Article

pubs.acs.org/est

Unifying Prolonged Copper Exposure, Accumulation, and Toxicity

from Food and Water in a Marine Fish
Fei Dang,† Wen-Xiong Wang,*,† and Philip S. Rainbow‡
†
Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong
‡
Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom
*
S Supporting Information

ABSTRACT: The link between metal exposure and toxicity is complicated by

numerous factors such as exposure route. Here, we exposed a marine fish (juvenile
blackhead seabream Acanthopagrus schlegelii schlegelii) to copper either in a commercial
fish diet or in seawater. Copper concentrations in intestine/liver were correlated
linearly with influx rate, but appeared to be less influenced by uptake pathway
(waterborne or dietary exposure). Influx rate best predicted Cu accumulation in the
intestine and liver. However, despite being a good predictor of mortality within each
pathway, influx rate was not a good predictor of mortality across both exposure
pathways, as waterborne Cu caused considerably higher mortality than dietary Cu at a
given influx rate. We show that the use of gill Cu accumulation irrespective of the
exposure route as a model for observed fish mortality provided a clear relationship
between accumulation and toxicity. Investigation of gill Cu accumulation may shed
light on the different accumulation strategies from the two exposure pathways. This
correlation offers potential for the use of branchial Cu concentration as an indicator of
long-term Cu toxicity, allowing for differences in the relative importance of the uptake
pathways in different field situations.

■ INTRODUCTION
Copper contamination of estuarine environments in southwest
England1 and China2 raises concern about Cu bioaccumulation
and toxicity in marine fish. Environmental risks of metals
depend greatly on exposure, but the linkage between exposure
and toxicity varies with metal, species, and exposure route.3−5
Currently, daily dose better correlates Cu exposure and toxicity
rather than dietary metal concentrations.6 However, assessment
of potential metal toxicity works on the premise of accurate
estimation of metal bioavailability leading to accumulation.
Therefore, further consideration of metal bioavailability on the
basis of daily dose may provide a more precise measure of metal
exposure and a better link among metal exposure, bioaccumu-
lation, and toxicity. Specifically, given the simultaneous
exposure of fish to waterborne and dietary Cu, it is useful to
seek a universal predictor for Cu bioaccumulation and toxicity,
which is applicable to both exposure routes, and finally to
produce a simplified tool for the evaluation of concurrent
exposure.
The biokinetic model has been used to forecast successfully
metal concentrations in field-collected organisms, based on
measured biokinetic parameters, i.e., metal influx rate,
elimination rate, and organism growth rate.3 Influx rate
characterizes the physiological processes of bioavailable metal
delivery via membrane transport and thus links metal exposure
and bioaccumulation. Elimination rate could also modify metal
bioaccumulation by metal loss, but it is usually less variable than
influx rate under various environmental conditions.7 The
control of growth rate on Cu accumulation by growth dilution
appears less important relative to elimination rate.8 Therefore,
influx rate could potentially serve as a more appropriate
indicator of Cu bioaccumulation for both exposure routes.
Acute dissolved metal toxicity in fish is manifested when a
critical amount of metal binds to a physiological target site on
the surface of the fish gill.9 Numerous data of metal acute
toxicity are available for a variety of metals in aquatic organisms
across different water chemistries. For instance, the physio-
logical mechanism of acute dissolved Cu toxicity involves
disrupting Na+ and Cl− uptake in fish.10,11 Conversely, a
growing number of different toxic effects of laboratory-derived
long-term dietary Cu exposure has also been observed in fish
(summarized in refs 6 and 12), including digestive physiology
changes,13−15 hepatic biochemical effects,16,17 behavior re-
sponses,17 and growth inhibition.13,18 At present, compared to
the well-known dissolved acute toxic mechanism, the
physiological mechanisms of long-term dietary Cu toxicity
remain less well understood.12
In this study, we have employed biokinetic principles to
investigate relationships among Cu exposure, time-dependent
Received: November 5, 2011
Revised: February 20, 2012
Accepted: February 28, 2012
Published: February 28, 2012

© 2012 American Chemical Society 3465 dx.doi.org/10.1021/es203951z | Environ. Sci. Technol. 2012, 46, 3465−3471
Environmental Science & Technology
Cu accumulation (gill, intestine, liver, and muscle), and
consequent toxicity in a marine fish, the blackhead seabream
Acanthopagrus schlegelii schlegelii, during 4-weeks laboratory
exposure. This study offers a comparison of accumulation and
toxicity arising from dietary or waterborne Cu exposure.
Specifically, we attempt to integrate both uptake pathways into
a common framework in terms of exposure, accumulation, and
toxicity, which may produce a simple but realistic tool for
environmental monitoring and risk assessment.
■ MATERIALS AND METHODS
Fish Diet and Experimental Design. Field-collected
juvenile blackhead seabream Acanthopagrus schlegelii schlegelii
(7−8 cm in length, initially approximately 1.2 μg Cu g−1 dw)
were obtained from a fish farm in Sai Kung, Hong Kong, which
is a pristine area close to Clear Water Bay. After transportation
to the laboratory in Clear Water Bay, they were reared in sand-
filtered Clear Water Bay seawater (pH 8.0, salinity = 33 psu,
DOC = 4.2 mg L−1, dissolved Cu = 2.93 ± 1.37 μg L−1) at 23
°C under a 14 h light:10 h dark regime. The same aerated sand-
filtered seawater was used in all the experiments. Fish were fed
fish diet pellets (purchased from a company in Xiamen, China)
three times a day at a ratio of 0.035 wet weight per wet weight
of fish per day. Fish could consume the diet rapidly within 1 h.
The measured Cu concentration in the diet was 7.73 ± 0.14 μg
g−1 with macronutrient contents of 42% crude protein, 3% fat,
and 16% crude ash.
To eliminate the effects of other metals on Cu toxicity, we
chose Cu-spiked commercial fish diet rather than a naturally
contaminated live diet containing multiple metals. Three
environmentally relevant Cu concentrations were selected
(50, 250, and 1000 μg g−1 nominally, but see Table 1) as Cu
Table 1. Measured Cu Concentrations in Fish Diet (n = 5)
and Seawater (n = 8)a
waterborne estimated
dietary concn concn influx rate
treatment (μg g−1) (μg L−1) (μg g−1 d−1)
control 7.73 ± 0.14 2.93 ± 1.37 0.11
50 μg g−1 66.9 ± 2.61 2.93 ± 1.37 0.94
250 μg g−1 273 ± 11.4 2.93 ± 1.37 3.82
1000 μg g−1 997 ± 39.4 2.93 ± 1.37 6.98b
100 μg L−1 7.73 ± 0.14 99.7 ± 17.1 0.62
500 μg L−1 7.73 ± 0.14 525 ± 104 3.28
a
The estimated influx rate (μg g−1dw d−1) was calculated by
multiplying Cu assimilation efficiency (AE, 10%), ingestion rate (IR,
0.14 g ww g−1 dw d−1) and dietary Cu concentration (Cf, μg g−1 ww)
for dietary exposures, or by multiplying dissolved uptake rate constant
(ku, 6.24 L kg−1 dw d−1) and dissolved Cu concentrations (Cw, μg L−1)
for waterborne exposures. For both dietary and waterborne exposures,
the contribution to Cu accumulation from the other route was
negligible (see text). Values are means ± SD. bThe ingestion rate was
only 7% of 1000 μg Cu g−1 and 7% control diet was also supplemented
to fish.
concentrations from 45 to 662 μg g−1 have been measured in
the polychaete Nereis diversicolor (a common prey item of fish)
collected from a range of estuaries in southwest England (Dang
et al. unpublished data). To obtain the Cu-contaminated fish
diets, 800 g fish diet pellets were soaked in 800 mL of Cu-
spiked solution (as CuCl2, 52, 260, and 1040 mg Cu L−1 in
nanopure water for each treatment, respectively) for 6 h and
remained intact. Then those pellets were dried at 50 °C
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overnight to decrease the moisture content to the original value
(6.2(±1.4)%). The control fish diet (8 μg g−1) was prepared in
the same way as above but with only nanopure water added. All
the prepared fish diets were kept in plastic bags at −20 °C for
fish feeding and Cu analysis.
Dietary influx rate varies as a function of assimilation
efficiency (AE, %), ingestion rate (IR, g g−1 dw d−1) and food
concentration (Cf, μg g−1, 4, 7). Conversely, waterborne influx
rate is a product of dissolved uptake rate constant (ku, L kg−1
dw d−1) and dissolved metal concentration (Cw, μg L−1, 7).
Dang et al.8 reported for A. schlegelii that Cu ku was 6.24 L kg−1
d−1 and Cu AE from brine shrimp and clam were comparable
(9−11%) but 2% when the fish were fed with oyster tissue. In
the current study a mean AE value of 10% from brine shrimp
and clam was used to represent Cu bioavailability in the
commercial fish diet. This assumption seems reasonable
because most Cu accumulated in oysters is bound with cellular
debris and metal-rich granules (as high as 90%, 2), resulting in
abnormally low AE in oysters. The ingestion rate was 0.14 g ww
g−1 dw d−1 except at the highest dietary Cu treatment (see
below), and a factor of 4 (ww/dw) was used to correct fish
mass. Metal concentrations in fish diet and the estimated influx
rates are presented in Table 1.
To obtain similar influx rates (μg g−1 dw d−1) from water and
food, the target dissolved Cu concentrations in seawater were
calculated as:
AE × IR × C f
Cw =
ku (1)
The calculated dissolved Cu concentrations were therefore 100
and 500 μg L−1, respectively, producing waterborne Cu uptake
rates equivalent to the daily influx rates for the nominal 50 and
250 μg g−1 dietary treatments, respectively (Table 1). The
dissolved Cu concentrations were slightly higher than those
found in the field (e.g., 0.1−176 μg L−1, 1)
Few fish could survive the 4-week exposure at high dissolved
Cu concentration (1119 μg L−1), equivalent to 1000 μg g−1
dietary treatment in a preliminary experiment. Thus, there was
a total of six treatments (control, three dietary levels, and two
waterborne levels), each group with three replicate tanks. We
defined toxicity as a decrease in either growth rate or survival
over the period of exposure.
Fish Exposure. Fish were randomly divided into 18 tanks
(size of 60 × 29 × 43 cm3, 16 fish per tank) and acclimated
under the same conditions as described above for another week
until exposure began. The dietary-exposed fish were fed one of
the three Cu-spiked fish diets, whereas the waterborne-exposed
and control fish were fed control fish diet without Cu spiking.
All the fish were fed three times daily, and the ingestion rate
was controlled to 0.14 g ww g−1 dw d−1 by the amount of fish
diet. One exception was 1000 μg Cu g−1 exposed fish, which
were first fed 7% of 1000 μg g−1 diet. After these diets were
almost consumed, 7% of control diet was supplemented. This
reduced the estimated influx rate to 6.98 μg Cu g−1 d−1 and
thus the differences of estimated influx rates between exposure
concentrations (50, 250, and 1000 μg g−1) were comparable.
The fish diet floated in seawater without sinking during the 1 h
feeding regime allowing the fish to feed easily. Fish feeding
behavior was monitored in all treatments during the feeding
regime and all diet pellets were found to be eaten except in the
highest dissolved and dietary exposure groups; uneaten food
and feces were then siphoned off. Our calculation based on the
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biokinetic equation ( f = [ku × Cw]/[AE × IR × Cf + ku × Cw],
the same variable values as eq 1) and data in preliminary
experiments (i.e., <10% of spiked Cu was released from the fish
diet into the 75 L seawater during 1 h) implied that the
contribution of dissolved uptake (f < 2%) was negligible
compared to dietary uptake in the dietary treatments. Indeed,
our measurements before and after feeding did not show
significant differences in dissolved Cu concentrations. DOC
released from food (4.2 ± 0.1 increased up to 5.6 ± 0.2 mg/L
in 75 L seawater without fish) was unlikely to reduce Cu
bioavailability in our system. When the background DOC level
exceeded 2.4 mg L−1 in Clear Water Bay, the further increase in
DOC levels would not affect Cu ku.19 For waterborne
exposures, 5/6 of the exposure seawater was renewed after
the last meal each day and new Cu stock (as CuCl2, 1 mg Cu
mL−1) was added to keep a relatively constant dissolved Cu
concentration. High concentration of DOC in the seawater
would complex most Cu and sustains high dissolved Cu
levels.20 We did not quantify Cu levels of uneaten diet in
waterborne-exposed treatments. Food rejection in the dissolved
500 μg L−1 groups (see Results), together with the short
feeding regime (1 h) and the relatively small surface-volume
ratio of diet pellets (2 mm in diameter) probably indicated that
any adsorption of dissolved Cu onto the fish diet would have
little effect on overall Cu accumulation in these waterborne-
exposed fish.
After 14 or 28 days exposure, all food remnants in the gut of
fish were depurated completely in clean seawater for 36 h,
before a subsample of fish (Table S1 in the Supporting
Information) was removed, euthanized in 100 mg L−1 tricaine
methanesulfonate (MS-222) for several minutes, rinsed in
deionized water, blotted dry, weighed, and measured for
standard length. To minimize the possibility of biased sampling,
i.e., the nonrandom capture of the easiest fish to catch, a fish
net of size comparable with the width of the experimental tank
was used to capture fish, ensuring most fish could not escape
and thus fish could be randomly chosen for sampling. All fish
were frozen in liquid nitrogen and then kept at −80 °C until
further analysis. The condition factor (CF = weight (g)/length3
(cm)) and liver somatic index (LSI = liver wet weight (g)/fish
wet weight (g) × 100%) were calculated for each individual
fish. Specific growth rates [SGR = (In(final weight/initial
weight)/number of days × 100%] over 4-weeks exposure were
calculated by fish mean weights.
Measurements. Fish were dissected for liver, intestine, gill,
and muscle samples which were rinsed in 0.9% NaCl to remove
blood, and oven-dried at 80 °C for 48 h. After weighing, fish
tissues were digested in concentrated nitric acid (70%,
analytical reagent grade, Fisher Scientific) at 110 °C for 1
day and diluted for Cu analysis by inductively coupled plasma-
optical emission spectrometry (ICP-OES, PerkinElmer
7000DV). The fish diet was weighed and fed to the fish; Cu
concentrations in fish diet were thus measured on a wet weight
basis (n = 5) rather than a dry weight basis. Standard reference
material (Oyster tissue 1566b, NIST) was concurrently
digested for Cu measurement. The recovery rates ranged
from 94% to 103%. Additionally, samples of exposure seawater
before or after feeding were collected every 3 days, filtered
through a 0.45 μm filter (Millipore), acidified to 3% by nitric
acid, and analyzed for Cu concentrations. The Cu background
concentration in natural seawater was measured by inductively
coupled plasma-mass spectrometry (ICP-MS, Agilent 7700x).
Standard Cu solutions (CuCl2 in 2% HNO3, Perkin-Elmer Pure
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Atomic Spectroscopy) were simultaneously measured after
every five samples and the recovery rates were 90−96%. Actual
measured Cu concentrations in spiked fish diets and seawater
samples are listed in Table 1.
Data Analysis. A one-way analysis of variance (ANOVA)
followed by Tukey posthoc tests was used to examine replicate
effects (statistical differences among experimental tanks) within
each treatment and showed no replicate effects with p > 0.10.
Thus data from replicates within each treatment were
combined and used to seek statistically significant differences
among treatments by ANOVA (p < 0.05). All measurements
were given as means ± SD.
■ RESULTS
The intestinal and hepatic Cu concentrations increased linearly
over a range of dietary and waterborne influx rates from 0.11 to
6.98 μg Cu g−1 d−1 after 2- or 4-weeks exposure (Figure 1). At
Figure 1. Copper tissue concentrations in A. schlegelii exposed for 2 or
4 weeks to dissolved or dietary Cu. Each regression line represents
data from both exposure routes at a time point (solid line for 2-weeks
exposure or dashed line for 4-weeks exposure). Note that D2 and D4
represent dietary exposure for 2 weeks and 4 weeks, respectively, while
W2 and W4 represent waterborne exposure for 2 weeks and 4 weeks.
Estimated influx rate is listed in Table 1. Values are means ± SD (n =
6).
similar influx rates, Cu burdens in intestine and liver after
waterborne exposure were comparable to those after dietary
exposure. Furthermore, hepatic and intestinal Cu concen-
trations were significantly correlated (p = 0.02 for 2-weeks
exposure and p < 0.001 for 4-weeks exposure). On the basis of
measured Cu levels in gill, muscle, intestine, and liver and tissue
weights, Cu contents in intestine and liver accounted for more
than 70% of the total Cu burden in exposed fish irrespective of
exposure routes, indicating the importance of intestine and liver
during Cu accumulation.
When both exposure routes were considered, no relation-
ships with influx rate were observed in gill and muscle (Figure
1). Copper concentrations in the gill upon 28-day exposure to
dissolved Cu were 1.1- or 4.9-fold (i.e., 0.8 vs 0.7 μg g−1, 8.8 vs
1.8 μg g−1, respectively) higher than those of dietary exposure
with comparable influx rates. Over the entire exposure period,
Cu in the muscle fluctuated from 0.7 to 1.5 μg g−1 and changed
little with increased influx rate.
The dietary-exposed blackhead seabream (6.98 μg Cu g−1
−1
d ) first showed evidence of sublethal toxicity in terms of
decreased consumption rates after 6 days exposure: a small
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amount of the contaminated diet and most of the control diet
was uneaten. But the ingestion rate was not corrected for
uneaten food as this was not quantified. Meanwhile, most fish
at the higher dissolved Cu influx rate (3.28 μg Cu g−1 d−1)
reduced their consumption rates on day 2 and rejected food
completely on day 4.
At a similar rate of Cu influx, fish exposed to either food or
water for 14 days showed comparable physiological responses
(e.g., wet weight, length, CF, LSI and SGR) with the exception
of a higher mortality rate (10%) at a dissolved influx rate of
3.28 μg g−1 d−1 (Table S1 in the Supporting Information).
After 28-day exposure, low influx rates (0.94 vs 0.62 μg g−1 d−1)
did not produce significant differences in any physiological
parameter between exposure pathways. However, in contrast to
the dietary exposure counterpart (3.82 μg Cu g−1 d−1, Table S1
in the Supporting Information), 11%, 12%, and 83% reductions
in mean condition factor (CF), liver somatic index (LSI), and
specific growth rate (SGR) were observed at the dissolved
influx rate (3.28 μg Cu g−1 d−1), partially due to food rejection
in this waterborne group. Furthermore, mortality rate increased
by 5-fold to 31%.
Under dietary Cu exposure, the influence of influx rate did
not follow a constant trend for all physiological parameters
(e.g., wet weight, length, condition factor, liver somatic index,
and specific growth rate) except for an increasing trend of
mortality rate. The highest dietary influx rate of 6.98 μg Cu g−1
d−1 was associated with a 36% decrease in SGR and an induced
8.3% mortality rate compared to the control. Interestingly,
there appeared to be a time lag for toxicity manifestation, as
reflected in SGR and mortality over the last 14 days, suggesting
that the exposed fish suffered long-term toxicity at high Cu
influx rates rather than acute toxicity. Overall, no relationship
between dietary influx rate and physiological parameters (wet
weight, length, CF, LSI, and SGR) was observed, except for a
significant correlation with the mortality rate observed (0−
8.3%, Figure 2).
Figure 2. Relationship between Cu influx rate and mortality rate in A.
schlegelii exposed to dissolved or dietary Cu for 4 weeks.
■
DISCUSSION
Influx Rate and Accumulation. Conceptually, influx rates
result from the interaction between bioavailable metal
concentrations and mechanistic characteristics (e.g., binding
affinity and capacity) of biological transport systems,7 and thus
provide a plausible link between exposure and accumulation. Its
strong correlations with intestinal/hepatic Cu concentrations
(Figure 1) suggest that influx rate as tested here can provide an
acceptable model of whole body fish Cu accumulation, as Cu in
liver and intestine accounted for more than 70% of total Cu
burden in exposed fish at the whole body level. Moreover, the
predictive capability of influx rates to Cu accumulation is likely
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to be independent of exposure pathway. In particular, regardless
of exposure routes, intestinal and hepatic Cu accumulation
remained comparable (p > 0.05) at similar influx rate, although
only two pairs of comparable influx rates from food and
seawater were examined (Figure 1).
The intestinal Cu concentrations derived from food and
seawater were comparable at similar influx rates examined,
highlighting the importance of the intestine as an organ for
dissolved Cu uptake in marine fish, which drink a substantial
volume of seawater for osmotic homeostasis.21 Indeed the
dissolved uptake rate via the intestine has been shown to be
approximately 10-fold higher than that via the gills in juvenile
blackhead seabream.21 Although dose-dependent dietary Cu
accumulation in the intestine was observed, Cu is likely to be
stored in the intestinal mucosa rather than be transferred into
the intestine, because mucus acts to effectively trap Cu in the
gut lumen and transportation across intestinal cells appears to
be a limiting step for Cu uptake by internal tissues.22
Subcellular fractionations may provide insights on intestinal
Cu accumulation.
After intestinal uptake, dietary-derived Cu is probably
delivered directly to the liver by the hepatic portal system,23
as evidenced by the significant relationship between hepatic and
intestinal Cu concentrations. Thereafter, Cu is distributed
throughout the body (e.g., gill and muscle), resulting in high
Cu concentrations in intestine and liver, followed by gill and
muscle (Figure 1).
Copper concentrations in gill or muscle are not strongly
influenced by dietary influx rate. The gill is directly exposed to
waterborne Cu, while dietary-derived Cu accumulated in the
gill would result from Cu redistribution after intestinal uptake.
Therefore, waterborne exposure appears to result in higher Cu
levels in gills compared to its counterpart. Finally, between the
two exposure routes banchial Cu levels did not show a
consistent relationship with influx rates (Figure 1). Muscle Cu
concentrations remained comparable among dietary influx
rates, as shown here and in earlier studies.14,24 When both
exposure pathways are considered, influx rate could not predict
Cu levels in gill and muscle, a different scenario from the
intestine and liver.
Influx Rate and 28-Day Toxicity. Copper influx rate from
water or food has previously been shown to predict toxicity in
acute exposures.4,25 This is consistent with our observation for
a single uptake pathway during prolonged exposure (Figure 2).
However, when both uptake pathways are considered, Cu
bioavailability estimated by influx rate is not an accurate
reflection of Cu toxicity. Between both exposure routes there
was no consistent relationship of mortality with influx rate
(Figure 2). This could be due to Cu regulation by intestine and
liver, which is not taken into account in measures of influx rate.
In addition, Cu elimination and detoxification rate (e.g.,
metallothionein induction) are crucial to toxicity prediction.3
For instance, Dang et al.8 reported a high Cu elimination rate
constant (0.091 d−1) and demonstrated the significance of Cu
elimination in a long-term exposure.
The incorporation of metal assimilation efficiency into daily
dose (i.e., influx rate) provides a more accurate estimation of
metal accumulation. This is particularly important when
summarizing metal accumulation from different food sources
because metal assimilation efficiency varies among food types
(e.g., copepods, barnacles, clams, mussels, fish viscera, 26).
Additional experimental work is, however, required to quantify
the Cu AE of the commercial fish diet and the fish ingestion
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Environmental Science & Technology
rate during exposure; in any case, the influx rates could be
different from those estimated in this study. Variation in metal
AE of the commercial fish diet does not affect the observed
relationships between mortality/accumulation and dietary
influx rate (Figure S1 in the Supporting Information). The
relationship between Cu accumulation and influx rate for both
exposure pathways is significant, when AE varies within normal
range (5−20%, excluding the abnormally low Cu AE in oyster,
Table S2 in the Supporting Information). Additionally, AE
differences among exposure concentrations within an experi-
ment are likely too small to significantly influence influx rate.4
As a result of reduced feeding behavior at the highest dietary
Cu level, dietary influx rate is slightly overestimated and the
relationships between mortality/accumulation and dietary
influx rate (Figures 1 and 2) may be modified. Future work
is required to evaluate these estimated influx rates and to
determine if the relationship holds over a wider range of influx
rates.
As mentioned previously, it is difficult to directly link
exposure and toxicity in the presence of multiexposure routes.
Only a portion of bioaccumulated Cu appears to be responsible
for long-term toxic effects. As a consequence, a better
understanding of internal Cu distribution from both uptake
pathways is required.
Tissue Cu Concentrations and 28-Day Toxicity. For
dietary exposure, our results show that Cu levels in intestine,
liver, and gill of blackhead seabream were linearly related to
mortality (0−8%, Figure 3). Specifically, the slightly larger
correlation coefficient for branchial Cu (e.g., 4.5 in gill >0.06 in
liver or 0.04 in intestine after 4-weeks exposure) implies that
Figure 3. Relationship between mortality rate after 4-weeks exposure
and tissue-specific Cu concentration (intestine, liver, and gill) at 2 or 4
weeks. The inset shows the relationship between mortality rate after 4-
weeks dietary exposure only and gill Cu levels. Values are means ± SD
(n = 6).
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gill is more susceptible to Cu (Figure 3, inset). It does not
support the general proposal that the gut is the major target
organ of toxicity in dietary exposure.12 Furthermore, when both
waterborne and dietary exposure are considered, the observed
mortality correlated linearly with gill Cu levels, suggesting that
gill Cu burden may serve as a good predictor of Cu toxicity
(Figure 3).
When compared to influx rate, gill Cu accumulation reveals
the different metal distribution patterns between uptake
pathways. The observed significant correlation between mortal-
ity and gill Cu concentrations in the present study indicates a
possibility of the extension of a fish gill modeling approach
from waterborne to dietary exposure in future study, but raises
questions about the mechanism of dietary Cu toxicity.
Predicting dietary Cu toxicity by gill Cu levels in the present
study is somewhat akin to the prediction of dissolved metal
toxicity by gill metal concentrations in the framework of the
biotic ligand model (BLM) demonstrated in previous studies,
i.e., higher metal concentrations in gill predicted greater
toxicity. In previous studies lethal accumulation (LA50),
expressed as gill metal concentrations (e.g., in 3 or 24 h),
could predict 50% mortality in acute (e.g., 96 h, 27) or chronic
(e.g., 30 d, 28) dissolved exposure. And a slight increase of gill
Cu concentration (i.e., 13 ng Cu g−1 ww) could result in 50%
mortality in rainbow trout during acute dissolved exposure,29
suggesting that the gill is extremely sensitive to Cu exposure.
Although the dietary exposures only resulted in mortality rates
from 0% to 8% and up to 31% for dissolved Cu exposures, our
results suggest that higher branchial Cu concentrations cause
greater mortality. Moreover, fish mortality observed here shows
a linear correlation across the range of gill Cu burdens
examined irrespective of exposure route. If the target site for
dietary Cu toxicity is the gill, then this relationship should be
expected. This was, for instance, demonstrated in tilapia
Oreochromis mossambicus exposed to dietary Cd with increased
apoptosis in the gill.30 Further work is, however, warranted to
evaluate this relationship over a wider range of mortality rates
and a better overlap between dissolved and dietary exposures.
The toxic effect of dietary Cu in marine fish remains less
clear, but the plausible relationship between mortality and gill
Cu levels is on par with the trend summarized from the few
available studies (i.e., increasing sublethal toxicity of dietary Cu
with branchial Cu levels). For example, a dietary Cu exposure
for 3 months, resulting in less than 6 μg Cu g−1 gill
concentration, did not significantly increase the gill MT
concentration, but increased the gill surface area leading to
increased respiration efficiency in rainbow trout Oncorhynchus
mykiss.17 No morphological change in gill of tilapia Oreochromis
niloticus was observed with 11 μg g −1 branchial Cu
concentrations after dietary Cu exposure.31 But 30-day
exposure resulted in a 19 μg g−1 gill Cu concentration,
significant lipid peroxidation, and an increase trend of total
glutathione concentration in the gill of African walking catfish
Clarias gariepinus, suggesting that the fish suffered oxidative
stress, although the gill showed a normal histology.15
The fundamental differences in gill characteristics between
marine and freshwater fish may constrain the extrapolation of
the current understanding of dietary Cu effects on freshwater
fish gill to marine fish. Marine fish exhibit significant
osmoregulatory disturbance on prolonged Cu exposure,32 but
probably as a result of inhibition of components other than
Na+/K+-ATPase in the gill,33 a situation different from that in
freshwater fish. Despite dietary exposure being recognized as an
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important pathway for Cu accumulation in fish,8,34 the
underlying mechanism of dietary Cu toxicity remains unclear.
Further investigation of various toxic end points in gills after
chronic dietary exposure and use of in situ perfusion procedures
will provide insights in marine fish. Nevertheless, gill Cu
concentrations could be indicative of chronic toxicity due to
differences in the relative importance of uptake routes in
different field settings.
To conclude, the present study has provided a first step
toward the integration of dietary and waterborne exposure into
a framework in terms of prolonged exposure, accumulation, and
toxicity. Estimated influx rate quantified Cu exposure and
served to forecast Cu accumulation. However, no direct link
between influx rate and toxicity was established, probably due
to the complication of Cu elimination, detoxification rate, and
tissue regulation within the fish. We have proposed a simple
approach to predict long-term Cu toxicity, centering on the
relationship between gill Cu concentrations and mortality rate,
which might offer the opportunity for a detailed mechanistic
explanation of Cu toxicity, in particular dietary Cu toxicity, to
marine fish. It may also offer potential as a realistic direct tool
for environmental monitoring and risk assessment. This
relationship, however, remains to be tested in other marine
and freshwater organisms using experimental methods allowing
accurate measurement of ingestion rate and assimilation
efficiency.
■
*
ASSOCIATED CONTENT
S Supporting Information
Table giving the exposure concentrations, estimated influx rate,
wet weight, standard length, condition factor, liver somatic
index, specific growth rate, and mortality rate of the blackhead
seabream Acanthopagrus schlegelii schlegelii, table giving the
relationships between intestinal/hepatic Cu concentrations and
estimated influx rates at various Cu AE levels, and a figure
giving the relationships between intestinal Cu concentrations
and dietary influx rate estimated from various AE values. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: wwang@ust.hk
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank the anonymous reviewers for their helpful comments
on this work. This study was supported by a General Research
Fund from the Hong Kong Research Grants Council (662610).
■
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