Ecotoxicology and Environmental Safety 79 (2012) 163–169

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Ecotoxicology and Environmental Safety

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Cadmium toxicity assessment in juveniles of the Austral South America

amphipod Hyalella curvispina
Anabella Giusto a, Lucas A. Somma a, Lucrecia Ferrari a,b,n
a
Applied Ecophysiology Program, Basic Sciences Department, Institute of Ecology and Sustainable Development (INEDES), National University of Luján, Casilla de Correo 221,
B6700ZBA-Luján, Argentina
b
Scientific Research Commission (CIC), La Plata, Buenos Aires, Argentina

a r t i c l e i n f o abstract

Article history: Hyalella curvispina is representative of zoobenthic communities in Austral South America. It is
Received 11 August 2011 taxonomically close to Hyalella azteca and abundant in fresh water bodies of the pampasic region of
Received in revised form Argentina. It is usually used as test organism in ecotoxicological studies at a regional level, and there is
27 December 2011
a strong concern to increase the knowledge of both their biology and their sensitivity to different toxic
Accepted 28 December 2011
agents. The aim of the present work was to assess the effects of cadmium on H. curvispina juveniles
Available online 24 January 2012
exposed to concentrations expected in the water bodies of the distribution area of the species. Survival,
Keywords: growth and cadmium body burden were evaluated in aqueous and solid matrices under fixed
Hyalella curvispina experimental conditions. Animals were exposed in ten-day static toxicity bioassays to 2.5, 5.25 and
Cadmium
11.25 mg Cd/L for the aqueous-phase bioassays, and to 0.85, 2.8 and 5.6 mg Cd/Kg dry sediment for the
Spiked sediment
solid-phase bioassays. In water only assays, the animals exposed to 11.25 mg Cd/L showed a significant
Body burden
Toxicity assessment decrease in survival and growth. In the sediment bioassays, no effect was observed on survival, and
weight was significantly reduced at the highest concentration. Cadmium uptake was concentration
dependent for both assay matrices and one order of magnitude higher in the aqueous medium than in
the solid matrix. Results indicate that juveniles of H. curvispina are sensitive to cadmium concentrations
expected in their environment and thus confirm their importance as test organisms for ecotoxicity
assessment in water bodies within the distribution area of the species.
& 2012 Elsevier Inc. All rights reserved.

1. Introduction 2005; Borgmann et al., 2005; Alves et al., 2008). H. azteca is

Invertebrates include the largest number of species generally
found at the lower trophic levels. The comparison of the
responses of invertebrates to toxicants is a priority issue in
aquatic ecotoxicology (Gourmelon and Ahtiainen, 2007). Particu-
larly, amphipods play an important role in benthic communities
due to their abundance, their detritivorous–herbivorous habits,
and the place they occupy as important links between producers
and higher trophic levels, being involved in the flow of energy
through food chains.
Hyalella azteca is the freshwater amphipod most widely used
in ecotoxicology. Its use as a test organism is based on a knowl-
edge of both their biology and their response to several toxics in
different test media and conditions. This information is the result
of decades of study (Borgmann and Munawar, 1989; Ingersoll,
1995; Ingersoll et al., 1998; Wang et al., 2004; Bartlett et al.,
n
Corresponding author at: Programa de Ecofisiologı́a Aplicada, Departamento
de Ciencias Básicas, Universidad Nacional de Luján, Casilla de Correo 221,
B6700ZBA-Luján, Argentina. Fax: þ54 23 23425795.
E-mail address: lucreciaferrari@gmail.com (L. Ferrari).
usually found in lentic habitats, such as ponds, and occasionally in
lotic habitats, such as streams, especially those with fine bottom
sediments (EPS, 1997). In addition this species is distributed in
the Northern Hemisphere from Central Mexico to the borders of
Canada and Alaska. However, there is a global tendency to
evaluate the impact of polutants on particular aquatic systems
through ecotoxicological monitoring of native species (Rand et al.,
1995; Ferrari et al., 1998; Castro et al., 2006; King et al., 2006;
Dutra et al., 2008; Giusto and Ferrari, 2008). The need of using
native species becomes evident because, among other reasons,
these species, adapted to the environmental characteristics, may
provide more realistic results than those obtained from exotic
species.
The species used in this work, Hyalella curvispina, is represen-
tative of zoobenthic communities in Austral South America. It is
distributed from Cangallo and Rı́o de Janeiro in the North to Punta
Arena and Malvinas Islands in the South (Garcı́a et al., 2010). It is
taxonomically close to H. azteca and abundant in fresh water
bodies of the pampasic region. Taking into account these con-
siderations, efforts to asses biological effects of sediments and
water contamination on H. curvispina have begun years ago in

0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2011.12.020
164 A. Giusto et al. / Ecotoxicology and Environmental Safety 79 (2012) 163–169
Argentina (Di Marzio et al., 1999; Di Marzio et al., 2005; Jergentz
et al., 2004; Doyle, 2007; Garcı́a, 2009; Garcı́a et al., 2010; Peluso,
2011; Peluso et al., 2011; Mugni et al., 2011). Thus, for several
reasons, including wide geographical distribution, easy laboratory
breeding, and their being native fauna to the area, there is an
increasing concern to expand the knowledge of both their biology
and their sensitivity to different toxic agents.
Concentrations of cadmium in freshwater environments have
increased substantially as a consequence of anthropogenic activ-
ities. Cadmium is more mobile in aquatic environments than most
other metals, and it is regarded as one of the most toxic elements
for aquatic organisms; it has a relatively long residence time in
aquatic systems; for example, the mean residence time calculated
in Lake Michigan is of 4–10 years (USEPA, 2007). In addition,
cadmium is one of the recommended substances as reference
toxicant (USEPA, 1993).
The quality criteria for the protection of freshwater aquatic life
allows concentrations in shallow waters between 0.2 and 4 mg Cd/L
for water hardness from 0 to 60 and above 180 mg CaCO3/L,
respectively (National Law No. 24051 of Hazardous Wastes, 1991).
Regarding freshwater sediment quality values, different guideline
categories (SQGs) have been established. In general terms there are
two threshold levels, one below which effects on the biota rarely
occur [e.g. the lowest effect level (LEL), threshold effect level (TEL),
minimal effects threshold (MET), effects range low (ERL) with a
value of consensus level (TEC)], and one above which effects are
likely to occur [e.g. the toxic effect threshold (TET), severe effect
level (SEL), probable effect level (PEL), effect range median (ERM)
with a value of consensus level (PEC)]. For cadmium, TEC is
0.99 mg Cd/Kg ds and PEC is 4.98 mg Cd/Kg ds (Burton, 2002;
MacDonald et al., 2000). Cadmium concentration in the polluted
water bodies of peri-urban areas of Argentina may reach values as
high as 700–1700 mg Cd/L (Salibián, 2006) in aqueous media, and
between 2.4–3.2 mg Cd/Kg ds in sediments (Ronco et al., 2008).
The sensitivity of H. curvispina to cadmium in function of age
and exposure time was recently published by Garcı́a et al. (2010).
They conclude that H. curvispina has a high sensitivity to water-
borne Cd, with LC50 values close to the guideline levels for aquatic
biota protection and they suggest that guidelines for cadmium in
surface water should be reconsidered. Therefore, further studies
are of major importance.
The aim of the present work was to increase the knowledge on
the sensitivity of H. curvispina juveniles to cadmium concentrations
expected in the water bodies of the distribution area of the specie.
Survival, growth and cadmium body burden were evaluated in
aqueous and solid matrices under fixed experimental conditions.
2. Material and methods
2.1. Tests organisms
For each assay, juveniles of H. curvispina (3–4.5 mm in length) were sorted
by sieving from lab cultures maintained in dechlorinated tap water (hardness
80–90 mg CaCO3/L). They were placed in a plastic container with moderately hard
reconstituted water (MHW) (NaHCO3, 96 mg/L; CaSO4  2H2O, 60 mg/L; MgSO4,
60 mg/L; KCl, 4 mg/L; pH, 7.4–7.8; hardness, 80–100 mg CO3Ca/L) (USEPA, 1993)
into an experimental chamber with programmed temperature and photoperiod
(237 1 1C, 16/8 h Light/Dark). Animals were fed crushed fish food (TetraFins) ad
libitum and went through this acclimation process during the four days prior to
the assay. All assays were conducted in this chamber under the same temperature
and photoperiod regime, using MHW as dilution medium.
2.2. Assays
Two series of experiments were carried out: water only and spiked sediment
assays.
2.2.1. Water-only bioassays
Two 10-day static assays were performed to evaluate the effects of waterborne
cadmium (MHW) on Hyalella curvispina according to the guidelines of EPS (1997)
and Garcı́a et al. (2010) with minor modifications.
The nominal test concentrations assayed were 0 (control), 5, 10 and 20 mg Cd/L.
These were prepared by dilution from a stock solution of 1 g/L Cd which was
prepared by dissolving CdCl2  2(1/2)H2O (analytical grade) in bidistilled water. The
concentrations range was selected based on values of LC 50–10 day, of 14.52 mg Cd/
L—for this species in similar conditions—which are, in addition, within the range of
cadmium concentrations detected in different regional water bodies (Garcı́a, 2009;
Garcı́a et al., 2010). Thus, the concentration of Cd assayed in this work may be
considered as environmentally relevant.
The treatments were conducted in triplicate (except controls, which were
conducted in quadruplicate) in polypropylene bakers of 400 ml. Each replicate
contained 10 animals, 200 ml of the corresponding test solution and a piece of
gauze of 5 cm2 for substrate, and was supplied with aeration. The bakers were
covered to prevent evaporation. The acceptability criterion of the assays was a
survival Z 90 percent in the control.
2.2.2. Sediment bioassays
The sediment used in spiked sediment toxicity testing was dredged from the
bottom (up to a depth of 10 cm) of Las Flores Stream (591 070 W and 341 290 S),
a non polluted water body which is used as control sediment (Giusto and Ferrari,
2008; Peluso et al., 2011). Heavy metal content in the sediment was undetectable
or below SQGs and presence of sulfites, cianides or hidrocarbures was not found.
Determinations of organochlorine, organophosphate and pyrethoid pesticides
reached values lower than detection levels (2 mg/Kg) (Ronco et al., 2008).
At the laboratory, the sediment was homogenized manually, left to dry,
crushed, sieved (1000 mm), homogenized once more and then stocked dry at
room temperature until use.
For the spiked assays, the stocked dry sediment was rehydrated (1 L
MHW:1 Kg sediment) and allowed to settle for 24 h. After removal of the super-
natant, subsamples from the sediment were taken to calculate dry weight/wet
weight (dw/ww) ratio (Pasteris et al., 2003), organic matter content and granulo-
metric composition (with previous organic matter removal) through Robinson
pipette method (Depetris, 1995).
Two 10-day static assays were performed following the protocol of USEPA
(2000) with minor modifications. The concentration nominals tested in the first
assay were 0 (control), 1 and 4 mg Cd/Kg ds. The second assay also included a
concentration of 8 mg Cd/Kg ds. The range of concentrations was selected accord-
ing to values of TEC, PEC and SEL for cadmium of 0.99, 4.98 and 10 mg/Kg
(MacDonald et al., 2000), and cadmium levels measured in freshwater contami-
nated sediments of the region ranged between 1 and 3.2 mg/Kg (Ronco et al.,
2008). The different concentrations were prepared from the cadmium stock
solution used for water only assays. Spiking was performed according to Giusto
and Ferrari (2008), by mixing the required weight of wet sediment (corresponding
approximately to 1.5 L), with 2 L of MHW and the required volume of Cd stock
solution in a 4-L jar. The contents of each jar were manually stirred for 5 min, using
a stainless steel spoon. Jars were then placed in a refrigerator at 4 1C for 14 day.
Stirring was repeated every 3 day. Afterwards, the supernatants from the control
and treatments were discarded and the remaining material was divided into five
replicates, each one with 100 ml of treated sediment and 175 ml of MHW (over-
lying water). The replicates were placed in the experimental chamber under
controlled conditions as described above, covered with glass plates to minimize
evaporation and were supplied with aeration. The assay bakers were allowed to
equilibrate for a week before the beginning of the experiment.
Four replicates were performed per treatment, each of which contained 10
animals, and a fifth replicate was also prepared without animals (abiotic control)
that was used for measured water and sediment cadmium concentrations. The
acceptability criterion of the assays was a survival Z 80 percent in the control.
2.2.3. Common experimental procedure for water-only and sediment bioassays
At the initial time of the assays, individuals randomly selected from the pool
subjected to acclimation were added to each replicate. The average length and dry
weight were determined from randomly selected individuals of the acclimation
group just before the beginning of each assay (initial group).
Each replicate was supplied with 2.5 mg of crushed fish food on days 4 and
8 of the experiment. The administered amount of food is based on previous
experiences.
At the end of the assays all surviving individuals in each replicate were put
24 h in EDTA 50 mM prepared in clean media for purging the gut (Borgmann et al.,
1991). Then they were fixed by freezing at  20 1C and stored until further
processing.
The endpoints of effect considered at the end of the bioassays were survival,
growth (biomass and length) relative to those in the control and initial groups and
Cd tissue accumulation. For water-only bioassays the survival was checked at 1, 2,
4, 7 and 10 day; in the sediments assays survival was checked at final time.
The length of survivors, defined as the distance from the base of the first pair
of antennae to the tip of the telson, was measured with a digital caliper (to the
 A. Giusto et al. / Ecotoxicology and Environmental Safety 79 (2012) 163–169 165

nearest 0.01 mm) under a stereoscopic microscope, holding the animal in a
stretched position (Doyle and Momo, 2009). Surviving animals were individually
dried at 60 1C for at least 48 h and weighed in a Mettler Analytical Balance
(accuracy Z0.01 mg). The biomass was expressed as dry weight. These dried
samples were used for determination of body cadmium content, according to the
protocol of Borgmann et al. (1991), with modifications. Pooled individuals of the
same replicate (approximately 4 mg of mass) were placed in 100 ml HNO3 for a
week, followed by the addition of 65.5 ml of H2O2; after 24 h, the solution was
brought up to volume 5 ml with Milli-Q water and destined for analytical
procedure.
2.3. Analytical procedure
The following variables were measured in the matrices assays: dissolved
oxygen, pH and conductivity with sensors (oxymeter Hanna [ 7 0.1 mg/L],
pH meter Mettler [ 7 0.01 mg/L], conductimeter Consort C532 [ 70.1 mS/cm]
respectively); ammonia concentration using a Spectroquant test kit (Merck) with
a measuring range 0.03–3 mg/L NH4þ ; and water hardness, estimated by the
volumetric method, with the Aquamerck test kit (Merck, sensitivity of 1 mg/L
CaCO3).
For determining cadmium concentrations in aqueous matrix and overlying
water, a sample from each concentration was preserved with 0.15 percent nitric
acid and refrigerated until processing. For sediment assays, a sample of each
treatment was subjected to digestion by multi-acid attack prior to the determina-
tion of the analytical cadmium concentration. All cadmium determinations were
done using inductively coupled plasma atomic emission spectrometry (ICPES,
Perkin-ElmerOptima-3100 Icp-XL) with a method detection limit o 1 mg Cd/L
(APHA, 1995) in water and 0.25 mg/kg in sediment.
2.4. Statistical analysis
The statistical significance of differences between treatments for survival and
growth was evaluated. The Shapiro–Wilk test and the Levene test were used to
assess normality and homoscedasticity, respectively. Only weight data in sedi-
ment fulfilled the normality and homoscedasticity assumptions and were ana-
lyzed through one-way ANOVA followed by Tukey’s test. Remaining data were
analyzed with the Kruskal–Wallis test followed by multiple comparisons
(Zar, 1999). Statistical analyses were performed with Infostat Program. Signifi-
cance was set at p o0.05.
3. Results
3.1. Water-only bioassays
Physico-chemical parameters and measured concentrations of
cadmium at initial and final times of the assays media are shown
in Table 1. Since there is no significant variation between treat-
ment and exposure time for any of the registered parameters
except for cadmium concentration, it is reasonable to assume that
the results obtained were due to cadmium. The measures of
cadmium from lowest to highest concentration were, 60, 70 and
80 percent of the nominal values at initial experimental time and
show a decrease of 1.5, 2 and 2.5 times, respectively, at exposure
final time (Table 1). Besides the Cd uptake by animals, this ‘‘loss’’
of cadmium is expected in a water static system and is probably
related to its adsorption onto the substrate and/or particulate
matter and also as precipitation of otavite (CdCO3) (Mastrangelo
et al., 2011). Based on this, the exposure values were estimated as
the average of the mean effective concentrations between the
initial and final times. Accordingly, henceforward the nominal
concentrations, 5, 10 and 20 mg Cd/L, are referred as 2.5, 5.25 and
11.25 mg Cd/L, respectively.
Under experimental conditions, a significant decrease of the
survival was observed only at the highest concentration tested
(11.25 mg Cd/L) from seven days exposure onward (Fig. 1A).
Animals increased their length and biomass in all treatments,
but increase was smaller in the highest Cd exposure group, of
which length and biomass differ significantly from the other
groups (Fig. 1B and C). At final exposure time the length and
biomass of surviving animals from 2.5 and 5.25 mg Cd/L treated
groups did not show a significant decrease with respect to those
of the control group (Fig. 1B and C).
3.2. Sediment bioassays
The grain size distribution analysis showed: Sand (61 percent),
Clay (12 percent) and Silt (27 percent) with 6.9 percent of organic
matter.
The measured concentrations of cadmium in the sediment
(Table 2) remained stable during the assay period but were from
15 to 30 percent lower than the nominal values. On this basis, the
exposure values corresponding to the nominal concentrations
1, 4 and 8 mg Cd/Kg ds were estimated as the average of the mean
analytical concentrations between the initial and final times, so
the exposure concentrations are referred as 0.85, 2.8 and 5.6 mg
Cd/Kg ds.
The physico-chemical parameters at initial and final times of
the overlying water are shown in Table 2. In all cases, these
parameters were within the range expected under standard
bioassay conditions and within the acceptable values according
to the standard guide for determination of the bioaccumulation of
sediment-associated contaminants by benthic invertebrates
(Van Geest et al., 2010). The concentrations of cadmium of the
overlying water at initial and final exposure times were not
detectable in all treatments (Table 2).
The group treated with 5.6 mg Cd/Kg ds showed a tendency to
decrease survival, but it did not differ significantly from the
control (Fig. 2A). The control and the three treated groups showed
a significant increase in length compared to the initial group. No
significant differences in length were found between treatments
(Fig. 2B). In relation to biomass the animals exposed to the
highest concentration were affected: their weight was signifi-
cantly lower to that of control group and of animals exposed to
0.85 and 2.8, but did not differ from the initial group (Fig. 2C).

Table 1
Water-only bioassays. Cadmium concentrations measured in solution, physicochemical parameters and cadmium body burden measured in the survival animals.

Mean measured concentration 0 lg Cd/L (Control) 2.5 lg Cd/L 5.25 lg Cd/L 11.25 lg Cd/L

Parameters registered Ti Tf Ti Tf Ti Tf Ti Tf

Measured Cd (mgCd/L) o 1.07 0.0 (2) o 1.07 0.0 (2) 3.07 0.2 (2) 2.07 0.1 (2) 7.0 71.0 (2) 3.5 70.7 (2) 16.0 71.0 (2) 6.57 0.7 (2)
pH 8.27 0.0 (2) 8.07 0.3 (8) 8.1 70.0 (2) 8.1 70.3 (6) 8.1 7 0.0 (2) 8.1 70.2 (6) 8.1 70.0 (2) 8.07 0.3 (6)
OD (mg/L) 8.37 0.5 (2) 6.67 1.2 (8) 8.3 70.4 (2) 6.7 71.3 (6) 8.2 7 0.4 (2) 6.9 70.9 (6) 8.3 70.3 (2) 6.77 1.3 (6)
Hardness (mmol/L) 0.97 0.1 (2) 1.07 0.1 (3) 1.07 0.1 (2) 1.07 0.1 (3) 1.0 70.1 (2) 1.07 0.1 (3) 1.07 0.1 (2) 1.07 0.1 (3)
Conductivity (mS/cm) 5087 15 (2) 538 730 (8) 506 715 (2) 530 736 (6) 500 79 (2) 535 732 (6) 501 79 (2) 5407 29 (6)
NH4þ (mg/L) 0.0 (2) 0.27 0.1 (3) 0.0 (2) 0.27 0.1 (3) 0.0 (2) 0.27 0.1 (3) 0.0 (2) 0.37 0.0 (3)

Cadmium body burden

Body burden (mgCd/g dry weight) ND (6) 80 7 20 (6) 140 740 (6) 219 740 (3)

Values are expressed as mean 7 SD both assays; n between brackets. Ti: initial time; Tf: final time; ND: not detected.
166 A. Giusto et al. / Ecotoxicology and Environmental Safety 79 (2012) 163–169

body burden (  220 mg Cd/g dry weight) represents the Cd body

120 burden of 55 percent of the surviving animals (see Fig. 1A). Thus,
under the experimental conditions assayed, it could be equated to
100 the expected Cd load for the LC50 of this species at this age. As for
assayed concentrations, in spiked-sediment assays body cadmium
80 concentration was one order of magnitude lower than in water
Survival (%)

only assays and shows the same tendency to increase with the
60 exposure concentration. It should also be noted that cadmium
∗ body burden of 20 mg Cd/g dry weight is associated to a survival
40 a rate similar to control. (Table 2 and Fig. 2A).

20
Control 2.5μgCd/L 5.25μgCd/L 11.25μgCd/L
4. Discussion
0
0 1 2 4 7 10
In order to establish guide levels for cadmium for the protec-
Day
tion of aquatic life, the sensitivity of 21 genera of aquatic
organisms to chronic exposure was determined and Hyalella
6 ranked within the four most sensitive genera (Mebane, 2006).
The toxicity of cadmium depends on the free Cd ion concentration
a a a a,b
5 (i.e., bioavailable Cd), which varies according to the composition
of the exposure media. In this sense, a complex variety of abiotic
4 and biotic processes affect the cadmium speciation and its
environmental distribution, including adsorption onto and deso-
Lenght (mm)

rption from mineral surfaces, incorporation in precipitates or

3
coprecipitates, release through the dissolution of minerals, and
interaction with plants and microorganisms. Some of these
2
processes can effectively isolate heavy metals from the media,
whereas others can release them or transform into different
1 species that may be more or less bioavailable, thus affecting their
toxicity to organisms (Sorensen et al., 1985; Brown et al., 1999;
0 Onyatta and Huang, 1999). Consequently, the dilution media,
Initial group Control 2.5 5.25 11.25 assay conditions tested (e.g. water hardness, pH, temperature,
Treatments (μgCd/L) feeding), and also, the species, life stage of the organisms, health
status, etc. result in a great variability of the toxic effects of
0.8 cadmium. For freshwater invertebrates, if the LC50 is considered
as endpoint, the most sensitive species seems to be Hyalella
0.7 a a a a,b curvispina at the neonate stage, (LC50 96 h of 0.00171 mg/L), while
0.6 the most resistant seems to be Artemia salina (LC50 96 h of
Dry Weight (mg/ind)

0.5
0.4
0.3
0.2
0.1
0 with LC50 of 29.99 mg Cd/L (96 h) and 14.52 mg Cd/L (10 day). It is
Initial group Control 2.5
Treatments (μgCd/L)
Fig. 1. Values mean 7 SEM of survival (A), length (B) and dry weight (C) of H.
curvispina exposed to different concentrations of cadmium in MHW. ‘‘n’’ indicates
significant difference of group exposed at 11.25 mg Cd/L respects to the control and the
others group; ‘‘a’’ indicates significant difference from the initial group and ‘‘b’’
indicates significant difference from the control group and 2.5 mg Cd/L.
3.3. Cadmium body burden
The body cadmium content for water only and spiked-sedi-
ment assays are shown in Tables 1 and 2, respectively. Results of
cadmium body burden in animals revealed that the exposure
period was long enough to accumulate cadmium in the tissue of
animals exposed to contaminated water and sediment in detect-
able concentrations. In water only assays, cadmium uptake
increased significantly with the exposure concentration. It is
important to note that at 11.25 mg Cd/L exposure, the registered
160 mg/L) (Achiorno et al., 2010). So, the wide variability in the
LC50 values reported in the literature is related not only to the
interspecific variability, but also to the differences in assay
conditions, not always reported correctly. This background of
variability and uncertainty makes comparisons more difficult.
Garcı́a et al. (2010) reported the differential sensitivity to Cd in
aqueous matrix for neonates, juveniles and adults of H. curvispina
at identical exposure conditions. They suggest juveniles are the
most appropriate stage for their use in environmental monitoring
5.25 11.25
important to note that quantifiable effects on survival and growth
were found only at cadmium concentration levels near those of
LC50 10 day for this species at this stage in a comparable dilution
medium. Therefore in aqueous-phase under these experimental
conditions, survival and growth will be similarly sensitive indi-
cators of cadmium toxicity for H. curvispina (Fig. 1).
The process of metal accumulation by an invertebrate depends
on a variety of intrinsic and extrinsic factors, but the functional
anatomy of digestive organs and gill epithelia, as well as the
physiology of an organism, play dominant roles in controlling
accumulation processes (Dallinger, 1994). In invertebrates such as
Eisenia fetida, Nereis virens, Gammarus zaddachi and G. salinus and
H. azteca, cadmium in aqueous phase showed a rapid uptake,
which increased with time and exposure concentration
(Shuhaimi-Othman and Pascoe, 2007; Shuhaimi-Othman et al.,
2002). H. azteca adults (CL50-312 h:5.8 mg Cd/L) exposed for
120 h to 4.4 mg Cd/L in hard water showed a cadmium body
burden near 50 mg Cd/g dw (Shuhaimi-Othmann and Pascoe,
 A. Giusto et al. / Ecotoxicology and Environmental Safety 79 (2012) 163–169 167

Table 2
Sediment bioassays. Cadmium concentrations measured in the sediment. Physicochemical parameters and analytical cadmium concentration in the overlying water.
Cadmium body burden measured in the survival animals.

Mean measured concentration 0 mgCd/Kg (Control) 0.85 mgCd/Kg 2.8 mgCd/Kg 5.6 mgCd/Kg

Parameters registered Ti Tf Ti Tf Ti Tf Ti Tf

Sediment
Measured Cd (mgCd/Kg) o0.25 (2) o0.25 (2) 0.957 0.07 (2) 0.757 0.07 (2) 2.99 70.01 (2) 2.547 0.09 (2) 5.5 (1) 5.7 (1)

Overlying water
Measured Cd (mgCd/L) o1.0 70.0 (2) o1.0 7 0.0 (2) o 1.07 0.0 (2) o 1.07 0.0 (2) o 1.07 0.0 (2) o 1.07 0.0 (2) o1.0 70.0 (1) o1.0 7 0.0 (1)
pH 8.5 7 0.0 (8) 8.4 7 0.0 (8) 8.47 0.1 (8) 8.47 0.0 (8) 8.57 0.0 (8) 8.37 0.1 (8) 8.5 7 0.0 (8) 8.3 7 0.1 (8)
OD (mg/L) 7.4 7 0.9 (8) 6.7 7 0.4 (8) 7.07 1.0 (8) 6.77 0.3 (8) 7.57 0.5 (8) 6.27 0.5 (8) 7.2 7 0.1 (8) 6.6 7 0.0 (8)
Hardness (mmol/L) 1.8 7 0.2 (6) 1.6 7 0.0 (3) 1.97 0.2 (6) 1.87 0.1 (3) 1.87 0.1 (6) 1.97 0.0 (3) 2.1 7 0.2 (3) 2.1 7 0.2 (2)
Conductivity (mS/cm) 917 7 41 (8) 788 7 15 (8) 9787 11 (8) 817 729 (8) 9507 28 (8) 8377 35 (8) 968 7 49 (8) 905 7 35 (8)
NH4þ (mg/L) 5.9 7 0.3 (4) 0.2 7 0.0 (2) 5.67 0.3 (4) 0.17 0.0 (2) 5.97 0.5 (4) 0.27 0.0 (2) 4.3 7 0.8 (2) 0.2 (1)

Cadmium body burden

(mgCd/g dry weight) ND (2) ND (2) 10 7 2 (2) 21 7 4 (2)

Values are expressed as mean 7 SD both assays; n between brackets. Ti: initial time; Tf: final time; ND: not detected.

2007). The accumulated body burden of cadmium registered in H.
curvispina juveniles was two to three times greater than those
seen for H. azteca exposed to comparable concentrations (80 and
140 mg Cd/g dw for 2.5 and 5.25 mg Cd/L, respectively). Despite
the high bioaccumulation found at these concentrations, the
mean Cd body burdens in H. curvispina (219 mg Cd/g dw) asso-
ciated with survival diminution was observed only at a higher
mean concentration exposure of 11.25 mg Cd/L. So, in function of
the results obtained, it could be assumed that in H. curvispina
juveniles body burden values Z200 mg Cd/g dw may be related to
high mortality (Table 1, Fig. 1).
This is the first report of bioaccumulation of cadmium in
H. curvispina and it can be inferred from this study that
H. curvispina posses the ability to concentrate large amounts of
cadmium in their tissues.
Sediments are ecologically important because they mediate
chemical exchange among the particulate, dissolved and biological
phases. They provide a valuable indication of overall environmental
contamination (Borgmann et al., 2005). In the last decades, impor-
tant efforts to determine sediment ecotoxicity have been made by
scientists and authorities. Thus, many bioassays have been devel-
oped with benthic species, being amphipods usually chosen as test
organism (ASTM, 1995; USEPA, 2000). Most of the numerous
sediment quality guidelines (SQGs) were developed to assist reg-
ulators in dealing with contaminated sediments based on data
obtained from North America (Burton, 2002). The specific applic-
ability of the SQGs to a particular geographical region is uncertain
due to differences in geography (e.g. temperature, salinity, geological
formation). This study presents the first results of sediment Cd
exposure effects in a freshwater amphipod species of the Southern
Hemisphere.
H. curvispina exposed to the metal for 10 day were not affected
for survival nor length; only animals exposed to the highest
assayed concentration (5.6 mg Cd/Kg ds) showed a reduction of
approximately 20 percent of their biomass, and it is important to
note that this response was obtained at concentrations close to
the consensus based PEC of 4.98 mg Cd/kg (Burton, 2002). These
results could be useful for the validation of the use of these
guidelines at a regional level. Milani et al. (2003) studied the
effect of cadmium on H. azteca individuals exposed to contami-
nated sediments for 28 day and found that 10 mg Cd/Kg sediment
caused a growth inhibition of 25 percent while the LC50 was of
33 mg Cd/Kg. Comparison of metal toxicity in contaminated
sediments is difficult because of differences between the sedi-
ments used, i.e., granulometry, TOC and AVS, among other
parameters relevant to the equilibrium partitioning and potential
toxicity of metals in sediments. In this sense, it should be
considered that the granulometric composition used by Milani
et al. (2003) contained a lower proportion of sand than the one
used in this study, thus possibly reducing the metal availability
for the animals.
Cadmium uptake by H. curvispina was concentration-depen-
dant and quantitatively lower in the whole sediment assay
compared to the water-only assay. Taking into account that there
was no cadmium uptake from the overlying water (see Table 2),
it must be considered that the uptake route of the metal is from
the sediment and significantly lower than that from the aqueous
matrix. In those animals exposed to the lowest assay concentra-
tion in water (2.5 mg Cd/L), body burden was higher than all the
concentrations assayed in sediment. Therefore, the lower increase
in biomass registered in animals exposed to 5.6 mg Cd/Kg ds
(Fig. 2C) would be a consequence of other factors such as a
reduction in the intake and/or a metabolism increase and not of
the metal uptake. As higher levels of enzymes are synonymous
with higher detoxification capability, higher levels of metals can
be stored inside organisms without deleterious effects (Pastorinho
et al., 2009). It is worthwhile to study whether the large amounts of
cadmium accumulated in the tissues of H. curvispina would be
transferred through the food chain to higher trophic levels, posing a
potential ecotoxicological risk.
5. Conclusion
Under the experimental conditions juveniles of H. curvispina
proved to be sensitive to ecologically relevant Cd concentrations,
in both the solid and aqueous matrix. In sediment assays,
response was quantifiable in values close to guidelines, thus
possibly validating the interim use in this region of the consen-
sus-based SQGs for Cd developed in the Northern Hemisphere.
Animals showed a significant capacity of Cd accumulation in their
tissue. These results reassert the importance of juveniles of
H. curvispina as a species suitable for assays of ecotoxicity assess-
ment in water bodies within the distribution area of the species.
Acknowledgments
This work was supported by grants from the National
University of Lujan, CIC-BsAs. Argentina and MINCyT/SPU/PPUA
168 A. Giusto et al. / Ecotoxicology and Environmental Safety 79 (2012) 163–169

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