Environmental Toxicology and Chemistry—Volume 37, Number 5—pp.

1367–1377, 2018
Received: 7 August 2017 | Revised: 5 October 2017 | Accepted: 4 January 2018 1367

Environmental Toxicology

Trophic Transfer of Cd from Duckweed (Lemna minor L.)

to Tilapia (Oreochromis mossambicus)
Yan Xue,a,* Willie J.G.M. Peijnenburg,b,c Jin Huang,a Dengjun Wang,d and Yan Jine
a
Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control, School of Environmental Sciences and Engineering, Nanjing University of
Information Science and Technology, Nanjing, China
b
National Institute of Public Health and the Environment, Center for the Safety of Substances and Products, Bilthoven, The Netherlands
c
Institute of Environmental Sciences, Leiden University, Leiden, The Netherlands
d
National Research Council Resident Research Associate, US Environmental Protection Agency, Ada, Oklahoma, USA
e
Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware, USA

Abstract: The transfer of the toxic heavy metal Cd from duckweed (Lemna minor L.) to the freshwater fish tilapia (Oreochromis
mossambicus) was investigated. Concentrations of Cd in different chemical forms in duckweed and in different tissues (gut,
edible muscle, and remnants or residual) of tilapia (i.e., ethanol-extractable fraction [FE], HCl-extractable fraction [FHCl], and
residual fraction [FR]) were quantified, and the bioaccumulation factors (BAFs) of Cd in the tilapia body were calculated. Simple
linear regression analysis was used to unravel the correlation and accumulation mechanisms of Cd along the short food chain.
Our results showed that with increasing exposure concentrations of Cd (0–50 mM for duckweed and 0–10 mM for tilapia), the
total, FE (Fe,d)-, FHCl (Fh,d)-, and FR (Fr,d)-Cd concentrations in duckweed and different tissues of tilapia increased progressively.
The Cd sources (aqueous or dietary) influenced the BAF for Cd accumulation in the whole body of tilapia. Furthermore,
regression analyses yielded significant positive correlations (R2 > 0.96) between the Cd concentration in duckweed and in both
the 3 parts and the whole body of tilapia. This finding suggests that Cd transfer from duckweed to tilapia can be quantitatively
evaluated when tilapia is exposed only to duckweed. In addition, the linear regression between Cd accumulation in whole tilapia
and Fe,d-, Fh,d-, and Fr,d-Cd showed that particularly the correlation with Fe,d-Cd is statistically significant (p < 0.001). The
accumulated Cd concentrations and chemical forms in tilapia tissues also positively correlated with Cd sources (solution or
duckweed). Compared with waterborne exposure only, duckweed especially increased the accumulation of Cd in the gut of
tilapia. Taken together, our findings support a strong dependence of Cd accumulation and transfer from duckweed to tilapia on
its chemical forms, especially on Fe,d-Cd. This knowledge may expedite more accurate risk assessment of heavy metals through
aquatic food chain ecosystems. Environ Toxicol Chem 2018;37:1367–1377.
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Keywords: Cadmium; Duckweed; Tilapia; Bioaccumulation; Trophic transfer

INTRODUCTION 2016) and fishes (Farombi et al. 2007; Wu et al. 2017) and then be
transferred to animals of higher trophic levels (e.g., fish and
The past few decades have seen aggravating water quality
shrimp; Erdogrul and Erbilir 2007), thereby bioaccumulating
deterioration and contamination in aquatic environments
along the aquatic food chain (Sofyan et al. 2006). Because fish
caused in part by the ever-increasing loading of a wide spectrum
are close to the top of the aquatic food chain and can
of toxic heavy metals including, but not limited to, arsenic (As),
bioconcentrate heavy metals from contaminated waterbodies,
lead (Pb), chromium (Cr), mercury (Hg), and cadmium (Cd; Chen
consumption of fish by organisms of a higher trophic level or by
et al. 2000; AltaSs and B€uy€ukg€ung€
or 2007). Once these toxic
humans increases the potential risks to ecosystem safety as well
heavy metals are released into the aquatic environment, they can
as to human health (Sinnett et al. 2010).
be directly or indirectly absorbed by plants (e.g., Salvinia
Heavy metals occur ubiquitously in natural systems with
herzogii and Phragmites australis; Maine et al. 2004; Vymazal
varying concentrations and chemical forms, which in turn
determine their transport efficiency, accumulation pathway,
* Address correspondence to xueyan@nuist.edu.cn and thus phytotoxicity in plants (Wu et al. 2005; Zhao et al. 2015).
Published online 5 January 2017 in Wiley Online Library
(wileyonlinelibrary.com). Using Cd as an example, Zhao et al. (2015) found that specific
DOI: 10.1002/etc.4076 chemical forms of Cd controlled the translocation inside plant

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tissues. Soluble (e.g., acid-soluble and exchangeable) Cd
fractions that inherently exhibit high transport efficiency are
bioavailable to plants and can induce high phytotoxicity (Chavez
et al. 2015). Wang et al. (2015) found that Cd tended to
accumulate in insoluble forms (e.g., as precipitate with
phosphate and pectate- or protein-bounded), thus reducing
Cd mobility and toxicity in watercress.
In comparison, accumulation of heavy metals in organisms of
a higher trophic level such as fish is thought to be more complex,
depending on the interplay of multiple parameters such as type
of heavy metal, concentration, as well as fish species (Chen et al.
2000). For example, Chen et al. (2000) identified different
accumulation pathways and modes for 5 heavy metals (Hg, Zn,
Cd, As, and Pb) in aqueous food chains. Mercury and Zn were
found to biomagnify from the lower trophic level plankton to
macrozooplankton and then to the higher trophic level fish,
whereas Cd bioaccumulated (rather than biomagnified) in fish
when fed Cd-contaminated food for a short-term period
(Ruangsomboon and Wongrat 2006). In addition, species, age
(growth stage), and size of fish were also found to affect the
bioaccumulation of heavy metals among different tissues of fish
(e.g., Tilapia nilotica; Amundsen et al. 1997; Al-Yousuf et al.
2000). Wang et al. (2010) reported that concentration enrich-
ment of Cd in aquatic organisms was affected by the exposure
route (waterborne vs dietary exposure). Furthermore, metal
speciation is another key factor determining the trophic transfer
of metals from the aquatic environment to biota (Kosolsaksakul
et al. 2014; Sungur et al. 2014; Chavez et al. 2016).
Although previous studies have examined heavy metal
accumulation and toxicity in both plants (Maine et al. 2004;
Vymazal 2016) and fish (Mansour and Sidky 2002) and through
the plant–fish food chain (Chen et al. 2000; Mendoza-Carranza
et al. 2016), little attention has been paid to unraveling how
different chemical forms (or speciation) of heavy metals impact
the bioaccumulation and trophic transfer of Cd from plant to fish.
Compared with other metal toxicant pollution, Cd contamina-
tion is one of the pressing and challenging global issues (Vidovic
et al. 2005) that has received immense interest all over the world,
primarily because Cd is highly mobile and transferable within
terrestrial ecosystems and beyond (Vig et al. 2003). For example,
Cd has been shown to transfer and accumulate along the
soil–rice–human food chain (Simmons et al. 2008), posing great
risks to the human health—particularly in Asia (e.g., China) where
rice is the most important food crop and dietary staple. Also,
evidence has indicated that Cd is easily transferable in aquatic
food chains (Ruangsomboon and Wongrat 2006) and Cd toxicity
and migration behaviors are highly related to its chemical forms
(Chavez et al. 2016; Wang et al. 2015). It is reasonable to expect
that diverse forms of Cd would also behave differently in the
transfer of Cd from plant to fish.
In the present study, we investigated the transfer of Cd from
duckweed to tilapia in the laboratory by monitoring different
chemical forms of Cd (i.e., ethanol-extractable fraction [FE],
HCl-extractable fraction [FHCl], and residual or remnant fraction
[FR]). Duckweed (Lemna minor L.) and tilapia (Oreochromis
mossambicus) were chosen as representative plant and fish
species, respectively, that are commonly found in aqueous


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Environmental Toxicology and Chemistry, 2018;37:1367–1377—Y. Xue et al.
environments. Another reason for selecting duckweed was
because of its known capacity of Cd uptake (Hou et al. 2007) and
tilapia often eat duckweed as their primary food. Specific
objectives of the present study were to: 1) measure accumulation
of Cd in duckweed after short-time exposure to different
concentrations and chemical forms of Cd, 2) investigate the
influence of 2 types of exposure (water only and via duckweed)
on Cd accumulation and chemical forms in tilapia, 3) quantify the
relationships among diverse chemical forms of Cd and Cd
concentrations in these chemical forms in duckweed and Cd
concentrations in dissimilar tissues of tilapia, and 4) identify the
mechanisms responsible for Cd transfer and accumulation from
duckweed to tilapia.
MATERIALS AND METHODS
The tested Cd concentrations were in most cases higher than
realistic environmental concentrations of Cd. The high Cd
concentrations were used to test the responses for duckweed
(L. minor L.) and tilapia, which mimic the scenario (acute
exposure) of Cd accidental spill/release in duckweed and tilapia
in aquatic environments.
Cd accumulation in duckweed
Duckweed was chosen as a representative plant for Cd
exposure experiments. Duckweed (L. minor L.) seedling samples
were collected from Yilian Garden in Nanjing, China. The
seedlings were transferred in Hoagland medium (KNO3 224 mg
L1, Ca(NO3)2  4H2O 235 mg L1, MgSO4  7H2O 62 mg L1,
K2HPO4 160 mg L1, KCl 1.77 mg L1, H3BO3 0.27 mg L1,
MnSO4  H2O 0.11 mg L1, Na2MoO4 0.05 mg L1 Mo, CuSO4
 5H2O 0.03, mg L1 Cu NaFe- ethylenediaminetetraacetic acid
[EDTA] 2.00 mg L1 Fe, and ZnSO4  7H2O 0.13 mg L1 Zn) and
cultivated under natural light conditions in a greenhouse. Before
cultivation the pH of the Hoagland medium was adjusted to
5.5 using 0.1 M NaOH and HCl. The medium was replaced every
2 d during seedling cultivation. Experimental cultures were
initiated by inoculation of a single colony with 2 to 3 fronds.
Duckweed plants having similar frond numbers (2.0  0.1 g,
fresh wt, each concentration) were selected and transferred to
Cd-containing Hoagland medium at Cd concentrations of 0, 0.1,
0.5, 1, 5, 10, 20, and 50 mM CdCl2 (i.e., 0, 0.011, 0.056, 0.112,
0.562, 1.12, 2.25, and 5.62 mg L1 CdCl2), and grown in the
greenhouse at 25  2 8C for 7 d. Triplicate experiments
were performed for each concentration test. At day 7 the plants
were harvested, washed with distilled, deionized water, and then
dried at 70 8C to constant weight. The resulting dry samples were
ground into powder using a stainless steel mill that was also
utilized for tilapia feeding and for analyses of total Cd
concentration and speciation.
Cd accumulation in tilapia from Cd-containing
water
Preliminary experiment to confirm the median lethal
concentration of Cd. Tilapia (O. mossambicus) purchased
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Cd trophic transfer from duckweed to tilapia—Environmental Toxicology and Chemistry, 2018;37:1367–1377
from an unpolluted fishery farm (the water samples were analyzed for
heavy metal analysis: Cd, Pb, and Hg were undetectable and the
concentration of Cu was 0.001 mM, as determined by atomic
absorption spectrophotometer [AAS; TAS-986; Purkinje General
Instrument]) in Nanjing, China (328030 N, 1188460 E), were used as the
representative fish in the present study. Before Cd accumulation
experiments, the tilapia were acclimated to laboratory conditions in
aerated laboratory tanks at optimal water temperatures of 20 to
25 8C (pH 7.4–7.7) for 1 wk under natural photoperiod conditions.
During the acclimation period, the fish were fed with commercial,
unpolluted fish food (Cd free) twice a day (morning and afternoon) at
a daily rate of 5% body weight (fresh wt).
After 1 wk of acclimatization, tilapia (6 tilapia per aquarium) in
good health and having similar sizes (body wt of 20.2  2.4 g
fresh wt; total length of 12.4  0.6 cm) were transferred to
experimental indoor aquaria (0.5 m  0.5 m  0.5 m) containing
50 L of diluted (medium:distilled water, 1:25, v/v) MA medium
(Ca(NO3)2  4H2O 50 mg L1, KNO3 100 mg L1, NaNO3
50 mg L1, Na2EDTA 5 mg L1, bicine 500 mg L1, disodium
b-glycerophosphate 100 mg L1, CoCl2  6H2O 5 mg L1,
FeC13  6H2O 0.5 mg L1, Na2MoO4  2H2O 0.8 mg L1,
1 1
MgC12  6H2O 50 mg L , MnCl2  4H2O 5 mg L , ZnCl2 0.5 mg
L1, H3BO3 20 mg L1, and Na2SO4 40 mg L1; Maeda et al. 1993)
and different Cd concentrations containing 0, 0.5, 1, 2, 4, 6, 8, 10,
20, and 50 mg L1 of Cd (added as CdCl2). The aquaria were
operated under a 12:12-h light:dark cycle. Fish were fed using a
controlled, Cd-free diet twice a day. Triplicate experiments were
carried out for each concentration treatment. The MA medium
was replaced every 2 d. The trial tests were performed for 7 d. The
results showed that 10 mg L1 (89 mM) of Cd were the median
lethal concentration, which is often used as the highest
concentration for acute toxicity testing experiments (Maeda
et al. 1993).
Formal experiment. The process of the experiment was the
same as in Preliminary experiment to confirm the median lethal
concentration of Cd, in the Materials and Methods section.
The Cd concentrations of 0, 0.5, 1, 2, 4, 6, 8, and 10 mg L1 were
added as CdCl2. When the culture was finished, tilapia were
harvested, washed with deionized water, and separated into 3
parts (Maeda et al. 1993): edible muscle, gut, and the residual
fraction including bones, scales, fins, and skin. Each part was cut
into small pieces and freeze-dried using a freeze-dryer vacuum-
concentrator (model FD-1A-50; Shanghai Jingxue Science
Apparatus CO., LTD, China). The dried samples were weighed
and then ground into powder using a stainless steel mill that was
then used for analyses of total Cd concentration and speciation.
The dry mass ratio of edible muscle:guts:remnants was
approximately 5:1:6.
Transfer of Cd from duckweed to tilapia
To simplify and strengthen the results of the Cd transfer, the
present study used 2 experimental designs according to Maeda
et al. (1993). In one of the designs the duckweed was cultured in
Cd-contaminated water (2.1), whereas in the other design the
tilapia were fed dried duckweed.
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In the transfer experiment, 2 tilapia (20 g fresh wt; 12 cm in
length) were fed with duckweed that had accumulated Cd from
the previous stage. Each fish was placed in an indoor aquarium
(0.5 m  0.5 m  0.5 m) filled with 50 L of aerated, diluted
(medium:distilled water, 1:25, v/v) MA medium and main-
tained under a 12:12-h light: dark cycle. First, to acclimate all
fish to the experimental conditions, they were fed a controlled
Cd-free diet (commercial fish food) twice a day at 9:00 AM and
5:00 PM for 3 d. The fish were able to consume the given diet
within 10 to 15 min after diet delivery. They were fed for 7 d
with the dry Cd-dosed duckweed powder containing different
chemical forms of Cd ( 11 mg of dry wt duckweed per fish per
day; 154 mg total for the entire experiment duration). The
major reason for employing the dried duckweed for feeding
the tilapia was that this procedure allowed us to obtain Cd
concentrations that were sufficiently high enough to accurately
assess bioaccumulation for tilapia. Each treatment was
performed in triplicate, and the medium in the aquaria was
renewed once every 2 d. As previously described, the fish were
harvested, washed, and separated into 3 parts for Cd
concentration and chemical speciation analyses.
Sample digestion
The Cd concentrations in the duckweed were analyzed
according to the method of Wang et al. (2003) with some
modifications. Briefly, 0.4  0.01 g of dry-powder plant samples
were combined with 10 mL of a mixture of concentrated HNO3
(8.7 mL) and HClO4 (1.3 mL). The resulting mixture was digested
first at 180 8C for 6 h and then at 220 8C for 2 h. During digestion,
additional concentrated HNO3 and/or HClO4 were added, if
needed, to digest the sample until no visible plant tissues
remained in the digestion solution. The digested solution was
then transferred to a flask, and deionized water was added to
obtain a final volume of 25 mL for Cd analysis.
Samples of fish tissues were digested using the method of Yi
et al. (2011) with minor modification. In brief, 0.4  0.01 g of
dry-powder samples of different fish tissues were put directly
into acid-washed Teflon digestion vessels. Five mililiters of
concentrated HNO3 was added into each vessel and evapo-
rated until all HNO3 was gone at the temperature of 100 8C
using XT-9800 pretreatment heater (Shanghai Jingxue Science
Apparatus CO.). Afterward, 5 mL of concentrated HNO3:H2O2
(4:1, v/v) mixture solution were added to each vessel and
digested in a microwave. To check the digestion efficiency,
each digestion experiment was conducted in parallel with
control treatments that included a reagent blank, a reference
standard material (yellow croaker, Pseudosciaena crocea,
GBW08573, from the National Research Center for Standards,
Beijing, China), and a sample without addition of the mixture of
concentrated acids. Specifically, microwave digestion included
3 steps: digestion at 0.5 MPa for 2 min, 1.0 MPa for 2 min, and
1.5 MPa for 3 min. After microwave digestion, the samples were
cooled down at room temperature for at least 1 h before being
transferred to plastic test tubes. Deionized water was added to
each tube, reaching a final total volume of 100 mL before Cd
concentration measurements (Yi et al. 2011).


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Sequential extraction of different Cd forms in
duckweed and tilapia
Samples of duckweed and different tissues of tilapia were
sequentially extracted using the methods of Wu et al. (2005) and
Wu et al. (2012) with modification, to examine the different
chemical forms of Cd. The Cd species in duckweed and tilapia
samples were chemically divided into FE-Cd, FHCl-Cd, and FR-Cd
fractions, respectively. Specifically, a 3-step sequential extrac-
tion procedure was carried out: 1) 80% ethanol was used to
extract soluble Cd (FE-Cd), primarily including soluble Cd and
aminophenol Cd; 2) 0.6 M of HCl were utilized to extract
insoluble CdHPO4 and Cd3(PO4)2, other Cd-phosphate com-
plexes, and Cd integrated with pectates and proteins (FHCl-Cd);
and 3) either a concentrated HNO3-HClO4 or HNO3-H2O2
mixture was used to release residual Cd (FR-Cd) that cannot be
extracted by ethanol or HCl from duckweed and tilapia.
The 3-step sequential Cd extraction procedure was as follows:
Dry-powder samples of duckweed or tilapia (4 g) were introduced
into a 50-mL centrifugal tube containing 25 mL of extractant (80%
ethanol). The mixture was then placed in a 30 8C water bath for
18 h, and then centrifuged at 5000 g for 10 min. The resulting the
supernatant (first extraction, containing FE-Cd) was collected. The
extraction procedure was repeated again by adding another
20 mL of extractant (0.6 M HCl) to the residual sample, which was
again placed in the water bath at 30 8C for 2 h, and then
centrifuged at 5000 g for 10 min. The supernatant (second
extraction, containing FHCl-Cd) was collected. The first and
second extractions were evaporated on an electric plate at
70 8C until a constant weight was achieved. The evaporated
extraction containing FE-Cd or FHCl-Cd was performed by using
HNO3-HClO4 (87:13, v/v, for duckweed) or HNO3-H2O2 (4:1, v/v,
for fish), respectively. The solid sample (containing FR-Cd) in the
centrifugal tube after second extraction was directly digested
using the same mixtures of HNO3-HClO4 or HNO3-H2O2 for
determining the concentration of residual Cd. The chemical forms
of FE, FHCl, and FR in duckweed were expressed as Fe,d, Fh,d, and
Fr,d, respectively. The chemical forms of FE, FHCl, and FR in tilapia
were expressed as Fe,t, Fh,t, and Fr,t, respectively.
Cd concentration measurement
The concentrations of Cd extracted using different extrac-
tants (described in Sequential extraction of different Cd forms in
duckweed and tilapia) in both duckweed and in different tissues
of tilapia were determined using an AAS (TAS-986; Purkinje
General Instrument) or a graphite furnace if the Cd concentration
was low (at ppb level). Reagent blanks, analytical duplicates, and
standard reference materials were measured in parallel for
quality assurance and quality control and to determine extrac-
tion efficiency. The recovery rates for the standard reference
materials were determined to be > 95%.
Bioaccumulation factors
When exposed to the aqueous phase Cd, the bioaccumu-
lation factor (BAFa) was calculated using the formula BAFa ¼
Ct/Cs L kg1, where Ct represents the mean Cd concentration in


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Environmental Toxicology and Chemistry, 2018;37:1367–1377—Y. Xue et al.
the whole body of tilapia and Cs represents the Cd concen-
trations in the solution to which tilapia are exposed. When
exposed to dietary Cd (duckweed), the bioaccumulation factor
(BAFd) was calculated as BAFd ¼ Ct/Cd kg duckweed kg1 (YW.
Wang et al. 2008), where Ct represents the mean Cd
concentration in the whole body of tilapia and Cd represents
the mean Cd concentration (mg kg1) in duckweed.
Statistical analyses
All experimental data were reported as mean values 
standard deviations. Bivariate correlation analyses were per-
formed to determine the relationship among different forms of
Cd in duckweed and Cd concentration in different tissues of
tilapia using Excel 2013. Significant differences were identified
by one-way analysis of variance using the least significant
difference test with a 95% confidence limit (p < 0.05).
RESULTS
Cd accumulation in duckweed (L. minor) from
exposure to Cd in solution
Table 1 presents the accumulation of Cd in duckweed
(L. minor) as a function of the Cd concentration in aqueous
solution. With increasing aqueous Cd concentrations, the total
accumulated Cd concentration in duckweed increased signifi-
cantly (p < 0.05). For example, when the Cd concentration in the
exposure medium was increased from 0.1 to 50 mM, the Cd
concentration in duckweed was elevated monotonically from 5.8
to 530 mg/kg dry weight. Similar trends were observed for Cd
accumulation in different chemical forms (i.e., the concentrations
of Fe,d, Fh,d, and Fr,d increased significantly [p < 0.05] on
increasing the Cd concentration in the aquatic exposure
medium; Table 1). Interestingly, regardless of the external Cd
concentration, Cd accumulated in duckweed was mostly in the
form of Fh,d, ranging from 76.2 to 88.1% of the total accumulated
Cd concentration (Table 1). In comparison, much lower
percentages, 8.1 to 16.4% and 3.9 to 11.3% Cd, were found
in the forms of Fe,d and Fr,d, respectively.
Cd accumulation and its chemical forms in tilapia
from exposure to Cd in solution
Total accumulated concentrations and concentrations in
different chemical forms of Cd in tilapia gut, edible muscle,
and remnants as a function of exposure concentration are shown
in Figure 1. In agreement with the results shown for duckweed
(Table 1), Cd accumulation in each part of the tilapia body
increased significantly (p < 0.05) with increasing Cd concentra-
tion in solution. Furthermore, among the different tissues of
tilapia, Cd concentrations in the gut were the highest. However,
because of its small total mass, the gut was not the dominant
tissue for Cd bioaccumulation overall, accounting for only 22.6
to 28.7% of total accumulated Cd. The percentage of Cd
accumulation in the different tissues followed the order of
remnants (61.7–65.2%) > gut (22.6–28.7%) > edible muscle
(8.1–11.2%).
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TABLE 1: Accumulation and chemical forms of cadmium (Cd) in duckweed (Lemna minor L.)

Cd concentration in duckweed (mg Cd/kg dry wt [%])

Cd concentration in water (mM) Total Fe,d-Cda Fh,d-Cdb Fr,d-Cdc

0 ND ND ND ND
0.1 5.8  0.55A 0.82  0.091A (14.1C,D) 5.1  0.039A (87.9A) 0.23  0.019A (3.9A)
0.5 23.9  2.47B 3.9  0.39B (16.4C,D) 20.3  2.44B (85.0A) 1.4  0.15B (5.9C)
1 49.0  5.20C 8.00  0.92C (16.3D) 37.4  4.21C (76.3A) 2.6  0.30C (5.3C)
5 147  19.6D 16.4  1.48D (11.2C) 112  12.6D (76.2A) 12.2  1.33D (8.3D)
10 270  30.3E 26.3  3.11E (9.8A,B) 231  28.9E (85.6A) 17.9  1.77E (6.6C)
20 420  40.1F 34.1  3.90F (8.1A) 370  41.7F (88.1A) 19.4  2.01F (4.6B)
50 530  56.6G 56.3  6.02G (10.6B) 429  52.4G (80.9A) 59.7  6.42G (11.3E)
Values in parentheses are relative % contents.
a
Fe,d-Cd—extracted by 80% ethanol; soluble Cd including nitrate, chloride, and aminophenol Cd.
b
Fh,d-Cd—extracted by 0.6 M HCl, insoluble CdHPO4 and Cd3(PO4)2, other Cd-phosphate complexes, and Cd integrated with pectates and proteins.
c
Fr,d-Cd—in the residuals.
Significant differences at p < 0.05 (analysis of variance).
ND ¼ not detectable.

With respect to the different chemical forms, Cd accumula-
tion in tilapia varied in a concentration-dependent manner
(Figure 1). For example, Cd accumulated in the gut was
predominately in the form of Fh,t, accounting for 78.1 to
85.8% of the total amount of Cd that had accumulated, followed
by Fr,t (9.8–11.7%) and Fe,t (5.6–10.3%), respectively (Figure 1A).
In contrast, accumulation of the various forms of Cd showed a
different pattern in the edible muscle (Figure 1C). Specifically,
Fh,t and Fe,t had almost equal percentages and accounted for
39.2 to 49.3% and 41.1 to 48.8%, respectively, of total
accumulated Cd, whereas Fr,t (10.8–17.0%) was approximately
3 times lower than the other 2 forms. In remnants (Figure 1B),
however, Fh,t (48.3–57.7%) was significantly higher than Fe,t
(31.3–43.1%) and Fr,t (10.8–15.6%).
Transfer and bioaccumulation of Cd from the
duckweed to tilapia
Table 2 shows Cd accumulation in 3 different tissues of
tilapia fed with duckweed powder containing diverse amounts
of Cd (Table 1). Accumulation of Cd in tilapia increased
significantly from 0.17 to 12.0 mg kg1 dry weight with
enhanced Cd content (from 5.8–530 mg kg1 dry wt) in
duckweed. Consistent with the findings of direct exposure of
tilapia to Cd in solution (see text under the heading Cd
accumulation and its chemical forms in tilapia from exposure to
Cd in solution, in the Results section), Cd concentrations in the
gut were the highest, followed by that in remnants and then in
edible muscle.
However, the percentage of Cd in different tissues of tilapia
was different from the findings of direct exposure of tilapia to Cd
in solution (Cd accumulation and its chemical forms in tilapia
from exposure to Cd in solution); the gut of tilapia, through food
transfer, accumulated greater amounts of Cd than the other
2 tissues (edible muscle and remnants). For example, 55.8 to
64.7% of Cd accumulated in the guts, whereas edible muscle
and remnants accumulated 1.1 to 1.2% and 32.0 to 37.2%,
respectively, of the total amount of Cd collected. Linear
regression analyses confirmed a significantly positive correlation
(with R2  0.96) between Cd content in duckweed and Cd uptake
by tilapia tissues (Figure 2).
BAFs for Cd in the whole body of tilapia via
different exposure routes
Figure 3 shows BAFa and BAFd as functions of the Cd
concentrations in the exposure mediums (either water or dried
duckweed). The BAFa decreased with an increase of Cd
concentration in water first and then remained steady. The
BAFd increased at first and then decreased with the increase of
Cd concentration in duckweed. The BAFs for Cd in the whole
body of tilapia varied with Cd sources. When exposed directly to
water, BAFa values ranged between 2.82 and 5.46 L kg1,
whereas the BAFd values were in the range of 0.035 to 0.019 kg
duckweed kg1 via duckweed.
Relationship between chemical forms of Cd in
duckweed and tilapia
The Cd concentration accumulated in whole tilapia was
significantly correlated with the chemical forms of Cd in
duckweed, as shown by the high linear regression constants
(Figure 4). The correlation with Fe,d-Cd (Figure 4a; R2 ¼ 1.00) was
the best of all, at a p < 0.001 significant level, followed by Fh,d-
Cd (Figure 4b; R2 ¼ 0.95), and Fr,d-Cd (Figure 4c; R2 ¼ 0.90).
Figure 5 shows the collections of different chemical forms of
Cd in different tissues of tilapia exposed directly to water and via
duckweed. The concentrations of Fh,t and Fr,t accumulated in
tilapia followed the same order of gut > remnants > edible
muscle, regardless of Cd source or exposed concentration
(Figure 5C–F). However, the concentration of Fe,t-Cd varied with
the Cd source. When exposed to aqueous phase Cd (Figure 5A),
the order of Fe,t-Cd concentration in tilapia was remnants > gut
> edible muscle, whereas Fe,t-Cd concentration was the highest
in the gut when the Cd source was duckweed (Figure 5B).
Overall, all chemical forms of Cd accumulated in different tilapia
tissues increased significantly (p < 0.05) with increasing Cd
concentrations in the exposure medium (solution or duckweed).

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FIGURE 2: Regression analyses of accumulated Cd concentration (mg

Cd/kg dry wt) between Lemna minor and Oreochromis mossambicus
whole body, and between L. minor and the 3 parts of O. mossambicus.
Tilapia, n ¼ 30. R2, the coefficient of determination, is significant at
p < 0.001.

For example, when the Cd concentration rose from 0.5 to

10 mg/L in the aqueous solution, Fe,t-Cd accumulation in the gut,
remnants, and edible muscle increased from 0.67 to 10.97, 1.23
to 16.87, and 0.31 to 3.44 mg kg1, respectively. Similarly, Fe,t-,
Fh,t-, and Fr,t-Cd in all parts of tilapia increased monotonically
with increasing Cd content in duckweed.

FIGURE 1: Accumulation and chemical forms of Cd in different tissues
of tilapia (Oreochromis mossambicus) from Cd-containing water.
(A) Gut, (B) remnants, and (C) edible muscle. Different lower case
letters at tops of bars indicate significant dissimilarities at p < 0.05
(analysis of variance) within the same forms of Cd with Cd concentration
in water. One-half of the tilapia did not survive. #Because duckweed
and tilapia have differing Cd endurance capacities, diverse Cd exposure
concentrations were selected in the present study. –The analytical data
are negative or near 0 when Cd concentration was 0 mM. Fh,t-Cd, Fe,t-
Cd, and Fr,t-Cd express the Cd chemical forms of HCl-extractable
fraction, ethanol-extractable fraction, and residual or remnant fraction,
respectively, in tissues of tilapia.
DISCUSSION
Cd chemical forms
The chemical forms of Cd affect its uptake pathways and thus
the Cd bioaccumulation and transfer through food chains. In
other words, the chemical forms of Cd determine the
bioaccumulation efficiency and toxic effects of Cd for each
trophic-level organism along a food chain (X. Wang et al. 2008;
Sungur et al. 2014). For instance, soluble Cd in inorganic form
and organic form (extracted by 80% ethanol, FE-Cd) exhibits
remarkably stronger migration ability, compared with the
undissolved Cd phosphate and Cd oxalate (extracted by 0.6 M
HCl, FHCl-Cd). This is because soluble Cd can easily penetrate
the symplasm system and it is thus highly transferable among
different tissues (Fu et al. 2011), resulting in more severe toxic
effects compared with those of other Cd species (X. Wang et al.

TABLE 2: Bioaccumulation and transfer of cadmium (Cd) in the duckweed–tilapia short food chain

Organisms Cd in organisms (mg Cd kg1 dry wt)

Lemna minor L.a (Cd in water, mM) 5.8  0.55 (0.1) 49.0  5.2 (1) 270  30.3 (10) 420  40.1 (20) 530  56.6 (50)
Oreochromis mossambicus whole bodyb 0.17  0.017A 1.7  0.23B 5.6  0.53C 7.9  0.79D 12.0  1.5E
O. mossambicus gut 0.76  0.052A 7.3  0.88B 21.5  1.9C 30.7  3.3D 49.4  6.4E
O. mossambicus edible muscle 0.0043  0.0003A 0.29  0.018B 1.6  0.17C 2.1  0.25D 2.9  0.32E
O. mossambicus remnants 0.22  0.024A 2.0  0.19B 6.3  0.60C 8.9  0.82D 13.4  1.6E
Values in parentheses are Cd concentrations in the medium culturing duckweed.
a
L. minor containing different Cd was chosen from Table 1.
b
Whole body Cd of the tilapia was calculated from the Cd concentration in each of the organ parts and the weight ratios of those parts.
Significant differences at p < 0.05 (analysis of variance).
Lemna minor L. ¼ duckweed; Oreochromis mossambicus ¼ tilapia.

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FIGURE 3: Bioaccumulation factors (BAFs) for Cd from the solution and from dietary (duckweed) in the whole body of tilapia. The whole body of the
tilapia was calculated from the weight ratios of these parts and Cd concentration (mg/kg dry wt) in each of the 3 parts shown in Table 2. One-half of
the tilapia did not survive. BAFa denotes Cd in the whole body of tilapia exposed to the solution. BAFd represents Cd in the whole body of tilapia via
duckweed.
2008; Mwamba et al. 2016). In comparison, FHCl-Cd integrat-
ing with proteins and pectic acids had low or no phytotoxicity,
which is responsible for the adaptation of plants to Cd stress
(Fu et al. 2011). In the present study, the majority of Cd in
duckweed was in the form of Fh,d-Cd, likely complexed with
pectates and proteins (Table 1), followed by Fe,d-Cd. Similar
results have been found in previous studies. For example, the
largest portion of Cd accumulated in roots and shoots of
cabbage was found to be integrated with proteins and
pectates (extracted by 1 M NaCl; Qiu et al. 2011). Similar
findings were reported as well for Porphyra yezoensis and
Laminaria japonica (Zhao et al. 2012). In addition, Wu et al.
(2005) found that Cd-resistant barley genotypes had higher
concentrations of pectate- and protein-integrated Cd and
lower concentrations of Cd in water-soluble and inorganic
forms, compared with Cd-sensitive genotypes. Further studies
are needed to decipher whether pectate- and protein-
integrated Cd (Fh,d-Cd in the present study) in duckweed
play an important role in alleviating the toxicity of Cd.
Cd concentrations in aquatic organisms
Our findings clearly show accumulation of Cd in duckweed as
a result of exposure to Cd in aqueous solution. Accumulation
was found to increase with increasing Cd concentration in the
exposure medium (Table 1). These results support previous
reports that duckweed can absorb Cd from surrounding
environments (Hou et al. 2007).
Fish can accumulate heavy metals from aquatic environments
(Kibria et al. 2016). For example, Hook and Fisher (2001)
reported that the body burden of Cd of the copepod Acartia
hudsonica increased 2-fold more than the background concen-
tration after a short-term exposure at 5 nM Cd. Furthermore, the
heavy metal accumulation capacity of fish varies among types of
fish as well as among the different tissues of each fish (Lwanga
et al. 2003). Al-Yousuf et al. (2000) observed that Cd enrichment
in Lethrinus lentjan fish tissues varied in the order gut > muscle
> skin. In the present study, Cd accumulation in different tilapia
tissues was affected by the Cd source (i.e., solution or diet).
When exposed to solution, the order of Cd accumulation in
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different tilapia tissues was remnants > gut > edible muscle.
Whereas when the Cd source was diet, the order was
gut > remnants > edible muscle. We further observed that the
contents of Cd in all tilapia tissues increased with boosting the
solution Cd concentrations, whereas Cd enrichment in the gut
was greater than enrichment in muscle and remnants (Figure 1).
The results indicate that diverse tissues have different abilities to
accumulate heavy metals (e.g., Cd). Karadede et al. (2004)
reported that in the gut metallothioneins can be produced to
alleviate the toxicity of heavy metals. Other studies also showed
that muscle was not an active tissue for heavy metal accumula-
tion (Mansour and Sidky 2002; Yilmaz et al. 2007). Thus the fish
gut, rather than other fish tissues, is more often used to indicate
the extent of water pollution (Karadede et al. 2004). Further
studies are needed to improve our current knowledge in terms of
which types of proteins or peptides are responsible for binding
and detoxification of Cd in the gut tissue of tilapia.
Biotransformation of Cd from duckweed to
tilapia
There have been reports that dietary exposure to Cd might
cause Cd biomagnification, a process whereby chemical
concentrations increase in aquatic organisms at each successive
trophic level because of increasing dietary exposures in aquatic
food webs (Wang et al. 2010). Other studies have also shown
that Cd accumulation by aquatic organisms varies largely with
the exposure route of Cd (Sofyan et al. 2007). More specifically,
Sofyan et al. (2007) found that compared with co-exposure to
both aqueous and dietary sources, the individual dietary
exposure pathway was less harmful to Ceriodaphnia dubia.
Our results indicate that tilapia (both whole fish and individual
tissues) bioaccumulated Cd (no biomagnification) when ex-
posed via diet (Table 2). Table 2 shows that the Cd concen-
trations in both whole fish and all 3 tissues are significantly lower
than those in duckweed, the dietary source of Cd. However,
feeding duckweed containing higher Cd concentrations to the
tilapia resulted in growth in the accumulated Cd concentrations
in tilapia and the 3 tissues (i.e., the gut, edible muscle, and
remnants). Furthermore, a positive correlation (R2 > 0.96) was


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FIGURE 4: Relationship between Cd chemical forms (Fe,d, Fh,d, and Fr,d)
in duckweed and accumulated Cd concentration in different tissues of
tilapia fed with the Cd-dosed duckweed for 7 d. The whole body of the
tilapia was calculated from the weight ratios of these parts and Cd
concentration (mg/kg dry wt) in each of the 3 parts shown in Table 2. Fe,d-
Cd, Fh,d-Cd, and Fr,d-Cd express the Cd chemical forms of ethanol-
extractable fraction, HCl-extractable fraction, and residual or remnant
fraction, respectively, in duckweed.
observed between Cd concentrations in duckweed and Cd in
the tilapia whole body (Figure 2). These results clearly suggest
that diet represents a direct pathway for Cd transfer from
duckweed to tilapia. Similar findings were reported previously
(Notten et al. 2005).
As shown in Table 2, regardless of the Cd source (aqueous or
dietary), Cd concentration was highest in the gut of tilapia,
consistent with the findings reported elsewhere (Roesijadi 1992).
According to Roesijadi (1992), Cd exposure resulted in Cd
accumulation in the gut because the gut plays a major role in
detoxification and excretion of metals through the induction of


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Environmental Toxicology and Chemistry, 2018;37:1367–1377—Y. Xue et al.
metal-binding proteins. Moreover, Chen et al. (2017) found that
heat-stable protein could bind most of the accumulated Cd in 2
different earthworms (Metaphire guillelmi and Eisenia fetida);
E. fetida had a higher proportion of proteins, resulting in a better
capacity to detoxify Cd and a greater tolerance of the metal than
in M. guillelmi. Al-Yousuf et al. (2000) found that the main
location of Cd accumulation varied strongly with fish species.
Several studies reported that Cd accumulation occurred mainly
in the liver, as in carp (Suresh et al. 1993) and in L. lentjan
(Al-Yousuf et al. 2000). This is not consistent with our results.
Nevertheless, the present study shows that the exposure route
of Cd (aqueous or dietary) influences the percentage of the total
Cd in different tissues of tilapia. The gut accumulated a higher
percentage of Cd via the diet than via the solution (Table 2).
BAFs for Cd in the whole body of tilapia
Bioaccumulation factors of fish for trace elements (Cd, Co, Cr,
Cu, Mn, Pb, and Zn) have been reported to be affected by the
metal concentration in the environment (Alhashemi et al. 2012).
Figure 3 shows that BAFa and BAFd varied with the Cd
concentration in the water and in the duckweed, respectively,
consistent with previously documented findings (Alhashemi
et al. 2012). Julian (2013) supposed that the BAF of Hg was
related primarily to the metal source. The BAF of Cd as obtained
in our study was in line with the hypothesis. Figure 3 indicates
that the Cd source influenced the whole-body accumulation of
Cd by tilapia. Arnot and Gobas (2006) controlled laboratory
conditions in which the diet was either included or not. It was
detected that BAF (diet included) was different from the
bioconcentration factor (diet not included), which agreed with
our findings. It was found that BAF was related not only to the
ambient concentration but also to growth dilution, elimination
and uptake, fecal egestion, and metabolic biotransformation
(Arnot and Gobas 2006). However, there was no direct evidence
supporting the influence of Cd chemical forms in different Cd
sources on the BAF. Further studies are needed to improve our
current knowledge of potential mechanisms responsible for the
metal transfer in the food chain.
Migration of different Cd chemical forms from
duckweed to tilapia
As discussed in section Cd chemical forms, different chemical
forms of Cd can result in different degrees of toxicity to
organisms and migration efficiency along food chains (X. Wang
et al. 2008; Chavez et al. 2016). For instance, Su et al. (2014)
reported a positive exponential relationship between peanut
shoot Cd concentration and FE-Cd concentration. Chavez et al.
(2015) found that the accumulation of Cd in cacao beans was
significantly correlated with Cd chemical forms in soils. As clearly
shown in Figure 4 in our study, the Cd concentration in tilapia
was closely correlated with the Cd chemical forms in their dietary
source (duckweed). The best linear correlation was observed
between Cd concentration in the whole tilapia body and Fe,d-Cd
in duckweed, whereas the correlation was weaker with Fr,d. In a
similar study, Prankel et al. (2005) fed sheep with a diet
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Cd trophic transfer from duckweed to tilapia—Environmental Toxicology and Chemistry, 2018;37:1367–1377 1375

FIGURE 5: Concentration of different Cd chemical forms in different tissues of tilapia exposed to water phase and via food (duckweed). One-half of the
tilapia did not survive. (A) Fe,t-Cd exposed to medium, (B) Fe,t-Cd exposed to duckweed, (C) Fh,t-Cd exposed to medium, (D) Fh,t-Cd exposed to
duckweed, (E) Fr,t-Cd exposed to medium, and (F) Fr,t-Cd exposed to duckweed. Fe,t-Cd, Fh,t-Cd, and Fr,t-Cd express the Cd chemical forms of ethanol-
extractable fraction, HCl-extractable fraction, and residual or remnant fraction, respectively, in tissues of tilapia.

containing organic Cd forms (Cd from plants fertilized with
sewage sludge) and inorganic Cd forms (CdCl2 added to their
diet), respectively. It was found that the chemical forms of Cd
(both organic and inorganic) significantly influenced the rate of Cd
accumulation in sheep. However, such correlation was not taken
into account in their study. In comparison, our research provides
clear new evidence on the important role that Cd chemical forms
played in the accumulation and transfer of Cd along the food
chain, which suggests that the chemical forms of heavy metals
(e.g., Cd) may be a reliable indicator that could be used to predict
their food-chain bioaccumulation and transfer potentials.
It should be mentioned that the concentrations of the
different Cd chemical forms, especially FE-Cd, were affected by
the route of exposure. Figure 5 shows that when exposed to
different Cd sources (solution or duckweed), Cd was enriched in
different forms among different tissues of the fish. Similarly,
Chavez et al. (2016) reported that the Cd concentration in cacao
beans was strongly correlated with exchangeable and acid-
soluble Cd pools. Our study extends the current understanding
that the Cd source and the chemical form of Cd collectively
determine Cd accumulation, translocation, and its chemical
forms in higher trophic organisms along the food chain.
Nevertheless, whereas the chemical form may provide a
convincing mechanistic explanation for observed metal toxicity,
translocation, and migration in the food chain, various potential
interferences could complicate the interpretations and should
be taken into consideration under more complex conditions.
CONCLUSIONS
The bioaccumulation and transfer of Cd were investigated
using a typical short freshwater food chain of duckweed–tilapia,
taking into account different Cd sources. In addition, Cd
accumulation in tilapia, representing the higher trophic level
considered, was studied in detail by measuring Cd accumulation
in 3 tilapia tissues. Bioaccumulation factors of Cd in the whole
body of tilapia were also analyzed. The Cd source is an important
factor influencing the bioaccumulation of Cd in the present
study. Taking all evidence together, we conclude that the
extent of Cd accumulation and transfer in the duckweed–tilapia

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food chain strongly depends on the chemical forms of Cd. In
particular, Fe,d-Cd and Fh,d-Cd showed a significantly positive
correlation (R2 ¼ 0.9995 and 0.9454, respectively) with Cd
accumulation in the whole tilapia, indicating that FE-Cd and
FHCl-Cd present in duckweed play a dominant role in
accumulation of Cd within tilapia and in Cd transfer within the
duckweed–tilapia food chain.
Acknowledgment—The present study was funded by the
National Natural Science Foundation of China (51109109,
41505113, and 41301581), fully supported by an open fund of
the Jiangsu Key Laboratory of Atmospheric Environment
Monitoring and Pollution Control, a project funded by the
Priority Academic Program Development of Jiangsu Higher
Education Institutions. We are also grateful to the Jiangsu
Government Scholarship Program for Overseas Studies.
Data Availability—Data, associated metadata, and calculation
tools are available from the corresponding author (xueyan@nu-
ist.edu.cn).
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