at 60°C for 24 h and U content determined by
nitric/hydrochloric digest (method G316M, APHA et al.
2005). The remaining solution was subsampled for total
and filtered (0.45 lm) U. Test tubes were air dried and
filled with 65% HNO3 to remove U adsorbed to the glass.
Detection of ROS
On two separate occasions, following exposure to U for
96 h, controls and various U treatments were selected to
assess E. gracilis for oxidative stress. Concentrations of up
to 1 mg L-1 U in the background medium, and up to
4.5 mg L-1 U in the SRFA medium, were chosen in order
to target concentrations at which toxic effects were
observed in each of the media. Each selected treatment was
first concentrated from 10 mL down to a 200 lL suspension by centrifugation at 1,500 rpm (780 g) for 1 min. The
method used to detect ROS was adapted from manufacturer
guidelines (Invitrogen 2006) and previous work (Watanabe
and Suzuki 2002; Periyakaruppan et al. 2007). Dihydrofluorescein diacetate (HFLUOR-DA), also known as
chloromethyl-dichlorodihydrofluorescein diacetate, acetyl
ester (CM-H2DCFDA, Invitrogen 2011) was used as the
probe for detecting the presence of intracellular oxidants
Dissolved organic carbon reduces uranium toxicity
(Hempel et al. 1999; Watanabe and Suzuki 2002). CMH2DCFDA was purchased from Invitrogen (Australia) and
stored in a dark, sealed container at -20°C. Immediately
prior to use, a 50 lg sample of the material was thawed in
the dark for 10 min. Then, in dim light, to reduce
decomposition of the probe, 86 lL of 100% ethanol was
added to the aliquot to produce a 1 mM stock solution. A
50 lM working solution was then prepared in a 0.5 mL
microtube by adding 20 lL of probe stock to 380 lL of
2 mM HEPES buffer (known to have minimal influence on
U toxicity at this concentration, Franklin et al. 2000).
20 lL of working solution was then added to 80 lL of
E. gracilis cell suspension, mixed for 1 min. by inverting
the microtube, and allowed to stand at *28°C in the dark
for 1 h. A positive control was run to confirm the CMH2DCFDA was effective in detecting ROS in E. gracilis.
Preparation of this control involved adding 10 lL of 1 mM
H2O2 stock to a 100 lL mixture of control E. gracilis and
CM-H2DCFDA. A Leica Letiz Laborlux S fluorescence
microscope with an I2 filter cube (450–490 nm excitation
wavelength and 505 nm emission) was used to observe the
fluorescence of the fluorescein dye. Photographic images
were captured with a Canon Powershot S70 camera.
Geochemical speciation modeling of U
The speciation of U in the test solutions was calculated
using the HARPHRQ geochemical speciation code (Version 1.02, Brown et al. 1991), with input parameters based
on physico–chemical data measured in the test solutions
(pH, temperature, DO and total cation and anion concentrations; Table 3). A conditional stability constant for UO2SRFA (log K = 6.8 at pH 6) was calculated from Glaus
et al. (2000). Stability constants for UO2-aspartate species
were taken from Gianguzza et al. (2005). Stability constants for the complexation of U with MES and major
inorganic anions (sulfate, carbonate, chloride, nitrate and
phosphate) were taken from Markich and Brown (1999).
1017
Table 3 Physico–chemical composition of E. gracilis media (background medium and medium containing 20 mg L-1 dissolved organic
carbon (DOC) in the form of Suwannee River fulvic acid)
Physico–chemical
variable (units)
E. gracilis
mediaa
pHb
6.0 (5.94–6.06)
-1 b
EC (lS cm )
267 (254–280)
Temperature (°C)c
28 (27–29)
Dissolved oxygen (%)b
100 (90–107)
Alkalinity (mg L-1 CaCO3)d
50 ± 9
Hardness (mg L-1 CaCO3)e
0.7
Chemical parameter
Limit of
reporting
f
Ca (mg L-1)
\0.1
K (mg L-1)
0.45
0.1
Mg (mg L-1)
\0.1
0.1
Na (mg L-1)
64 ± 5
0.1
0.1
-1
0.1
SO4 (mg L )
198 ± 15
Cl (mg L-1)
8.4
0.1
Al (lg L-1)
Cd (lg L-1)
3.1 ± 0.6
0.3 ± 0.06
0.1
0.02
Cr (lg L-1)
0.10 ± 0.01
0.1
Cu (lg L-1)
2.1 ± 0.3
0.01
Fe (lg L-1)
\20
Mn (lg L-1)
0.12 ± 0.03
Ni (lg L-1)
0.40 ± 0.07
0.01
Pb (lg L-1)
0.16 ± 0.05
0.01
U (lg L-1)
1.0 ± 0.30
0.001
-1
20
0.01
Zn (lg L )
3.2 ± 1.0
0.1
NO3 (lg L-1)
15,800 ± 1,430
5
PO4 (lg L-1)
210 ± 10
5
a
Physical parameters are the median (10–90th percentile) of both
mediums, n = 47
b
Physical parameters measured on 0 and 96 h water
c
Mean (range), n = 7
d
Mean (±SE), n = 3
e
Hardness by calculation (APHA et al. 2005), n = 1 (value not used
in modelling)
f
Statistical analyses
Toxicity data (presented as a function of the control
response) were pooled from five tests for the background
DOC treatment and data from two tests were pooled for
the 20 mg L-1 DOC (as SRFA) treatment. Non-linear
(3-parameter logistic) regression was used to generate U
concentration-response curves for each treatment (SigmaPlot 11.0). U concentrations at which there was 10%
inhibition of growth (IC10) and 50% inhibition of growth
(IC50) and their 95% confidence limits (CLs), were determined from the equation of the curve fits. All U species
that were predicted by HARPHRQ to be present in proportions greater than 0.8% of total U were incorporated,
n = 7 for all analytes except for K and Cl (n = 1) and NO3 and PO4
(n = 3)
together with DOC and U toxicity (growth inhibition,
doublings d-1) in a stepwise multiple linear regression
analysis (Minitab 15.0), to identify which, if any, of the U
species had a significant relationship with U toxicity.
Results and discussion
Development of a low nutrient test medium
Table 1 shows, for each medium tested, the DOC content,
the growth rates and sensitivity of E. gracilis to U (IC50).
123
1018
M. A. Trenfield et al.
Test acceptability and water chemistry
The control growth rate of E. gracilis was 0.43–0.64
doublings d-1 across all tests. A summary of measured
physicochemical data across all experiments is shown in
Table 3. There was minimal fluctuation in physico–chemical variables within each treatment over 96 h (pH 6.0 ±
0.1, DO ±10%, conductivity \10% and temperature 28 ±
1.0°C). While the concentration of sulfate in test solutions
was high (*180 mg L-1) due to the addition of sulphur
(through the use of MES buffer), sulfate was predicted to
have minimal influence on U complexation (1–8% of U
was present as UO2SO4).
Toxicity of U to E. gracilis and the influence of DOC
Percentage growth rate
(as % of control)
Growth rate was found to be a sensitive endpoint for
monitoring U toxicity to E. gracilis. A concentration of
57 lg L-1 U (95% CLs: 40–82 lg L-1 U) resulted in 50%
inhibition of growth (IC50) of E. gracilis in the background
medium (Fig. 1a). However, as little as 5 lg L-1 U (95%
CLs: 1–12 lg L-1 U) resulted in a 10% decline in growth
(IC10). In background medium, growth was completely
(a) Background medium
100
80
60
40
20
0
0
200
400
600
800
Uranium (µg L-1)
Fig. 1 Concentration response plots for Euglena gracilis exposed to
uranium (0.45 lm filtered) for 96-h in a 150 lM aspartic acid
medium at background dissolved organic carbon (DOC: 10 mg L-1)
and b medium containing an additional 20 mg L-1 DOC (as
123
inhibited at *700 lg L-1 U. Toxicity can be attributed
primarily to the chemical toxicity of U rather than its
radiological toxicity (Mathews et al. 2009). The sensitivity
of E. gracilis to U appeared to be equivalent to that of the
most sensitive species previously studied under similar
physicochemical conditions (sub-lethal IC50s range from
30–1,200 lg L-1 U for a cladoceran, green alga, fish,
hydra and mussel species; Markich et al. 2000; Hogan et al.
2005; Zeman et al. 2008; Trenfield et al. 2011a).
Shape change (polymorphic forms) of E. gracilis in
response to metal exposure has been previously described
in the presence of Cd (at 0.1–1.6 mg L-1, Watanabe and
Suzuki 2001) and tributyltin chloride (5.6 mg L-1, Ohta
et al. 2001). In the present study, many cells changed shape
from a healthy, long spindle form to a ‘tear-drop’ or a cystlike spherical shape at concentrations greater than
300 lg L-1 U in background medium. Cells also occasionally became V-shaped, star-shaped or hypertrophic at U
concentrations of 300 lg L-1 U or greater (Fig. 2). Previous studies involving exposure of E. gracilis to Cd, Cu and
Zn have resulted in elevated cellular concentrations of
proteins, lipids and chlorophyll, and this has been linked to
the failure of E. gracilis to complete cell division under
these conditions (Einicker-Lamas et al. 1996; EinickerLamas et al. 2002). Watanabe and Suzuki (2002) observed
hypertrophic cells following exposure of E. gracilis to Cd,
linking the cell enlargement with the suppression of cytokinesis. In the current study, shape change was observed
only at U concentrations at which the growth rates of
E. gracilis were reduced to less than 50% of the controls.
Shape change was therefore not considered to be a sensitive
indicator of U toxicity. Danilov and Ekelund (2001) also
found E. gracilis cell shape (and motility) to be insensitive
to short-term exposure (24 h) to Cu, Ni, Pb and Zn.
In the presence of 20 mg L-1 DOC, which is typical of
higher DOC concentrations found in floodplain environments
Percentage growth rate
(as % of control)
Growth rates were higher in the higher nutrient media, but
were acceptable (i.e. 0.4 ± 0.2 doublings d-1) across all
media. The DOC concentration was lowest, and U toxicity
highest, in the aspartic acid medium. Aspartic acid medium
was predicted (through speciation modelling) to have
minimal complexation with U (complexing *6–36% of U
over the total U range 0.03–6.6 mg L-1). Consequently,
the aspartic acid medium was selected as the test medium
for the U toxicity experiments (Table 1) and the authors
are of the view that U toxicity data from this study can
be considered to be relevant to natural freshwater
environments.
(b) 20 mg L-1 DOC (as SRFA)
100
80
60
40
20
0
0
2000
4000
6000
-1
Uranium (µg L )
Suwannee River fulvic acid—SRFA). Curves 3-parameter logistic
fit with five pooled tests for background DOC (r2 = 0.84, n = 27,
p \ 0.0001) and two pooled tests for 20 mg L-1 DOC (r2 = 0.75,
n = 14, p = 0.0001). Each point mean of two replicates ±SE
Dissolved organic carbon reduces uranium toxicity
1019
(Mladenov et al. 2005), there was a marked reduction in U
toxicity to E. gracilis (Fig. 1b), with the IC50 of E. gracilis
increasing 4 to 5-fold to 254 lg L-1 U (95% CLs:
100–670 lg L-1 U). The IC10 in medium with additional
20 mg L-1 DOC increased 3 to 4-fold from that of the
background medium; 17 lg L-1 U (95% CLs: 1–77 lg L-1
U). An analysis of covariance on the regressions of each of
the DOC data sets shown in Fig. 1 (regression of ln[growth
rate] vs U) showed a significant interaction between DOC
and U (p = 0.000, a = 0.05, r2 = 0.55 for background DOC
and 0.51 for 20 mg L-1 DOC). Thus there was a significant
difference in the response of growth rate to U concentration
between the two DOC treatments.
Speciation of U
The proportion of the major U species predicted to be present
at the IC50 concentration for each of the DOC treatments is
shown in Table 4. In the presence of 20 mg L-1 DOC (as
SRFA), 84% of U was predicted to complex with SRFA.
Speciation calculations show a 6 to 7-fold decrease in the
proportion of inorganic U species with the addition of SRFA
(Fig. 3). The concentration of UO22? present at the IC50
concentration in the presence and absence of SRFA was
found to be similar (2.2 and 3.1 lg L-1 respectively,
Table 4), providing support that toxicity is linked to this
species, in particular, and that the amelioration of toxicity is a
result of complexation of U by SRFA.
Stepwise multiple linear regression analyses, incorporating the inorganic U species shown in Table 4, indicated
that only UO22? had a significant (p B 0.01) relationship
with E. gracilis growth, explaining 51% of the variation in
U toxicity. By comparison, total U explained 38% of U
toxicity. The toxic response of E. gracilis in terms of the
UO22? concentration is shown in Fig. 4.
Distribution of U in the test system and uptake of U
by E. gracilis
Fig. 2 Various cell morphologies of Euglena gracilis following 96 h
uranium exposure: a healthy control cell with flagella, b abnormal
V-shaped cell (exposed to 300 lg L-1 U), and c abnormal hypertrophic cell (exposed to 1 mg L-1 U) exhibiting fluorescence (indicating
oxidative stress) as detected by the CM-H2DCFDA fluorescence
probe. Bar 20 lm
Figure 5 shows the distribution of U within the test system
at 48 and 96 h for the background medium and medium
containing SRFA. A greater proportion of the total U was
adsorbed to the glass in tubes containing the background
medium (*30%) compared with the tubes holding the
SRFA medium (*3%) at both 48 and 96 h. The proportion
of U adsorbed to the glass at 24 h from the background
medium and the SRFA medium was *15 and 2%
respectively. There was also greater U uptake by E. gracilis cells in the background medium (mean = 36 and
31% of initial U at 48 and 96 h, n = 9) compared with
the SRFA medium (mean = 3 and 2% of initial U at 48
and 96 h, n = 9). Looking at the data on U uptake or
123
1020
M. A. Trenfield et al.
Table 4 Predicted speciation and toxicity (IC50) of uranium (U) to E. gracilis in background medium (150 lM aspartic acid) and medium
containing dissolved organic carbon (DOC) in the form of Suwannee River fulvic acid (SRFA)
Medium type
DOC
(mg L-1)
IC50
(lg L-1 U)a
% of total U
UO22?
UO2OH?
UO2(OH)2
UO2SO4
UO2HPO4
UO2ASP
UO2OHASP
Backgroundb
10
57
5.5
32
3.6
8.8
6
7.3
31.8
Background ? 20 mg L-1
DOC
30
254
0.9
5
0.6
1.4
1
1.2
5.1
UO2SRFA
na
84.3
Species comprising \1% of total U have been excluded for clarity
na Not applicable
a
U concentration at which there was 50% reduction in growth of E. gracilis over 96 h
b
See Table 2 for details of medium composition
UO2OH+
(a) Background
100
30
Growth rate (as% of control)
% of inorganic U species
35
25
20
15
10 UO2SO4
5
0
UO2
2+
UO2OH2
Background medium
+ 20 mg L-1 DOC (as SRFA)
80
60
40
20
0
0
200
400
600
800
1000
1200
1400
Uranium (µg L-1)
7
(b) 20 mg L-1 DOC
UO2FA
90
85
80
5
UO2OH+
75
4
70
3
65
2
60
UO2SO4
1
2+
UO2
UO2OH2
55
0
50
0
2000
4000
6000
Uranium (µg L-1)
Fig. 3 The proportion of uranium (U) species present (as predicted
by the HARPHRQ speciation model) in a 150 lM aspartic acid
medium and b medium containing 20 mg L-1 DOC as Suwannee
River fulvic acid. UO2HPO4, UO2ASP and UO2OHASP made up the
remainder of the U species not shown here
adsorption more closely (see Table S1), the percentage
uptake or adsorption of U by E. gracilis increased with
increasing U exposure concentration in both media (at both
48 and 96 h). In the background medium at 20, 180, and
1,000 lg L-1 U, cellular or adsorbed U was 15, 28 and
50%, respectively, of the initial U exposure concentration
(n = 3 for each U concentration). This pattern was also
123
10
20
30
40
50
60
70
UO22+ (µg L-1)
% UO2FA
% inorganic U species
6
0
Fig. 4 The toxicity of uranium to Euglena gracilis expressed in
terms of the concentration of UO22? and its influence on growth rate
(shown as a percentage of the control growth rate). UO22? values
were calculated by the HARPHRQ speciation model. Data are shown
for background medium containing 10 mg L-1 DOC and also for the
medium containing an additional 20 mg L-1 DOC in the form of
Suwannee River fulvic acid (SRFA)
observed for E. gracilis exposed to U in the SRFA medium, albeit a much smaller increase (cellular and adsorbed
U contributed 2.5, 3 and 4%, respectively, of the three U
concentrations tested).
The differences in U distribution between the background medium and the medium containing SRFA, suggests the SRFA was effectively competing for U with
binding sites on the glass and the cell surface of E. gracilis.
The lower uptake of U in the presence of SRFA also
supported the assumption that U bound to the FA was no
longer bioavailable to E. gracilis. Studies of the influence
of DOC on metal uptake by phytoplankton cells have also
found the sorption of DOC by the cell surface, and that this
interaction is likely to retard metal diffusion to uptake sites
on the cell membrane, thus reducing the bioavailability of
the metal (Parent et al. 1996; Campbell et al. 1997).
The greater proportion of U bound to the glass in the
background medium corresponded with less potentially
Dissolved organic carbon reduces uranium toxicity
1021
100
30
Background medium
20 mg L-1 DOC (as SRFA)
25
% Fluoresced cells
% of U at 0 h
80
60
40
20
15
10
5
20
0
h
96
h
0
D
O
C
96
1000
2000
3000
4000
Uranium (µg L-1)
20
m
g/
L
nd
ro
u
B
ac
kg
m
g/
L
20
B
ac
kg
ro
u
nd
D
O
C
48
48
h
h
0
Euglena treatments
U adsorbed to glass
U adsorbed to/absorbed by Euglena
Particulate (>0.45 µm) U in solution
Fig. 6 The percentage of Euglena gracilis cells exhibiting signs of
oxidative stress (fluorescing in the presence of CM-H2DCFDA
molecular probe) following 96 h uranium exposure. 100 cells were
observed from a single replicate at each concentration of uranium
(0.45 lm filtered)
Dissolved (<0.45 µm) U in solution
Fig. 5 Distribution of uranium (U) in the Euglena gracilis test
system at 48 and 96 h in 150 lM aspartic acid test medium at
background dissolved organic carbon (DOC: 10 mg L-1) and background medium with an additional 20 mg L-1 DOC (as Suwannee
River fulvic acid), expressed as a percentage of the total U measured
at the start of the test (0 h)
bioavailable U being present in solution in this medium,
and as such, the U exposure concentrations shown in Fig. 1
have been adjusted for the loss of U to the glass. No
adjustment was made for the difference in U uptake by
cells in each of the treatments, as this study did not address
the bioavailability of the U that was in or on the cell.
Previous studies demonstrating metal uptake by E. gracilis
(Navarro et al. 1997; Einicker-Lamas et al. 2002), have
shown the vacuoles of E. gracilis can play a detoxification
role by accumulating metals. Thus, not all of the U stored
within the cell may necessarily be bioavailable.
100 lM of Cd2? (*11 mg L-1) for 1 h doubled the
intensity of cell fluorescence compared to that shown by
control cells (fluorescence intensity increased from 25 to
55 U). Watanabe and Suzuki (2002) also noted intense
fluorescence occurred in E. gracilis exposed to 10 lM Cd2?
(*1 mg L-1) for 5 days. However, no data were provided
concerning the proportion of cells exhibiting fluorescence.
Preliminary results suggest the presence of SRFA may
reduce the generation of ROS, which could be a result of
lower uptake of U in this medium. However, more data are
required to refine this relationship. The lack of fluorescence
occurring even at concentrations of 150 lg L-1 U suggests
either there is a mechanism of toxicity occurring other than
the generation of ROS or that the method used in this study
for detecting oxidative species in E. gracilis requires
refinement. The effectiveness of the method used in this
study may have been improved by monitoring ROS sooner
following the start of U exposure rather than after 96 h.
U toxicity and the generation of ROS
The fluorescence of E. gracilis following 96 h U exposure
and inoculation with the CM-H2DCFDA probe was quite
varied, but did increase somewhat with exposure to
increasing U (Fig. 6). Based on the response using CMH2DCFDA, cells did not appear to exhibit any fluorescence
(oxidative stress) until they were exposed to U concentrations of 60 lg L-1 or greater. Generally, the proportion of
fluorescing cells was quite low even in treatments where cell
proliferation had ceased. For example, at 1 mg L-1 U (in
medium without SRFA), 23% of cells exhibited fluorescence. Watanabe and Suzuki (2002) using the same probe
(formerly known as HFLUOR-DA) found that exposure to
Conclusions
Uranium concentrations of 5 and 57 lg L-1 resulted in 10
and 50% declines in growth of E. gracilis, respectively,
while concentrations of 700 lg L-1 U completely ceased
cell proliferation. The addition of 20 mg L-1 DOC (as
SRFA) reduced the toxicity of U to E. gracilis by three- to
five-fold, with 17 and 254 lg L-1 resulting in 10 and 50%
declines in growth of E. gracilis, respectively. Speciation
modelling indicated 84% of U in the 20 mg L-1 DOC (as
SRFA) medium was complexed by SRFA. This reduction
in the bioavailability and toxicity of U in the presence of
123
1022
SRFA, corresponded with a 15-fold decrease in the cellular
uptake/adsorption of U at 96 h. While the addition of
SRFA resulted in a decrease in the predicted concentrations
of all inorganic U species, UO22? provided the best relationship with toxicity (r2 = 0.51). Preliminary studies
suggested that although exposure to C60 lg L-1 U induces
oxidative stress in E. gracilis, this method may not be a
sensitive indicator of toxicity. This and other biological
endpoints require further research.
Acknowledgments The authors would like to thank Professor
Tetsuya Suzuki, Professor Mari Ohta and Dr. Masumi Watanabe at
the Graduate School for Creation of New Photonics Industry, Japan
for providing the E. gracilis Z strain and culturing advice and Dr.
David Jones at eriss for providing comments on the manuscript. This
project was funded by an ARC-Linkage grant (LP 0562507). EnTox
is a partnership between Queensland Health and The University of
Queensland.
References
Antunes SC, Pereira R, Goncalves F (2007) Acute and chronic
toxicity of effluent water from an abandoned uranium mine.
Arch Environ Contam Toxicol 53:207–213
APHA, AWWA, WEF (2005) in: Eaton AD, Clesceri LS, Rice EW,
Greenberg AE (eds) Standard methods for the examination of
water and wastewater. American Public Health Association,
American Water Works Assocation, Water Environment Federation, Washington DC
Barillet S, Adam C, Palluel O, Devaux A (2007) Bioaccumulation,
oxidative stress, and neurotoxicity in Danio rerio exposed to
different isotopic compositions of uranium. Environ Toxicol
Chem 26:497–505
Barillet S, Larno V, Floriani M, Devaux A, Adam-Guillermin C
(2010) Ultrastructural effects on gill, muscle, and gonadal tissues
induced in zebrafish (Danio rerio) by a waterborne uranium
exposure. Aquat Toxicol 100:295–302
Brown P, Haworth A, Sharland S, Tweed C (1991) HARPHRQ: an
extended version of the geochemical code PHREEQE. NSS/R
188. UK Atomic Energy Authority, Oxford
Buet A, Barillet S, Camilleri V (2005) Changes in oxidative stress
parameters in fish as response to direct uranium exposure.
Radioprotection 40:S151–S155
Campbell PGC, Twiss MR, Wilkinson KJ (1997) Accumulation of
natural organic matter on the surfaces of living cells: implications for the interaction of toxic solutes with aquatic biota. Can J
Fish Aquat Sci 54:2543–2554
Danilov RA, Ekelund NGA (2001) Responses of photosynthetic
efficiency, cell shape and motility in Euglena gracilis (Euglenophyceae) to short-term exposure to heavy metals and pentachlorophenol. Water Air Soil Pollut 132:61–73
dos Santos Ferreira V, Roccetta I, Conforti V, Bench S, Feldman R,
Levin M (2007) Gene expression patterns in Euglena gracilis:
insights into the cellular response to environmental stress. Gene
389:136–145
Einicker-Lamas M, Soares MJ, Soares MS, Oliveira MM (1996)
Effects of cadmium on Euglena gracilis membrane lipids. Braz J
Med Biol Res 29:941–948
Einicker-Lamas M, Meziana GA, Fernandesa TB, Silva FLS, Guerra
F, Miranda K, Attias M, Oliveira MM (2002) Euglena gracilis as
123
M. A. Trenfield et al.
a model for the study of Cu2? and Zn2? toxicity and
accumulation in eukaryotic cells. Environ Pollut 120:779–786
Franklin NM, Stauber JL, Markich SJ, Lim RP (2000) pH-dependent
toxicity of copper and uranium to a tropical freshwater alga
(Chlorella sp.). Aquat Toxicol 48:275–289
Gajdosova J, Reichrtova E (1996) Different growth response of
Euglena gracilis to Hg, Cd, Cr and Ni compounds. Anal Bioanal
Chem 354:641–642
Gianguzza A, Pettignano A, Sammartano S (2005) Interaction of the
dioxouranium(VI) ion with aspartate and glutamate in NaCl(aq)
at different ionic strengths. J Chem Eng Data 50:1576–1581
Glaus M, Hummel W, Van Loon L (2000) Trace metal-humate
interactions. I. Experimental determination of conditional stability constants. Appl Geochem 15:953–973
Good NE, Winget GD, Winter W, Izawa S, Singh RMM (1966)
Hydrogen ion buffers for biological research. Biochemistry
5:467–477
Hempel SL, Buettner GR, O’Malley YQ, Wessels DA, Flaherty DM
(1999) Dihydrofluorescein diacetate is superior for detecting
intracellular oxidants: comparison with 29,79-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-29,79- dichlorodihydrofluorescein diacetate and dihydrorhodamine 123. Free Radic Biol
Med 27:146–159
Hogan AC, van Dam RA, Markich SJ, Camilleri C (2005) Chronic
toxicity of uranium to a tropical green alga (Chlorella sp.) in
natural waters and the influence of dissolved organic carbon.
Aquat Toxicol 75:343–353
Hynes TP, Schmidt RM, Meadley T, Thompson NA (1987) The
impact of effluents from a uranium mine and mill complex in
Northern Saskatchewan on contaminant concentrations in receiving waters and sediments. Water Pollut Res J Can 22:559–569
International Atomic Energy Agency (2008) Energy, electricity and
nuclear power estimates for the period to 2030. Reference Data
Series No. 1. Vienna
International Humic Substances Society (2008) Source materials for
IHSS samples. http://www.humicsubstances.org/sources.html.
Accessed Jan 2011
Invitrogen (2006) Reactive oxygen species (ROS) detection reagents.
http://probes.invitrogen.com/media/pis/mp36103.pdf. Accessed
Jan 2011
Invitrogen (2011) CM-H2DCFDA (general oxidative stress indicator). http://products.invitrogen.com/ivgn/product/C6827. Accessed 7 Apr 2011
Kaddissi SA, Legeay A, Gonzalez P, Floriani M, Camilleri V,
Gilbin R, Simon O (2011) Effects of uranium uptake on
transcriptional responses, histological structures and survival
rate of the crayfish Procambarusclarkii. Ecotoxicol Environ Safe
74:1800–1807
Koren LE, Hutner SH (1967) High-yield media for photosynthesizing
Euglena gracilis Z. J Protozool 14 (suppl):17
Lacky J (1968) Ecology of Euglena. In: Beutow DE (ed) The biology
of Euglena, vol I. Academic Press, New York, pp 27–44
Landa ER, Gray JR (1995) US geological survey research on the
environmental fate of uranium mining and milling wastes.
Environ Geol 26:19–31
Malcolm RL (1985) Geochemistry of stream fulvic and humic
substances. In: Aiken GR, McKnight DM, Wershaw RL (eds)
Humic substances in soil, sediment, and water. Wiley, New York,
pp 181–209
Markich SJ, Brown PL (1999) Thermochemical data (log K) for
environmentally-relevant elements. Commonweath of Australia,
Sydney
Markich SJ, Brown PL, Jeffree RA, Lim RP (2000) Valve movement
responses of Velesunio angasi (Bivalvia: Hyriidae) to manganese
and uranium: an exception to the free ion activity model. Aquat
Toxicol 51:155–175
Dissolved organic carbon reduces uranium toxicity
Mathews T, Beaugelin-Seiller K, Garnier-Laplace J, Gilbin R,
Adam C, Della-Vedova C (2009) A probabilistic assessment of
the chemical and radiological risks of chronic exposure to
uranium in freshwater ecosystems. Environ Sci Technol 43:
6684–6690
Matsuo AYO, Playle RC, Val AL, Wood CM (2004) Physiological
action of dissolved organic matter in rainbow trout in the
presence and absence of copper: sodium uptake kinetics and
unidirectional flux rates in hard and softwater. Aquat Toxicol
70:63–81
Meybeck M (2003) Global occurrence of major elements in rivers. In:
Drever JI (ed) Treatise on geochemistry, surface and groundwater, weathering and soils. Elsevier, London, pp 207–223
Mladenov N, McKnight DM, Wolski P, Ramberg L (2005) Effects of
annual flooding on dissolved organic carbon dynamics within a
pristine wetland, the Okavango Delta, Botswana. Wetlands 25:
622–638
Navarro L, Torres-Marquez ME, Gonzalez-Moreno S, Devars S,
Hernandez R, Moreno-Sanchez R (1997) Comparison of physiological changes in Euglena gracilis during exposure to heavy
metals of heterotrophic and autotrophic cells. Comp Biochem
Physiol C 116:265–272
Nechay BR, Thompson JD, Palmer Saunders J (1980) Inhibition by
uranyl nitrate of adenosine triphosphatases derived from animal
and human tissues. Toxicol Appl Pharm 53:410–419
Ohta M, Okazaki S, Hiramatsu M, Suzuki T (2001) Comparative
studies on the cellular response to chemical loading in unicellular eukaryote, Euglena gracilis, strains Z and SMZ. Nutr Res
18:83–89
Overbaugh JM (1985) Initial observations on the role of glutathione
peroxidases in euglena. Free Radic Biol Med 1:187–193
Parent L, Twiss M, Campbell P (1996) Influences of natural dissolved
organic matter on the interaction of aluminum with the
microalga Chlorella: a test of the free-ion model of trace metal
toxicity. Environ Sci Technol 30:1713–1720
Periyakaruppan A, Kumar F, Sarkar S, Sharma CS, Ramesh GT
(2007) Uranium induces oxidative stress in lung epithelial cells.
Arch Toxicol 81:389–395
1023
Pyle GG, Swanson SM, Lehmkuhl DM (2002) Toxicity of uranium
mine receiving waters to early life stage fathead minnows
(Pimephales promelas) in the laboratory. Environ Pollut 116:
243–255
Ribera D, Labrot F, Tisnerat G, Narbonne JF (1996) Uranium in the
environment: occurrence, transfer and biological effects. Rev
Environ Contam Toxicol 146:53–89
Semaan M, Holdway DA, van Dam RA (2001) Comparative
sensitivity of three populations of the cladoceran Moinodaphnia
macleayi to acute and chronic uranium exposure. Environ
Toxicol 16:365–376
Trenfield MA, Ng JC, Noller BN, Markich SJ, van Dam RA (2011a)
Dissolved organic carbon reduces uranium bioavailability and
toxicity. 2. Uranium[VI] speciation and toxicity to three tropical
freshwater organisms. Environ Sci Technol 45:3082–3089
Trenfield MA, McDonald S, Kovacs K, Lesher E, Pringle JM, Markich
SJ, Ng JC, Noller BN, Brown PL, van Dam RA (2011b) Dissolved
organic carbon reduces uranium bioavailability and toxicity. 1.
Characterization of an aquatic fulvic acid and its complexation
with uranium[VI]. Environ Sci Technol 45:3075–3081
Watanabe M, Suzuki T (2001) Cadmium-induced abnormality in
strains of Euglena gracilis: morphological alteration and its
prevention by zinc and cyanocobalamin. Comp Biochem Phys C
130:29–39
Watanabe M, Suzuki T (2002) Involvement of reactive oxygen stress
in cadmium-induced cellular damage in Euglena gracilis. Comp
Biochem Physiol C 131:491–500
Yazzie M, Gamble SL, Civitello ER, Stearns DM (2003) Uranyl
acetate causes DNA single strand breaks in vitro in the presence
of ascorbate (Vitamin C). Chem Res Toxicol 16:524–530
Zeman FA, Gilbin R, Alonzo F, Lecomte-Pradines C, GarnierLaplace J, Aliaume C (2008) Effects of waterborne uranium on
survival, growth, reproduction and physiological processes of
the freshwater cladoceran Daphnia magna. Aquat Toxicol 86:
370–378
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