Arch Environ Contam Toxicol (2011) 61:280–291
DOI 10.1007/s00244-010-9620-4
Trophic Transfer of Lead Through a Model Marine Four-Level
Food Chain: Tetraselmis suecica, Artemia franciscana, Litopenaeus
vannamei, and Haemulon scudderi
M. F. Soto-Jiménez • C. Arellano-Fiore •
R. Rocha-Velarde • M. E. Jara-Marini •
J. Ruelas-Inzunza • F. Páez-Osuna
Received: 3 May 2010 / Accepted: 20 October 2010 / Published online: 17 November 2010
Ó Springer Science+Business Media, LLC 2010
Abstract The objective of this investigation was to
assess the transfer of lead (Pb) along an experimental, fourlevel food chain: Tetraselmis suecica (phytoplankton) ?
Artemia franciscana (crustacean, brine shrimp) ? Litopenaeus vannamei (crustacean, white shrimp) ? Haemulon
scudderi (fish, grunt). T. suecica was exposed to a sublethal
dose of Pb in solution and then used as the base of a marine
food chain. Significant differences in Pb concentrations
were found between exposed organisms of the different
trophic levels and the control. Particularly, Pb concentrations in fish of the simulated trophic chain were two-to
three times higher in the exposed specimens than in the
control. Levels of Pb in phytoplankton showed a substantial increase with respect to the solution (level I), with
bioconcentration factors averaging from 930 to 3630. In
contrast, a strong decrease in Pb concentration from phytoplankton to zooplankton (level II) and from zooplankton
to shrimp tissues (level III) was evidenced by bioaccumulation factors \1. Despite the decrease in the
M. F. Soto-Jiménez (&) F. Páez-Osuna
Instituto de Ciencias del Mar y Limnologı́a, Universidad
Nacional Autónoma de México, Apartado Postal 811, 82040
Mazatlán, Mexico
e-mail: [email protected]
C. Arellano-Fiore J. Ruelas-Inzunza
Instituto Tecnológico de Mazatlán, 82000 Mazatlán, Mexico
R. Rocha-Velarde
Mazatlán Aquarium, Mazatlán, Mexico
M. E. Jara-Marini
Centro de Investigación en Alimentación y Desarrollo,
Hermosillo, Mexico
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assimilation efficiency of metal transfer observed in these
two predators, Pb concentration in the grunt fish (level IV)
was higher than in the shrimp (level III) (bioaccumulation
factor [1.0). Some of the added Pb is transferred from the
phytoplankton along the food chain, thus producing a net
accumulation of Pb mainly in fish and, to a lesser extent, in
shrimp tissues. Because Pb is one of the most pervasive
contaminants in coastal ecosystems, its transference by
way of diet and potential net accumulation in higher predators is of ecologic importance for marine life. In addition, because shrimp and adult Haemulon scudderi are
commercially important resources, this issue is of particular relevance to the safety of marine products.
Recent studies have remarked the importance of diet in the
contribution of metals to overall metal body burden in
marine organisms (Schlekat et al. 2002; Wang and Ke
2002; Zhang and Wang 2006) and as a major route for the
transfer of metals in marine food webs (Amiard-Triquet
et al. 1993; Fisher and Reinfelder 1995; Rainbow 2002;
Wang 2002; Mathews and Fisher 2008). However, the
mechanisms that regulate metal transfer through the food
web are still little known (Szefer 1998; Dietz et al. 2000;
Gray 2002; Barwick and Maher 2003). Moreover, understanding the trophic transfer of metals in the most productive marine food webs is even more complex (Wang
2002). The prediction of metal concentrations in aquatic
animals living in tropical and subtropical coastal systems is
more difficult because they involve numerous species and
linking alternatives. In addition, these unique ecosystems
are characterized by wide variations (spatial and seasonal)
in the tidal regime, temperature, solar radiation, evapotranspiration, input of underground and surficial freshwater, strong droughts, and incidence of tropical storms etc.
Arch Environ Contam Toxicol (2011) 61:280–291
(Lankford 1977), all of which might influence the specific
patterns of metal transfer. Thus, the questions regarding the
processes involved in the transfer of metals along food web
components, as well as measurements of bioconcentration,
bioaccumulation, and eventually biomagnification factors,
are difficult to solve in the natural environment of tropical
and subtropical ecosystems.
Experimental studies to understand the trophic transfer
of metals in aquatic food chains have been developed
during the past four decades (e.g., Bryan 1979; Fowler
1982; Besser et al. 1993; Fisher and Reinfelder 1995;
Wang et al. 1996; Nott 1998; Fisher et al. 2000; Evans
et al. 2000; DeForest et al. 2007; Watanabe et al. 2008).
Bioassays have been used to study the transfer processes of
a metal through a marine food chain under controllable
conditions, which is much less complex than what happens
in nature (Luoma 1996). In particular, a laboratory feeding
study is a reasonable approach to understand how metals
move through a realistic food chain and to eventually
predict metal concentrations in the aquatic animals. Laboratory experiments with a defined marine food chain, with
typical species and defined environmental conditions,
decrease the complexity of studying metals passing
through food chains. Through such experiments, it is easier
to quantify concentration, accumulation, and magnification
factors. In addition, they provide data to validate mathematical models to predict metal concentrations in aquatic
organisms. Such studies can be particularly useful in
tropical and subtropical coastal ecosystems because there
are multiple alternatives for metal transfer and unique and
changing environmental characteristics.
The purpose of this research was to examine the consequences of high Pb exposure to phytoplankton and
subsequent Pb accumulation by shrimp and fish by way of
a modeled food chain. The pattern of trophic transfer of
Pb was examined through a simplified four-level food
chain in a marine ecosystem (phytoplankton, zooplankton,
shrimp, and fish). We considered Pb doses in laboratory
experiments that represent Pb concentrations from coastal
environments with moderate (1–3 lg L-1) to increased
(10–50 lg L-1) Pb pollution (Sadiq 1992; El-Moselhy
and Gabal 2004; Cuong et al. 2008). This range of concentrations is typically targeted by most environmental
assessments. To understand Pb movement through the
marine food chain, the following questions were formulated: How much Pb is concentrated by phytoplankton
from seawater? How much of the Pb concentrated by
phytoplankton cells is transferred to the primary consumer
(zooplankton), then to the secondary consumer (crustacean), and then to the tertiary consumer (fish) by way
of diet? Does Pb concentration increase with trophic
level (i.e., fish [ crustacean [ zooplankton [ phytoplankton) in the marine food chain?
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Methods
Experimental Design
To understand Pb movement through the marine food chain
(i.e., trophic transfer), first we exposed cells of the marine
prasinophyte Tetraselmis suecica to 20 lg L-1 Pb. This is
equivalent to 2.5 times the criterion continuous concentration (CCC) given by the national recommended ambient
water quality criteria (CCC = 8.1 lg L-1) (United States
Environmental Protection Agency 2006). The CCC represents the concentration of dissolved Pb that would protect
95% of the species in an aquatic community. It is intended
to be a good estimate of this threshold of unacceptable
effect; however, the derivation of criteria value considers
only the dissolved Pb and completely ignores dietary
exposure routes.
Simultaneously, control batches of microalgae were
exposed to ambient concentrations of Pb in Mazatlán Bay
seawater, which was used as a water source for the
experiments. Phytoplankton biomass exposed to Pb was
used as the only source of food for the entire marine food
chain. Pb passed from phytoplankton to zooplankton
(a model crustacean), then from zooplankton to a benthic
crustacean, and finally from crustacean to fish (metal
transfer by way of diet). The exposure time in each
experiment was enough to allow each consumer to eat
enough biomass of the exposed prey (several times its
own body weight). Although prey items were reared on a
Pb-exposed diet before being offered to the predator, the
entire components of the food chain were exposed to dissolved Pb in seawater from Mazatlán Bay (metal uptake
from seawater).
The structure and composition of the marine food chain
was derived from the availability of cultured organisms and
from a review of previous studies that have examined the
gut contents, feeding strategies, and habitat preferences of
organisms commonly residing in the coastal waters of the
southeastern Gulf of California. An exception was Artemia
franciscana, which is not a coastal species.
Trophic Level I: T. suecica
Tetraselmis suecica was grown in f medium in five 160-L
transparent glass fiber cylindrical tanks (Guillard and
Ryther 1962). Filtered seawater (temperature 28.2°C ±
2.0°C, salinity 34.6 ± 1.2 ppt, and pH 8.1 ± 0.1) was
enriched with essential nutrients (nitrates, phosphates),
trace metals (cobalt, copper, manganese, molybdenum, and
zinc) and vitamins (cyanocobalamin, thiamine, and biotin)
by adding sterile nutrient solutions. Bioassay exposure
concentration of approximately 20 lg L-1 was reached by
adding the exact amount of dissolved Pb(II) from a
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1000 mg L-1 standard solution of Pb(NO3)2. Pb(II) is the
most stable ionic species present in the environment and is
thought to be the form in which most Pb is bioaccumulated
by aquatic organisms (Nussey et al. 2000).
The exposure period was at least 3 days and a maximum
of 4 days, which was estimated by preliminary experiments
as being the time required to accumulate a detectable
concentration of metal. Culture growth was estimated on a
daily basis by the measurement of cell numbers using a
Bürker haemocytometer and every other day by dry biomass concentration (Chini-Zittelli et al. 2006). Included
controls were seawater plus 20 lg Pb L-1 (no algae) and
seawater plus algae (no Pb). Ten lots of 3 annular columns
were cultivated outdoors (in a roof shed) with T. suecica
exposed to Pb and controls. Light was provided with a
14:10-h light-to-dark photoperiod. Aseptic conditions were
kept in the phytoplankton cultures by using closed columns
and filtered air.
Trophic Level II: A. franciscana
For the primary consumer, we used nauplii of A. franciscana obtained from INVE Aquaculture. The eggs were
hydrated, disinfected (in 0.4% aqueous sodium hypochlorite solution for 2 h) and washed extensively with filtered
seawater until all traces of chlorine were removed. Then
the eggs were hatched in filtered seawater (at 26°C 29%
salinity, pH 7.9) under gentle aeration and constant white
illumination. A. franciscana nauplii were harvested after
24 h and transferred to the experimental vessels (fiberglass
tanks 3000 L) containing approximately 2500 L filtered
seawater. The density of A. franciscana nauplii was kept at
350–400 individuals L-1. Organisms were fed on a high
cell concentration of Pb-exposed phytoplankton
(2–4 9 106 cells L-1). Feeding of A. franciscana was
performed two times per day for 17–20 days. Three and 6
replicates were performed in 3 separate sets of experimental runs for the control and test treatments,
respectively.
Trophic Level III: L. vannamei
Approximately 600 juveniles (1.0–1.5 g and 5.3–5.7 cm
weight and length, respectively) of white shrimp L. vannamei were used for this experiment. Shrimp were acclimatized for 3 days in fiberglass tanks (3000 L) with filtered
seawater. During this period, shrimp were fed to satiation
twice a day with nonexposed Artemia biomass. After
acclimatization, the specimens were randomly separated,
30 specimens each, into 16 aquariums (100 L). Twelve
aquariums were used for the specimens exposed to high
doses of Pb, and 4 were used for controls. Shrimp in
exposed aquariums were fed twice a day to satiation with
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adult A. franciscana which had previously been fed with
Pb-exposed microalgae.
To guarantee enough quantity of food to stimulate the
growth of the specimens and to minimize cannibalism,
feeding habits were carefully observed during and after
feeding. Excess food was removed after 1 h (to apparent
satiation). Specimens that were attacked by other specimens during molting stages were removed from the
aquariums and were given as food to the grunt fish. During
the experimental period, three to four molting events were
observed. Biometric characteristics of the shrimp specimens were monitored during the experiments by measuring
length and weight.
Trophic Level IV: H. scudderi
Specimens of grunt H. scudderi were captured at Mazatlán
Bay and immediately transported to the Mazatlán Aquarium. A total of 120 fish of similar total length and weight
(7.2 ± 1.2 cm and 4.6 ± 1.7 g, respectively) were selected
and acclimated for 3 days in fiberglass tanks (3000 L) with
filtered seawater (at 26°C, salinity 35%, and pH 7.9). A
preventive treatment to avoid diseases and parasites was
given with Malaquite green/formalin solution. Subsequently, the specimens were removed and placed, 15
specimens each, into 8 experimental vessels (300 L). Five
vessels were used for the high Pb-exposure fish, and three
for control fish. During the 6 days from capture to the
beginning of the experiment, the fish were fed daily to
satiation with non-Pb-exposed shrimp. Food excess was
removed after 1 h. One day before the feeding experiments,
the fish were not fed to allow complete gut depuration; then
they were fed to satiation with Pb-exposed shrimp. Biometric characteristics of fish specimens were monitored
during the experiments by measuring length and weight.
The seawater used in the experiments was pumped from
the Mazatlán Bay to the Mazatlán Aquarium, and then
filtered with 2-lm filters followed by ozone disinfection
and ultraviolet light exposure. Factors such as salinity,
temperature, and pH were monitored daily and controlled
in the seawater during the experiments. Dissolved oxygen
([7 mg L-1) was kept to 95% to 100% of saturation by
continuous aeration in all vessels and aquariums. Seawater
samples for analysis of dissolved Pb were collected once
per week. Aseptic conditions were maintained as much as
possible in the phytoplankton cultures and the experimental
vessels.
Sampling
We followed protocols of clean techniques for collection
and analysis of the samples to minimize potential contamination (Soto-Jiménez et al. 2008). Figure 1 shows the
Arch Environ Contam Toxicol (2011) 61:280–291
design of the experiment and the program to collect samples and perform analysis. To quantify Pb concentrations in
T. suecica and the dissolved fraction in water, one sample
of harvested phytoplankton cells was collected every
3–4 days, rinsed, divided into replicates, and analyzed for
Pb. Seawater samples were also collected in 1-L low
density polyethylene (LDPE) bottles (LDPE; Nalgene),
which had previously been cleaned for metal analysis. In
the laboratory, seawater and phytoplankton samples were
filtered (200–1000 mL) through a precombusted (at 500°C
for 4 h) and trace-metal precleaned (2 M HCl) glass fiber
filter (GF/F, 0.45 lm) with a low-pressure vacuum pump.
Filtered water samples were transferred into acid-cleaned
polyethylene bottles and acidified to pH approximately1.5
using 6 N HCl Optimum Grade (Fisherbrand) and stored
for at least 1 month. Phytoplankton cells collected in the
filters were carefully rinsed with high purity water to
remove the salt excess and then air dried at 55°C. The total
amount was determined by comparing filter weights before
and after filtration of a known amount of water.
Specimens of A. franciscana nauplii (4–5 g), and randomly selected shrimp and fish specimens (4–12 and 3–5
organisms, respectively) were taken from low and high Pbexposure experiments every week. The collection was
made before the cleaning up of vessels. Organisms were
collected after several hours (6–12) of being fed, so no
depuration was necessary. One composite sample (by culture) of phytoplankton cells (every 3–4 days) and one
sample of whole-body A. franciscana specimens (each
week) were collected for Pb analyses. The fecal pellets
produced by A. franciscana, white shrimp, and fish were
siphoned and filtrated into a 40-lm filter cup and rinsed
with filtered seawater and high purity water. The collection
was made every 3–4 days before the cleaning up of vessels,
but only one composite sample per week was processed for
analysis. Analysis of fecal matter in this study allowed the
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generation of additional information not usually available
in a field study.
Shrimp specimens were dissected to get muscle, exoskeleton (including antennules, carapace, pleopods, and
pereiopods), hepatopancreas, and remaining tissues of cephalotorax (gills, stomach and its content). Fish were dissected for muscle, skeleton (including skull and dorsal and
anal fin-rays), skin and scales, gills, liver, and viscera and
remaining tissues (stomach and its content, kidney, and
other internal organs) (Schmitt and Finger 1987). Samples
of tissues and fecal wastes were frozen and lyophilized
(133 9 10-3 mBar and –40°C) for 72 h. Dried samples
were ground, homogenized, and stored into polypropylene
containers hermetically closed until analysis.
Analysis
All of the samples were processed and analyzed in a HEPA
(class 1000) air filtered, trace-metal clean laboratory using
high-purity reagents and high-purity (18.2 MX cm) water.
Solid aliquot samples (100–200 mg powder) were digested
using 8–10 mL trace metal grade HNO3:HCl (3:1 v/v) into
Teflon vessels (30 mL) and heated at 130°C overnight on a
Mod Block unit. Digested samples were transferred to
cleaned polypropylene vials and diluted to 25 mL with
high purity water. Blanks and certificated reference materials (SLEW-2 estuarine water [n = 5], DORM-2 dogfish
muscle [n = 6], and NIST 1566b oyster tissue [n = 4])
were included in each digestion batch to verify the accuracy of the extraction method. Concentrations of dissolved
Pb in acidified water and in biologic samples were determined using graphite furnace-atomic absorption spectroscopy (Varian SpectrAA 220). Results of the Pb analysis
of the certificated reference material were 0.008 ±
0.001 lg L-1 for SLEW-2, 0.0611 ± 0.012 lg g-1 for
DORM-2 and 0.32 ± 0.02 lg g-1 for NIST 1566b (certified values 0.009 ± 0.001, 0.065 ± 0.007, and 0.31 ±
0.01 lg g-1, respectively) (Soto-Jiménez et al. 2008).
Data Processing
Fig. 1 Schematized experimental procedure to study the trophic
transfer of Pb in a simulated marine food chain
Weight-normalized concentration of Pb in the different
organs and tissues were calculated by multiplying the Pb
concentrations in each analyzed organ tissue by its percentage contribution to total dry body weight. Then the Pb
concentration in the whole individual shrimp and fish was
estimated as the sum of weight-normalized concentrations
in each organ and tissue. One-way analysis of variance
followed by Tukey’s Honestly Significant Difference test
was performed to compare Pb concentrations among
groups and treatments. Linear and nonlinear regression
models were constructed and tested for equality of slopes
and intercepts by analysis of covariance (Sokal and Rohlf
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2003). The level of significance in all statistical tests was
0.05. Statistica 7.0 (Statsoft, Tulsa, OK) and Excel 2007
(Microsoft, Redmond, WA) software were used for all
statistical analyses.
BCF, BAF, and BMF were estimated in the whole-body
burden to evaluate the trophic transfer of Pb through the
marine food chain (Gobas and Morrison 2000). BCF was
determined as the ratio of Pb concentration in the phytoplankton cells to initial Pb concentration in the experimental seawater. BAF was determined by dividing Pb body
burden in predator organisms by Pb concentration in
whole-body burden prey organisms (Bryan 1979). BMF
was calculated by dividing Pb concentration in the wholebody burden in fish by Pb concentration in phytoplankton.
Pb concentrations in the organisms at the end of the
exposure time were used for these factors.
Results
Pb in T. suecica (Level I)
Total Pb concentration in seawater from Mazatlán Bay
varied from 1.80 to 4.43 lg L-1 (average 2.93 ± 1.80
lg L-1) in summer 2006. Dissolved Pb concentrations in
the seawater represented approximately 5% to 10% of total
Pb averaging 0.29 ± 0.12 lg L-1 (0.18–0.50 lg L-1).
Cultures of the tropical marine microalgae T. suecica
(Fig. 2) in this seawater from Mazatlán Bay averaged Pb
concentrations between 2.1 and 7.6 lg g-1 dry weight,
Fig. 2 Averaged Pb concentrations in control (open squares) and
Pb-treated (approximately 20 lg L-1) (filled squares) phytoplankton
(T. suecica) cells. Cells were exposed for 3 to 4 days in each of 10
batches. Data represent the mean of 3 replicate samples per batch,
with error bars representing the SDM. Cell counts ranged from 1.3 to
1.8 9 108 in control samples and 0.7 to 1.2 9 108 in treated samples
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whereas phytoplankton cells exposed to doses approximately 2.5 times higher than the CCC (high Pb-exposed or
test) averaged Pb values from 12.5 to 22.0 lg g-1. Large
variations in the Pb concentrations in the microalgae
depended mainly on the Pb concentrations in the dissolved
phase and cell growth rates in the different batches. A
depletion of the dissolved Pb concentrations was observed
during exposure time, with final concentrations representing 60% to 85% of the initial value. Metal loss in the
control with Pb but no algae was estimated between 5%
and 15% of the added doses (?20 lg L-1), which can be
related to organic complexion and/or precipitation of Pb.
Considering Pb concentration in phytoplankton and the
number of cells by a given volume, the uptake rate at
20 lg L-1 of Pb was 5.7 9 10-7 lg h-1 cell-1 (ranging
2.9–17.1 9 10-7 lg h-1 cell-1) and 5.1 9 10-8 lg h-1
cell-1 (ranging 2.6–15.3 9 10-8 lg h-1 cell-1) for control cells. This Pb concentration is 11 times higher in
exposed cells than in the control, which showed the
capacity of T. suecica to concentrate Pb. High metal
accumulation in marine phytoplankton was confirmed by
high BCF values (1090–1570) in exposed batches respect
to the controls (170–570) (Table 1).
Pb in A. franciscana (Level II)
Figure 3 shows the concentration of Pb (lg g-1 dry
weight) in the organisms and feces of A. franciscana
exposed to high and low doses of Pb as a function of time.
Pb concentrations in whole-body A. franciscana increased
from 1.3 to 15.0 lg g-1 in exposed batches, whereas Pb
accumulation in the organisms from control batches averaged 1.13–1.77 lg g-1. The wide variability is attributed
to differences in the growth stages of A. franciscana during
the sampling and variations in the Pb concentrations in the
microalgae.
Because Pb accumulation in zooplankton results from
the net balance between metal uptake by way of the diet
(and water) and elimination of unassimilated Pb mainly by
excretion into fecal pellets, this material was also collected
and analyzed. During the experiments, Pb in feces
increased from 0.8 to 3.5 lg g-1 and from 1.06 to
11.2 lg g-1 in control and exposed organisms, respectively. A relatively constant Pb concentration was observed
in feces of control A. franciscana, whereas in test organisms the concentrations increased along with the exposure
days. This behavior is related to the variation of the content
of Pb in whole-body live A. franciscana during the same
period (r = 0.66; p \ 0.05).
Transfer of Pb from phytoplankton to the primary consumer A. franciscana was evaluated by calculating the
BAF (Table 1).The calculated values were lower than unity
(Pb in A. franciscana \ Pb in T. suecica) for exposed and
Arch Environ Contam Toxicol (2011) 61:280–291
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Table 1 BCF, BAF, and BMF estimated for a simulated marine food chain (±SD)a
Relations
Factor
Control
n
Test
n
Seawater ? phytoplankton T. suecica
BCF
3630 ± 1140a
Phytoplankton T. suecica ? brine shrimp A. franciscana
BAF
0.48 ± 0.27
30
930 ± 170
30
9
0.81 ± 0.52
Brine shrimp A. franciscana ? white shrimp L. vannamei
BAF
18
0.53 ± 0.45
4
0.24 ± 0.13
12
White shrimp L. vannamei ? fish H. scudderi
Entire food chain T. suecica ? H. scudderi
BAF
1.31 ± 0.27
3
1.40 ± 0.11
5
BMF
0.34 ± 0.24
0.17 ± 0.12
a
BCF was estimated using the initial concentration of dissolved Pb in seawater and the weighted averages of Pb in phytoplankton. BAF and
BMF were estimated by using only the averaged Pb concentrations in organisms at end of the exposure time
Fig. 3 a Averaged Pb concentration in whole brine shrimp A.
franciscana fed control (open squares) and Pb-treated (approximately20 lg L-1) (filled squares) phytoplankton (T. suecica) cells.
(b) Averaged Pb concentration in fecal matter. Data represent the
mean of 9 and 18 replicates/time point for control (open squares) and
treated (filled squares) samples, respectively, with error bars representing the SDM. Sample masses ranged from 4 to 5 g (wet weight)
control organisms, with nonsignificant differences in the
bioaccumulation ability. These results indicate that A.
franciscana does not efficiently accumulate Pb.
Pb in L. vannamei (Level III)
Results of average Pb concentrations in whole-body nonexposed shrimp varied from 0.2 to 1.1 lg g-1 (0.8 ± 0.3)
Fig. 4 a Averaged Pb concentration in whole white shrimp
L. vanammei fed with brine shrimp (A. franciscana) fed control
(open squares) and Pb-treated (approximately 20 lg L-1) treated
(filled squares) phytoplankton (T. suecica) cells. b Averaged Pb
concentration in fecal matter. Data represent the mean of 4 and 12
replicates/time point for control and treated samples, respectively,
with error bars representing the SDM
without a tendency to increase or decrease (Fig. 4). Pb
concentrations in exposed organisms ranged from 0.2 to
3.5 lg g-1 (1.9 ± 1.1 at the beginning and end of the
exposure period, respectively). A linear tendency to
increase the concentration of Pb in the whole-body burden
with the time of exposure was observed. A significant
difference in Pb accumulation rates was observed in
exposed (0.081 lg Pb g-1 day-1) compared with control
specimens (0.017 lg Pb g-1 day-1).
Figure 4 also shows the concentration of Pb (lg g-1 dry
weigh) in the feces of L. vannamei as a function of time.
Concentrations of Pb in feces from control shrimp varied
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from 2.5 to 6.2 lg g-1 with a discrete but significant
(p \ 0.05) tendency to increase with time. Feces samples
from high Pb–exposed shrimp showed low concentrations
during the first 2 weeks of the experiment (3.7–4.6 lg g-1)
but increased until reaching a maximum concentration of
113–116 lg g-1 after 30 days of exposure. The role of
zooplankton (brine shrimp) in the transfer of Pb to shrimp
was evaluated by calculating the BAF (Table 1). For the
second step, from primary to secondary consumer (Artemia–shrimp relation), the BAF values were also lower
than unity in Pb-exposed and control batches.
Pb in H. scudderi (Level IV)
Pb transfer was also evaluated on the four levels of the
marine food chain by analyzing Pb concentrations in fish
and their feces (Fig. 5). Nonexposed fish showed concentrations that ranged from 0.6 to 1.3 lg g-1 (1.0 ± 0.2),
with nondefined tendency regarding time of exposure. Pb
concentrations in the whole-body burden of exposed fish
Fig. 6 Variation of Pb concentration in whole-body burden
H. scudderi as a function of Pb concentrations in L. vanammei used
as feed. Filled and open squares represent control and treated
samples, respectively, with error bars representing the SDM
increased from 0.5 to 0.8 lg g-1 in the first days of the
experiment up to 2.9–3.4 lg g-1 after 35 days of exposure.
Exposed organisms showed a tendency for metal concentration to increase with time. Pb unassimilated by fish was
determined by analyzing fecal pellets. Pb concentration in
feces of control fish was within a narrow range from 1.2 to
1.9 lg g-1 throughout the experiment, whereas in exposed
fish the concentration increased from 1.7 ± 0.2 lg g-1 at
day 1–7.1 ± 2.1 lg g-1 at day 16 and relatively constant
onward (Fig. 5).
Figure 6 shows how Pb concentration in the whole-body
burden in fish increased with Pb concentrations in shrimp.
The relation between concentration of Pb in shrimp (diet)
and fish (consumer), as evaluated by regression analysis
(r2 = 0.95, p \ 0.05), evidenced that Pb concentration in
the predator strongly depend on the metal concentration in
the whole-body burden prey. Considering the slope value,
Pb accumulation in fish is lower than in shrimp. For the
shrimp–fish relation (from secondary to tertiary consumer),
BAF values were higher than unity (Table 1),without significant differences (p \ 0.05) between high Pb-exposed
(1.3 ± 0.3) and control (1.4 ± 0.1) organisms.
Pb Concentrations in Organs and Tissues of Shrimp
and Fish
Fig. 5 a Averaged Pb concentration in whole grunter fish H. scudderi
fed white shrimp, (L. vanammei) which were fed brine shrimp
(A. franciscana), which were fed control (open squares) and Pb-treated
(approximately20 lg L-1) (filled squares) phytoplankton (T. suecica)
cells. b Averaged Pb concentration in fecal matter. Data represent the
mean of 4 and 12 replicates/time point for control and treated samples,
respectively, with error bars representing the SDM
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The dry-weight normalized Pb concentrations (mean ±
SD), calculated for each organ and tissue of shrimp and
fish, are shown in Fig. 7. Significant (p \ 0.05) differences
were observed in most of the weight-normalized Pb concentrations for the individual tissues and organs of shrimp
and fish between days 0 and 42. In addition, significant
Arch Environ Contam Toxicol (2011) 61:280–291
287
Discussion
Pb Uptake by Phytoplankton
Fig. 7 a Weight-normalized concentration of Pb in the different
organs and tissues of white-shrimp L. vanammei. b Weight-normalized Pb concentrations in the different organs and tissues of grunt fish
H. scudderi. The percentage of contribution of each organ and tissue
to the whole-body organism (dry weight) is underlined with a dashed
line
(p \ 0.05) differences were found in all tissues and organs
from exposed and control specimens. In the case of shrimp,
Pb contents in all tissues and organs increased 2–6 times in
exposed specimens compared with controls. The final
destination of Pb in exposed shrimp was muscle, exoskeleton, and remaining tissues of cephalotorax (with 30% total
Pb in each one). Muscle and exoskeleton made up
approximately 60% and 30% of body weight, respectively,
whereas the remaining internal organs and tissues in
cephalotorax represented \5%.
Similar to shrimp results, the dry weight of liver and
viscera and remaining internal organs and tissues made up
approximately 12% of body weight in fish; however, these
organs accounted approximately 30% of Pb accumulated in
fish. Pb transferred along the food chain to the exposed fish
was also accumulated in skin/scales, skeleton, and muscle.
Skin/scales, which made up approximately 10% of fish
body weight (dry weight), muscle (50% to 55% of total
body weight) and bones (22% to 25%) showed equal distribution (with 20% to 25% of whole-body total Pb). The
liver (\5% of fish body weight) accumulated approximately 15% of transferred Pb. The remaining internal
organs and tissues (\10%) accounted for 10% to 12% of
whole-body total Pb.
The concentration of dissolved Pb in seawater from Mazatlán Bay (0.18–0.50 lg L-1) was within the typical concentrations found in the coastal and estuarine waters with
low anthropogenic impact, which range from 0.02 to
\1 lg L-1 (Bryan and Langston 1992; Sadiq 1992; Cuong
et al. 2008). The range of dissolved Pb concentration in
moderate to highly polluted coastal areas varies from 1.0 to
50 lg L-1 (Sadiq 1992; El-Moselhy and Gabal 2004; Cuong
et al. 2008). The present range of concentrations of dissolved
Pb in Mazatlán Bay was significantly lower than in those
values found in the middle 1980s (0.24–0.77 lg L-1)
(Osuna-López et al. 1989) when leaded gasoline was
being highly consumed, but it is still significantly higher
than those reported for seawater in open oceans (dissolved
Pb 1–14 ng L-1; Bryan and Langston 1992). Recently, a
research by Soto-Jiménez and Flegal (2009), based on a
mixing model using Pb isotopes data, showed that Pb in the
port of Mazatlán is predominantly derived from natural
weathering and past leaded gasoline combustion with the
later influence of inputs from a more radiogenic source
related to anthropogenic lead of North American origin.
However, urban effluents and industrial emissions are still an
additional source of local Pb input for seawater from
Mazatlán Bay.
Results of the phytoplankton Pb exposure showed the
high capacity of T. suecica cells to take up dissolved Pb
from seawater (BCF [1). The effect of direct metal uptake
from seawater was evident by the high Pb concentrations of
the phytoplankton batches exposed to high Pb doses
compared with the control and with most other published
reports in marine plankton (0.15–7.4 lg g-1; Martin and
Knauer 1973; Michels and Flegal 1990). Previous experiments with Tetraselmis sp. also showed that it has high
uptake rates of cadmium and copper, which results in the
accumulation of high concentrations of these metals into
cells compared with other species (Pérez-Rama et al. 2002;
Levy et al. 2007, 2008). The high uptake rates of metal
may be related to the large surface area of the cells of
Tetraselmis sp. (240 ± 60 lm2).
Although the results of this study under controlled conditions (e.g., characterized by a nutrient-enriched medium
and high doses of dissolved Pb) may not be applicable to
conditions prevailing in the field, these are relevant considering that eutrophication and metal contamination due to
human activities are occurring simultaneously in many
coastal and estuarine waters. Because coastal eutrophication may impact metal trophic transfer in aquatic systems,
the interaction between metal contamination and eutrophication remains to be further investigated.
123
288
Pb Transference Through the Food Chain
Despite T. suecica being efficient at taking up dissolved Pb
from the water column, consumer organisms occupying
different trophic levels are relatively inefficient at transferring it through the food chain. Except for the relation of
white shrimp to grunt fish, data showed that Pb concentrations retained in the whole-body burden predators were
lower than in their preys (BAF \ 1.0). Thus, Pb transfer
from phytoplankton to zooplankton (T. suecica–A. franciscana relation) and from zooplankton to shrimp
(A. franciscana–L. vannamei relation) is restricted.
Low BAF values in A. franciscana and L. vannamei
suggest that these species have mechanisms to detoxify and
self-regulate metal uptake. The mechanisms may include
the production of metallothioneins and phosphate granules
(Wang 2002; Ahearn et al. 2004; Rainbow et al. 2006). In
addition, Pb appears to be metabolized by way of the
calcium metabolic pathways and therefore accumulates in
the exoskeleton and is excreted by way of molting (Fowler
1977; Rainbow 1997; Abd-allah and Moustafa 2002;
Rainbow et al. 2006). Pb-detoxified forms in phosphate
granules, molts, and/or excretions make the metal less
trophically available (insoluble) in crustaceans (Rainbow
et al. 2006).
In this study, approximately 30% of total Pb in exposed
shrimp was accumulated in exoskeleton. Considering that
three to four molting events were observed during the
experiments, we concluded that the molting mechanism
constituted a main route of Pb detoxification for shrimp.
However, the high Pb concentrations observed in hepatopancreas and remaining tissues of cephalotorax and, to a
lesser extent, in muscle evidenced their important role in
the trophic transfer process of Pb. High Pb concentrations
found in the fecal wastes of zooplankton and shrimp (2–4
times higher to the whole-organism concentrations)
revealed that fecal wastes from predators also constitute a
route of detoxification and self-regulation of metals.
Field studies have evidenced Pb accumulation by way of
dietary uptake in marine fish species, with differences in
the patterns of distribution of the metal in organs and tissues among fish species (e.g., Canli and Atli 2003; Usero
et al. 2003). Those differences are related to a number of
factors that affect the exposure and accumulation of metal
in fish, including tissue-specific differences and individual
specific differences in species life history (diet and
behavior) and physiologic state (e.g., age, sex, body condition, reproductive status, and quality of health) (Roach
et al. 2008).
In the fish H. scudderi used in this study, most of the
transferred Pb was distributed in skeleton, skin/scales, and
liver. The preferential placement of Pb in skeleton and
scales suggested that it follows the Ca metabolic pathway
123
Arch Environ Contam Toxicol (2011) 61:280–291
(Patterson and Settle 1977). However, Pb was concentrated
to a greater extent in liver (16.8 ± 5.2 lg g-1) compared
with others body compartments, which evidenced that the
liver acts as the main organ for the detoxification and
elimination of metals in fish (Sorenson 1991; Roach et al.
2008). Remaining internal organs, including kidney
grouping, also showed higher concentrations of Pb compared with muscle tissue. Despite that gonad is reported as
a sensitive tissue to detect contaminant gradients and thus
is very useful in studies on the distribution of contaminants
(Roach et al. 2008), in this study we did not collect sufficient samples of this tissue for analysis because we used
only juvenile specimens of H. scudderi for our
experiments.
Fish feces showed also higher Pb concentration (2
times) than in whole-body burden, evidencing that fish can
detoxify or self-regulate metal intake. There is no evidence
for the induction of Pb-binding proteins in fish tissues
(Roesijadi 1992; Hansen et al. 2004), such as occurs in
crustaceans. However, the large amount of Pb accumulated
in their bones, scales, and internal organs decreases the Pb
concentration in muscle tissue, consequently decreasing Pb
bioavailability for higher trophic levels, including humans.
In fact, shrimp and fish showed similar strategies in handling transferred Pb, such as excreting the metal or storing
it in less biologically available pools (Rainbow 2002;
Zhang and Wang 2007).
Pb Biomagnification
Although some Pb is transferred through the marine food
chain, from phytoplankton to shrimp, the Pb concentration
did not increase systematically. In fact, the BAF from
phytoplankton (level I) to zooplankton (level II) and then to
shrimp (level III) was estimated to be lower than unity for
Pb-exposed and control organisms. Only for the shrimp–
fish relation BAF values were higher than unity. According
to Barwick and Maher (2003), two links in the trophic
chain have positive transference (i.e., metal increased from
seawater to phytoplankton and from shrimp to fish), but
they are separated by two links that have negative transference (i.e., the Artemia and the shrimp had lower tissue
concentrations of Pb than in their prey). Considering that
biomagnification is defined as the tendency of pollutants to
increase body burden throughout the food chain, this process did not occur in our simulated marine food chain. The
strategies of metal sequestration in the prey decrease the
availability of accumulated Pb to upper trophic levels
(Rainbow 2002; Zhang and Wang 2006). Therefore, Pb did
not biomagnify in our modeled food chain.
If Pb is not being biomagnified along successive trophic
levels of the food chain, then the rates of passive or/and
active excretion of Pb lead to a diminution of the metal.
Arch Environ Contam Toxicol (2011) 61:280–291
Our results are consistent with the literature on the nobiomagnification process occurring with nonessential
metals, such as Pb. Previous studies have documented that
the trophic transfer of Pb along the food webs is reasonably
inefficient and that a biodiminution effect in Pb concentration occurs with increasing trophic position (Amiard
et al. 1980; Michels and Flegal 1990; Szefer 1991; van
Hattum et al. 1991; Dietz et al. 2000; Barwick and Maher
2003; Rainbow et al. 2006; Ruelas-Inzunza and PáezOsuna 2008; Watanabe et al. 2008). Unlike the case for
organic pollutants or methyl mercury, biomagnification of
trace metals along food chains is not a general phenomenon
(Amiard-Triquet et al. 1993; Wang 2002).
We concluded that the transference, accumulation, and
magnification process depends, in addition to the metal, of
the involved species of a trophic chain. The physiologic
accumulation pattern of a species for a particular metal is a
determinant factor in the existence or not of biomagnification processes. Thus, different comparisons among
potential trophic relations can yield different scenarios.
Implications for Human Health
Despite the observed restriction on Pb transfer, our results
provide evidence that Pb-contaminated seawater is transferred through a dietary exposure pathway, thus producing
a net accumulation of Pb in higher trophic-level species.
Some dissolved Pb added to phytoplankton (level I) was
transferred across the food chain and reached the shrimp
(level III) and grunt fish (level IV). Pb concentrations in the
whole-body burden of shrimp and fish ([3 lg g-1) and in
particular the muscle can pose a hazard for their consumers. Despite that most of transferred Pb is accumulated
in the noncomestible portions of exposed shrimp and fish,
Pb in the muscle of both species (1.6 ± 0.23 and
1.4 ± 0.27 lg g-1, respectively) reached health-risk concentrations for humans. In fact, concentrations in muscle
were higher than the maximum residue limits of Pb permitted in fish and seafood of 0.3 lg g-1 (FAO/WHO 1972;
Commission Regulation 1881/2006) and [1.0–1.3 lg g-1
(FDA 1993; Secretarı́a de Salud in México 1995).
Considering the importance of shrimp and adult
H. scudderi as commercial food resources, high concentrations of dissolved Pb in seawater represents a risk for the
human health. Because Pb is not an essential trace element
in humans, its presence in the body, even at low concentrations, can have deleterious effects. This metal can cause
serious neurodevelopmental effects, particularly in developing fetuses, babies, and young children, because their
brains and nervous systems are still developing, and the
blood–brain barrier is incompletely developed (Agency for
Toxic Substances and Disease Registry 1999).
289
Acknowledgments The authors thank J. F. Ontiveros-Cuadras,
A. Nuñez-Pastén, and S. Rendón-Rodrı́guez for help in bioassays;
H. Bojórquez-Leyva for laboratory analysis; and V. Montes,
C. Ramı́rez-Jáuregui, C. Suárez-Gutiérrez, and G. Ramı́rez-Reséndiz
for manuscript preparation. Special thanks to personal of Mazatlán
Aquarium for their support. We are grateful to two anonymous
reviewers for the truly helpful comments. Financial support was
provided by the grant SEP-CONACYT 60215, UNAM-PAPIIT
IN206409, IN210609, and IN217408-3.
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