Ecotoxicology and Environmental Safety 95 (2013) 83 90
Contents lists available at SciVerse ScienceDirect
Ecotoxicology and Environmental Safety
journal homepage: www.elsevier.com/locate/ecoenv
The toxicity of cadmium to three aquatic organisms (Photobacterium
phosphoreum, Daphnia magna and Carassius auratus) under
different pH levels
n
R.-J. Qu, X.-H. Wang, M.-B. Feng, Y. Li, H.-X. Liu, L.-S. Wang, Z.-Y. Wang
State Key Laboratory of Pollution Control and Resources Reuse, School of Environment, Xianlin Campus, Nanjing University, Nanjing 210023,
Jiangsu, PR China
a r t i c l e i n f o a b s t r a c t
Article history: This study investigated the effect of pH on cadmium toxicity to three aquatic organisms: Photobacterium
Received 27 February 2013
phosphoreum, Daphnia magna and Carassius auratus. The acute toxicity of Cd2+ to P. phosphoreum and
Received in revised form
D. magna at five pH values (5.0, 6.0, 7.0, 8.0, and 9.0) was assessed by calculating EC50 values.
15 May 2013
We determined that Cd2+ was least toxic under acidic conditions, and D. magna was more sensitive to
Accepted 16 May 2013
the toxicity of Cd than P. phosphoreum. To evaluate Cd2+-induced hepatic oxidative stress in C. auratus at
Available online 12 June 2013
three pH levels (5.0, 7.25, 9.0), the activity of antioxidant enzymes (superoxide dismutase, catalase and
Keywords:
glutathione peroxidase), the level of glutathione and the malondialdehyde content in the liver were
Cadmium
measured. Oxidative damage was observed after 7 d Cd exposure at pH 9.0. An important finding of the
Toxicity
current research was that Cd2+ was generally more toxic to the three test organisms in alkaline
pH
environments than in acidic environments.
Photobacterium phosphoreum
& 2013 Elsevier Inc. All rights reserved.
Daphnia magna
Carassius auratus
1. Introduction http://www.epa.gov/caddis/ssr_ph4s.html). Fluctuations in pH may
lead to changes in cadmium speciation, thereby influencing its
The global increase in freshwater contamination by numerous bioavailability and toxicity to exposed organisms. Thus, it is of
natural and industrial chemical compounds is a major environ- significance to study cadmium toxicity to different aquatic species
mental problem in the world (Schwarzenbach et al., 2006). Heavy as a function of pH.
metals are important contaminants of aquatic environments In aquatic toxicological studies, bacteria, daphnids and fish are
worldwide. Among these metals, cadmium has received consider- the most frequently used test species (Farre and Barcelo, 2003).
able attention in recent years because its concentration in water These organisms represent different trophic levels in the aquatic
body has been markedly increased by human activities such as food chain and are capable of reflecting the water quality.
sewage treatment, production of pulp and paper, and processing A bioluminescence inhibition assay using the marine bacterium
of metals (Hare, 1992). As a nonessential element, cadmium Photobacterium phosphoreum is often chosen as the first toxicity
may endanger the growth and development of aquatic life. For screening method in a test battery because it is fast and cost
example, cadmium may inhibit the bioluminescence of bacteria effective (Davoren et al., 2005; Pandard et al., 2006; Girotti et al.,
(Ishaque et al., 2006), cause limited activity even death in 2008). In the assay, light production is directly proportional to the
daphnias (Canizares-Villanueva et al., 2000), and induce oxidative metabolic activity of the bacterial population, and inhibition of
stress in fish (Livingstone, 2001). The toxicity of cadmium in enzymatic activity causes a corresponding decrease in lumines-
contaminated ecosystems depends not only on the concentration cence intensity. Recently, this simple and sensitive biotest has
of this metal but also on the water chemistry. An important been widely used to investigate the toxicity of various inorganic
environmental stressor that affects most chemical and biological and organic compounds in water samples (Ren and Frymier, 2005;
processes in water is pH. The pH of aquatic systems can be Trang et al., 2005; Gueune et al., 2009; Katritzky et al., 2010).
decreased or increased by a variety of anthropogenic sources, Acute toxicity testing using freshwater daphnids, particularly
including agriculture, urbanization, industry, and mining (USEPA, Daphnia magna, is a popular bioassay used internationally for
toxicity screening of chemicals and for monitoring of effluents and
contaminated waters (Persoone et al., 2009). D. magna has been
n
recommended as a standard test organism by many international
Corresponding author. Fax: [phone].
E-mail address: [e-mail] (Z.-Y. Wang). organizations (e.g., ISO and OECD). The use of D. magna has many
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ecoenv.2013.05.020
84 R.-J. Qu et al. / Ecotoxicology and Environmental Safety 95 (2013) 83 90
2071 1C for 10 days. The water used for acclimation and subsequent experiments
advantages, such as a high sensitivity and a short reproductive
had a pH of 7.2570.25, conductivity of 340.6716.4 źs/cm, total hardness of
cycle. Since its initial application in 1928, D. magna has been used
135.579.3 mg CaCO3/L, and alkalinity of 40.775.2 mg CaCO3/L. The following
routinely in toxicological studies (Biesinger and Christensen, 1972;
ion levels were measured: Na+, 11.270.2 mg/L; K+, 2.3470.07 mg/L; Mg2+,
Hermens et al., 1984; De Schamphelaere et al., 2004).
7.7470.02 mg/mL; Ca2+, 41.0770.82 mg/L and Cl-, 28.371.2 mg/L. The aquaria
were aerated with air stones attached to an air compressor to saturate with oxygen
Fish are an indispensable component of integrated toxicity
(6.7670.84 mg O2/L). The fish were fed twice a day with commercial pellets. The
testing of aquatic environments. The prominence of fish in
experiments were initiated when the total mortality fell to below 1%.
environmental risk assessment is demonstrated by several toxicity
tests in the OECD guidelines. In particular, the fish acute toxicity
2.3. Experimental design
test is a mandatory component of the base set of data require-
ments for ecotoxicity testing (Lammer et al., 2009). However, in
2.3.1. P. phosphoreum
routine acute tests with mortality as the endpoint, it is difficult to
The test was carried out according to the National Standard Method of China
evaluate the physiological changes that occur in experimental fish.
(Water quality  Determination of the acute toxicity  Luminescent bacteria test.
Thus, in the present study, we evaluated contaminant-induced GB/T15441-1995). The metal stock was prepared from 3CdSO4 8H2O by dissolving
6.016 g in 3.0% NaCl solution, resulting in 2.637 g Cd/L. Based on the preliminary
oxidative stress in fish, which may ultimately lead to cell death.
test, six gradient concentrations at each pH value (i.e., 1.319, 2.637, 13.185, 26.370,
Several oxidation-related biomarkers of the liver, including the
131.85, and 263.70 mg Cd/L for pH 5.0 and 6.0; 0.264, 1.319, 2.637, 13.185, 26.370,
activity of antioxidant enzymes such as superoxide dismutase
and 131.85 mg Cd/L for pH 7.0; and 0.132, 0.264, 1.319, 2.637, 13.185, and 26.370 mg
(SOD), catalase (CAT) and glutathione peroxidase (GPx), the level
Cd/L for pH 8.0 and 9.0) were used to determine the EC50 values. The concentration
of nonenzymatic antioxidant glutathione (GSH), and the concen- series in octuplicate and eight controls were arranged in a 96-well (8 rows 12
columns) black flat-bottom microplate (GRE, USA.). First, each well in the first
tration of malondialdehyde (MDA), were measured in the goldfish
column of the microplate was filled with 180 źL of 3.0% NaCl solution to serve as
Carassius auratus. This freshwater fish was chosen as the test
the control group. Second, the same volume of HgCl2 standard solution was added
animal due to its extensive distribution in China and the wide-
to each of the eight wells in the second column to serve as a reference to verify the
spread use in ecotoxicological researches (Sun et al., 2006; Zhu reliability of the experimental results. The third column which was injected with
180 źL of the pH-adjusted 3.0% NaCl solution was set as the pH-control group. Next,
et al., 2008; Zhao et al., 2011).
180 źL of pH-adjusted metal solutions was added to the wells from the fourth
To the best of our knowledge, previous toxicity testing con-
column to the ninth column in order of increasing concentration. Then, 20 źL of
cerning the influence of pH on cadmium toxicity was either
bacterial suspension was added into each test well to get a total volume of 200 źL.
performed within a narrow pH range or was limited to only a
The metal solutions and controls were adjusted with HCl (0.12 mol/L and
single species. These shortcomings prompted us to conduct the 0.012 mol/L) and NaOH (0.05 mol/L and 0.005 mol/L) to obtain final pH values of
approximately 5.0, 6.0, 7.0, 8.0, and 9.0. These five pH values were selected because
current study. By investigating the toxicity of cadmium to three
the previous research has shown that within this range, the pH has no effect on
aquatic organisms (P. phosphoreum, D. magna and C. auratus)
light emission by luminescent bacteria (Fulladosa et al., 2004). The non-complexing
across a relatively wide pH range (5.0 9.0), we were able to
buffer MOPS was used at a concentration of 2 mM to stabilize the pH.
characterize the contribution of pH to cadmium toxicity in
To accurately determine Cd toxicity to P. phosphoreum at different pH levels, it
was necessary to adjust the pH without significantly altering Cd concentrations in
different organisms. The results may provide useful information
solution. The pH of the bacterial suspension was approximately 7.58. Repeated
for evaluating the toxicological effects of Cd in various environ-
trials indicated that the pH of the metal solutions should be adjusted to 4.90, 5.92,
ments with different pH.
6.95, 8.10, and 9.16 to achieve the desired pH of 5.0, 6.0, 7.0, 8.0, and 9.0,
respectively, after the addition of the bacterial liquid. All the final pH values were
adjusted to be within 70.1 of the desired value.
2. Materials and methods For example, to prepare 263.70 mg Cd/L solution (pH 4.90), the following steps
were performed. First, the pH of the 3.0% NaCl solution was adjusted to 4.90 with
HCl (0.12 mol/L and 0.012 mol/L). Next, 2.5 mL of the metal stock, 10 mL of 3.0%
2.1. Chemicals and instruments
NaCl solution (pH 4.90), and 2.5 mL of the buffering agent (2 mM) were added to a
50 mL glass beaker. After readjustment to pH 4.90, the mixture was transferred to a
Chemicals: Cadmium sulfate, hydrochloric acid and sodium hydroxide, bought
25 mL volumetric flask. The beaker was washed three times with the 3.0% NaCl
from Sinopharm Chemical Reagent Co., Ltd., are of analytical grade. 3-(N-morpho-
solution (pH 4.90), which was also transferred to the volumetric flask. Additional
lino) propanesulfonic acid (MOPS) with a purity of 99% was supplied by Aladdins
3.0% NaCl solution was added until the liquid level reached the scale line on the
Reagent. The kits for the analysis of oxidative stress biomarkers were purchased
volumetric flask. Before use, the solution was poured into the pipetting reservoir,
from Nanjing Jiancheng Bioengineering Institute.
and the pH was remeasured and adjusted to 4.90 if needed. This pH adjustment did
Instruments: METTLER-TOLEDO S20 SevenEasy pH Meter (METTLER-TOLEDO,
not significantly alter the Cd concentration in solution because the amount of HCl
China), Tecan Infinite 200s PRO multimode microplate reader (Tecan, Switzerland),
or NaOH added was negligible (less than 0.03 mL). After mixing in a 9:1 volume
PRX-250B Intelligent Artificial Climate Chamber (SafeÿChina), Eppendorf 5417R
ratio with the bacterial suspension, the pH and the metal concentration were
centrifuge (Eppendorf, Germany), IKA T10 homogenizer (IKA, Germany), TU-1800
5.0 and 237.33 mg Cd/L, respectively. Due to dilution by the bacterial liquid, all the
UV vis spectrophotometer (Persee, China), and Atomic absorption spectrophoto-
concentrations were scaled by a factor of 0.9 for EC50 calculation. The exposure
metry (SOLLAR M6, Thermo, USA).
solution was used immediately after preparation. The bioluminescence of various
treatments and controls was determined using a Tecan Infinite 200s PRO multi-
mode microplate reader after exposure of 15 min at 25 1C. The toxicity of each
2.2. Test species and corresponding treatments
treatment was expressed as the relative light rate (RLR, %), which is calculated as
follows:
Freeze-dried powder of P. phosphoreum (T3 mutation) was obtained from the
RLRð%Þ ÅºL=L0 100%;
Institute of Soil Science, Chinese Academy of Sciences (Nanjing, China). After
injection of 0.5 mL of cold sterilized 2.0% NaCl solution into a vial containing 0.5 g
where L0 and L are the average light units of the controls and the treatments,
of freeze-dried powder, the solution was mixed thoroughly by shaking.
respectively.
After 2 min, P. phosphoreum was revived, and 10 źL of the bacterial liquid was
By fitting a straight line between the RLR values falling within the 10 90%
diluted with 2 mL of 3.0% NaCl solution to serve as the working fluid for
range and the corresponding concentrations with the linear regression method, the
subsequent tests.
regression equations were obtained and used to calculate EC50 values (i.e., the
The D. magna strain was supplied by the Research Center for Eco-
concentration at which RLR is 50%).
Environmental Sciences, Chinese Academy of Sciences (Beijing, China). Tap water
that had been adsorbed by activated carbon and aerated for more than 48 h was
used as culture water. Daphnids were kept in the culture water (pH 7.2570.25) in a 2.3.2. D. magna
14 h: 10 h light: dark cycle at 20 1C, and were fed daily with green algae, The acute toxicity of the metal-spiked samples to D. magna was determined in
Scenedesmus obliquus. Juvenile fleas that had undergone three generations of accordance with the National Standard Method of China (Water quality Determi-
parthenogenesis (6 24 h old) were used in the experiment. nation of the acute toxicity of substance to Daphnia (D. magna straus) GB/T 13266-
C. auratus (weight: 30.1574.35 g; length: 13.870.9 cm) were purchased from 1991). Preliminary experiments were performed to investigate the effect of pH on
a local aquatic breeding center. Before the experiments, the goldfish were D. magna. The experiments revealed that the activity of the organism was not
acclimatized in tanks containing 150 L dechlorinated and aerated water at reduced by 24 h exposure to culture medium of pH 5.0, 6.0, 7.0, 8.0 or 9.0,
R.-J. Qu et al. / Ecotoxicology and Environmental Safety 95 (2013) 83 90 85
indicating that the effect of pH in this range is negligible. Consequently, toxicity test adjustments will definitely alter Cd concentration in solution. Due to the difference
was performed at these pH values. The pH was adjusted using the test procedure in the dropping speed of HCl and NaOH solution, changes in the concentration of
for P. phosphoreum, except that the 3.0% NaCl solution was replaced with standard Cd2+ were different. In pH 5.0 water, Cd2+ was measured as 0.0094670.00005 mg/L
dilution water. The most suitable ranges of Cd2+ concentrations for toxicity testing and 0.093470.00053 mg/L, whereas in pH 9.0 water, the concentration was
were 0.44 2.19 mg Cd/L for pH 5.0 and 6.0, 0.04 2.19 mg Cd/L for pH 7.0, and 0.04 0.009470.0001 mg/L and 0.09270.0021 mg/L, respectively. To facilitate the dis-
0.44 mg Cd/L for pH 8.0 and 9.0. Five or six concentrations within each range were cussion, the nominal values of 0.01 and 0.1 mg/L were used in the following
used to determine EC50 values. Glass culture dishes (120 mm) were used as test paragraphs.
vessels. Ten daphnids were placed in each vessel containing 100 mL of test solution,
and the experiment was replicated four times. Toxicity tests were conducted at
2.5. Modeling of metal speciation
2072 1C with a 14 h: 10 h light: dark photoperiod in an illuminating incubator. No
food was provided during the experimental period. The number of immobilized D.
magna was recorded after 24 h exposure. Each test was accompanied by a control In the acute toxicity tests for P. phosphoreum and D. magna, Cd speciation at the
test with standard dilution water. several studied pH values was calculated using the chemical equilibrium model
The EC50 values and their 95% confidence intervals were calculated using Visual MINTEQ (http://www.lwr.kth.se/English/OurSoftware/vminteq/index.htm)
Spearman Karber software (the 1978 version of the Trimmed Spearman Karber for the interpretation of the laboratory results. For each analysis, the EC50 value
method) developed by Montana State University. was used as the total Cd concentration. It was assumed that the pH was fixed
during computations. We used the corresponding water quality parameters for
each test as the input variables.
2.3.3. C. auratus
Nine glass tanks (28 cm 60 cm 36 cm) were used for the experiment. Each
tank contained ten acclimated fish that were randomly selected. The group of fish
2.6. Statistical analysis
was exposed to control (no cadmium addition in natural aerated water, pH 7.25),
low pH-acid medium (pH 5.0), high pH-alkaline medium (pH 9.0), 0.01 mg Cd/L in
Experimental data were presented as the mean7standard deviation (SD). The
acid medium (pH 5.0), 0.01 mg Cd/L in natural medium (pH 7.25), 0.01 mg Cd/L in
values of oxidative stress biomarkers (SOD, CAT, GPx, GSH and MDA) were checked
alkaline medium (pH 9.0), 0.1 mg Cd/L in acid medium (pH 5.0), 0.1 mg Cd/L in
for normality using the Shapiro Wilk test and for homogeneity of variance using
natural medium (pH 7.25), or 0.1 mg Cd/L in alkaline medium (pH 9.0). A final pH of
the Levene test. One-way analysis of variance (ANOVA) followed by Duncan s test
5.0 or 9.0 was achieved through adjustment with 1.2 mol/L HCl or 2.0 mol/L NaOH
was used to determine the significance of differences (Po0.05 or Po0.01) between
solution. Because the pH of the test medium is difficult to control, we only studied
individual treatments and controls. All statistical analyses were performed using
three representative pH values for the biomarker evaluation. The two Cd doses we
the SPSS statistical package (ver. 17.0, SPSS Company, Chicago, USA).
evaluated were selected based on a series of toxicity tests, particularly those
involving oxidative stress responses of fish to Cd exposure (Atli and Canli, 2010;
Cao et al., 2010; Jia et al., 2011). During the experimental period, 0.001 mol/L HCl or
2.7. Integrated biomarker response
0.05 mol/L NaOH solution was added dropwise at an appropriate rate to set the pH
to within 70.1 of the desired value. The experimental fish were fed twice a day
The Integrated Biomarker Response (IBR) (Beliaeff and Burgeot, 2002), a
with commercial pellets during the toxicity tests but were fasted 24 h prior to
method for combining all the measured biomarker responses into one general
biochemical analysis. Food residue was removed daily by an automatic water-
stress index, was applied to assess the potential toxicity of different exposure
changing system to minimize contamination from metabolic waste. Half of the
protocols to fish. The procedure of IBR calculation is described here briefly. Data
dirty water was released and an equal volume of experimental water was
were first standardized to allow direct visual comparison of the biomarker
replenished. Afterward, metal solution was added and the pH was adjusted to
responses under the test conditions. The standardized data (Y) were calculated as
maintain the test conditions. Five fish were randomly sampled at 1 d and 7 d for
Yź(X-m)/s, where X is the value of each biomarker response, m is the mean value
analysis.
of the biomarker, and s is the standard deviation of the biomarker. Next, we
The fish were killed by a sharp blow to the head at the end of the exposure
computed ZźY in the case of activation or Zź Y in the case of inhibition. The
period. Liver tissues were carefully dissected, washed with cold physiological saline
minimum value (Min) was obtained for each biomarker. Finally, the score (S) was
(0.9 percent NaCl solution), weighed, and homogenized (1:10, w/v) in cold
computed as SźY+|Min|, where Se"0 and |Min| is the absolute value of Min.
physiological saline using an IKA T10 homogenizer (IKA, Germany). The homo-
Star plots were used to display the biomarker results. A star plot radius
genates were centrifuged (Eppendorf, Germany) at 4000 g for 15 min at 4 1C. The
coordinate represents the score of a given biomarker. When Si and Si+1 are assigned
supernatants were used as an enzyme source for biochemical analysis.
as two consecutive clockwise scores of a given star plot, n is assigned as the number
The enzyme (SOD, CAT, GPx) activity, GSH level, MDA content and protein
of radii corresponding to the biomarkers. Thus, the area Ai obtained by connecting
concentration of the supernatants were measured using the Diagnostic Reagent
the ith and the (i+1)th radius coordinates can be calculated as
Kits according to the manufacturer s instructions. SOD activity was measured
at 550 nm using the xanthine oxidase method (McCord and Fridovich, 1969). Si
Ai ź sin ²ðSi cos ² þ Siþ1 sin ²Þ;
CAT activity was determined by monitoring residual H2O2 absorbance at 405 nm 2
(Goth, 1991). GSH level was determined at 420 nm through the reaction between
where
5,5-dithiobis-2-nitrobenzoic acid (DTNB) and thiol-containing compounds. GPx activity,

Siþ1 sin Ä…
estimated by the rate of GSH oxidation, was measured at 412 nm (Hafeman
² ź Arc tan ;
Si-Siþ1 cos Ä…
et al., 1974). The MDA content, a biomarker for lipid peroxidation, was determined
at 532 nm by the thiobarbituric acid reactive species (TBARS) assay, which
Ä… is 2Ä„/n radians, and Sn+1 is S1.
measures the amount of MDA that reacts with thiobarbituric acid (Livingstone
The total area corresponding to a given situation (IBR value) was obtained as
et al., 1990). The protein concentration was measured at 595 nm by the Coomassie
n
Brilliant Blue dye binding technique (Bradford, 1976), with bovine serum albumin
IBR ź " Ai;
as a standard. The absorbances were recorded using a TU-1810 UV vis spectro-
i ź 1
photometer (Persee, China). The specific activity of enzymes is expressed as U/mg
where n is the number of biomarkers.
protein, while GSH level and MDA content are denoted by źmol/g protein and
nmol/mg protein, respectively.
3. Results
2.4. Chemical measurements
3.1. Metal speciation
For the toxicity tests with P. phosphoreum and D. magna, the stock solution was
acidified with 10 volume of HNO3 then measured by a flame-atomic absorption
spectrophotometer (SOLLAR M6, Thermo, USA) to check the concentration of Cd
For P. phosphoreum, there was almost no change in cadmium
actually present. The measured concentration was 97.1% and 97.9% of the nominal
speciation when the pH increased from 5.0 to 8.0 (Fig. 1A). At these
value, respectively. Therefore, the nominal concentrations were used in the toxicity
pH values, the dominant species were the two chloro-complexes
assessments.
CdCl+ and CdCl2, which resulted from the complexation of cadmium
For the toxicity test with C. auratus, the dissolved cadmium concentration in
the glass tanks was determined at the start and at the end of the test using a flame- ion with chloride ions that are abundant in the medium (3.0% NaCl
atomic absorption spectrophotometer (SOLLAR M6, Thermo, USA). Similarly, prior
solution). As pH increased to 9.0, the percentage of Cd(CO3)22-
to analysis, the collected water samples were acidified with 10 volume of HNO3.
increased appreciably to 12%, while CdCl+ and CdCl2 exhibited
The measurements were done in triplicate. In the mediums with Cd addition,
approximately the same level of dominance. Throughout the whole
the initial Cd concentration was measured to be 0.009570.00029 and
0.09470.0018 mg/L. The increase in the volume of exposure water caused by pH pH range, the free ion form of cadmium was least abundant.
86 R.-J. Qu et al. / Ecotoxicology and Environmental Safety 95 (2013) 83 90
Fig. 1. Species distribution diagram of Cd at various pH levels for P. phosphoreum
(A) and D. magna (B).
For D. magna, the species distribution of cadmium changed
Fig. 2. Effects of pH on Cd toxicity to P. phosphoreum (A) and D. magna (B). Error
markedly with pH (Fig. 1B). The four major species, Cd2+, CdHCO3+,
bars represent 95% confidence interval.
CdCO3, and Cd(CO3)22-, accounted for more than 98% of the total
dissolved cadmium in solutions with pH in the 5.0 9.0 range.
However, the relative abundance of each species varied at different from 5.0 to 6.0, the EC50 value decreased slightly from 1.21 mg Cd/L
pH levels. At pH 5.0, Cd2+ was predominant, comprising 95% of the to 1.16 mg Cd/L. Then, it dropped drastically to 0.42 mg Cd/L
total species. At pH 6.0, Cd2+ was still in dominance but its at pH 7.0. As the pH further increased, the EC50 value declined
proportion was reduced to 77%. In contrast, the percentage of slightly until a minimum value of 0.35 mg Cd/L was obtained
CdHCO3+ increased to 23%. Under neutral pH conditions (pH 7.0), at pH 9.0. Additionally, when the Cd2+EC50 was expressed as a
Cd2+ level was still higher than CdHCO3+ level, and CdCO3 cannot function of pH (Fig. 3B), a positive relationship was observed. The
be negligible. As the water became alkaline, the dominant species correlation equation was Cd2+EC50ź3.075-0.376 pH (R2ź0.913).
changed. At pH 8.0, CdCO3 became more abundant than the other
three species. At pH 9.0, CdCO3 and Cd(CO3)22- were the two major
3.2.3. C. auratus
species. In the pH range we evaluated, the percentage of Cd2+ 3.2.3.1. Biochemical measurements. Control values for the biochemical
decreased dramatically with increasing pH.
parameters SOD, CAT, GPx, GSH and MDA at day 1 were measured as
87.677.5 U/mg protein, 24.473.6 U/mg protein, 121712 U/mg
protein, 14.071.3 źmol/g protein, and 2.8170.42 nmol/mg protein,
3.2. Acute toxicity
respectively. At day 7, control values for these parameters were
90.375.5 U/mg protein, 28.572.8 U/mg protein, 119711 U/mg
3.2.1. P. phosphoreum
protein, 13.471.4 źmol/g protein, and 2.6470.39 nmol/mg protein,
In general, the EC50 values presented a gradient descent trend,
respectively (Table 1).
i.e., the toxicity to P. phosphoreum increased with increasing pH
Antioxidant enzymes: Generally, no significant changes in SOD,
(Fig. 2A). The largest EC50 value was 19.3 mg Cd/L at pH 5.0, and
CAT and GPx activity were observed in any of the test groups after
the smallest was only 1.03 mg Cd/L at pH 9.0. Moreover, when the
1 d exposure. However, a longer exposure duration resulted in
EC50 was expressed on a Cd2+ basis as Cd2+EC50, a linear relation-
significant decreases (Po0.05 or Po0.01) in the activity of
ship between Cd toxicity and pH was evident (Fig. 3A).
antioxidant enzymes in several groups compared to controls. The
The regression equation describing this trend is Cd2+EC50ź
greatest changes appeared in the two groups exposed to Cd at pH
1.805-0.214 pH (R2ź0.961).
9.0, which had significant (Po0.05) and occasionally very sig-
nificant (Po0.01) decreases in the activity of all three indicators.
3.2.2. D. magna Specifically, 0.78-fold and 0.77-fold differences in SOD activity,
A general downward trend in EC50 values was observed 0.71-fold and 0.53-fold differences in CAT activity, and 0.58-fold
across the pH range we studied (Fig. 2B). When the pH increased and 0.63-fold differences in GPx activity were observed in the
R.-J. Qu et al. / Ecotoxicology and Environmental Safety 95 (2013) 83 90 87
pH(9)-Cd(0.01) group and pH(9)-Cd(0.1) group, respectively. However, a significant 1.38-fold increase (Po0.01) occurred in
The effects of Cd were less pronounced in other groups, such that the pH(7.25)-Cd(0.1) group.
only the pH(5)-Cd(0.1) group had significant SOD depletion (Po0.05). Lipid peroxidation: For MDA content, no test groups exhibited
Nonenzymatic antioxidant: There were no significant changes in significant changes relative to control after 1 d exposure. As exposure
GSH level in any test group relative to control after 1 d exposure, time increased, MDA content was significantly increased by 1.90-fold
but significant GSH differences (Po0.01) were observed with (Po0.05) in the pH(7.25)-Cd(0.01) group and by 3.05-fold (Po0.01)
prolonged exposure. Compared with the control, GSH was sig- in the pH(9.0)-Cd(0.1) group.
nificantly reduced by 0.69-fold (Po0.05) in the pH(9)-Cd(0.01)
group and by 0.63-fold (Po0.01) in the pH(9)-Cd(0.1) group.
3.2.3.2. Integrated biomarker response. The IBR values were different
among the various exposure protocols after 7 days, ranging from 1.19
in the pH(9) group to 12.50 in the pH(9)-Cd(0.1) group (Fig. 4).
The change in IBR caused by the addition of Cd is smaller at pH
5.0 than at pH 9.0. Moreover, for any given waterborne Cd
concentration, the IBR at pH 9.0 is larger than the corresponding
value at pH 5.0. IBR calculations were not performed at day 1 due to
the short exposure duration.
4. Discussion
4.1. P. phosphoreum
In general, the toxicity of Cd to P. phosphoreum was inversely
related to pH. It has been reported that the effect of pH on metal
toxicity is twofold: the hydrogen ion may exert its effect either
directly by affecting metal uptake or indirectly by affecting the
chemical speciation and bioavailability of the dissolved metal pool
(Peterson et al., 1984). There was an approximately 13-fold
increase in the 15 min-EC50 value when the pH was decreased
from 8.0 to 5.0, whereas the distribution of metal species was
Fig. 3. The Cd2+EC50 value (with 95% confidence interval) as a function of pH for P. Fig. 4. Integrated biomarker response (IBR) values for different exposure protocols
phosphoreum (A) and D. magna (B). after 7 days.
Table 1
Effects of short-term exposure to Cd2+ under different pH on enzyme (SOD, CAT and GPx) activity, GSH level and MDA content in liver of C. auratus.
Biomarkers Duration (d) No Cd addition Cd (0.01 mg/L) Cd (0.1 mg/L)
pHź5.0 pHź7.25 pHź9.0 pHź5.0 pHź7.25 pHź9.0 pHź5.0 pHź7.25 pHź9.0
SOD 1 96.3713.9a 87.677.5a 82.174.5a 91.0715.8a 81.979.9a 67.677.7a 87.079.2a 67.778.1a 70.774.8a
7 80.474.1bc 90.375.5bc 86.276.7bc 91.0711.1bc 101.2713.0c 70.778.3a 72.374.7a 78.877.2ab 69.976.1a
CAT 1 17.071.2a 24.473.6a 25.972.3a 25.672.9a 21.373.7a 22.772.1a 28.073.8a 21.171.8a 22.171.1a
741.672.7cd 28.572.8cd 36.874.6cd 28.773.6cd 32.473.8d 20.571.5ab 24.172.6bc 26.372.3bcd 15.271.6an
GPx 1 126.8723.4abc 121712abc 114.977.8abc 14077c 97.7710.4a 135711bc 107714ab 106 714ab 115713abc
7 114.274.5cd 119711cd 109.6719.6cd 10878bcd 138 710d 69.876.8an 94.3715.3abc 94.1720.8abc 75.8712.1abn
GSH 1 18.071.2ab 14.071.3ab 15.370.76a 16.971.9ab 18.571.4b 12.371.6a 18.571.1ab 13.771.5a 13.171.9a
7 12.873.4c 13.471.4c 14.371.7c 12.471.6bc 11.371.2abc 9.2671.03ab 9.8171.58abc 18.672.2dn 8.4971.41an
MDA 1 3.9271.2ab 2.8170.42ab 3.0570.40ab 2.5770.32ab 2.9470.24ab 2.4470.20a 2.7070.28a 3.9570.42b 2.8370.26ab
7 2.5770.72a 2.6470.39a 2.8070.54a 4.2170.45ab 5.0270.22bc 3.9470.35ab 4.9170.29ab 3.9470.35ab 8.0870.59cn
Data are presented as means7standard deviation (SD), n is 5 for each data point.
a d
indicate significant differences from control for different exposure conditions during the same exposure time (Po0.05).
n
Denotes very significant differences from control for a test group in the same exposure period (Po0.01).
88 R.-J. Qu et al. / Ecotoxicology and Environmental Safety 95 (2013) 83 90
almost unchanged in this pH region. This inconsistency suggests H2O and O2. The decreased SOD and CAT activity in fish may be
that the protective effect of low pH against Cd toxicity is probably caused by the interactions between Cd and essential trace ele-
the consequence of decreased uptake of Cd into the cell, which is ments. Cd has an adverse effect on the enzymatic systems of cells
likely due to the competitive exclusion of Cd2+ binding to the cell due to its substitution for divalent metals (Zn2+, Cu2+, and Mn2+)
surface by H+. The partial inhibition of Cd bioaccumulation by H+ in metalloenzymes and its very strong affinity for sulfhydryl-
was also found in the clam Corbiculafluminea (Graney et al., 1984) containing biological macromolecules such as proteins, enzymes
and the mussel Unio pictorum (Pynnonen, 1990). At pH 9.0, the and nucleic acids (Lionetto et al., 2000; Liu et al., 2011; Dabas et al.,
EC50 decreased to a minimum value, and the cadmium species 2012). The competition between Cd and Zn, Cu or Mn in SOD
distribution was changed considerably. Interactions of the three molecules could explain the decrease in SOD activity, which may
major species, Cd(CO3)22-, CdCl+, and CdCl2, might be responsible imply a reduction in H2O2 production. Because the activity of CAT
for the high toxicity (Villaescusa et al., 1996). Moreover, at this pH, is directly proportional to the substrate H2O2 level which is
the decline in proton concentration weakens the competition of assumed to be produced by SOD (Kono and Fridovich, 1982;
H+ with Cd2+, thereby enhancing Cd toxicity. Over the studied pH Aitken and Roman, 2008) the CAT activity is decreased. The Cd-
range (5.0 9.0), the linear correlation between Cd2+EC50 and pH induced reduction in hepatic CAT activity may reflect a reduced
also indicated that there was possibly a modest competition capacity of the liver to eliminate H2O2.
between H+ and Cd2+. As the second-line defenses against oxidative damage
(Cnubben et al., 2001), GSH and GSH-related enzymes play a
4.2. D. magna major role in cellular metabolism and free radical scavenging
(Pena-Llopis et al., 2003; Liu et al., 2008). GSH is a major none-
According to the free-ion activity model (FIAM) proposed by nzymatic low-molecular-weight antioxidant and functions as a
Campbell (1995), metal toxicity is governed by the activity of the direct free radical scavenger that quenches oxyradicals through its
free hydrated metal ion. Consequently, Cd should be more toxic to sulfhydryl group (Zhang et al., 2004). GSH also serves as an
D. magna in acidic environments than in neutral and alkaline ones available co-substrate for GPx activity, and it is conjugated in
due to the predominance of Cd2+ (as confirmed by our metal xenobiotic reactions (Griffith, 1999; Sies, 1999; Lu, 2009). Our
speciation analysis results). However, our results show the oppo- experimental results are consistent with earlier studies (Chatterjee
site trend, i.e., the acute toxicity of Cd2+ was decreased by a lower and Bhattacharya, 1984; Cirillo et al., 2012) that showed decreased
pH. This finding is consistent with that of Clifford and McGeer levels of GSH in tissues exposed to Cd. Pandey et al. (2008) stated
(2010), who reported a general trend of increasing EC50 values for that decreased intracellular GSH levels and simultaneous inhibi-
Cd in Daphnia pulex when pH was decreased from 8.02 to 6.10. tion of GSH-related antioxidant enzymatic activity could result in
Although H+ has a dual effect on the toxicity of metals, changes in oxidative imbalance and subsequent cell death. There are two
Cd toxicity to D. magna largely reflect changes directly related to possible explanations for lowered GPx activity observed in our
H+ concentration. Hence, the toxicity test results in the present study. First, reduced GSH might cause the inactivation of GPx
study may be better explained by the competition between because GPx is highly dependent on GSH concentration. Second,
protons and the metal ions at the cell surface. The competition the interaction between Cd and Se in the GPx molecule could lead
of protons with free cadmium ions at the biotic ligand was to the inhibition of GPx activity.
confirmed by the linear relationship between Cd2+EC50 and pH. Moreover, MDA content was generally increased in fish liver
In a previous research work, Playle (2004) found a similar after 7 d exposure, indicating an elevation of lipid peroxidation in
competitive interaction, reducing the binding of Cd to the gills of this organ. Lipid peroxidation is one of the main manifestations of
fathead minnows (Pimephales promelas) as pH decreased to 4.8. oxidative damage. Enhanced peroxidation of lipids in intra- and
Furthermore, low pH may trigger some physiological reactions extracellular membranes causes damages to the cells, tissues and
within organisms, which are involved in weakening the toxicity of organs because the reactive carbonyls produced during lipid
Cd. Guan and Wang (2006) proposed that the induction of peroxidation may spread damage far from the original site of
metallothionein by low pH is the major Cd detoxification mechan- radical production (Cheeseman, 1993). The enhanced lipid perox-
ism in D. magna. idation in the Cd-treated fish in our study might result from the
decrease in the liver activity of antioxidants and the generation of
4.3. C. auratus radicals during normal metabolism.
In summary, suppressed activity of antioxidant enzymes,
The fact that no test groups showed statistically significant decreased GSH level, and enhanced lipid peroxidation in the liver
changes in SOD, CAT and GPx activity, GSH level, or MDA content of fish after 7 d exposure to Cd2+ (particularly at pH 9.0) confirmed
after 1 d exposure seems to indicate that the exposure duration the occurrence of oxidative stress arising from insufficient neu-
was too short for the two concentrations of Cd to produce tralization of ROS generated. It is noteworthy that at pH 5.0, the
oxidative stress in the liver. However, with prolonged exposure, two concentrations of Cd2+ we investigated almost triggered no
significant changes in several biochemical indexes were observed oxidative stress, although few indices exhibited statistically con-
in certain experimental groups, suggesting that cadmium induced firmed changes. Under alkaline conditions, especially in pH
oxidative stress. Particularly, the antioxidants were most strongly 9.0 water, significant changes in the measured oxidative stress
inhibited in the groups kept in an alkaline environment (pH 9.0). biomarkers were commonly observed in both high-dose and low-
Also, reductions were observed in the groups at pH 5.0, but the dose Cd2+ exposure groups after 7 days. This demonstrates that
vast majority of these reductions were not statistically significant. the toxicity of Cd2+ to the aquatic organism is increased by an
In all, there was little induction of antioxidants in this study. One alkaline environment, to a certain extent.
reason for this could be the combined action of Cd and pH. Of the The IBR index, a simple tool for visualizing biological effects,
antioxidant enzymes, SOD and associated CAT are considered as also confirmed the findings presented above. Generally, the higher
the vital first-line defenses against oxygen toxicity (Yu, 1994). They the IBR value is, the more stressful the environment is. According
can protect organisms from oxidative damage by partially elim- to the IBR index, the relative toxicity of the exposure conditions in
inating reactive oxygen species (ROS). Under physiological condi- this study is as follows: pH(9)opH(5)-Cd(0.01)opH(5)opH(5)-
-
tions, SOD catalyzes the dismutation of superoxide radical (O2 ) Cd(0.1)opH(7.25)-Cd(0.1)opH(9)-Cd(0.01)opH(7.25)-Cd(0.01)o
into hydrogen peroxide (H2O2), whereas CAT converts H2O2 into pH(9)-Cd(0.1). As waterborne Cd concentration is increased, the
R.-J. Qu et al. / Ecotoxicology and Environmental Safety 95 (2013) 83 90 89
change in IBR index is larger at pH 9.0 than at pH 5.0. In addition, Chatterjee, S., Bhattacharya, S., 1984. Detoxification of industrial pollutants by the
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