Ecotoxicology and Environmental Safety 123 (2016) 60–64

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Lethal and sublethal endpoints observed for Artemia exposed to two

reference toxicants and an ecotoxicological concern organic compound
Loredana Manfra a,n, Sara Canepa b, Veronica Piazza c, Marco Faimali c
a
Institute for Environmental Protection and Research, Via Brancati 60, 00144 Rome, Italy
b
University of Genoa, Corso Europa, 16146 Genoa, Italy
c
Institute of Marine Science, National Council of Researches (CNR), Via De Marini 6, 16149 Genoa, Italy

art ic l e i nf o a b s t r a c t

Article history: Swimming speed alteration and mortality assays with the marine crustacean Artemia franciscana were
Received 4 May 2015 carried out. EC50 and LC50 values after 24–48 h exposures were calculated for two reference toxicants,
Received in revised form copper sulphate pentahydrate (CuSO4
5H2O) and Sodium Dodecyl Sulphate (SDS), and an ecotox-
28 July 2015
icological concern organic compound, Diethylene Glycol (DEG).
Accepted 17 August 2015
Different end-points have been evaluated, in order to point out their sensitivity levels. The swimming
Available online 3 September 2015
speed alteration (SSA) was compared to mortality values and also to the hatching rate inhibition (lit-
Keywords: erature data). SSA resulted to be more sensitive than the mortality and with a sensitivity comparable to
A. franciscana (or even higher than) the hatching rate endpoint.
Swimming speed alteration
& 2015 Elsevier Inc. All rights reserved.
Hatching rate inhibition
Mortality

1. Introduction to automate in order to be useful for a wide range of applications

Aquatic invertebrates are widely used in ecotoxicological stu-
dies, because they are relatively easy to maintain under test con-
ditions, their use is not (as yet) restricted by ethical concerns, and
they are generally more sensitive to a range of pollutants than
vertebrates or plants (Piazza et al., 2012).
Several bioassays using larval stages of marine crustaceans
have been proposed to investigate the biological effects of con-
taminants and environmental matrices on primary consumers, i.e.
with the cirriped Amphibalanus amphitrite (Greco et al., 2006),
with the copepods Tigriopus fulvus (Faraponova et al., 2003) and
Acartia tonsa (Gorbi et al., 2012), with the brine shrimp Artemia
franciscana (Manfra et al., 2015).
Among the endpoints mainly observed on larval stages, beha-
vioural parameters are accurate, sensitive and reliable indicators of
stress. The behaviour of an organism is the endpoint of a sequence
of complex neurophysiological events (stimulation of neurons via
the release of chemical messages and muscular contractions)
(Thiéry et al., 2012).
The swimming of aquatic organisms is a behavioural response
well defined and practical to measure, sensitive to a wide range of
contaminants and adaptable to different species, ecologically re-
levant and has representation across species. Moreover, it is simple
n
Corresponding author.
E-mail address: loredana.manfra@isprambiente.it (L. Manfra).
(Rand, 1985). Changes in locomotor behaviour can therefore be
used as a stress indicator in ecotoxicological studies, thus obtain-
ing a realistic picture of the effects of contaminants at the eco-
system level (Tahedl and Häder, 2001).
Mostly in the last years, changes in motion behaviour as a re-
sponse to exposure to organic or inorganic pollutants, have been
observed in a range of aquatic invertebrates such as Artemia salina,
A. Amphitrite, Brachionus calyciflorus, Corophium volutator, Gam-
marus fossarum and other copepods (Charoy and Janssen, 1999;
Faimali et al.; 2006, Rao et al., 2007; Kienle and Gerhardt, 2008;
Xuereb et al., 2009; Seuront, 2011).
In particular, Faimali et al. (2006) developed a Swimming
Speed Alteration (SSA) recording system, by applying a video-
camera tracking system to detect linear swimming speed as be-
havioural end-point. This system has been already used on larvae
of A. amphitrite (Crustacea Cirripedia), on the brine shrimp Artemia
sp. and the rotifer Brachionus plicatilis (Garaventa et al., 2010),
demonstrating that alterations in swimming speed can be
detected.
Taking into account these previous information, aims of this
study were: (a) to measure the swimming speed alteration and
mortality of A. franciscana exposed to two reference toxicants i.e.
copper sulphate pentahydrate (CuSO4
5H2O) and Sodium Dodecyl
Sulphate (SDS); (b) to record the swimming speed alteration and
mortality of Artemia exposed to an ecotoxicological concern ad-
ditive i.e. Diethylene Glycol (DEG); (c) to compare the response, in

http://dx.doi.org/10.1016/j.ecoenv.2015.08.017
0147-6513/& 2015 Elsevier Inc. All rights reserved.
 L. Manfra et al. / Ecotoxicology and Environmental Safety 123 (2016) 60–64
term of sensitivity, between swimming speed, mortality (data in
this study) and hatching capability (Rotini et al., in press; Manfra
et al., 2015).
(CuSO4
5H2O) and SDS were selected because commonly used
in Artemia biotests (Persoone et al., 1993; Guzzella, 1997; Manfra
et al., 2012). Regards to DEG, it is used to prevent hydrate for-
mation during the gas production process and since it may be
discharged into the sea from the gas platforms, its ecotoxicological
characterisation is required (M.D. 28.07.1994). The most previous
results showed lethal and sub-lethal (i.e. bioluminescence inhibi-
tion for bacteria, growth rate inhibition for algae, development
inhibition for mussels) toxic effects at DEG concentration higher
than 9 g/L (Tornambè et al., 2012). Fish biomarker outcomes also
did not show significant toxic effects up to 5 g/1, with the only
exception of a slight genotoxic damage (Gorbi et al., 2009). The
hazard assessments on aquatic organisms indicated Predicted No
Effect Concentrations for marine waters of 1–10 mg/L (European
Chemicals Agency Database) and 5.9–59 mg/L (Manfra et al.,
2015), for constant or intermitted release of DEG. These above-
mentioned concentrations are higher than quantities really mea-
sured in the Adriatic Sea (o2 mg/L) (Cianelli et al., 2008).
2. Materials and methods
A control sample (Synthetic Sea Water SSW, 0.22 mm filtered, at
35‰ salinity) was obtained by adding the marine salt mixture
Instant Ocean (Aquarium Systems Mentor, Ohio, USA and Sarre-
borg, France) to deionized water. Solutions for treatments were
prepared by diluting CuSO4
5H2O (Sigma Aldrich, Z98%, CAS#
7758-99-8), SDS (Sigma Aldrich, Z99%, CAS# 151-21-3) and DEG
(Carl Roth GmbH, Z99%, CAS# 111-46-6) in the SSW to obtain the
following concentrations: 2–4–8–16–32 mg/l CuSO4
5H2O, 3–6–
12–24–48 mg/l SDS and 10–20–40–80–160 g/lDEG. These intervals
have been based on previous results obtained in hatching rate and
mortality tests (Guzzella, 1997; Manfra et al., 2015; Rotini et al., in
press). Preliminarily, the stability of the compounds (24 h mea-
sured concentrations) was evaluated under the toxicity test con-
ditions. The copper was measured according to Clescerl et al.
(1999); for SDS and DEG the ISO no. 7875 method (ISO, 1984) and
the UNICHIM no. 1367 method (UNICHIM, 1999) were applied,
respectively. The 80–90% of the measured initial concentration at
24 h was maintained (Table 2, Supplemental Data).
Dehydrated cysts of A. franciscana (certified from the Labora-
tory for Biological Research in Aquatic Pollution, University of
Ghent, Belgium) were used for the experiments. To obtain Instar
II–III stage larvae (less than 48 h old), the hatching was performed
as described in Manfra et al. (2015). Larvae were added into the
multiwell-plates with 24 compartments (Barloworld Scientific
Ltd.). Ten individuals were put in each well/replicate containing
1 ml of test solution; the control was also analyzed. The plates
were kept at 25 °C in the dark.
The test solutions and the control were analyzed in three re-
plicates and three test repetitions were performed for each toxic
compound. The average of the linear speed of 10 test organisms
and the dead individual number were observed for each replica.
The swimming speed and mortality were recorded after 24 and
48 h of exposure, according to Garaventa et al. (2010) and Guzzella
(1997), respectively. The swimming speed alteration was recorded
using the SSA recorder described in Faimali et al. (2006), consist-
ing of a video camera with a macroobjective for recording the
larval swimming paths. The larvae were dark-adapted for 2 min
before the video recording (time fixed by preliminary tests, to
reach steady speeds and a uniform spatial distribution) and the
images were analyzed using advanced image processing software
to reconstruct the individual paths/tracks (Gambardella et al.,
61
2014).
Larvae that were completely motionless were counted as dead
organisms, and the percentage of mortality was calculated com-
pared to the control. The term “motionless” means organisms that
do not change their own barycentre position and do not move any
appendages in 5 s.
The data referred to the swimming alteration have been nor-
malised to the mean swimming speed (S) of the control, where:
alteration (%) ¼[(Streated- Scontrol/Scontrol)]*100.
Median effective concentration on swimming alteration (24 h
EC50 and 48 h EC50) and mortality (24 h LC50 and 48 h LC50) were
calculated using Trimmed Spearman-Karber analysis (Finney,
1978).
The tests were considered acceptable when the control mor-
tality was r 10% (Guzzella, 1997, Garaventa et al., 2010).
Significant differences between the exposure times (24 h EC50s
vs. 48 h EC50s and 24 h LC50s vs. 48 h LC50s) and also between le-
thal and sublethal endpoints (24 h EC50s vs. 24 h LC50s and 48 h
EC50s vs. 48 h LC50s) were calculated by statistical analysis (t-test).
3. Results and discussion
The tests resulted acceptable because the death rate of the
controls was o10%, according the methods used in the
experiments.
The swimming speed alteration and mortality percentages of A.
franciscana after 24 and 48 h of exposure to CuSO4
5H2O, SDS and
DEG are shown in Fig. 1. These were slightly higher after 48 h than
24 h of exposure. The EC50 and LC50 values are summarised in
Table 1.
The results indicated the lowest toxicity of DEG compared to
the toxic effects of the reference toxicants. DEG LC50 values were
within 80–160 g/l concentration range. For CuSO4
5H2O and SDS,
48 h EC50 values were lower (about half) than 24 h results. The
difference was less marked for DEG. These differences between the
two exposure times were always statistically significant (p o0.05)
for SDS and DEG but not for CuSO4 LC50 values (see Table 3, Sup-
plemental Data).
The differences between lethal and sub-lethal end-points were
significant (po 0.05) for SDS but not for CuSO4 (see Table 4, Sup-
plemental Data).
Our LC50 values were comparable to literature results: 12.57
and 10.71 73.13 mg/l for CuSO4 (Persoone et al., 1993; Manfra
et al., 2015), 25.60 75.50 mg/l for SDS (Guzzella, 1997).
The swimming test is a short-chronic bioassay and it may give
results similar to long-term exposures. In fact, our SDS 48 h EC50s
(7.49 71.33) appeared comparable to 14 d LC50s (8.50 73.34 mg/l)
(Manfra et al., 2015).
Studies on the 48 h hatching rate inhibition of Artemia exposed
to CuSO4
5H2O (Manfra et al., 2015), SDS and DEG (Rotini et al., in
press) are available in literature. We compared our swimming
speed alteration results (48 h EC50) to these data. For CuSO4
5H2O,
we observed a mean value (2.517 0.37 mg/l) 1.5–2 times lower
than 48 h hatching EC50 (4.95 72.26 mg/l). For SDS, the mean
value (7.49 71.33 mg/l) was about 2 times lower than hatching
value ( 415 g/l). Similar results were measured for DEG (swim-
ming: 64.56 73.42 g/l; hatching: 450 g/l). Generally, the swim-
ming speed alteration and the hatching rate inhibition resulted
more sensitive than the acute (24–48 h) mortality. In addition, our
results showed a swimming test sensitivity comparable or higher
than hatching test.
The results obtained selecting these sublethal endpoints are
comparable to the response of other marine organisms (as algae,
rotifers, amphipods, crustaceans, urchin and fish) that give an EC50
range of [1.30–17.40] mg/l for CuSO4
5H2O, [2.36–7.42] mg/l for
62 L. Manfra et al. / Ecotoxicology and Environmental Safety 123 (2016) 60–64

A. franciscana -24h swimming speed alteration % mortality A. franciscana -48h swimming speed alteration % mortality
100 100
80 80
60 60
40 40
20 20
0 0

%
%

-20 -20
-40 -40
-60 -60
-80 -80
-100 -100
0 2 4 8 16 32 0 2 4 8 16 32
CuSO4 5 H2O( mg/l) CuSO4 5 H2O (mg/l)

A. franciscana -24h swimming speed alteration % mortality A. franciscana -48h swimming speed alteration % mortality
100 100
80 80
60 60
40 40
20 20
0 0

%
%

-20 -20
-40 -40
-60 -60
-80 -80
-100 -100
0 3 6 12 24 48 0 3 6 12 24 48
SDS (mg/l) SDS (mg/l)

A. franciscana -24h swimming speed alteration % mortality A. franciscana -48h swimming speed alteration % mortality
100 100
80 80
60 60
40 40
20 20
0 0
%
%

-20 -20
-40 -40
-60 -60
-80 -80
-100 -100
0 10 20 40 80 160 0 10 20 40 80 160
DEG (g/l) DEG (g/l)

Fig.1. Mean percentages of swimming speed alteration and mortality for A. franciscana after 24–48 h of exposure to (a and b) CuSO4
5H2O (mg/l), (c and d) SDS (mg/l) and (e
and f) DEG (g/l). Results are the mean of the three test repetitions (mean 7 SE; n¼ 3). Each test repetition provided three replicates per concentration, with 10 individuals per
replicate.

Table 1 insights into various levels of biological organisation (Scott and

EC50/LC50 mean values (s.d.) after 24–48 h of exposure to CuSO4
5H2O (mg/l), SDS Sloman, 2004). How might the contaminants interact on Artemia
(mg/l) and DEG (g/l).
to produce the reduction in swimming velocities (compared to
24 h EC50 48 h EC50 24 h LC50 48 h LC50 controls)?
Davenport and Healy (2006) assessed the relationship between
CuSO4
5H2O (mg/l) 5.03 (0.58) 2.51 (0.37) 14.21 (10.63) 2.51 (0.22) physical parameters (medium salinity, body density, buoyancy)
SDS (mg/) 16.15 (0.32) 7.49 (1.33) 19.41 (1.00) 15.60 (0.27)
and swimming in Artemia franciscana larvae. They found that the
DEG (g/l) 80.37 (3.95) 64.56 (3.42) 80–160 80–160
horizontal swimming speed was unaffected by salinity and visc-
osity (over the range 8.5–100‰) but the observed differences in
SDS, [5.90–90.40] g/l for DEG (Nipper et al., 1993; Ribelles et al., vertical swimming rates are solely due to relatively constant body
1995a, 1995b; Rosety et al., 2001; Verbruggen et al., 2005; Blan- density. Larsen et al. (2008) also suggested that changes in
chard and Grosell, 2006; Mariani et al., 2006; Tornambè et al., swimming velocity of Artemia are due to change in kinematic
2012). viscosity. Previously, Williams (1994a, 1994b) published an ana-
Several locomotor responses (distance travelled, travel velocity, lysis of locomotion in Artemia, showing that larvae have a single
frequency of direction change, time spent active, response to light, pair of antennae and exhibit jerky, discontinuous swimming in
and tail-flip escape response) have been assessed on various test which the animal’s body moves to and fro with each rowing
organisms. Swimming speed is perhaps the most frequently used stroke. No information seems be available in literature on the
behavioural measurement of the physiological status of an aquatic swimming speed alteration of Artemia exposed to CuSO4
5H2O,
organism (Faimali et al., 2006). Behaviour is a determinant that SDS and DEG, except a mobility study of Kokkali et al. (2011). They
results from molecular, physiological and ecological aspects of quantified the Cu2 þ EC50 value (7.6 mg/l), which resulted to be
toxicology (Little et al. 1990). Therefore, behaviour can provide 2.5 times lower than the LC50 (19.5 mg/l) but did not explain the
 L. Manfra et al. / Ecotoxicology and Environmental Safety 123 (2016) 60–64
toxic effect mechanisms. Moreover, some authors studied the ef-
fects of copper and SDS on aquatic organisms, proving to explain
the toxicity mechanisms. Blanchard and Grosell (2006) studied to
copper effects on fish: gills become frayed and lose their ability to
regulate transport of salts such as sodium chloride and potassium
chloride into and out of fish. These salts are important for the
normal functioning of the cardiovascular and nervous systems and
their uncorrected transport may cause fish death. In addition,
copper can affect the activation of the olfactory receptor neurons
or intracellular signalling in the neurons. Sublethal copper ex-
posure alters a number of behaviours in invertebrates. It reduces
the ability of decapods to detect and orient to food odours in a
water plume. It produces adverse effects on locomotory and sen-
sory systems and these can also be correlated with adversely af-
fected predator avoidance behaviours. It induces a slowing of heart
rate in mussels and it adversely affects the ability of worms to
produce body reversal or helical swimming behaviours following
tactile stimulation (O’Gara et al., 2004).
Barbieri et al. (1998) revealed that increasing concentrations of
SDS affect fish metabolism (the oxygen consumption increases)
and locomotor capacity (swimming capacity decreases). These
authors observed that at the highest concentration (10 mg/l),
swimming capacity was reduced 5 times and oxygen consumption
increased 2.8 times in comparison to the control. SDS is char-
acterized by high amphilicity and adsorption ability. The basis of
its toxicity seems to be mainly related to the alteration of the
cellular ionic balance caused by cellular membrane permeability
alterations and to the induction of oxidative stress (which, in turn,
can generate other physiological and biochemical stresses (Mes-
sina et al., 2014).
Hymela et al. (2002) observed variation in fish swimming
performance after exposure to glycols (ethylene glycol). The
buildup of metabolic products and increased metabolic cost of
removing ethylene glycol from the body may have caused or
contributed to the decrease in swimming performance of exposed
fish.
Earlier reports indicated that the decrease in velocity in other
organisms could be caused by food deprivation, anoxic conditions
or inhibition of acetylcholine (ACh) enzyme (Westerterp, 1977;
Nilsson et al., 1993). In short-time tests (24–48-h) Artemia is not
facing either anoxic conditions or deficiency of food (Guzzella,
1997). In the past, Artemia species have been referred as organisms
that accumulate toxic compounds with no effect on their life cycle
(Sarabia et al., 2002). After that, Rao et al. (2007) observed that the
(ACh) enzyme activity may be inhibited by the toxicant (i.e., pes-
ticides), resulting in accumulation of acetylcholine at neuromus-
cular junctions. The accumulation of this neurotransmitter (ACh)
may alter the locomotion behaviour of the organism, interrupting
the coordination between the nervous and muscular junctions.
Toxicity testing of copper-contaminated sediments to amphipods
(Hyalella azteca) and daphnids (Daphnia magna) using techniques
of enzyme inhibition and growth rate showed that these variables
are more sensitive in accurately predicting copper sensitivity than
LC50 (48 h) values (Eisler, 1998). Recently, this neurotoxic effect has
been investigated in Artemia salina nauplii exposed to organic
compounds (i.e., Eserine) (Garaventa et al., 2010) but there are not
clearly evidences for copper, SDS and DEG exposures.
For these toxic mechanisms, the swimming speed alteration
may potentially deliver more sensitive indicator of stress than
conventional mortality assay for Artemia, as already observed for
behavioural responses compared to endpoints such as survival,
growth, and reproduction (Gerhardt et al. 2005; Dell’Orno, 2002).
4. Conclusion
To promote alterative endpoints for Artemia, the sensitivity of
63
the swimming speed and the mortality have been evaluated by
using two well-known reference toxicants and a potentially en-
vironmental hazardous compound (Manfra et al., 2015).
This study has proved that an high sensitivity was detected for
Artemia by tracking naupliar swimming and estimating their
average speed using digital image processing. This endpoint seems
to be sensitive, ecologically relevant and comparable to another
end-point: the hatching rate. Both of them showed to be prefer-
able to mortality in order to point out a toxic effect at low con-
centrations. The swimming speed alteration is of great ecological
importance as the alteration of behaviour represents an integrated
whole-organism response that can link the physiology and ecology
of an organism to its environment (Little and Brewer, 2001). In
addition, changes in swimming behaviour of invertebrates may
have important indirect effects on marine and freshwater com-
munities, such as alteration in predation pressure, zooplankton
prey pressure, and phytoplankton abundance (Charoy and Janssen,
1999).
All applicable international, national, and/or institutional
guidelines for the care and use of animals were followed. This
article does not contain any studies with human participants
performed by any of the authors.
Informed consent was obtained from all individual participants
included in the study.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.08.017.
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