Biol Trace Elem Res (2014) 159:241–253
DOI 10.1007/s12011-014-9975-x
Environmental Cadmium Exposure Impacts
Physiological Responses in Manila clams
Liqiang Zhao & Yu Zhang & Jian Liang & Xian Xu &
Hua Wang & Feng Yang & Xiwu Yan
Received: 24 March 2014 / Accepted: 8 April 2014 / Published online: 29 April 2014
# Springer Science+Business Media New York 2014
Abstract The physiological responses of marine bivalves to
chronic cadmium (Cd) exposure at sub-lethal concentrations
have been well documented. As of now, few studies have
examined the effect of Cd exposure and subsequent recovery
period at environmentally realistic concentrations. In this
study, environmentally, Cd exposures were performed to as-
sess the physiological responses of the Manila clam Ruditapes
philippinarum. The clams were exposed to waterborne Cd at
two environmentally realistic concentrations (4 and
40 μg L−1) for 35 days and then allowed to recover for another
35 days. The accumulation and elimination of Cd in
R. philippinarum were tissue-specific and dose- and time-
dependent. Cd accumulation increased sharply in the digestive
gland, and Cd elimination was rapid in the gill. Major phys-
iological responses, including clearance rate, absorption effi-
ciency, respiration rate, excretion rate, oxygen to nitrogen
ratio, and scope for growth, were significantly affected by
Cd exposure. Yet, the clams exposed to 4-μg L−1 Cd were
able to quickly recover their normal physiological processes
and clearly exhibited catch-up growth once they were trans-
ferred to clean seawater. Hence, R. philippinarum can exhibit
good physiological plasticity when confronted with moder-
ately environmental Cd exposure. All physiological responses
measured exhibited a highly significant and generally predict-
able correlation with tissue Cd concentration, which in turn,
reflected environmentally realistic exposure conditions. Our
results further confirm that the measurement of physiological
responses is a sensitive method for assessing stress at envi-
ronmentally realistic metal concentrations.
L. Zhao : Y. Zhang : J. Liang : X. Xu : H. Wang : F. Yang (*) :
X. Yan (*)
College of Fisheries and Life Science, Dalian Ocean University,
Dalian 116023, China
e-mail: yangfeng@dlou.edu.cn
e-mail: yanxiwu@dlou.edu.cn
Keywords Ruditapes philippinarum . Physiological
responses . Scope for growth . Cadmium
Introduction
Estuarine and coastal areas throughout the world are under
increasing pressure from intensive human activities.
Environmental quality and ecosystem health in these regions
have been decreasing dramatically as a consequence of an-
thropogenic inputs from domestic and industrial effluents,
urban and agricultural runoff, and ship wastewater discharge
[1, 2]. Over the last decade, the widespread occurrence of
metal pollution in estuarine and coastal ecosystems has be-
come increasingly serious and has attracted much attention
from researchers and governmental regulatory agencies.
Especially, some toxic metals such as cadmium and mercury
can be biomagnified progressively through the aquatic food
chain, and eventually threaten aquatic wildlife and human
health [3, 4]. Various physiological disorders and serious
human diseases, including immunodeficiency, Minamata dis-
ease, and Itai-itai disease, have been attributed to aquatic metal
pollution [5–8]. As a consequence, how to stop aquatic metal
pollution and how to prevent these metals from being trans-
ferred to humans through the food chain are hot research
topics throughout the world.
Biomonitoring is an effective technique for assessing metal
pollution in estuarine and coastal environments [9]. Bivalve
mollusks are of particular significance for such purposes: their
ability to continually filter the surrounding water, coupled
with their widespread occurrence, sedentary lifestyle, ease of
collection and identification, and sensitivity to a wide range of
environmental factors make them ideal sentinel species for
many pollution monitoring programs [10]. Measures of metal
burden on bivalves are likely to reflect the potential ecosystem
damage caused by metal contaminants. Over the years,
242
numerous biomonitoring approaches from local to global
scales have been conducted using a variety of bivalve species,
including Crassostrea gigas, Crassostrea hongkongensis,
Mytilus edulis, Mytilus galloprovincialis, Perna viridis,
Ruditapes philippinarum, Ruditapes decussates, and
Cerastoderma edule [1, 11–15].
Awareness is growing that quality assessment of estuarine
and coastal ecosystems cannot be based principally on deter-
mination of physical and chemical parameters alone, as this
approach does not reflect the effects of pollutants on the biota
[16]. So, it is necessary to assess both the presence of metals
accumulating in the biota and the biological/ecological effects
to which they give rise [17, 18]. Biomarkers, which include
physiological, biochemical, and cellular responses to exposure
to pollutants or the effects of pollutants in the environment
[19], have proven to be quite useful as indicators of the health
status of aquatic species and the ecosystem [20–22].
Generally, when the pollutant level is transiently or chronical-
ly enhanced, aquatic organisms exhibit a wide variety of
adaptive responses to neutralize the adverse effects of pollut-
ants on their physiological functions. If these adaptive re-
sponses cannot counteract these effects, the functioning of
biological systems may be affected and ultimately result in
deleterious effects on survival, growth, reproduction, and
metabolism [23].
In recent years, interest in the use of physiological
measurements of bivalves (e.g., clearance rate, absorp-
tion efficiency, respiration rate, and excretion rate) as
indicators of their health status has been on the rise [24,
25]. Scope for growth (SFG), which integrates these
measures, represents the balance between energy acqui-
sition (from ingestion and digestion) and energy expen-
diture (from respiration and excretion) [26] and conse-
quently provides an instantaneous measure of the energy
status of bivalves. It is, therefore, an important index of
the energy available for growth and reproduction after
energy use and loss [27]. Exposure to pollutants would
likely disturb this energy balance, making SFG an ac-
curate and practical indicator of the underlying
pollution-induced stress [28]. For example, bivalves that
are not stressed by environmental conditions (e.g., tem-
perature, salinity, metals, etc.) consistently show positive
and high SFG. However, those living in environmentally
stressful conditions tend to show low or even negative
SFG [29]. In particular, at the level of individual organ-
isms, SFG combined with the analysis of pollutants
such as metals in bivalves therefore can provide ecolog-
ically meaningful data for interpretation of the detrimen-
tal biological effects of certain pollutants [30]. For this
reason, SFG has been successfully used as a biomarker
in numerous laboratory and field studies designed to
detect, quantify, and identify the ecotoxicological effects
of ubiquitous pollutants [31–35].
Zhao et al.
Cadmium (Cd) is a non-essential metal that is not
involved in the majority of enzymatic processes or other
biological activities. In estuarine and coastal waters, it is
predominantly released by anthropogenic activities such
as mining, smelting, electroplating, and production and
use of batteries, pigments, and plastics [36]. Cd can exert
diverse toxic effects on almost all physiological process-
es, especially in bivalves [37]. For many bivalves, pre-
viously reported data show that Cd exposure, even at
quite low concentrations, has a strong inhibitory effect
on the bioenergetic function of mitochondria, leading to
dysfunction in mitochondrial activity [38]. Cd accumula-
tion in mitochondria therefore has the potential to cause
disturbance of energy metabolism [39] and ultimately
result in impairment of animal growth and reproduction
and long-term population survival [40]. Cd can also
stimulate oxidative stress by producing reactive oxygen
species, which may cause cellular and genotoxic damage
[41].
The Manila clam, R. philippinarum, is widely distributed in
estuarine and coastal waters of the Indo-Pacific region and has
been successfully commercialized for human consumption
[42]. R. philippinarum is one of the most exploitable bivalves,
especially in China, where its annual production has reached
over 3.5 million tons, representing 97.4 % of total world
production [43]. Due to its considerable economical impor-
tance, R. philippinarum has become the preferred indicator for
estuarine and coastal monitoring purposes [44–46]. A number
of recent studies have demonstrated the impact of Cd exposure
on the toxicity, bioavailability, and biomarker responses of
R. philippinarum [47–50]. However, very few studies, have
established an integrated relationship between external levels
of Cd exposure, internal levels of tissue Cd accumulation, and
chronic physiological disturbance. Furthermore, the recovery
profile from Cd exposure and the extent to which physiolog-
ical recovery may be closely related to the elimination of
tissue Cd are still poorly understood. Moreover, environmen-
tal concentrations of metals are not necessarily constant but
fluctuate over time. Generally, periods of intense exposure
might be followed by episodes of relatively low or no expo-
sure, in principle, allowing exposed organisms to recover from
toxic injury.
The objectives of the current study were to (i) quantify the
accumulation and elimination of Cd in the gill and digestive
gland of R. philippinarum during exposure to environmentally
realistic concentrations of Cd and after subsequent recovery;
(ii) examine physiological changes in clams in response to Cd
exposure; (iii) determine whether clams have the ability to
recover from these disturbances; (iv) identify the relationship
between tissue Cd burden and physiological responses; and
(v) assess the suitability of using R. philippinarum as a senti-
nel species to monitor metal pollution in estuarine and coastal
waters.
Environmental Cadmium Exposure Impacts Physiological Responses
Materials and Methods
Clam Collection and Maintenance
Manila clams (mean shell length 37±3.26 mm) were collected
from an intertidal shore (39° 38′ 56.46″ N, 123° 1′ 20.04″ E)
located close to Zhuanghe Bay, Liaoning Province, northeast
China. Approximately 1,000 individuals were removed from
the sediment using a rake during low tide and subsequently
transported on ice to the laboratory within 2 h of collection.
They were acclimatized in 500-L tanks filled with continu-
ously aerated seawater under a 12-h light/12-h dark regime for
at least 2 weeks and were fed with the diatom Nitzschia
closterium. The water conditions were recorded daily and
maintained as follows: temperature 19–20 °C, salinity 28.2–
29.5, pH 7.8–8.1, and dissolved oxygen 7.8–8.6 mg L−1. No
mortality was observed prior to experimentation.
Chemicals
Unless otherwise indicated, all chemicals used were pur-
chased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China) and were of analytical reagent grade (purity
>99.9 %). A stock solution of Cd (1,000 mg L−1) was pre-
pared by dissolving 2.032 g of CdCl2 ·2.5H2O in 1 L of
distilled water.
Cadmium Exposure and Recovery
After acclimatization, the clams were exposed to waterborne
Cd for 35 days. Briefly, 18 groups of 50 clams each were
randomly divided and placed in acid-washed polyethylene
containers with 30 L of filtered natural seawater. Six groups
were exposed to 4 μg L−1 Cd, and six groups were exposed to
40 μg L−1 Cd. Additionally, the residual six groups without
exposure to Cd were used as the blank group. Thus, three
treatments with six replications were obtained in this study.
Following a 35-day exposure period, Cd-exposed clams were
allowed to recover in clean seawater for a further 35 days. In
this study, the Cd concentrations used during the exposure
phase are environmentally realistic, as evidenced by the ample
literature on these topics [51–53].
The rearing conditions of each group were main-
tained identically but separately. Seawater was renewed
every 24 h to minimize contamination from metabolic
wastes, and Cd concentrations were re-established dur-
ing the exposure period. Seawater temperature, salinity,
pH, dissolved oxygen, photoperiod, and the food ration
were identical to those used during the acclimation
period. Samples were taken at weekly intervals on days
7, 14, 21, 28, and 35 (during the exposure period) and
on days 42, 49, 56, 63, and 70 (during the recovery
period). At each time point, two clams were randomly
243
selected from each group (total 12 clams for each treat-
ment) and used for physiological measurements; an ad-
ditional two clams from each group were used for tissue
metal analysis.
Physiological Measurements
All physiological measurements were made on randomly
selected individual clams according to the well-established
procedure [26].
Clearance Rate
Clearance rate (CR) is an estimate of the volume of water
cleared of particles per unit time. Prior to measurement, the
clams were depurated for 2 h to empty their guts. Seawater
was previously filtered through Whatman No. 1 filters to
remove all particulate matter. Each clam was placed in a 5-L
polyethylene beaker filled with 4 L of filtered seawater. After
acclimation for 30 min, N. closterium were added to reach an
initial algal concentration of 60,000 cells mL−1, which is the
threshold concentration at which no pseudofeces were pro-
duced in our preliminary experiment. A beaker without clams
was used as the blank. To measure the decline in algal con-
centration, seawater samples were taken from each beaker
every 20 min for 1 h. The algal concentration was determined
using a hemocytometer. CR was calculated using the follow-
ing equation:
lnC 0 −lnC t
CR ¼ V 
W t
where CR is the clearance rate (L g−1 h−1), V is the volume of
filtered seawater used (L), C0 and Ct are the algal concentra-
tions (cells L−1) at the initial and final time points of the
experiment, respectively, W is the dry tissue weight of the
experimental clam (g), and t is the time elapsed (h).
Absorption Efficiency
Absorption efficiency (AE) was determined using the ratio
method [54]. AE represents the efficiency with which organic
material is absorbed from the ingested food. A volume of
N. closterium equal to that used in the CR experiment was
filtered onto washed, ashed (at 450 °C), and pre-weighed by
47-mm Whatman GF/C filters. These filters were rinsed with
distilled water, dried at 110 °C for 24 h, and weighed in order
to calculate the dry weight of the total particulate matter
(TPM). They were then ashed at 450 °C for 6 h in a muffle
furnace and reweighed in order to calculate the dry weight of
particulate organic matter (POM). Feces produced by clams at
the end of each CR measurement were collected using a
244
pipette and filtered onto the filters. Subsequently, the organic
content of the feces was determined in the same manner used
for algal samples. AE was calculated using the following
equation:
F−E
AE ¼  100
ð1−E Þ  F
where AE is the absorption efficiency (%), F is the POM/TPM
ratio in the food, and E is the POM/TPM ratio in the feces.
Respiration Rate
After CR and AE measurements were taken, the rate of
oxygen consumption of individual clams was measured
by closed respirometry. Each replicate of clams was put
in a 500-mL respirometer filled with previously filtered
and oxygen-saturated seawater at the same temperature
(20 °C). A blank without clams served as the control.
The decline in partial pressure of oxygen (pO2) inside
each respirometer was recorded using an oxygen meter
(YSI 58) at the start and after a 1-h incubation period.
To ensure that the clams had resumed respiration, the
record of pO2 started 30 min later when the siphons of
each clam had protruded. Respiration rate (RR) was
calculated using the following equation:
½C ðt 0 Þ−C ðt 1 ފ  V  60
RR ¼
W  ðt 1 −t 0 Þ
where RR is the respiration rate (μmol O2 g−1 h−1), C(t) is the
concentration of oxygen in the water (μmol O2 L−1) at time t,
V is the volume of water in the respirometer, W is the dry tissue
weight of the experimental clam (g), and t0 and t1 are the initial
and end times (in minutes) of the measurement period,
respectively.
Excretion Rate
At the end of the RR measurement, the same clams were
placed individually in 500-mL polyethylene beakers
filled with filtered and oxygen-saturated seawater at
20 °C to measure the excretion rate (ER). A beaker
without clams was used as the control. Following a 2-h
incubation period, 10-mL volume of seawater was taken
from each beaker, and ER was estimated by analyzing
the concentration of ammonia present using the phenol-
hypochlorite method [55]. ER was expressed in terms of
micromole NH4-N g−1 h−1.
Zhao et al.
Oxygen to Nitrogen Ratio
RR and ER values measured for individual clams were used to
calculate the atomic ratio of oxygen utilized to nitrogen ex-
creted (O/N) using the following equation:
mgO2 h−1 =16
O=N ¼
mgN H 4 −N h−1 =14
Scope for Growth
After the physiological measurements were completed, the
soft tissues of each clam was dissected and dried at 60 °C
for 48 h to measure the dry tissue weight. CR, RR, and ER
were converted into their energy equivalents and expressed in
terms of joule per gram per hour using constant values given
by Widdows et al. [26]. SFG was then calculated using the
following equation:
SFG ¼ A‐ðR þ U Þ
where SFG is the scope for growth (J g−1 h−1), A is the
absorbed ration (J g−1 h−1), R is the energy lost in respiration
(J g−1 h−1), and U is the energy lost in ammonia excretion
(J g−1 h−1).
The component parameters incorporated in the equation
above were determined as follows:



A ¼ CR Lg‐1 h‐1
 POM mg L‐1
 23:5 J mg‐1 POM  AR ð%Þ



R ¼ RR μmol O2 g‐1 h‐1  0:456 J μmol‐1 O2



U ¼ ER μmol NH4− N g‐1 h‐1  0:34 J μmol‐1 NH4− N
Water and Tissue Cd Analysis
Cd concentrations in seawater and clam tissue samples during
and after metal exposure were analyzed using an inductively
coupled plasma atomic emission spectrometer (ICP-AES,
IRIS intrepid 11XSP, USA). All tubes used for metal analysis
were soaked in 10 % HNO3 for 96 h, washed three times with
deionized water, and dried at 80 °C prior to use. Seawater
samples were filtered (0.45 μm) and acidified with 10 %
HNO3 before analysis. The clam samples were dissected
individually using a plastic knife, and the gills and digestive
gland were excised, rinsed, and freeze dried to constant
weights. For analysis, 0.5 g of dried tissue samples was
digested in 6 mL of HNO3 (65 %) and 2 mL of H2O2
(30 %) using a microwave digestion system (MARS-X,
Environmental Cadmium Exposure Impacts Physiological Responses 245

CEM, Mathews, NC, USA) for 30 min. All completely

digested samples were diluted using deionized water to a final
volume of 50 mL. A certified mussel tissue (GBW08571,
National Research Center for Certified Reference Materials,
Beijing, China) was routinely digested, diluted, and measured
in the same manner as the samples to ensure internal quality
assurance/quality control (QA/QC) practices. The recovery
for Cd was restricted within 91.1–103.4 %.

Statistical Analysis

Data were checked for normal distribution (Shapiro-Wilk’s

test) and homogeneity of variances (Bartlett’s test). Results
were compared using a two-way analysis of variance
(ANOVA) followed by a post hoc test (Duncan’s test) for
pairwise comparisons. All results were expressed as mean ±
standard deviation (SD). Relationships between tissue Cd
burden and physiological biomarkers were determined using
Pearson correlation analysis and the results were corrected
with a sequential Bonferroni test. The SPSS for windows
(version 17.0; SPSS, Chicago, IL, USA) was used for statis-
tical analyses. Statistically significantly difference was set at
p values of <0.05.

Results Fig. 1 Cd accumulation and elimination in Ruditapes philippinarum gill

Mortality over the course of the experiment was less than 4 %
and did not differ significantly over time in either the control
or the experimental treatments. Over the course of 24 h, there
was a small loss of Cd from the solution, which most likely
was due to adherence to the container walls and/or absorption
by R. philippinarum. An overall average of 0.61 % (4 μg L−1
Cd) and 4.14 % (40 μg L−1 Cd) was lost between each water
change. However, no significant difference was observed
between nominal and measured Cd concentration.
Consequently, nominal concentrations (4 and 40 μg L−1 Cd)
were used for data presentation and calculation in the present
study.
Cadmium Accumulation and Elimination
Figure 1a, b show the accumulation and elimination of Cd in
the gill and the digestive gland, respectively, of clams exposed
to two exposure doses (4 and 40 μg L−1) as a function of the
exposure and recovery time. Compared with control clams,
Cd concentrations in the tissues of Cd-exposed clams
depended significantly on exposure dose and time, and a
significant interaction between these two factors also was
found (Table 1). A general pattern of Cd accumulation and
elimination in the gill (Fig. 1a) and the digestive gland
(Fig. 1b) was evident for both treatments, although the degree
and time course varied. At each sampling time, exposure dose
(a) and digestive gland (b) during and after Cd exposure. Data are
expressed as mean ± SD. Asterisks denote significant differences between
the treatments and the control for the same experimental time.
resulted in significant tissue Cd concentrations following the
pattern 40>4 μg L−1 >control. Cd was continuously accumu-
lated over time in the gill and the digestive gland during the
exposure period. After 35 days of exposure, the tissue that
accumulated the most Cd was the digestive gland, which
contained 61.5- and 163.8-fold more Cd in the 4 and
40 μg L −1 groups, respectively, as compared to the
control. Conversely, Cd was eliminated much more slow-
ly from the digestive gland than from the gill following
35 days of recovery. By the end of 35 days of recovery,
the gill released an overall average of 77.8 and 72.1 %
of accumulated Cd after exposure to 4 and 40 μg L−1,
respectively, whereas in the digestive gland, eliminated
only 45.5 and 30.7 %.
Cd accumulation in the gill and the digestive gland showed
a significant linear increase with exposure dose at the end of
35 days of exposure (Table 2). The relationship between
exposure dose and Cd elimination was also significant and
linear for both tissues following the 35 days of recovery.
Whatever the exposure dose in the seawater, Cd concentra-
tions in the digestive gland was significantly higher than that
in the gill. A significant positive relationship between gill Cd
concentration and digestive gland Cd concentration was
246 Zhao et al.

Table 1 Two-way ANOVA results on the effects of exposure concentration and time on gill Cd concentration, digestive gland Cd concentration,
clearance rate, absorption efficiency, respiration rate, excretion rate, O/N ratio, and scope for growth during and after cadmium exposure

Treatment Source Gill Cd Digestive gland Cd Clearance rate Absorption efficiency

F(df1, df2) P F(df1, df2) P F(df1, df2) P F(df1, df2) P

Exposure Concentration F(2, 75)=3,514.20 <0.001 F(2, 75)=6,803.19 <0.001 F (2, 75)=623.00 <0.001 F(2, 75)=61.72 <0.001
Time F(4, 75)=11,953.86 <0.001 F(4, 75)=2,931.30 <0.001 F(4, 75)=3.22 <0.001 F(4, 75)=6.72 <0.001
Interaction F(8, 75)=626.36 <0.001 F(8, 75)=1,236.35 <0.001 F(8, 75)=0.718 0.639 F(8, 75)=5.04 <0.001
Source Respiration rate Excretion rate O/N ratio Scope for growth
F(df1, df2) P F(df1, df2) P F(df1, df2) P F(df1, df2) P
Concentration F(2, 75)=113.10 <0.001 F(2, 75)=877.21 <0.001 F(2, 75)=18.041 <0.001 F(2, 75)=638.25 <0.001
Time F(4, 75)=8.09 <0.001 F(4, 75)=55.58 <0.001 F(4, 75)=29.24 <0.001 F(4, 75)=7.17 <0.001
Interaction F(8, 75)=2.58 0.015 F(8, 75)=1.867 0.078 F(8, 75)=1.341 0.237 F(8, 75)=3.99 0.001
Recovery Source Gill Cd Digestive gland Cd Clearance rate Absorption efficiency
F(df1, df2) P F(df1, df2) P F(df1, df2) P F(df1, df2) P
Concentration F(2, 75)=7,074.70 <0.001 F(2, 75)=2,2350.13 <0.001 F(2, 75)=300.21 <0.001 F(2, 75)=71.62 <0.001
Time F(4, 75)=456.98 <0.001 F(4, 75)=480.03 <0.001 F(4, 75)=2.05 0.097 F(4, 75)=12.53 <0.001
Interaction F(8, 75)=265.43 <0.001 F(8, 75)=176.04 <0.001 F(8, 75)=1.53 0.201 F(8, 75)=6.21 0.306
Source Respiration rate Excretion rate O/N ratio Scope for growth
F(df1, df2) P F(df1, df2) P F(df1, df2) P F(df1, df2) P
Concentration F(2, 75)=19.42 <0.001 F(2, 75)=291.71 <0.001 F(2, 75)=16.96 <0.001 F(2, 75)=418.39 <0.001
Time F(4, 75)=2.80 0.032 F(4, 75)=20.11 <0.001 F(4, 75)=3.35 0.014 F(4, 75)=5.97 <0.001
Interaction F(8, 75)=3.12 0.004 F(8, 75)=0.70 0.689 F(8, 75)=1.35 0.235 F(8, 75)=1.85 0.081

Exposure concentration=0, 4, and 40 μg L−1 ; Exposure time=7, 14, 21, 28, and 35 days; Recovery time: 42, 49, 56, 63, and 70 days. Significant effects
are highlighted in bold.

subsequently established after 35 days of exposure followed
by 35 days of recovery (Table 2).
Clearance Rate
Figure 2 shows the time course and pattern of the CR in the
control and Cd-exposed clams during the exposure and sub-
sequent recovery periods. Compared with the control, a de-
cline in CR was observed at all time intervals in both exposure
treatments throughout the exposure period. In particular, the
clams exposed to 40 μg L−1 Cd showed a significantly lower
CR relative to the control at each analysis time point. In
contrast, significant inhibition of the CR was observed only
on day 21 in clams exposed to 4 μg L−1 Cd. A significant
effect of exposure dose and time was found, but no interaction
was noted between these two factors (Table 1). During the
recovery period, 40 μg L−1 Cd-exposed clams displayed a
tendency for increased CR, but it was not a statistically sig-
nificant change. The CR of clams in the 4 μg L−1 Cd treatment
was not significantly lower relative to the control during the
recovery period. Although there was a significant effect of
exposure dose on the CR during the recovery period, the
overall effect of time and the interaction between exposure
dose and time were not significant (Table 1).
Absorption Efficiency
As shown in Fig. 3, Cd exposure dose and time resulted in
significant changes in the AE. The clams exposed to Cd
displayed significant decline in the AE relative to the control
throughout the recovery period. At the end of 35 days of
recovery, these inhibitions in the AE were significantly lower

Table 2 Relationship between exposure dose and Cd concentration in the gill and digestive gland of R. philippinarum after exposure (35 days, 0–
40 μg L−1) and recovery (70 days, 0–40 μg L−1). Significant effects are highlighted in bold.

Treatment Tissue Equation of the line of best fit Correlation coefficient (R2) P value

Exposure Gill y=8.153x+30.823 0.969 <0.001

Digestive gland y=15.943x+103.79 0.917 <0.001
Recovery Gill y=2.308x+7.158 0.935 <0.001
Digestive gland y=11.478x+52.253 0.961 <0.001
Environmental Cadmium Exposure Impacts Physiological Responses
Fig. 2 Clearance rate measurements of Ruditapes philippinarum during
and after Cd exposure. Data are expressed as mean ± SD. Asterisks denote
significant differences between the treatments and the control for the
same experimental time.
than those observed in control clams. Two-way analysis of
variance (ANOVA) of the effects of Cd exposure on the AE of
clams over the course of exposure showed a significant effect
of exposure dose and time and a significant interaction be-
tween these two factors, but no significant interaction between
exposure dose and time was found following the 35 days of
recovery (Table 1).
Respiration Rate
The clams exposed to Cd exhibited significantly lower RRs
than control clams throughout the exposure period (Fig. 4).
RR was significantly impacted by exposure dose and time,
and an interaction between these factors was detected
(Table 1). Following the 35 days of recovery, the RR of
4 μg L−1 Cd-exposed clams was significantly inhibited from
days 42 through 49, but this inhibition dissipated thereafter.
Although the clams exposed to 40 μg L−1 Cd displayed an
increase in the RR during the recovery period, this increase
was not statistically significant. A significant effect of
Fig. 3 Absorption efficiency measurements of Ruditapes philippinarum
during and after Cd exposure. Data are expressed as mean ± SD. Asterisks
denote significant differences between the treatments and the control for
the same experimental time.
247
Fig. 4 Respiration rate measurements of Ruditapes philippinarum dur-
ing and after Cd exposure. Data are expressed as mean ± SD. Asterisks
denote significant differences between the treatments and the control for
the same experimental time.
exposure dose and time and an interaction between these
factors was also observed following the recovery (Table 1).
Excretion Rate
The clams exposed to 40 μg L−1 Cd exhibited a significant
increase in the ER compared to control clams at all time points
(Fig. 5). The ERs of 4 μg L−1 Cd-exposed clams were gener-
ally higher than that of the control throughout both the expo-
sure and recovery periods, but no significant differences were
observed. Significant effects of exposure dose and time were
observed for ER (Table 1). However, no significant effects of
the interaction between these factors were observed.
Oxygen to Nitrogen Ratio
The clams exposed to 40 μg L−1 Cd displayed a significant
decline in the O/N ratio compared to control clams at all time
points throughout both the exposure and recovery periods
Fig. 5 Excretion rate measurements of Ruditapes philippinarum during
and after Cd exposure. Data are expressed as mean ± SD. Asterisks denote
significant differences between the treatments and the control for the
same experimental time.
248
Fig. 6 O/N measurements of Ruditapes philippinarum during and after
Cd exposure. Data are expressed as mean ± SD. Asterisks denote signif-
icant differences between the treatments and the control for the same
experimental time.
(Fig. 6). At the end of the 35-day recovery period, the O/N
ratio was significantly lower than that in the other groups.
Although the O/N ratio of clams exposed to 4 μg L−1 Cd was
significantly lower than that of the control over the course of
the exposure period, this effect dissipated on day 56 of the
recovery period. Significant increases in O/N ratios were
observed in both Cd-exposed groups following the 35 days
of recovery. Two-way ANOVA of the effects of Cd exposure
on the O/N ratios of clams revealed significant exposure dose
and time effects (Table 1). However, the interaction between
exposure dose and time was not statistically significant.
Scope for Growth
The 4 μg L−1 Cd-exposed clams exhibited positive SFG
values at all time points during the exposure period, although
values significantly lower than those of control clams were
observed on days 21, 28, and 35 (Fig. 7). Conversely, the
40 μg L−1 Cd-exposed clams exhibited significantly de-
creased SFG, and negative values were recorded on days 21,
Fig. 7 Scope for growth measurements of Ruditapes philippinarum
during and after Cd exposure. Data are expressed as mean ± SD. Asterisks
denote significant differences between the treatments and the control for
the same experimental time
Zhao et al.
28, and 35. A significant effect of exposure dose, time, and a
significant interaction between these two factors were detect-
ed for SFG during the exposure period (Table 1). During the
recovery period, the SFG of the 4 μg L−1 Cd treatment was
greatly elevated on day 49 and was not significantly different
from the control, whereas a significantly lower SFG value was
observed for the 40 μg L−1 Cd treatment. Significant effects of
exposure dose and time were documented for SFG during the
recovery period, but the interaction between exposure dose
and time was not significant (Table 1).
Relationships between Tissue Cadmium Burden
and Physiological Biomarkers
Table 3 shows the relationship between Cd concentration in
the digestive gland and gills and physiological biomarkers of
Cd-exposed clams at the end of the 35-day exposure period
and the subsequent 35 days recovery period. Linear regression
analysis revealed a highly significant linear correlation be-
tween tissue Cd concentration and physiological biomarkers
relative to control clams. CR was positively and significantly
correlated with tissue Cd concentration after both the exposure
and the recovery periods. Similar positive and significant
correlations, but with reduced correlation coefficients, were
found for AE, RR, O/N ratio, and SFG. Conversely, the
relationship between ER and tissue Cd concentration was
negative and significant.
Discussion
Assessing the impact of Cd exposure on the physiology of the
clam, R. philippinarum requires knowing the accumulation
and elimination rates of this metal by target tissues. Although
recent studies describe the kinetics of Cd in other species of
clams, namely Ruditapes decussates [52], Mactra
veneriformis [48], and Macoma balthica [56], to our knowl-
edge, this is the first study to quantify the accumulation and
elimination of Cd in different tissues of R. philippinarum
exposed to environmentally realistic Cd concentrations (4
and 40 μg L−1) and to correlate tissue burden of this metal
to physiological responses of this species.
Cd has a strong binding constant to protein, and the rate of
accumulation is very fast [57]. Thus, in our study, Cd expo-
sure produced a significant accumulation response, with clams
at the higher exposure dose accumulating much more Cd in
their tissues (Fig. 1). The digestive gland had a higher capacity
to accumulate Cd compared to the gill. These findings are
consistent with results for other bivalves, such as the mussels
Mytilus galloprovincialis [58], Mytilus edulis [59], and Perna
canaliculus [35], the oysters Crassostrea virginica [39] and
Crassostrea gigas [60], and the scallop Chlamys nobilis [61];
Environmental Cadmium Exposure Impacts Physiological Responses 249

Table 3 Relationship between Cd concentration in the gill and digestive gland of R. philippinarum and physiological biomarkers after exposure
(35 days, 0–40 μg L−1) and recovery (70 days, 0–40 μg L−1). Significant effects are highlighted in bold.

Treatment Physiological biomarker Tissue Equation of the line of best fit Correlation coefficient (R2) P value

Exposure Clearance rate Gill y=−0.006x+1.029 0.892 <0.001

Digestive gland y=−0.003x+1.276 0.764 <0.001
Absorption efficiency Gill y=−0.08x+51.257 0.776 <0.001
Digestive gland y=−0.041x+53.133 0.832 <0.001
Respiration rate Gill y=−0.025x+11.731 0.780 <0.001
Digestive gland y=−0.013x+12.375 0.881 <0.001
Excretion rate Gill y=0.004x+0.814 0.885 <0.001
Digestive gland y=0.002x+0.740 0.917 <0.001
O/N ratio Gill y=−0.033x+12.293 0.717 <0.001
Digestive gland y=−0.018x+13.251 0.818 <0.001
Scope for growth Gill y=−0.018x+5.441 0.891 <0.001
Digestive gland y=−0.009x+5.742 0.885 <0.001
Recovery Clearance rate Gill y=−0.008x+1.230 0.829 <0.001
Digestive gland y=−0.004x+1.009 0.797 <0.001
Absorption efficiency Gill y=−0.158x+46.242 0.723 <0.001
Digestive gland y=−0.034x+47.140 0.789 <0.001
Respiration rate Gill y=−0.048x+10.692 0.659 <0.001
Digestive gland y=−0.009x+10.751 0.608 <0.001
Excretion rate Gill y=0.006x+0.532 0.737 <0.001
Digestive gland y=0.001x+0.506 0.798 <0.001
O/N ratio Gill y=−0.143x+18.497 0.590 <0.001
Digestive gland y=−0.030x+19.130 0.634 <0.001
Scope for growth Gill y=−0.035x+4.346 0.667 <0.001
Digestive gland y=−0.007x+4.545 0.757 <0.001

in these organisms, Cd accumulation is tissue specific and
dose and time dependent.
The uptake of metals by bivalves can occur via two major
routes: the gill, in the case of dissolved metal uptake, and the
digestive gland, in the case of dietary metal ingestion [47]. It
should be noted that the clams in our study were fed with the
diatom N. closterium, and consequently, the accumulation of
Cd in algal cells may have contributed to dietary Cd exposure.
It is likely that tissue Cd accumulation is the result of both
dissolved and dietary exposure. However, the Cd concentra-
tion in N. closterium was lower than the detection limit, which
indicates that the dietary contribution to the accumulation
pattern among the tissues was negligible in our study.
The gill is a physiologically complex and vulnerable organ
that is in direct contact with the external environment.
Following exposure to waterborne Cd, most metal uptake
occurs via the gill where Cd is likely to initially bind to
sequestered proteins such as metallothioneins or
metallothionein-like proteins for temporary storage [62]. Cd-
binding proteins may be incorporated into lysosomes for
degradation into insoluble residual bodies [63]. These insolu-
ble deposits are usually excreted by exocytosis or subsequent-
ly released into the blood plasma and circulating hemocytes
[64]. Thus, this homeostatic mechanism can allow
R. philippinarum to regulate the toxicity of dissolved Cd in
the gill. Once transferred to the digestive gland, however, Cd
is continuously accumulated and eventually internalized into
specific organelles in specific cell types for long-term storage
in order to prevent it from impacting sensitive cellular pro-
cesses [65]. This difference in the metabolic transformation of
Cd likely explains the difference in tissue Cd burden in the
present study. Cd accumulation in the digestive gland was
much greater than that in the gill. However, the rate of elim-
ination of Cd was significantly slower in the digestive gland.
Therefore, the measure of gill Cd burden in R. philippinarum
might be a sensitive and simple biological measure for mon-
itoring the short-term variables of environmental Cd levels,
whereas the concentration in the digestive gland could be
indicative of long-term environmental exposure.
The primary objective of ecotoxicological studies is to
predict and diagnose the causes of biological impacts resulting
from exposure to environmental stressors. Although there is
no single biological measurement that can satisfy all environ-
mental situations, quantifying cause–effect relationships be-
tween tissue pollutant levels and biological effects in terms of
physiological energetics may be well suited to such an
250
objective [66]. Under normal conditions, bivalves can obtain
enough energy through feeding and food absorption to main-
tain vital bodily functions, but they also expend energy
through respiration and excretion. Once maintenance of basal
physiological processes has been fulfilled, the residual energy
is available for growth and reproduction [26]. A general
response of a bivalve under stressed conditions is the utiliza-
tion of more nutrient reserves to meet a metabolic requirement
that may have been enhanced above normal values [67].
Consequently, measures of physiological energetics in many
bivalve species can provide information about the processes
of energy acquisition, energy expenditure, and energy avail-
able for growth and reproduction while also enabling predic-
tion of the biological effects of exposure to environmental
stressors.
CR is an important physiological factor that reflects the
energy acquisition of a bivalve. It represents the capacity of an
individual to filter algal cells from the water, and hence, it was
used in this study as a measure of feeding rate. This measure is
widely accepted to be very sensitive and to have a clear dose–
response relationship [47]; thus, bivalves subjected to stressful
conditions often exhibit decreased CRs. When CR is reduced,
the total amount of energy available to the organism is also
reduced, which provokes general metabolic depression [68].
The results of the current study showed that CR was consis-
tently inhibited throughout the course of the 35-days exposure
period at the exposure dose of 40 μg L−1, and it failed to
recover following a 35 days recovery period. This result
indicates a significant long-term impairment of the feeding
activity associated with high-level Cd exposure stress.
Interestingly, this depression in CR was not observed in the
4 μg L−1 Cd treatment, which indicates that the clams are
tolerant of moderately polluted conditions. Overall, in terms
of utility as a physiological indicator of environmental levels
of exposure, CR may be a particularly sensitive measure.
When exposed to an environmental stressor near or beyond
their tolerance limits, bivalves immediately close their shell
valves to reduce their exposure to the outside environment
[69]. Valve closure helps a bivalve protect its internal organs
from the stressor. In the case of Cd, valve closure reduces
metal accumulation in soft body tissues and thus limits the
toxic effects of exposure. However, this action can only pro-
vide short-term protection, as normal physiological processes
are also inhibited when the valves are closed. A consequence
of this behavior in the current study was that the CR was
significantly decreased. It is also likely that decreased CR
represents a physiological impairment of the gill. During
metal exposure, bivalves tend to secrete more mucus in order
to protect themselves from toxicity [70]. Although copious
mucus secretion may have a protective function, it also covers
the gill epithelium and clogs the gill pores, thereby affecting
the ciliary activity that is essential in the normal process of
feeding and oxygen consumption [71]. In addition, Cd can
Zhao et al.
cause structural damage to the gill. A number of studies have
demonstrated structural alterations of gills in a variety of
bivalves exposed to heavy metals such as Cd. These effects
include clubbing of gill filaments [72] and uncoupling of
interfilamentar junctions [73]. These alterations may result in
decreased CR.
It is reasonable to assume that decreased CR would be
accompanied by decreased AE, which represents how effec-
tively a clam can utilize its nutrient intake. As exposure
duration progressed, a significant decrease in AE was ob-
served in the current study. Therefore, this decreasing trend
may indicate that metal exposure not only affected the effi-
ciency of feeding but also the activity of enzymes. In contrast,
the organic content of feces increased, which illustrates that
the digestion process was less efficient, thus less energy was
available for physiological processes. Previous studies also
showed that AE was significantly affected by metal exposure
[74–76]. In these studies, energy acquisition in clams was
greatly reduced mainly because of decreased CR and AE.
Oxygen consumption and ammonia excretion, which are
measured in terms of RR and ER, respectively, can be used as
a measure of energy expenditure in clams. Fluctuations in
these physiological processes not only provide a good indica-
tion of the metabolic state of the organisms [77], but they also
serve as an early indicator of the stress resulting from exposure
to heavy metals [62]. In our study, clams exposed to Cd had
significantly decreased RRs, increased ERs, and low values of
the O/N ratio. Cd was previously reported to decrease RR and
increase ER in bivalves [35, 78], although these physiological
responses varied according to exposure dose, exposure time,
and species tested. These effects may be due to interference
with gaseous exchange and the inhibition of mitochondrial
respiration [79]. Nevertheless, in the current study, both RR
and ER in exposed R. philippinarum recovered quickly after
the clams were transferred to clean seawater. Bivalves are
known to have an excellent ability to regulate metabolic
activity and thus can quickly recover their normal physiolog-
ical processes once they are transferred from adverse to favor-
able conditions.
Under healthy conditions, bivalves rely heavily on carbo-
hydrates and lipids to fuel normal energy metabolism.
Because it is energetically costly to fuel stress effects, bivalves
under stressed conditions tend to utilize more nutrient reserves
to maintain basal metabolism and fuel any additional stress
responses. In general, proteins are an emergency supply, and
they are utilized when other nutrient reserves are not available.
Thus, the O/N ratio can be used as an indicator of the relative
utilization of protein in energy metabolism of bivalves. In
Mytilus edulis, for example, an O/N ratio value of 50 repre-
sents the exclusive use of carbohydrate and lipid as an energy
source, which is generally indicative of a physiologically
healthy condition; in contrast, values less than 30 represent a
heavy reliance on protein to provide energy, and a value of 7
Environmental Cadmium Exposure Impacts Physiological Responses
represents total reliance on protein as the sole source of energy
[80]. In the present study, the decreased O/N ratios in Cd-
exposed clams suggest that Cd exposure caused enough stress
in clams to deplete their carbohydrate and lipid reserves so
that they began to utilize protein as an energy source. This
switch in energy utilization may also indicate that the increase
in ER observed in stressed clams was primarily due to the
metabolism of nutrient reserves in the form of protein.
SFG represents the energy budget of an organism, as it
delimits the energy available for growth and reproduction by
integrating measures of major physiological responses (e.g.,
feeding, AE, respiration, and excretion) [67]. This index is
unspecific, but it provides a simple, rapid, and sensitive mea-
sure of stress induced by environmental conditions in marine
invertebrates, including bivalves [28]. SFG can range from
positive values, which indicate that energy is available for
growth and reproduction, to negative values, which indicate
that the animal is consuming its bodily reserves for metabo-
lism [30]. Thus, measurement of SFG can be used to detect
and quantify stress effects on the physiological status of
clams. The negative SFG values observed in the present study
indicate severe stress and show that stressed clams were
unable to obtain enough energy to fuel their metabolic re-
quirements. The decrease of SFG to negative values was most
likely due to the drastic decrease in CR and the increase in ER,
as these two parameters constitute the major sources of energy
acquisition and energy expenditure of a clam. It is, however,
important to note that because both of these measures showed
recovery, a significant recovery to a positive SFG value was
also observed. This result, coupled with the significant corre-
lation of SFG with tissue Cd concentration, confirm that
measurement of SFG is a reliable method to assess general
stress effects and that it provides an early warning signal of the
potential consequences of exposure to environmentally real-
istic concentrations of this metal.
Conclusion
The results obtained in this study highlight the utility of
employing R. philippinarum as a sentinel species for moni-
toring metal stress in estuarine and coastal ecosystems. Our
findings confirm that R. philippinarum possesses good phys-
iological plasticity towards Cd pollution. This ability was
observed through measurable physiological responses (i.e.,
CR, AE, RR, ER, O/N ratio, and SFG). All of these physio-
logical measurements exhibited a significant and predictable
correlation with tissue Cd accumulation when clams were
exposed to environmentally realistic Cd concentrations. Our
results show that the use of CR, a sensitive index of physio-
logical condition, would yield cost-effective information
about the health status of clams under natural conditions.
Negative SFG values are indicative of severe stress, during
251
which the clams are in a state of negative energy balance and
have to utilize bodily reserves for metabolism; however, under
slightly or moderately contaminated conditions, the clams are
still capable of normal growth (as demonstrated by the posi-
tive but low SFG values observed in the 4 μg L−1 Cd treat-
ment). These results indicate that R. philippinarum is relative-
ly tolerant to Cd exposure. As a corollary, this high physio-
logical plasticity and the high tolerance to pollution are likely
important contributory factors to the wide distribution and
high density of this species in estuarine and coastal waters.
Because the physiological responses measured herein are at
the organism level, they have the advantage of being readily
interpreted as being beneficial or deleterious. Furthermore,
they may be predictive of long-term consequences at the
population level, and therefore, they can provide a compre-
hensive view of the ecological consequences of exposure to
pollutants.
Acknowledgments The authors would like to acknowledge the follow-
ing people for technical assistance: Dawei Zhang, Wuting Lu, and Yinhou
Zhang. This project was financially supported by the earmarked fund for
Modern Agro-industry Technology Research System (CARS–48), the
Science and Technology Foundation of Education Department of Liao-
ning Province (L2011117), and the Public Science Research Funds Pro-
jects of Liaoning Province (2012005002)
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