biomonitoring toxicity of natural sediments using juvenile hyalella curvispina (amphipoda) as test...

Biomonitoring toxicity of natural sediments using juvenileHyalella curvispina (Amphipoda) as test species: evaluationof early effect endpoints

Anabella Giusto • Alfredo Salibian •

Lucrecia Ferrari

Accepted: 28 December 2013 / Published online: 11 January 2014

� Springer Science+Business Media New York 2014

Abstract The utility of early effect endpoints as bio-

markers of ecotoxicity of natural sediments in water–sed-

iment static system was investigated. The particular goal

was to evaluate the ecotoxicity of the sediment samples

from La Choza stream, located in upper basin of the Re-

conquista river, the second most polluted river of Argen-

tina. Native juveniles Hyalella curvispina were used as test

organisms evaluating survival, growth, oxidative stress

parameters (SOD; CAT, TBARS) and the electron trans-

port system (ETS) activity as early toxic effect. This study

used methodologies and techniques that allow the assess-

ment of sediment pollution with a native species as test

organism and provided data to discuss the viability of

sublethal endpoints as tools for freshwater sediment

assessment. In spring and in summer two ten-day series of

whole-sediment assays were conducted simultaneously:

(a) standard assays and (b) biomarkers assays. A control

sediment was ran simultaneously in which no––effect on

survival was measured. In summer there was a significant

increase in length and biomass in both exposed and control

groups. In spring an inhibitory effect on growth and an

increase in oxidative damage with a concomitant rise in

antioxidant defenses, was observed in animals exposed to

La Choza sediment. ETS measurement indicated a signif-

icant depression of metabolic activity of amphipods

exposed to contaminated sediments. The measured bio-

markers represent the first record for juvenile H. curvispina

exposed to polluted natural sediments under standardized

laboratory conditions. The used bioanalytical tools dem-

onstrated higher sensitivity and a more accurate assessment

of the effects than those obtained by the standard tests of

survival and growth. We propose their adoption in bio-

monitoring of freshwater sediment toxicity.

Keywords Polluted sediments � Bioassays � Oxidative

stress � Electron transport system (ETS) � Reconquista

river � Hyalella curvispina

Introduction

When wastes are discharged to watercourses, they are

distributed into different compartments of the ecosystem

with sediment being the final receptor. Sediments are

complex matrices that represent a mixture of materials and

can accumulate significant amounts of toxic elements and

substances. Contaminated sediment has a different chemi-

cal profile and/or toxicological quality in relation to geo-

chemical criteria of pristine sediment. They can have

adverse effects on the environment and/or on human health

(Burton 2002). The quality of the sediment is often deter-

mined by comparing the total concentration of contami-

nants with baseline or reference values. However, it has

long been accepted that the quantification of pollutants

alone is not sufficient to determine possible adverse effects

of sediments on organisms (Ingersoll 1995).

Along with the analytical quantification of contamina-

tion, the associated effects on biota must also be studied

since freshwater benthic invertebrates are intermediaries

between primary producers and higher consumers. In

A. Giusto � A. Salibian � L. Ferrari (&)

Applied Ecophysiology Program (PRODEA), Basic Sciences

Department, Institute of Ecology and Sustainable Development

(INEDES), National University of Lujan, P.O. Box 221,

B6700ZBA Lujan, Argentina

e-mail: [email protected]

L. Ferrari

Scientific Research Commission (CIC), La Plata, Buenos Aires,

Argentina

123

Ecotoxicology (2014) 23:293–303

DOI 10.1007/s10646-013-1173-7

addition, due to the short generation times, the morpho-

logical and biochemical changes in benthic organisms may

be evident in the short and medium term.

For the evaluation of sediment quality, the most widely

used bioassays are carried out using whole sediments

samples and different crustaceans (amphipods, cladocer-

ans), insects (dipterous) and annelids (polychaetes, oligo-

chaetes) as test organisms (Ingersoll 1995). Among

crustaceans, amphipods are widely used in laboratory

toxicity tests of sediments (Burton et al. 1996; Bat and

Raffaelli 1998; USEPA 2000). Standardized protocols are

commonly used to evaluate the effect on their growth and

mortality, assuming that the absence of effect indicates a

sediment ecotoxicologically acceptable. However, it is a

priority to develop testing protocols to incorporate early

effect endpoints that would reduce the gap between labo-

ratory and field results. In recent years, attention has

focused on the use of physiological and biochemical

parameters as indicators of toxic stress through exposure of

test species to different pollutants (Dutra et al. 2007, 2008).

The ecological significance of these parameters is based on

the fact that short exposures to different pollutants can have

long term effects on the life cycle of organisms even when

the pollutants are not persistent in the environment.

The rivers and streams running through urban and sub-

urban areas of the rolling Pampas region of Argentina

frequently receive contaminated discharges from agricul-

tural, livestock and industrial activities (Rodrıgues Capıt-

ulo 1984; Jergentz et al. 2005; Giusto and Ferrari 2008;

Ronco et al. 2008; Mugni et al. 2011). La Choza is one of

the streams that flow into the Roggero dam located in the

upper basin of the Reconquista river, which is one of the

most polluted peri-urban rivers in Argentina. It is 30 km

long and drains an area of approximately 440 km2. The

principal activity of the region is agriculture and farming;

in addition, La Choza stream also receives raw sewage

from a country club, a rural zone and a small industrial

park, all located upstream from the sampling site (Rigacci

et al. 2013).

The amphipod, Hyalella curvispina, is widely distrib-

uted in South America (Gonzalez 2001). In recent years, it

has been commonly used as a test organism in freshwater

toxicity and biomonitoring tests of sediments in laboratory

and field conditions (Anguiano et al. 2008, 2012; Di

Marzio et al. 2005, 2010; Garcıa et al. 2010, 2012; Giusto

et al. 2012; Graca et al. 2002; Jergentz et al. 2004; Mugni

et al. 2011, 2012; Peluso 2011; Peluso et al. 2013). How-

ever, the use of biomarkers as endpoints for ecotoxicity

evaluation are infrequently used (Anguiano et al. 2008,

2012; Dutra et al. 2009; Venturino et al. 2007).

The general toxicity pathway that can be induced by

many chemical contaminants is related to their capacity for

catalyzing oxidative reactions, which leads to the

production of reactive oxygen species (ROS) and oxidative

stress (Timbrell 2009). Two important antioxidant enzymes

are catalase (CAT) and superoxide dismutase (SOD).

Failure of these antioxidant defenses to stop excess ROS

production can lead to significant oxidative damage

including enzyme inactivation, protein degradation, DNA

damage and lipid peroxidation (Halliwell and Gutteridge

1999). In particular, lipid peroxidation is considered to be a

major consequence by which oxyradicals cause tissue

damage, impairing cellular function and physicochemical

properties of cell membranes, which in turn may disrupt

vital cellular functions (e.g. cellular permeability).

Among possible biomarkers of metabolic stress, the

activity of the electron transport system (ETS) is not

widely used. The ETS is a multi-enzyme complex located

in the inner membrane of mitochondria whose activity

indicates the amount of oxygen consumption that would

occur if all enzymes function maximally. An ETS assay

was first proposed by Packard (1971), and has since been

modified and improved by several authors, it has proven

useful for estimating the metabolic activity of different

organisms such as microplankton (Devol and Packard

1978), zooplankton (Owens and King 1975; Borgmann

1978; Simcic and Brancelj 2001), benthic macrofaunal

species (Cammen et al. 1990), sediments (Simcic and

Brancelj 2002) and biofilms (Blenkinsopp et al. 1991).

We investigated the utility of early effect biomarkers in

ecotoxicity assessments of natural sediments. We used an

ecologically relevant benthic species in sediment–water

static systems to evaluate the sublethal early effects as tools

for the toxicological assessment of sediment quality. The

aim of this study was to evaluate the ecotoxicity of sedi-

ment samples from the polluted La Choza stream. The

native amphipod, H. curvispina, was used as the test

organism and survival, growth, oxidative stress parameters

(SOD and CAT activity, lipid peroxidation) and electron

transport system (ETS) activity were the endpoints.

Materials and methods

Test organisms

The test organisms were juvenile H. curvispina (3–4 mm in

length) obtained from outdoor and indoor (laboratory)

cultures. The outdoor cultures, a mesocosmos system, were

maintained under a natural regimen of temperature and

photoperiod. The indoor cultures were maintained under

constant temperature (23 ± 1 �C) and a photoperiod regi-

men of 16L/8D. Both were cultured in tap water (hardness:

80–90 mg CaCO3/L) and had the aquatic macrophyte

Egeria densa as a substrate. The cultures were maintained

according to instructions in Somma et al. (2011). Animals

294 A. Giusto et al.

123

used in summer assays came from outdoor cultures and

those used in spring assays came from indoor cultures (first

generation).

For each assay, a pool of animals was selected from the

culture by sieving and acclimated for 7 days before the

beginning of the assay in aerated moderately hard water

(MHW) of the following composition (mg/l): NaHCO3, 96;

CaSO4�2H2O, 60; MgSO4, 60; KCl, 4; pH, 7.4–7.8; hard-

ness, 80–100 mg CaCO3/L (USEPA 1993) under con-

trolled temperature and photoperiod (23 ± 1 �C; 16L/8D)

conditions. Animals were fed daily ad libitum with crushed

fish food of the following composition (%): carbohydrates

30.0; proteins 42.7; fat 10.5; ash 10.5; moisture 6.3, and

gross energy value of 14.316 J/g. The survival rate in the

animals over the acclimation period was higher than 90 %.

Sediment treatments

Sediment was dredged from the outlet bottom (up to a depth

of 10 cm) of the mouth of the stream of La Choza (34�400S,

58�640W) (Fig. 1). Two samples were taken in both, sum-

mer and spring of 2010. Each sample was made of at least

ten sub-samples collected from the upper layers of the

sediment. In order to remove leaves and macro fauna, the

entire wet sediment sample was sieved using a broad pore

network (1,000 mm). Then, it was manually homogenized

and stored at 4 �C for 24 h. After this time, the supernatant

was removed and the sediment was homogenized again for

use in the assays within the next 24 h.

A control sediment was taken from Las Flores, an

influent stream of the Lujan river (59�070W, 34�290S)

(Fig. 1) using the same methodology. Subsamples of the

sediment were analyzed to chemical analysis. Sulfites,

cyanides or hydrocarbons were not detected. Determina-

tions of organochlorine, organophosphate and pyrethroid

pesticides reached values below the detection levels (2 mg/

Kg) (Ronco et al. 2008). Subsamples of the sediment were

homogenized manually, left to dry, crushed, sieved,

homogenized once more and then kept dry at room tem-

perature until use. For the assays, the stock dry control

sediment was rehydrated (1 kg sediment/L MHW) and

allowed to settle for 3 h. Then the supernatant was dis-

carded and the rehydrated sediment was used as test con-

trol. Although manipulations of sediments have been

shown to affect benthic invertebrate responses in whole-

sediment toxicity tests (Day et al. 1995), we have not

recorded any effect on growth and survival of H. curvisp-

ina in bioassays with rehydrated control sediment (Giusto

et al. 2012).

Before the beginning of each assay subsamples from

both sediments (Las Flores and La Choza) were taken to

determine moisture, organic matter content and granulo-

metric composition (with previous organic matter removal

by ignition loss at 550 �C for 5 h) through the Robinson

pipette method (Depetris 1995). In addition, As, Cd, Zn,

Cu, Cr and Pb content was determined by inductively

coupled plasma atomic emission spectroscopy (ICP-AES)

after digestion of the sample (weak oven-microwave

digestion) according to the USEPA Standard 3051 Method.

Three of the sediment samples were provided by National

Water Research Institute of Canada. Data were expressed

as mg/Kg dw.

Fig. 1 Geographical location of

the Reconquista river basin.

Asterik indicates the sampling

sites: the control sediment from

Las Flores stream (a tributary of

Lujan river) and the polluted

sediment at the mouth of La

Choza stream, tributary of the

Roggero dam

Biomonitoring toxicity of natural sediments 295

123

Bioassay procedure

For each sediment sample, two series of static ten-day

whole-sediment assays were conducted in parallel:

(a) standard assay of survival and growth, following pro-

tocol described in USEPA (2000) with modifications; and

(b) assays for the evaluation of biochemical biomarkers.

For the standard assays, the control sediment (from Las

Flores stream) and those from La Choza stream were

partitioned into three replicates of 100 ml sediment and

175 ml MHW (overlying water) each (USEPA 2000). For

the biomarkers assays, five replicates of each sample were

tested with 500 ml sediment and 875 ml MHW. Thus a

constant sediment/overlying water volume ratio was

maintained in both assays. Each polypropylene beaker

(diameter of 6.5 and 13 cm for standard and biomarker

assays, respectively) was covered to minimize evaporation.

All replicates were placed in a culture chamber under

controlled conditions as it was described above, supplied

with constant aeration and allowed to equilibrate for 2 days

before the beginning of the assays. For standard assays ten

individuals per replicate were used and 50 individuals per

replicate in biomarkers assays. The overlying water/sedi-

ment rate and the number of individuals for each replicate

followed the USEPA (2000) guidance for amphipod tests.

Each replicate was supplied with crushed fish food

flakes in a proportion of 5 mg/10 individuals/10 days

(Giusto et al. 2012). The amount of food given was higher

than that recommended in the USEPA standard method

(USEPA 2000) based on our previous experience (Giusto

et al. 2012). The survival acceptability criterion for both

experimental series was [80 % survival in the controls.

At the initial and final exposure times, the following

parameters were measured in the overlying water: dis-

solved oxygen, pH and conductivity (oxymeter Hanna, pH

meter Mettler, conductimeter Consort C532). Ammonium

nitrogen concentration was determined using a Spectro-

quant test kit (Merck; measuring range 0.05–3 mg/l total

NH4-N) and hardness estimated by the volumetric method

with the Aquamerck test kit (Merck; sensitivity 1 mg/l

CaCO3).

Endpoints in standard assays

At the beginning of the assay, 15–20 animals from the

acclimation stock group were separated and frozen at

-20 �C for processing at a later date (initial group). At the

end of the assays, all surviving individuals in each replicate

were also frozen at -20 �C. The endpoints measured at the

end of the assays were survival and growth (as body weight

and length) relative to those of the control and initial

groups. The length, defined as the distance from the base of

the first pair of antennae to the tip of the telson, was

measured under a stereoscopic microscope with a digital

caliper (nearest to the 0.01 mm) while holding the animal

in a stretched position (Doyle and Momo 2009). Surviving

animals were dried at 60 �C for 48 h and weighed on a

Mettler balance (accuracy[0.01 mg). The individual mass

was expressed as dry weight in mg.

Endpoints in biomarker assays

The evaluated endpoints were CAT and SOD activity, lipid

peroxidation level (TBARS) and electron transport system

(ETS) activity. At the end of the bioassays amphipods were

pooled and immediately frozen at -80 �C until they were

used to determine the biochemical parameters.

For each replicate (controls and exposed), four individ-

uals were taken to determine ETS and the remaining were

used to determine total protein content, quantification of

lipid peroxidation, and CAT and SOD activity. Biomarkers

were determined by spectrophotometric methods in quin-

tuplicate and the results were expressed as mean ± SEM.

Proteins were measured using commercial Labtest kit

based on Biuret’s method; the results were expressed in mg/

g. For lipid peroxidation level measurement, the pools were

extracted using the method of Llesuy et al. (1985), followed

by quantification as described Buege and Aust (1978); the

results were expressed as mean in nmoles of TBARS/mg of

protein. The CAT activity was determined using the method

described by Boveris et al. (1972) and the results expressed

as lmoles H2O2/mg protein/min. SOD activity was mea-

sured according the method described by Misra and

Fridovich (1972) and expressed as mean of U/mg protein.

The measurement of ETS activity method is based on

the spectrophotometric determination of tetrazolium oxi-

doreductase activity recorded as the electrochemical

equivalent of oxygen to INT (p-IodoNitroTetrazolium

Violet)-formazan produced. The cryopreserved animals

were placed individually on Parafilm and weighed (accu-

racy 0.01 mg). Then they were ground in ice-cold

homogenizing buffer [0.1 M Tris–HCl buffer pH 8.5, 15 %

(w/v) polyvinyl pyrrolidone, 153 lM MgSO4, 0.2 % (w/v)

Triton-X-100] in a proportion of 5 mg/ml buffer. The

homogenates were centrifuged at 4 �C and 3,000g for

10 min. After centrifugation, 300 lL of buffered substrate

(0.13 M Tris–HCl pH 8.5 and 0.3 % (w/v) Triton X-100)

and 100 lL of NADPH (1.7 mM NADH and 250 lL

250 lM of NADPH) were added to 100 ll of supernatant.

The reaction was induced by adding 100 lL INT (8 mM,

pH 7.5). After and incubation at 30 �C for 20 min, the

reaction was stopped with 100 lL of 1:1, formalin: H3PO4

solution. All chemicals were analytical grade.

ETS activity was calculated from INT-formazan

absorption at 490 nm against the blank (Kenner and

Ahmed 1975) as:


123

ETS ¼ Abs490nm� Vr � Vh � 60

Va � S � t � 1:42

where, Vr is the final volume of the reaction mixture (ml); Vh,

the volume of the homogenate (ml); Va, volume of the

homogenate aliquot (ml); S, sample size (mg wet mass); t,

incubation time (min); and 1.42, conversion factor to volume

of O2; ETS activity was expressed as ll de O2/mg ww/h.

Statistical analysis

For biomarkers all results were expressed as mean ± SEM.

Differences between treatments were checked by the Stu-

dent’s t test; the significance level was 5 %. For growth,

the statistically significant differences between treatments

and the initial group was evaluated through a one way

ANOVA followed by Tukey’s test (Zar 2010); the Shap-

iro–Wilk and the Levene tests were used to assess nor-

mality and homoscedasticity, respectively. The statistical

analyses were carried out with the Infostat or Statistical

Package (Di Rienzo et al. 2012) of the Social Science

software (SPSS 11.5) for Windows.

Results

Table 1 shows grain size, humidity and organic matter

content of the control (Las Flores stream) and La Choza

sediments. They can be classified as sandy loam (MESL

2001). In the control sediment the percentage of organic

matter was high and humidity was found within the

expected range according to our previous studies (Giusto

and Ferrari 2008; Giusto et al. 2012). The sediment from

La Choza exhibited higher water content and lower organic

matter than the control. Heavy metal concentrations in

sediments of both streams were found below the levels

stipulated by the CEQG Canadian Environmental Quality

Guidelines (2013) for sediment quality guidelines for the

protection of aquatic life even in summer samples of La

Choza (with the exception of As) (Table 2).

The physicochemical parameters of the overlying water

in the standard and biomarkers assays at initial and final

times are shown in Tables 3 and 4. These parameters did

not show remarkable variations between summer or spring

samples. With the exception of ammonium, they were

within acceptable range for standard bioassay conditions

(Borgmann 1994; Environment Canada 2013). The

ammonium concentrations showed variability even in the

controls, but with values within the acceptable survival

range for H. curvispina (Giusto et al. 2012).

Table 5 shows the measured endpoints at the end of the

standard bioassays. Percentages of amphipods survival in

the control sediment ranged between 83 and 90 %, meeting

the accepted criteria recommended by USEPA (2000) for

whole sediment bioassays.

Relative to the controls, the average survival in the

sediment from La Choza was slightly lower but the dif-

ference was not statistically significant. In terms of growth,

a differential response between the two trials was observed;

in the summer the Las Flores group (control) and the group

exposed to sediment from La Choza showed a significant

increase in length and biomass compared to the initial

values without significant differences between treatments.

In the spring test, control and exposed animals, did not

register a significant increase in length compared with the

initial group. However, while the biomass of the control

group significantly increased in relation to the initial group,

the one exposed to La Choza sediment did not differentiate

from the initial group, indicating an inhibitory effect on the

growth rate.

Table 6 shows the recorded biomarkers values and their

ratios between controls (Las Flores stream) relative to

those of La Choza group for both assays. There were dif-

ferences in the activities of CAT, SOD and ETS in the

control samples. However, although the differential

response between controls was evident, in the groups

exposed to the sediment from La Choza and their respec-

tive controls, a significant increase in the antioxidant

activity and oxidative damage was observed. There was

also a reduction of the ETS activity. Due to background

seasonal variations in the biochemical responses of

H. curvispina (Dutra et al. 2008) and the different stocks of

Table 1 Grain size composition, humidity and organic matter of

whole sediment samples

Sediment Sand Silt Clay Humidity Organic

matter

n

Las Flores

(control)

61 27 12 39.3 ± 0.9 6.9 ± 0.1 6

La Choza

(summer sample)

57 30 13 55.6 ± 0.7 4.0 ± 0.2 3

La Choza

(spring sample)

58 27 15 56.2 ± 0.0 5.4 ± 0.1 3

Data as percentage; mean ± SD; n number of determinations

Table 2 Heavy metals content in the assayed sediments

Sample As Cd Zn Cu Cr Pb

Las Flores (control) \2 \0.5 \2 4 19 3

La Choza (summer sample) 20 \2 106 25 26 \10

La Choza (spring sample) \2 \0.5 \2 7 22 \10

CEQGa 5.9 0.6 123 35.7 37.3 35

a CEQG Canadian sediment guidelines for protection of aquatic life;

data as mg/Kg dw


123

animal pools used, the evaluation of the response to La

Choza sediments was relative to their parallel controls. In

the summer and spring tests, the activity of CAT and SOD

and the level of TBARS increased in animals exposed to

sediment from La Choza in comparable magnitudes. The

CAT activity in La Choza samples showed an increase of

1.4–1.6 times over the control. SOD increased 9.7 times

(summer) and 5.3 times (spring), and TBARS was aug-

mented 3.2 and 3.4 times in spring and summer,

respectively. Relative to the control, ETS activity in La

Choza group decreased at a rate of 0.6. Except for SOD,

which was more active during the summer trial, there were

no significant differences between trials in biomarkers. An

increase in oxidative damage and in antioxidant defenses in

the groups exposed to sediment from La Choza was evi-

dent. In addition, changes in the ETS showed a depression

of metabolic activity in animals exposed to La Choza

sediments.

Table 3 Physicochemical parameters in the overlying water at initial and final exposure times in the standard assays

DO (mg/l) pH Conductivity (lS/cm) Hardness (mg CaCO3/L) NH4–N (mg/l) n

Summer assay

Initial time Control 6.5 ± 0.3 7.9 ± 0.1 611 ± 10 130 ± 0 0.8 ± 0.1 3

La Choza 7.2 ± 0.3 8.0 ± 0.1 551 ± 3 110 ± 10 8.7 ± 0.5 3

Final time Control 7.0 ± 0.2 8.7 ± 0.0 n m 240 ± 0 4.8 ± 0.5 2–3

La Choza 5.9 ± 0.2 8.1 ± 0.1 n m 110 ± 10 0.6 ± 0.2 2–3

Spring assay

Initial time Control 7.6 ± 0.4 8.4 ± 0.1 793 ± 17 170 ± 0 2.7 ± 0.0 2–3

La Choza 8.2 ± 0.3 8.2 ± 0.1 710 ± 16 90 ± 0 5.9 ± 0.0 2–3

Final time Control 8.2 ± 0.1 8.8 ± 0.0 1,084 ± 10 230 7 1–3

La Choza 6.2 ± 2.5 8.2 ± 0.3 1,115 ± 24 140 5 1–3

Data as mean ± SD; n number of determinations; n m not measured

Table 4 Physicochemical parameters in the overlying water at initial and final exposure times in the biomarkers assays

DO (mg/l) pH Conductivity (lS/cm) Hardness (mg CaCO3/L) NH4-N (mg/l) n

Summer assay

Initial time Control 7.7 ± 0.6 8.3 ± 0.1 580 ± 20 140 ± 10 0.2 ± 0.1 4–5

La Choza 6.6 ± 0.5 8.1 ± 0.1 504 ± 11 110 ± 0 6.9 ± 0.6 4–5

Final time Control 5.0 ± 1.0 8.4 ± 0.1 n m 300 ± 30 6.1 ± 1.0 4–5

La Choza 4.8 ± 0.4 8.5 ± 0.1 n m 170 ± 10 0.3 ± 0.1 4–5

Spring assay

Initial time Control 7.0 ± 0.8 8.0 ± 0.2 716 ± 17 160 ± 0.0 3.0 ± 0.3 2–5

La Choza 7.9 ± 0.1 8.2 ± 0.0 678 ± 19 90 ± 0 5.9 ± 0.5 2–5

Final time Control 8.2 ± 0.1 8.8 ± 0.0 1,048 ± 12 260 ± 20 6.5 ± 0.3 2–5

La Choza 8.0 ± 0.3 8.5 ± 0.0 1,051 ± 28 130 ± 10 5.9 ± 1.1 2–5

Data as mean ± SD; n number of determinations; n m not measured

Table 5 Endpoints measured in the standard assays at final exposure time

Bioassays Survival (%) Length (mm/individual) Dry weight (mg/individual)

Control La Choza Initial Control La Choza Initial Control La Choza

Summer 83 ± 11

(3)

72 ± 15

(3)

3.42 ± 0.38

(20)

4.08 ± 0.48a

(25)

4.05 ± 0.61a

(21)

0.34 ± 0.12

(20)

0.65 ± 0.21a

(25)

0.67 ± 0.24a

(21)

Spring 90 ± 10

(3)

77 ± 6

(3)

3.69 ± 0.26

(15)

3.94 ± 0.41

(27)

3.72 ± 0.32

(21)

0.40 ± 0.11

(15)

0.61 ± 0.17a

(27)

0.44 ± 0.12b

(23)

Data as mean ± SD; between brackets, number of replicates (survival) and number of individuals (length and dry weight)a Indicates significant difference with the initial groupb Indicates significant difference from the control group (p \ 0.05)


123

Discussion and conclusions

Located in the upper basin of the Reconquista river, La

Choza stream provides the greatest contaminant load to the

riverbed. This is mainly a consequence of discharge from a

sewage treatment plant and from point wastewater

unloading such as those generated by a small industrial

park located near the mouth of the stream. At this site there

is, among others, an agrochemical industry and poultry

slaughter, which continuously discharges effluent into the

stream. Studies at this site showed elevated Biochemical

Oxygen Demand (BOD5) values and suspended solid levels

indicating organic pollution (Rigacci et al. 2013).

The composition of the sediment of the Las Flores and

La Choza streams is comparable to that of other waterways

in the region because of the similarity of their geological

substrates. Exposure to La Choza stream sediment did not

significantly affect the survival of H. curvispina relative to

the controls. When survival was evaluated as an endpoint

in previous sediment toxicity tests, this species proved to

be sensitive to metal contamination (Garcıa et al. 2012;

Peluso et al. 2011). Although the La Choza sediment has

also an elevated microbiological pollution, particularly in

faecal coliforms (Lopez et al. 2013), sediments collected

for this study contained low levels of contamination by

metals (Table 2), which may partially explain its low lethal

effect. Organochlorines and organophosphates pesticides

content were below their detection limits. These results

suggest that under our experimental conditions the survival

of H. curvispina may not be a sensitive endpoint (Giusto

and Ferrari 2008; Giusto et al. 2012; Peluso 2011); in

response to environmental pollution-related stress it might

not be excluded the protective role of the antioxidant sys-

tem (CAT–SOD) (see Table 6). Even when standard assays

did not elicit statistically significant effects, sublethal

responses of the biomarkers may be detected.

The two seasonal trials (summer and spring) were con-

ducted with juvenile H. curvispina under identical experi-

mental conditions after an acclimation period, but the pool

of organisms used in each case differed in their origin. In

the spring test, organisms came from an indoor culture with

the same temperature and photoperiod regime as the

experimental period, while in the summer test animals

came from an outdoor culture with natural temperature and

photoperiod regimes. The only source of variation between

pools should be attributed to conditions of the original

culture (indoor/outdoor), although both cultures were

started with H. curvispina individuals from the same pop-

ulation (Las Flores stream).

In the summer assays the test animals (from the outdoor

culture) of the control and exposed groups increased their

length and biomass in a comparable manner. On the con-

trary, in spring assay (indoor culture) both, control and

exposed group did not increased in length suggesting the

absence of ecdysis during the experimental period; only the

controls gained weight (Table 5). These results suggest a

higher level of vulnerability of the indoor culture animals.

It can be concluded that, in this case, there was no sediment

effect on the growth of H. curvispina. Assuming that the

degree of contamination of La Choza sediment samples

were relatively comparable between the two assays

(Tables 1, 2, 3 and 4); the differential response in the

growth could be attributed to a consequence of changes

happened in the homeostatic condition of the animals.

Probably the increased vulnerability of animals grown in

controlled laboratory conditions (indoor) could overesti-

mate the recorded toxic effect.

Regarding the biomarkers of oxidative stress in the

summer test, the controls showed higher antioxidant

activity than in the summer controls but, according TBARS

values, no significant differences were observed in relation

to possible oxidative damage (see Table 6). These results

raise questions about the representativeness of the

responses at the population level and highlight the diffi-

culty in establishing reliable baselines for these biomark-

ers. Information about the effects of pollution on oxidative

stress in amphipods is not abundant. The works of Tim-

ofeyev et al. (2006a, b) with five species of amphipods

Table 6 Endpoints measured in the biomarkers assays at the end of

the exposure time

Control La Choza La Choza/

control

n

Summer bioassay

CAT

(lmolH2O2/

min/mg Prot)

29.64 ± 4.15 47.57 ± 5.61a ?1.6 5

SOD (U/mg

Prot)

15.37 ± 1.45 149.32 ± 53.39a ?9.7 5

TBARS

(nmol/

mg de Prot)

6.14 ± 0.55 19.68 ± 2.89a ?3.2 5

ETS (ll O2/

mgww/h)

2.37 ± 0.08 1.56 ± 0.60a -0.65 20

Spring bioassay

CAT

(lmolH2O2/

min/mg Prot)

36.30 ± 0.77 51.21 ± 1.1a ?1.4 5

SOD (U/mg

Prot)

6.69 ± 0.20 35.23 ± 1.85a ?5.3 5

TBARS

(nmol/

mg Prot)

5.09 ± 0.25 17.22 ± 0.39a ?3.4 5

ETS (ll O2/

mgww/h)

1.26 ± 0.11 0.89 ± 0.49a -0.7 20

Data as mean ± SD; n number of determinationsa Statistically significant differences from the control (p \ 0.05)


123

from Lake Baikal assessed the potential oxidative stress of

organic matter and demonstrated a significant increase in

lipid peroxidation and catalase activity. Another study

(Timofeyev and Steinberg 2006) evaluated the oxidative

stress response in species with divergent habits from the

same lake (shallow-water dwelling and the deep-layer

inhabitant species). They found a differential response

between littoral and the deep-layer inhabitants and con-

cluded that the stable environment of the deep zones does

not provide triggers for antioxidant or general defense

systems. They also assessed a possible specificity in anti-

oxidant systems between palearctic versus endemic species

concluding that the difference in enzyme activity most

likely depends on interspecific variation rather than on

specific environmental conditions (Timofeyev 2006). This

is an important topic that should be studied further.

On the other hand, Timofeyev et al. (2009) showed that

the duration of acclimation, may alter response patterns.

This must be considered when designing studies conducted

in the laboratory using animals taken from their natural

environment. Interestingly, exposure of amphipods to

pollutants may trigger anticipatory response to overcome

oxidative stress with SOD activity. These results empha-

size the importance of understanding interactions between

antioxidant responses to different stressors and physiolog-

ical mechanisms of oxidative damage (Gorokhova et al.

2013).

SOD activity for H. curvispina juveniles was compara-

ble to that obtained for juveniles of other amphipods,

polychaetes, bivalves, decapods and echinoderms (Correia

et al. 2002). The same authors reported much more vari-

ability between species for CAT activities with (up to two

orders of magnitude difference). H. curvispina CAT values

observed in this study are close to that published for other

crustaceans (Correia et al. 2003). In addition, Anguiano

et al. (2012) recently observed a significant increase of

CAT activity in H. curvispina exposed to the organo-

phosphate pesticide azinphos methyl.

It is important to take into account that ROS may result

from contaminant exposure but it is also produced during

aerobic respiration; therefore, high metabolic activity may

result in elevated ROS levels (Hoguet and Key 2008).

Regard to the variability of responses, seasonal variation of

biological parameters within populations should not be

dismissed. Even cultures reared for generations in the

laboratory are not completely ‘‘silenced’’. Dutra et al.

(2007, 2008) noted seasonal metabolic variations for

Hyalella.

The tests in this study were conducted during a meta-

bolically active stage and over the period of the year during

which the natural populations have maximum biomass

(Poretti et al. 2003; Garcıa 2009). It is possible, therefore,

that juvenile amphipods in this study were subjected to

major competition for food and substrate. Regardless of the

variability observed between both trials, the magnitude of

the difference in biomarker response relative to their con-

temporaneous controls was comparable (Table 6). Animals

exposed to sediment from La Choza showed high antiox-

idant activity due to the increased CAT and SOD activities,

which was accompanied by high levels of lipid

peroxidation.

The activity of the electron transport system (ETS) is a

biomarker rarely used in ecotoxicological studies (De Coen

and Janssen 1997, 2003; Smolders et al. 2004; Verslycke

et al. 2003). The trials in our study, reported a significant

decrease in the metabolic activity of the animals exposed to

test sediments. The ETS values observed in this study are

comparable to those recorded for other amphipods, Daph-

nia and anostracs (Simcic and Brancelj 2003, 2004; Simcic

et al. 2005, 2012). The determination of ETS is feasible in

small individual tissue samples and it is a promising

parameter to include as a biomarker of metabolic status in

standard bioassays with amphipods. The difference

between our two controls represents a clear indication of

variability unrelated to treatment and is probably the result

of the different fitness of the pool of organisms used in both

cases; the highest values of ETS from the summer test

animal pool would confirm this appreciation (Table 6).

It is important to note that sublethal parameters (length

and biomass) and biomarkers response showed significant

variability that may be attributed to other reasons than the

stressor exposure. Hence, there is a need for other param-

eters to objectively assess the quality of environmental

samples with unambiguous interpretation that can be used

in environmental monitoring assessment studies. Regard-

less of the possible quantification of different toxic agents,

it is difficult to establish adverse cause––effect relation-

ships for the test organisms. Thus, it is crucial to establish

monitoring programs that include parameters to reasonably

extrapolate the results of assays carried out in laboratory

experimental conditions to natural conditions.

In brief, amphipods have many factors that complicate

toxicity biomarker selection. In crustaceans, factors such as

phylogenetic differences among taxa, locomotive activity,

body size, morphogenesis and molting cycle have an

impact on metabolism. There is also the impact of natural

environmental variables, such as nutrition, pH, tempera-

ture, salinity, and osmotic pressure (Anger 2001). Never-

theless, biomarkers are important in toxicological research

because they aid in overall risk assessment and can be used

as an early warning signs (Hoguet and Key 2008).

According to the literature and the data provided by this

study, we propose that it should be standard practice to

express values from the biochemical results relative to their

contemporary controls. The measured biomarkers in this

study represent the first record for juvenile H. curvispina


123

exposed to contaminated sediments under standardized

laboratory conditions. While it is necessary to further

evaluate the sublethal responses in laboratory and in nat-

ural conditions biomarker parameter response in H. cur-

vispina exposed to contaminated sediments relative to

control exposures was more consistent and robust, showed

greater sensitivity and may be deemed to provide a better

evaluation of effects than the endpoints obtained by the

standard assay for survival and growth.

Acknowledgments This work was supported by grants from AN-

PCyT (PICT No 371/2007), the National University of Lujan and the

CIC-BsAs. Argentina. We thank to Guendalina Turcato Oliveira and

Bibiana Kaiser Dutra for the invaluable contribution to this work and

to Martın Da Silva for the technical assistance. We also thank the

anonymous reviewers for their valuable comments and suggestions,

which have greatly improved the manuscript.

Conflict of interest The authors declare that they have no conflict

of interest.

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