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557
ACTA ZOOLOGICA BULGARICA
Acta zool. bulg., 70 (4), 2018: 557-564
Aquatic Ecology
Research Article
Introduction
Cadmium (Cd) is non-essential and one of the most
toxic metals and poses a serious threat to aquatic or-
ganisms ( et al. 2011) because of its persis-
tent nature and slow elimination from the environ-
mental compartments ( et al. 2014). It is re-
leased naturally from weathering of minerals, forest
contamination results from anthropogenic sources,
of zinc, coal combustion, electroplating processes,
iron and steel production, pigment, fertilizer and
pesticide use ( et al. 2013). All the
cadmium ultimately deposits into the aquatic sys-
tems, and then creates potential hazards to aquatic
organisms ( 2014). Polycyclic aromatic hy-
drocarbons (PAHs) are known to result from the in-
complete combustion of organic matter, especially
fossil fuels (pyrolytic origin), from the discharge of
petroleum and its products (petrogenic origin), as
well as from the post-depositional transformation of
biogenic precedents (diagenetic origin). PAHs can
and volcanoes, or can be derived from biogenic pre-
cursors ( et al. 2017). Under this consideration,
the United States Environmental Protection Agency
(USEPA
Short- and Long-term Toxicity of Cadmium
and Polyaromatic Hydrocarbons on Zebra Mussel
Dreissena polymorpha (Pallas, 1771) (Bivalvia: Dreissenidae)
Vesela Yancheva1, Elenka Georgieva1, Stela Stoyanova1*, Vesela Tsvetanova2,
Kostadinka Todorova3, Ivelin Mollov1 & Iliana Velcheva1
1 Faculty of Biology, Plovdiv University, 24 Tsar Asen Street, 4000 Plovdiv, Bulgaria
2 Regional Accredited Laboratory, Executive Environment Agency, Ministry of Environment and Water, Plovdiv, Bulgaria
3 Plovdiv University, Lyuben Karavelov Branch, Kardzhali, Bulgaria
Abstract:
hydrocarbons (PAHs) could have on the lysosomal membrane stability in haemocytes of the invasive
mollusk zebra mussel (Dreissena polymorpha) by applying the neutral red retention assay (NRRA). The
(acute exposure) and 31 days (chronic exposure). These are considered as priority substances in surface
waters according to 2008/105/EC. We found lysosomal membrane destabilisation in all mus-
sels treated with Cd and PAHs, including concentrations, which were lower than the allowable ones. In
addition, we determined a trend of lower retention time in the mussels treated with Cd as compared to the
p
that the neutral red retention assay could be used as a cheap, fast and reliable biomarker for Cd and PAHs
water contamination. However, further studies are required in order to better understand the negative ef-
fects of Cd and PAHs on this bivalve species.
Key words: mussels, Cd, PAH, lysosomes, haemocytes
*Corresponding author: stela.st@bv.bg
558
Yancheva V., E. Georgieva, S. Stoyanova, V. Tsvetanova, K. Todorova, I. Mollov & I. Velcheva
( et al. 2006). Therefore, integration of chemi-
cal analyses with biomarker responses in organisms
is recommended for monitoring anthropogenic ac-
tivities, which could lead to PAHs contamination
( et al. 2008).
Sediment-associated organisms, such as marine
bivalves, have been used to monitor the health of
to accumulate and tolerate high concentrations of
pollutants, present across a very wide geographical
area, easy to collect, having a suitable size for chemi-
cal analyses and particularly abundant in coastal and
estuarine waters ( 1986). Furthermore,
mussels have the ability to accumulate organic pol-
lutants, especially PAHs and are, therefore, currently
used to monitor PAHs contamination in aquatic en-
vironments ( et al. 2013). Mytilus sp. have
been widely used since the 90s and have been shown
to be one of the most successful model organisms
for time-integrated responses to complex mixtures
of pollutants (UNEP/RAMOGE 1999). The zebra
mussel, Dreissena polymorpha (Pallas, 1771) can be
used as a freshwater substitute of Mytilus sp. (
et al. 2012, et al. 2015). The lysosomes in
the mussel cells function as a central site for seques-
tration and accumulation of toxic metals and organic
xenobiotics, but they also play a key role in detoxi-
compounds ( 2001).
Thus, the measurement of lysosomal membrane sta-
bility was proposed as a rational biomarker of gen-
eral stress in aquatic bivalves, both in laboratory and
in vivo studies ( et al. 1987).
et al. (2016) published preliminary
on zebra mussel, which directed us to an expanded,
inorganic and organic toxicants on this species.
-
ment was to study the negative impact, which Cd
and PAHs could possibly have on the lysosomal
membrane stability in haemocytes of zebra mussel
(Dreissena polymorpha) by applying the neutral red
retention assay (NRRA). The mussels were exposed
their permissible levels in inland waters according
to the EU legislation in laboratory conditions for 96
hours and 31 days. These toxicants are considered
as priority substances in surface waters according
to 2008/105/EC of the European parlia-
ment and the Council. The second objective was to
determine, which one, Cd or PAHs, could have more
Materials and Methods
About 150 specimens of the same size-group (mean
length 2.5 cm ± 0.5) were hand-collected in the au-
near Plovdiv City, Bulgaria (42° 30’ 07.81” N, 24°
80’ 49.75” E). After transportation on the same day
the mussels were moved in a 25 L glass aquarium
with chlorine-free tap water (by evaporation) to ac-
climatise for a week. The water was kept oxygen
saturated. During the entire duration of the experi-
ment, the mussels were maintained under a natural
light/dark cycle (12:12). They were not fed. After the
acclimatisation the mussels were divided into seven
groups (n = 20 in each experimental tank and n =
20 for control). The mussels were treated with dif-
ferent concentrations of cadmium nitrate tetrahydrate
(Cd(NO3)2.4H2
-
solved in cyclohexane for a total period of 96 hours
(acute exposure) and 31 days (chronic exposure).
Their permissible levels in inland waters according
to the law are presented in Table 1.
The toxicant concentrations were prepared as
50% above and 50% below the maximum allowable
concentration (MAC) set by the national law for 25
L tanks. Therefore, in the current experiment 2 µg
Cd (AA, 100%), 1 µg Cd (50% below MAC) and
4 µg Cd (50% above MAC), as well as 2 µg PAHs
(AA, 100%), 1 µg PAHs (50% below MAC) and 3 µg
PAHs (50% above MAC) were applied. The physical
and chemical characteristics of both the control and
contaminated water, such as pH, conductivity, tem-
perature and oxygen level, were measured once on
the 24th, 48th, 72nd and 96th hour, as well as on the 31st
The analytical procedure of neutral red retention
time (NRRT) assay was adapted from
(1994) and et al. (1995). It is based on the use
of a cationic probe neutral red, which is taken up into
trapped within the lysosomal compartment (
et al. 1991). Over time, the dye tends to leak out of
the lysosomes into the cytosol, which is then stained
by the dye (et al. 1997). The exposure to
toxic substances damages the lysosomal membrane
and hence, increases its permeability. About 0.5 mL
haemolymph was withdrawn from the posterior ad-
-
tal tank and treated as described by
(2012). In order to maintain the haemocytes alive a
calcium/magnesium free physiological saline was
prepared: 0.595 g/L HEPES, 3.215 g/L NaCl, 0.185
g/L KCl, 500 ml distilled H2O, pH adjusted to 7.3.
Short- and Long-term Toxicity of Cadmium and Polyaromatic Hydrocarbons on Zebra Mussel Dreissena polymorpha
559
The neutral red stock was prepared by dissolving 4
mg of neutral red into 2 mL DMSO and. 10 µL of the
stock solution was made up to 5 mL with the physi-
ological saline. Then 40 µL of the haemolymph was
applied on microscopic slides, which were contained
in a light proof humidity chamber, then treated with
40 µL of the neutral red and covered with cover slips.
The slides were examined using a light microscope
(Leica DM500). The time, at which in more than
50% of the lysosomes neutral red leaked out in the
cytosol was considered the end point parameter.
The average percent of cells with destabilised
lysosomes was calculated for each experimental mus-
sel and thereafter for each toxicant group. The aver-
using Student t-test (p<0.05) using STATISTICA
software, version 7.0 for Windows. The water physi-
cal and chemical data were analysed in the same way.
Results
Short-term exposure to Cd and PAHs
from each experimental tank are presented in Table
2. In general, the values were similar for the studied
test period (96 hours). The conducted statistical anal-
values of the control and test water on the 24th, 48th,
72nd and 96th hour (p>0.05). Thus, we consider that
the changes, which were observed in the lysosomal
membrane stability were not due to changes in the
environmental conditions.
The summarised average results on the lysoso-
mal membrane stability of the control mussels and
those subjected to the action of the tested toxicants
on the 24th, 48th, 72nd and 96th hour are presented in
Fig. 1. The average retention time of the control mus-
sels on the 24th hour was 90 min. This time represents
the accepted minimum, which shows lysosomal sta-
bility. Therefore, the lysososmes were considered as
healthy and non-stressed. On the other hand, on the
24th-
somal membrane stability were registered in the mus-
sels treated with Cd. It was observed that the lyso-
somal membrane stability changed when increasing
its concentration. The lowest retention time was 15
min in the mussels exposed to the highest Cd concen-
Bulgarian legislation) retained the dye 27 min, while
-
low AA) 30 min, respectively. The statistical analysis
p<0.05) be-
tween the retention time of the dye on the 24th hour
Table 1. List of PAHs used in the present study (n=16)
Name of substance Annual average (AA), inland surface
waters, µg/L
Maximum allowable concentration
(MAC), inland surface waters, µg/L
Cd 0.08 0.45
Acenaphthene* 3.8 50
Acenaphthylene* 0.64 not applicable
Anthracene 0.1 0.1
Benz(a)anthracene* 0.01 not applicable
** 0.017
** 0.017
Benzo(g,h,i)-perylene ** 0.017
Benzo(a)pyrene 1,7 × 10 –4 0.27
Dibenzo(a,h)anthracene* 0.02 not applicable
Fluoranthene 0.0063 0.12
Fluorene* 2,5 not applicable
Indeno(1,2,3-cd)pyren ** not applicable
Naphthalene 2 130
Phenanthrene* 1.3 not applicable
Pyrene* 0.012 not applicable
Chrysene 0.02 not applicable
* No data for annual average (AA) and maximum allowable concentration (MAC) in inland surface waters (µg/L) ac-
cording to the Bulgarian legislation based on the environmental quality standards 2008/105/
EC;
** For the group of priority substances of polyaromatic hydrocarbons (PAH) (No 28), the biota EQS and correspond-
ing AA-EQS in water refer to the concentration of benzo(a)pyrene, on the toxicity, of which they are based. Benzo(a)
pyrene can be considered as a marker for the other PAHs, hence only benzo(a)pyrene needs to be monitored for com-
parison with the biota EQS or the corresponding AAEQS in water.
560
Yancheva V., E. Georgieva, S. Stoyanova, V. Tsvetanova, K. Todorova, I. Mollov & I. Velcheva
in the lysosomes subjected to the action of all Cd
concentrations and the control mussels. The average
retention time in the control mussels on the 48th hour
was longer than in the control mussels on the 24th
hour – 120 min. Therefore, the lysosomes were con-
sidered also as non-stressed. The trend in the lysoso-
mal stability in the mussels after a 48-hour exposure
to Cd was similar to that of the mussels treated with
Cd for 24 hours, i.e lysosomal membrane destabilisa-
tion was established earlier at higher Cd concentra-
tions. The mussels treated with the highest concentra-
min, respectively; in those treated with the lowest Cd
p<0.05) between the retention time of the
dye in the lysosomes exposed to all Cd concentra-
tions and control was proved on the 48th hour. The av-
erage retention time of the dye in the lysosomes from
the control mussels on the 72nd hour was 90 min, thus
we assumed that they were relatively non-stressed.
Similarly to the trend, which was observed above, the
mussels treated with Cd for 72 hours showed lower
retention time at higher concentrations. The mussels
-
tained the dye 21 min (similarly to the mussels ex-
amined at the 48th hour). The mussels exposed to Cd
Fig. 2. Average results on the lysosomal membrane stability in control and experimental groups of zebra mussel ex-
Fig. 1. Average results on the lysosomal membrane stability in control and experimental groups of zebra mussel ex-
Short- and Long-term Toxicity of Cadmium and Polyaromatic Hydrocarbons on Zebra Mussel Dreissena polymorpha
561
destabilisation after 30 min, while those exposed to
-
(p<0.05) between the retention time of the dye in the
lysosomes of control mussels and those exposed to
all Cd concentrations on the 72nd hour. The control
mussels, dissected on the 96th hour showed stable lys-
osomal membranes, since destabilisation was not ob-
-
cant decreases in the indices of lysosomal membrane
stability were registered in the mussels treated with
those treated with the lowest Cd concentration of 1
Comparing the retention time of the dye in the
(100% AA, 50% above AA, 50% below AA) on the
24th, 48th, 72nd and 96th hour, we registered statisti-
-
th
hour (pth hour
(p<0.05).
As said before, the average retention time of the
control mussels on the 24th hour was 90 min. On the
above AA) held the dye only 18 min, those treated
concentrations under the Bulgarian legislation – 24
-
between the retention time of the dye in the control
group and all treated with PAHs concentrations mus-
sels (p<0.05). The average retention time of the dye
in the mussels from the control on the 48th hour was
120 min. As for the treated mussels, the retention time
of the dye of the lysosomes in the mussels treated
dye equally and little longer – 48 min. A statistically
p<0.05) between the retention
time of the dye in the control group and the groups
treated with all PAHs concentrations was found on
the 48th hour. The average retention time of the dye
in the control mussels on the 72nd hour was 90 min.
However, the mussels treated with PAHs showed a
similar trend in the dye retention time, which was
shorter. Lysosomal destabilisation was registered af-
neutral red leakage into the cytoplasm after 27 and
p<0.05)
was found only between the retention time of the dye
PAH (50% above AA). Analogous results were ob-
tained for the mussels on the 96th hour of exposure
to PAHs.
Comparing the retention time of the dye of the
-
tions, which were applied on the 24th, 48th, 72nd and
96th -
24th hour (p < 0.05), as well as between the groups
(50% below AA) on the 24th hour (p<0.05).
Comparison between the toxicity of Cd and PAHs
on the lysosomal membrane stability – short-term
experiment
We could not distinguish a clear trend regarding the
toxicity of both pollutants on the lysosomal mem-
brane stability of Dreissena polymorpha in the short-
term experiment (96 hours). Overall, the retention
time of the dye was similar in the groups exposed to
Cd and PAHs, i.e a shorter retention time at higher Cd
and PAHs concentrations. A longer retention time of
the dye was reported only in the mussels exposed to
th
AA) on the 72nd
on the 72nd hour as compared to those treated with
p<0.05),
Table 2. Physical and chemical properties of the water treated with Cd and PAHs – short-term experiment
Test concentrations, 25 l
tanks Temperature, ˚C pH Oxygen level, mg/l Conductivity, µS/cm
2 µg Cd (AA, 100%) 19.9±0.2 7.92±0.07 8.9±0.1 486.5±1.7
1 µg Cd (50% below AA) 20.1±0.08 7.91±0.07 8.8±0.1 487.5±1.8
4 µg Cd (50% above AA) 20.2±0.2 7.9±0.07 9±0.1 488.5±1.1
2 µg PAHs (AA, 100%) 20±0.07 7.89±0.06 9.1±0.1 476.25±0.8
1 µg PAHs (50% below AA) 20.1±0.2 7.84±0.1 9.2±0.1 477.25±0.8
3 µg PAHs (50% above AA) 19.9±0.08 7.9±0.06 8.85±0.06 481±4.9
Control 20.1±0.2 7.68±0.08 8.7±0.2 444.5±9.4
562
Yancheva V., E. Georgieva, S. Stoyanova, V. Tsvetanova, K. Todorova, I. Mollov & I. Velcheva
however, was proven for the reported retention time
th hour, as well as for the
48th hour. The time was longer for the mussels ex-
posed to PAHs as Cd caused destabilisation earlier,
-
ososmes.
Long-term exposure to Cd and PAHs
The physical and chemical properties for each ex-
perimental tank measured on the 31st day (Table 3)
demonstrated, in general, the values close to those
measured during the short-term experiment. The data
suggested that the physical and chemical properties
remained relatively unchanged, which gave us a rea-
son to consider that the observed changes in the lyso-
somal membrane stability were once again not due to
changes in the abiotic factors.
The summarised average results on the lysoso-
mal membrane stability in the mussels treated with
st day,
including the control, are shown in Fig. 2. The neu-
tral red retention time of the control mussels after the
long-treatment was 120 min. Therefore, we assumed
that they were non-stressed. On the other hand, sig-
-
ity were registered in the treated with Cd mussels.
below AA) held the dye 54 min, those treated with
-
brane destabilisation after only 30 min. The statistical
retention time of the dye between the mussels treated
with all test Cd concentrations and the control, as
p<0.05).
-
brane stability were also registered in the treated with
(50% below AA) held the dye 60 min, those exposed
-
-
bilisation of the lysosomal membranes after 30 min
(similarly to the mussels subjected to the action of
in the retention time of the dye were proven between
the control mussels and those undergoing all test
PAHs concentrations (100% AA), as well as for the
p<0.05).
Comparison between the toxicity of Cd and PAHs
on the lysosomal membrane stability – long-term
experiment
Similarly to the results obtained from the short-term
experiment, there was no clear trend regarding the
toxicity of Cd and PAHs on the stability of the lyso-
somal membranes of zebra mussel after the treatment
of 31 days (Fig. 2). The overall picture showed short-
er retention at higher concentrations of Cd and PAHs.
The retention time of the dye was similar in the tested
groups, but earlier destabilisation in the lysosomal
membranes occurred in the mussel exposed to Cd.
-
tween the mussels exposed to Cd and PAHs on the
31stp>0.05).
Figures 1 and 2 show that the lysosomal mem-
branes of the zebra mussels discharged the dye in the
-
cants. The lowest average time was recorded on the
24th hour in the mussels treated with the highest Cd
and PAH concentrations (50% over AA) – 15 and
18 min, respectively. On the other hand, the average
retention time of the dye on the 31st day was once
again lower in the mussels subjected to the higher
toxicant concentrations but was longer compared to
those reported on the 24th, 48th, 72nd and 96th hour. The
-
st day and 24th
st day and 96th
Cd on the 31st day and 24th hour (p<0.05). Regarding
-
tween the retention time of the dye in the mussels
st day and the 72nd / 96th
Table 3. Physical and chemical properties of the water treated with Cd and PAHs – long-term experiment
Test concentrations, 25 l
tanks Temperature, ˚C pH Oxygen level, mg/l Conductivity, µS/cm
2 µg Cd (50% above AA) 21.4 7.92 9.1 475
1 µg Cd (AA, 100%) 22 7.9 9 478
4 µg Cd (50% below AA) 21.6 7.91 8.9 477
2 µg PAHs (AA, 100%) 21.5 7.87 9.2 479
1 µg PAHs (50% below AA) 22 7.9 8.9 481
3 µg PAHs (50% above AA) 21.7 7.68 8.8 451
Control 20 7.5 8 435
Short- and Long-term Toxicity of Cadmium and Polyaromatic Hydrocarbons on Zebra Mussel Dreissena polymorpha
563
st day and the 24th
hour (p<0.05). In these cases, the retention time was
longer on the 31st day of exposure. Thus, we could
hypothesise that this result could indicate some initial
form of adaptation of the mussels to live in contami-
nated aquatic environment.
Discussion
Short-term exposure to Cd and PAHs
With regard to the toxicity of Cd, the shortest reten-
tion time of the dye was observed at the beginning of
the experiment. We assumed that this could be due to
stress, which the incorporated toxicant caused on the
mussels. They continued to experience stress, as the
retention time did not reach the 90-minute stability
standard, however it stayed relatively constant. For
example, on the 48th hour the mussels exposed to 4
nd and 96th hours after
33 min. Probably, in this case and the fact that there
were no dead individuals in the course of the whole
experiment, we could try to explain the given results
with an initial form of acclimatisation to the contami-
nated aquatic environment.
For the mussels exposed to PAHs the shortest
neutral red retention time was also registered in the
th hour), as well as
We believe that this could be once again a result of
the stress of the toxicant introduction and the follow-
ing contaminated aquatic environment. On the other
-
low AA) had a longer retention time of the dye on the
24th and 48th hour (48 min) compared to 72h and 96h
(30 min). Thus, we consider that mussels maintained
a relatively constant tolerance to PAHs when treated
was below 90 min. Furthermore, we might consider
that the mussels attempted to adapt to the polluted
to 30 min, at the end of the experiment.
Long-term exposure to Cd and PAHs
After 31 days no mortality was reported for the
treated with Cd and PAHs zebra mussels, which sup-
ported our conclusion from the short-term experi-
ment that the mussels could have tried to adapt to
pollution. In addition, the statistical analysis showed
comparing the short and long-term exposure to Cd
and PAHs; it was longer in the mussels observed after
31 days, but still lower than 90 min.
the lysosomal membrane stability on Dreissena pol-
ymoprha is relatively limited, we found that our re-
sults corresponded to those of other authors and con-
in bivalves and other invertebrates such as gastropods
is weakened as a result of contaminated aquatic envi-
ronment. Consequently, at lower toxicant concentra-
tions the retention time of the dye is longer, and vice
versa ( 2000, et al.
2001, et al. 2011, 2012,
et al. 2013, et al. 2014,et
al. 2016 a,b, et al. 2017).
Conclusions
test concentrations and exposure period, aiming to
compare the toxicity of both, inorganic and organic
toxicants, it can be concluded that Cd and PAHs have
Dreissena poly-
morpha, which leads to destabilisation of the cellular
organisation. Overall, we found that: (1) the observed
changes in the stability of the lysosomal membranes
of the mussels were dose-dependent and more pro-
nounced at Cd and PAHs concentrations, which rep-
resent 100% AA and 50% above AA; (2) faster cell
(24 h) as compared to the 48th, 72nd, 96th hours and
31 days; (3) changes in the stability of the lysosomal
membranes of the mussels subjected to lower concen-
trations (50% under AA) were also observed, con-
the treated with Cd mussels the lysosomal membrane
stability was impaired faster than with PAHs, although
red retention time; (5) the neutral red retention assay
-
-
Dreissena polymorpha could
be used as a bioindicator for contaminated freshwater
ecosystems, in particular with Cd and PAHs. Last but
not least, the results from this experiment can be used
as a base in future studies, as well as in monitoring
with inorganic and organic toxicants.
Acknowledgements: This study was supported by the NPD-
Plovdiv University under Grant No SP17-BF003 “Integrated
biomarkers for priority toxic substances in aquatic ecosystems
by using zebra mussel (Dressena polymorpha Pallas, 1771) as
bioindicator”.
564
Yancheva V., E. Georgieva, S. Stoyanova, V. Tsvetanova, K. Todorova, I. Mollov & I. Velcheva
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anodonta woodiana) under ex situ exposure: preliminary
data. Biharean Biologist 10 (1): 55-57.
stability and respiratory rate of two freshwater mollusks under
ex situ exposure: preliminary data. South-Western Journal of
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on the lysosomal membrane stability and respiration rate in
Zebra mussel (Dreissena polymorpha Pallas, 1771). Acta
Zoologica Bulgarica, Supplement 8: 85-90.
Received: 30.05.2017
Accepted: 28.10.2017
