Aquatic Toxicology 177 (2016) 98–105

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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Lethal and sub-lethal effects on the Asian common toad Duttaphrynus

melanostictus from exposure to hexavalent chromium
Vindhya A.K. Fernando a , Jagathpriya Weerasena b , G. Pemantha Lakraj c , Inoka C. Perera a ,
Chandima D. Dangalle a , Shiroma Handunnetti b , Sunil Premawansa a ,
Mayuri R. Wijesinghe a,∗
a
Department of Zoology and Environment Sciences, University of Colombo, Colombo 03, Sri Lanka
b
Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Colombo 03, Sri Lanka
c
Department of Statistics, University of Colombo, Colombo 03, Sri Lanka

a r t i c l e i n f o a b s t r a c t

Article history: Chromium discharged in industrial effluents frequently occurs as an environmental pollutant, but the
Received 19 February 2016 lethal and sub-lethal effects the heavy metal might cause in animals exposed to it have been insuffi-
Received in revised form 16 May 2016 ciently investigated. Selecting the amphibian Duttaphrynus melanostictus, we carried out laboratory tests
Accepted 21 May 2016
to investigate the effects of short and long term exposure to hexavalent chromium (Cr(VI)) in both tad-
Available online 24 May 2016
poles and adult toads. The concentrations used were 0.002, 0.02, 0.2, 1.0 and 2.0 mg/L, the first three
corresponding to field levels. In vitro exposures were also carried out using toad erythrocytes and Cr(VI)
Keywords:
concentrations of 0.0015, 0.003, 0.015, 0.03, 0.15 mg/ L. Mortality, growth retardation, developmental
Chromium
Genotoxicity delays and structural aberrations were noted in the metal-treated tadpoles, with increasing incidence
Growth corresponding to increase in Cr(VI) level and duration of exposure. Many of the sub-lethal effects were
Haematology evident with long term exposure to environmentally relevant levels of the toxicant. Changes in selected
Tadpoles blood parameters and erythrocyte morphometry were also detected in Cr(VI) exposed toads, indicating
Toxicity anaemic and leucopenic conditions. In the genotoxicity study, DNA damage indicated by comet assay and
increased micronuclei frequency, occurred at the low Cr(VI) concentrations tested. The multiple delete-
rious effects of exposure to chromium signal the need for monitoring and controlling the discharge
of chromium to the environment. The dose-dependency and genotoxic effects observed in this widely
distributed Asian toad indicates its suitability for monitoring heavy metal pollution in aquatic systems.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction World Health Organization, 2003). As opposed to Cr(III), Cr(VI) is

Natural habitats of animal species are being contaminated
increasingly due to the discharge of effluents from industries, con-
taining toxic heavy metals. Chromium (Cr) is a toxic heavy metal
used in industries such as leather tanning, chrome plating, paint
pigments, cement and wood preservatives. Hexavalent chromium
(Cr(VI)) may exist as water-soluble complexes which may persist
in the water or may react with organic matter in the environ-
ment and form trivalent chromium (Cr(III)) (Callahan et al., 1979;
∗ Corresponding author. Tel.: +94 714 406 277.
E-mail addresses: vink1025@gmail.com (V.A.K. Fernando),
jagath@ibmbb.cmb.ac.lk (J. Weerasena), pemantha@stat.cmb.ac.lk (G.P. Lakraj),
icperera@sci.cmb.ac.lk (I.C. Perera), cddangalle@gmail.com (C.D. Dangalle),
shiromah@gmail.com (S. Handunnetti), suviprema@gmail.com (S. Premawansa),
mayuri@sci.cmb.ac.lk (M.R. Wijesinghe).
extremely toxic to organisms as a result of its higher cellular uptake.
Hexavalent Cr readily crosses through the cell membrane via facili-
tated diffusion through non-specific anion channels whereas Cr(III)
is absorbed through passive diffusion or phagocytosis. Intracellu-
lar damage by Cr(VI) is mostly caused by reactive oxygen species
(ROS) such as superoxide, hydroxyl radicals, hydrogen peroxide
and Cr(III), generated through its reduction (IPCS, 2009). The final
product of Cr(VI) metabolism, Cr(III), forms stable coordination
complexes with nucleic acids and proteins. Therefore, inside the
cell, Cr(VI) reduction is the activation event that is responsible for
the generation of genotoxic damage and other forms of toxicity
(Salnikow and Zhitkovich, 2008).
Documented end points monitored for exposure to Cr in faunal
taxa include growth retardation, developmental delays and death,
as well as haematological, histological and genotoxic effects. Exam-
ples follow: rats – Elbetieha and Al-Hamood (1997); amphibians –
Khangarot and Ray (1987), Natale et al. (2000); fish – Pickering

http://dx.doi.org/10.1016/j.aquatox.2016.05.017
0166-445X/© 2016 Elsevier B.V. All rights reserved.
 V.A.K. Fernando et al. / Aquatic Toxicology 177 (2016) 98–105
(1980), Van der Putte et al. (1981), Vutukuru (2005), Velma and
Tchounwou (2010); sea urchins – Okubo and Okubo (1962); prawns
– Murti and Shukla (1983); dipterans – Trumble and Jensen (2004);
earthworms – Sivakumar and Subbhuraam (2005), Fernando et al.
(2015). Many of these studies have used high concentrations of Cr
which may not depict levels commonly found in the environment.
Among aquatic organisms, amphibians are known to be sensi-
tive to heavy metals (Niethammer et al., 1985) and are considered
to be sentinel species for monitoring the impact of these pol-
lutants (Boncompagni et al., 2004). Toxicity of heavy metals on
amphibians is commonly demonstrated using the larvae, where
mortality, growth impairment, developmental delays, malforma-
tions, and behavioural impairments have been reported, as effects
due to exposure to heavy metals (Herkovits and Helguero, 1998;
Ranatunge et al., 2012). However, only a handful of studies have
documented toxic effects of Cr on tadpoles (Khangarot and Ray,
1987; Natale et al., 2000). Evidence on adverse effects of Cr in
adult amphibians is also scarce. Two studies report on Cr induced
biochemical changes in Rana ridibunda (Loumbourdis et al., 2007)
and histological effects in R.lk esculenta (Boncompagni et al., 2004).
Although genotoxicity and haematotoxicity are well documented
for other taxa, little or no information is available regarding these
impacts on amphibians.
In this paper we investigate many aspects of Cr(VI) toxicity
in the Asian common toad (Duttaphrynus melanostictus) covering
several endpoints: mortality, behaviour, growth and development
impairment (for the tadpoles), and haematological alterations and
genotoxicity (for the toads). D. melanostictus was selected because it
is commonly found throughout Sri Lanka, within and outside urban
areas, and hence would be present in nearly all situations where
heavy metal contamination might occur. Moreover, it is one of the
few amphibians, among many present in Sri Lanka, where capture
for research purposes is permitted. Additionally, D. melanostic-
tus can be easily reared and handled in the laboratory. The wide
occurrence of this species in other south Asian countries (Global
Amphibian Assessment, 2004) makes the results specifically appli-
cable to developing countries where pollution is generally poorly
controlled.
On the adverse impacts on tadpoles of D. melanostictus,
Shuhaimi-Othman et al. (2012) have reported lethal toxicity of
metals (Cu, Cd, Zn, Pb, Ni, Fe, Al and Mn), not including Cr,
and Ranatunge et al. (2012) the lethal and sub-lethal effects of
Cd (growth, development and swimming). With regard to Cr,
Khangarot and Ray (1987) report on lethality in D. melanostictus
tadpoles resulting from exposure to high concentrations of this
metal. We have not found any studies focusing on effects such
as haematological, histological, biochemical or genotoxicity aris-
ing from exposure of this species to Cr(VI). To ensure relevance and
applicability of the generated results, the present study considers
ranges of the toxicant that represent recorded levels in the natu-
ral aquatic environment in a local context. We also expect that the
outcome of our investigation would indicate the suitability of this
widespread amphibian species for monitoring heavy metal pollu-
tion in aquatic ecosystems.
2. Material and methods
2.1. Test organism and chemicals
Newly hatched D. melanostictus tadpoles at Gosner stage 24–26
(Gosner, 1960) and adult toads (74.4 ± 7.1 g) were collected from
home gardens in the Colombo district and acclimatized to labora-
tory conditions for two weeks prior to use in the exposure trials.
Animals from different collection sites were assigned equally to the
various treatments and the control. Tadpoles were housed in glass
99
tanks that contained aged tap water and fed with ground commer-
cial fish food. Adult toads were maintained in glass tanks kept in a
slanted position to simulate a semi-aquatic environment, following
Allran and Karasov (2001). The toads were fed with maggots and
beetles collected from home gardens from where soil and banana
piths and water from ponds, previously tested, showed the absence
of Cr.
Analytical grade potassium dichromate (Sigma-Aldrich, USA,
99% purity) was used to prepare test concentrations. For expo-
sure trials with tadpoles and adult toads in order to monitor
changes in survival, growth, development, haematology and in vivo
genotoxicity, five test concentrations were used. Three, 0.002,
0.02 and 0.2 mg/L (3.8 × 10−8 , 3.8 × 10−7 , 3.8 × 10−6 mol/ L), were
based on field levels recorded in surface water bodies in Sri Lanka
(CEA/Euroconsult, 1993; Manage and Wijesinghe, 2009), and a fur-
ther two, 1 and 2 mg/L (1.9 × 10−5 and 3.8 × 10−5 mol/ L), to allow
for observations of dose-dependent trends. Measured concentra-
tions for each of the nominal concentrations were recorded using
atomic absorption spectrophotometry (AA 6650 Shimadzu Atomic
Absorption spectrophotometer [Duisburg, Germany]). Accord-
ingly, measured concentrations of 0.002 ± 0.001, 0.019 ± 0.001,
0.191 ± 0.004, 0.942 ± 0.016 and 1.899 ± 0.029 mg/L were obtained
for the nominal concentrations of 0.002, 0.02, 0.2, 1.0 and 2.0 mg/L,
respectively. Further, for genotoxicity trials conducted in vitro,
erythrocytes of toads were exposed to five Cr(VI) levels (0.0015,
0.003, 0.015, 0.03, 0.15 mg/ L) which were selected based on lev-
els recorded in fish and human blood (Knoll and Fromm, 1960;
Torra et al., 1999). We are unaware of any reported values of Cr
for amphibian blood.
2.2. Experimental setup for toxicity trials
Ethical clearance for procedures used in the present study
was obtained (Ref No. ERCIOB107/07/13) prior to the experi-
ments. Exposure trials with tadpoles were conducted in glass tanks
(25 × 15 × 15 cm) containing 2 l of aged tap water (Wijesinghe et al.,
2011). Eighteen tadpoles were placed in each tank and exposed in
triplicate to 2 l of the five concentrations of Cr(VI) (0.002–2 mg/L)
for 21 days. Controls without the test substance were also main-
tained in triplicate. Test solutions and the controls were renewed
every other day to avoid confounding effects of fouling (Ranatunge
et al., 2012). Experimental conditions were pH 7.3 ± 0.5, dis-
solved oxygen 7.1 ± 1.0 mg/L and temperature 27.1 ± 1.0 ◦ C, with no
significant differences between control and treatment tanks (one-
way ANOVA pH F5,30 = 0.75, p > 0.05; dissolved oxygen F5,30 = 0.35,
p > 0.05; temperature F5,30 = 0.55, p > 0.05 respectively). Tanks in
each case were maintained under a natural photoperiod (12L:12D).
During the trial, tadpoles were fed with commercial fish feed
(30 ± 1 mg per tank), with the amount of food provided being
increased as tadpoles grew, and adjusting for mortality (Wijesinghe
et al., 2011).
Adult toads were exposed singly in glass tanks (28 × 16 × 15 cm)
that were kept in a slanted position (Allran and Karasov, 2001)
with 350 ml of water treated with the five different levels of Cr(VI)
(0.002–2.0 mg/L). When slanted, water covered 50% of the tank
bottom surface. The tanks were taken off the slant for 8 h a day
(0830–1630 h) so that the toads experienced forced cutaneous
exposure to the treatment (Allran and Karasov, 2001). The control
tanks (without test substance) were also placed in a similar fash-
ion. Each treatment and the control were replicated six times and
the exposure was continued for 28 days. Tanks were cleaned and
the test solutions and water renewed every other day. The tanks in
each case were maintained under a natural photoperiod (12L:12D).
The toads were fed with maggots and beetles collected from home
gardens every other day during the trial.
100 V.A.K. Fernando et al. / Aquatic Toxicology 177 (2016) 98–105

2.3. Assessing endpoints of toxicity

2.3.1. Mortality, growth and development in tadpoles

Mortality and metamorphosis of the tadpoles were monitored
in each of the tanks on a daily basis. LC50 values were determined
using measured concentrations for short term (4 day) and long term
(21 day) exposure of the tadpoles. Initial, day 4 and weekly snout
vent lengths of tadpoles were measured using a digital vernier cal-
liper (Comecta, Barcelona, Spain). Tadpoles with spinal deformities
as a percentage of those alive on the day of assessment, was also
noted.

2.3.2. Adult toads

For observing alterations in haematological parameters and
in vivo genotoxicity, both treated and untreated adult toads were
assessed at days 4 (short term) and 28 (long term). Blood samples
from the unexposed toads were used for in vitro genotoxicity trials.
Genotoxic effects were studied (i) in vivo using the micronucleus
(MN) test where the frequency of micronuclei in erythrocytes of
control and treated toads at day 4 and day 28 were assessed (Feng
et al., 2004) and (ii) in vitro using the comet assay with toad erythro-
cytes exposed to Cr (Singh et al., 1988; Baragama-arachchi, 2013).
Toads were anaesthetized with MS-222, using the lowest recom-
mended concentration (Gordon, 2004), to obtain blood through
cardiac puncture (Allran and Karasov, 2001).
2.3.3. Haematological alterations and micronucleus test
To assess changes in haematological parameters in the toads,
blood (100 ␮l) was collected into tubes containing 0.1 M EDTA
at day 4 and day 28. Thin blood smears were prepared immedi-
ately after bleeding, air dried and fixed with methanol. Slides were
stained with 10% Giemsa solution. Micronuclei count, differential
leucocyte count and erythrocyte morphometry (cell length, cell
width, nucleus length and nucleus width) were recorded under
x1000 magnification using an eyepiece graticule. The total ery-
throcyte count (RBC) and total leucocyte count (WBC) (Neubauer
haemocytometer) were taken, and the haemoglobin concentra-
tion (Hb) (Sahli haemoglobinometer) and packed cell volume
(PCV) (micro-haematocrit) were measured. Three haematological
indices − mean cell volume MCV, MCV [␮m3 cell−1 ] = PCV [v/v
ratio] × 1000/RBC [106 cell ␮l−1 ], mean cell haemoglobin MCH,
MCH [pg cell−1 ] = Hb [gdl−1 ] × 10/RBC [106 cell ␮l−1 ], and mean cell
haemoglobin concentration MCHC, MCHC [gdl−1 ] = Hb [gdl−1 ]/PCV
[v/v ratio] – were calculated from RBC, Hb and PCV according to Lee
et al. (1998). Micronuclei were counted under oil immersion (×100
magnification) from thin blood smears, in 1000 erythrocytes per
slide, two slides per animal, and 6 animals per concentration (as in
previous studies e.g. Mouchet et al., 2007).
Fig. 1. Mortality of tadpoles exposed to different levels of Cr(VI) at day 4 and day
21. Arrow indicates maximum level of chromium recorded recently in urban water
bodies of Sri Lanka. *Indicates significant differences from the control (p < 0.05).
layer of LMPA was added. The prepared slides were immersed in
cold lysis buffer (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris-HCl,
10% DMSO, 1% sodium sarcosinate, 1% Triton X-100, pH 10) for
◦
2 h at 4 C, transferred to cold electrophoresis buffer (0.3 M NaOH,
◦
1 mM EDTA, pH > 13) and left for ½ hour for unwinding at 4 C. Elec-
◦
trophoresis was carried out for 45 min at 17 V and 300 mA at 4 C.
After electrophoresis, slides were neutralized by immersing in cold
neutralization buffer (0.4 M Tris-HCl, pH 7.5) for 5 min each, with
three changes of buffer. The cells were fixed with cold methanol
and allowed to air dry. Slides were stained with ethidium bro-
mide (5 ␮g/ ml) and observed under the fluorescence microscope
(BX51, Olympus, Japan) at ×200 magnification and comet images
were obtained. Images were analysed by Opencomet software. Each
assay was triplicated and 30 cells per slide × 6 slides per concentra-
tion were analyzed. The DNA damage was assessed in terms of tail
length and tail DNA percentage (Mouchet et al., 2007). PBS was used
as a negative control while 500 ␮M H2 O2 was used as a positive
control (Nacci et al., 1996).
2.4. Statistical analysis
Significant differences between the recorded end points of con-
trol and treated tadpoles/toads were determined using one-way
ANOVA or repeated measures ANOVA with Tukey’s multiple com-
parison post hoc test (Velma and Tchounwou, 2010). LC50 values

2.3.4. Comet assay

Blood was collected into sterile microcentrifuge tubes with
0.1 M EDTA and diluted in sterile 1X phosphate buffered saline (PBS,
pH 7.2). The cells were counted and tested for viability using Try-
pan blue method (Feng et al., 2004) and samples with ≥95% viability
were used for the next step. These blood cells were exposed to the
5 levels of Cr(VI) (0.0015–0.15 mg/ L) and incubated overnight in
5% CO2 at room temperature. This environment was maintained
by placing the samples within a candle jar at room temperature,
where the candle is lit while the cover is put on and the stop-
cock kept open; when the candle flame goes out, the stopcock is
closed (according to Nonaka et al., 1996). On the following day
the cells were centrifuged and re-suspended in 100 ␮l of sterile 1X
PBS. The cell suspensions were re-tested and those over 75% via-
bility were selected for the comet assay. The cells were mixed with
0.5% low melting point agarose (LMPA) and spread on slides pre- Fig. 2. Percentage inscrease in snout to vent length (mean ± SEM) of tadpoles
coated with 1% normal melting point agarose. Thereafter a third exposed to the different concentrations of Cr(VI).
 V.A.K. Fernando et al. / Aquatic Toxicology 177 (2016) 98–105 101

Table 1
Micronuclei frequencies in erythrocytes of the toads (D. melanostictus) exposed to
different levels of Cr(VI) in-vivo.

Level of Cr(VI) exposure (mg/ L) Micronuclei frequency (‰)

Short term Long term

exposure (4 days) exposure (21 days)

Control 1.0 ± 0.3 2.1 ± 0.3

0.002 1.8 ± 0.3 4.7 ± 0.8**
0.02 2.0 ± 0.2 7.8 ± 1.0**
0.2 2.3 ± 0.2* 11.0 ± 1.6**
1.0 2.5 ± 0.3* 12.2 ± 0.9**
2.0 2.7 ± 0.2* 13.5 ± 0.9**
*
p < 0.05 (significant differences between control and exposed groups as indi-
cated by one way ANOVA).
**
p < 0.001 (significant differences between control and exposed groups as indi-
cated by one way ANOVA).

Fig. 3. Percentage of tadpoles at two stages leading to metamorphosis at the end

of 21 days exposure to the different concentrations of Cr(VI). *Indicates significant
differences from control (p < 0.05).
were calculated using Probit analysis. The level of significance con-
sidered was p < 0.05.
3. Results
3.1. Mortality in tadpoles
In comparison to the control, short term exposure (4 days)
of tadpoles to Cr(VI) induced significantly higher mortality at
2 mg/L (Fig. 1) (One way ANOVA and Tukey’s pair wise compar-
ison F5,12 = 104.76, p < 0.05). With long term exposure to Cr(VI),
significantly higher mortality than in the control was observed at
1 and 2 mg/ L (One way ANOVA and Tukey’s pair wise compari-
son F5,12 = 77.62, p < 0.05;). Neither short nor long term exposure
to the field levels (≤0.2 mg/L) caused significantly greater levels of
mortality in comparison to the control. Based on the measured con-
centrations of Cr(VI), the LC5096 h for D. melanostictus tadpoles was
determined to be 2.1 mg/ L, while LC5021 days was 1.0 mg/ L (1.8–2.5
and 0.9–1.1, respectively, for LC5096 h and LC5021days at the 95% con-
fidence interval).
3.2. Growth and development
not affected significantly by the Cr(VI) concentration or the dura-
tion of exposure (split-plot design, repeated-measures ANOVA,
and Tukey’s post-hoc tests). None of the tadpoles exposed to 1
and 2 mg/ L Cr(VI) concentrations completed metamorphosis (i.e.
attaining Gosner 46) by the end of the long term exposure period
(21 days), whereas around 12% of those exposed to the lowest
concentration and those in the control completed metamorpho-
sis (one-way ANOVA and Tukey’s pairwise comparison F5,12 = 5.46,
p < 0.05). The percentage of tadpoles with forelimbs (Gosner stage
42 and above) was significantly lower in the groups exposed to
1 and 2 mg/ L concentrations in comparison to that of the con-
trol (one-way ANOVA and Tukey’s pairwise comparison F5,12 = 5.61,
p < 0.05) (Fig. 3).
3.3. Morphological abnormalities
Some of the metal exposed tadpoles exhibited abnormal curva-
tures in their tails i.e. kyphosis – abnormally convex/hunchback
spine, and scoliosis – lateral deviation from the typical straight
line in the spine, as described by Meteyer (2000) and David and
Kartheek (2015), at all Cr(VI) concentrations after day 15 (Fig. 4).
At the end of the long term exposure 25% of the surviving tad-
poles exposed to the highest concentration and approximately 15%
in each of 0.02 and 0.2 mg/ L, which are environmentally relevant
concentrations, had such malformations (Fig. 5).

The increase in body length, as a percentage of the initial
length, was compared among treatment and control groups (Fig. 2).
Although not statistically significant, the metal exposed tadpoles
were smaller at all tested levels except in the lowest (0.002 mg/L),
indicating growth retardation. The overall growth change was
3.4. Haematological alterations
Short term exposure to Cr(VI) resulted in marked alterations
in WBC (one-way ANOVA and Tukey’s pairwise comparison
F5,67 = 5.46, p < 0.05), but the other tested haematological param-

Fig. 4. Morphological abnormalities observed in the tails of D. Melanostictus exposed to Cr(VI), at Gosner stages: (a) 43, (b) 42 and (c) 29. Arrows indicate spinal abnormalities.
102 V.A.K. Fernando et al. / Aquatic Toxicology 177 (2016) 98–105
Fig. 5. Percentages of tadpoles showing spinal abnormalities i.e: (the number of
tadpoles with spinal abnormalities as a percentage of those surviving on a particular
day) at days 12, 15, 18 and 21, exposed to the different concentrations of Cr(VI).
Control did not exhibit malformations, hence not shown.
eters were not significantly affected (i.e. RBC, PCV, Hb, MCV, MCH
and MCHC). WBC levels significantly declined in toads exposed to
levels as low as 0.002 mg/L. Extended exposure to Cr(VI), however,
caused significant alterations in RBC, PCV, Hb, MCV, MCH and MCHC
in comparison to the control (one-way ANOVA and Tukey’s pair-
wise comparison– F5,67 = 19.14, p < 0.001; F5,67 = 34.70, p < 0.001;
F5,67 = 65.85, p < 0.001; F5,67 = 12.27, p < 0.05; F5,67 = 9.33, p < 0.001;
F5,67 = 12.72, p < 0.001, respectively) (Fig. 6 and Fig. 7). With regard
to erythrocyte morphometry, short and long term exposures to
Cr(VI) resulted in significant decline in erythrocyte length and
width, including at levels well below the recorded field level of
0.20 mg/L (one-way ANOVA and Tukey’s pairwise comparison –
short term F5,354 = 7.42, p < 0.05; F5,67 = 18.49, p < 0.001, respec-
tively; long term F5,354 = 6.45, p < 0.001; F5,67 = 23.99, p < 0.001,
respectively). In addition, significant decline was observed in
Fig. 6. Variation in haematological parameters (mean ± SE) of toads exposed to the different concentrations of Cr(VI) over 28 day exposure period. *Indicates significant
differences from the control (p < 0.05).
nuclear width at 0.002 mg/L in the short term exposure (one-way
ANOVA and Tukey’s pairwise comparison– F5,354 = 5.88, p < 0.05)
and in nuclear length at 0.002 and 0.02 mg/L with long term
exposure (one-way ANOVA and Tukey’s pairwise comparison–
F5,354 = 6.27, p < 0.05). The percentage of neutrophils dropped sig-
nificantly at 0.02 mg/ L and above (one-way ANOVA and Tukey’s
pairwise comparison test) from that of the controls at the end of
the long term exposure.
3.5. Genotoxicity
3.5.1. Micronucleus test
Genotoxicity was indicated by the significant increase in the fre-
quency of micronuclei in erythrocytes of the exposed toads after
both short and long term exposure to Cr(VI) at concentrations cor-
responding to environmental levels. The increases in comparison
to the control were significant after 4 days of exposure to the lev-
els higher than 0.02 mg/ L (one-way ANOVA and Tukey’s pairwise
comparison F5,67 = 9.98, p < 0.001), and after 28 days at all test levels
(one-way ANOVA and Tukey’s pairwise comparison F5,67 = 19.20,
p < 0.001) (Table 1).
3.5.2. Comet assay
Genotoxicity was also evident in the in vitro tests with ery-
throcytes. The comet assay showed that Cr(VI) exposure caused
significant changes in tail DNA length and tail DNA percentage
compared to the control indicating genotoxicity at 0.015 mg/L
and above (one way ANOVA and Tukey’s pairwise comparison
F5,1074 = 32.26, p < 0.05) (Fig. 8).
4. Discussion
A growing body of information suggests that aquatic fauna
could be detrimentally affected through direct mortality or through
sub lethal effects induced by exposure to toxic heavy metals.
 V.A.K. Fernando et al. / Aquatic Toxicology 177 (2016) 98–105
Fig. 7. Variation in haematological indices (mean ± SE) of toads exposed to the dif-
ferent concentrations of Cr(VI) over 28 days. *Indicates significant differences from
control (p < 0.05).
In support of this, the current study, through short and long
term exposure to a range of Cr(VI) concentrations, also includ-
ing environmentally-relevant levels, has provided further evidence
of lethal and sub-lethal effects on the Asian common toad (D.
melanostictus) distributed widely in Sri Lanka and many other Asian
countries. Cr(VI) was seen to cause mortality and growth retarda-
tion and to induce haematological and DNA damage.
Mortality of the tadpoles was significantly enhanced at Cr(VI)
of 1 mg/L and above. High concentrations of chromium do in fact
occur close to discharge points (1520 mg/ L has been recorded from
a tannery effluent in Sri Lanka – Qader et al., 2013; 100 mg/ L has
been recorded from waste waters elsewhere – Dakiky et al., 2002).
Mortality was dose-dependent with LC5096 h of 2.1 mg/ L, lower than
that reported for Cr by Khangarot and Ray (1987) for older stages of
the same species. There is no single identified mechanism by which
mortality is induced, although it is possible that the cumulative
action of chromium at various metabolic sites could be responsible
(Vutukuru 2005).
103
Fig. 8. Variation in the tail DNA length (mean ± SEM) and tail DNA percentage
(mean ± SEM) in the in vitro tests with toad erythrocytes exposed to the different
levels of Cr(VI). Values for negative and positive controls are also shown. *Indicates
significant differences from respective control (p < 0.05).
With the exception of the lowest level of Cr(VI) concentration
of 0.002 mg/L, growth retardation in the tadpoles occurred in all
the other treatments. However, the growth retardation was not
statistically significant, and this could be attributed to the “crowd-
ing effect” (Gromko et al., 1973), i.e. increased mortality favouring
the surviving individuals. Metal induced growth retardation in tad-
poles has also been observed with copper (Redick and La Point,
2004), lead (Chen et al., 2006) and cadmium (Ranatunge et al.,
2012).Growth retardation to any degree induced by exposure to
chromium would result in lowered fitness (Marian et al., 1983).
A matter of much concern is the effect of Cr(VI) in impairing the
process of metamorphosis evident at levels as low as 0.02 mg/ L. A
similar result has been previously demonstrated for cadmium in
the same species (Ranatunge et al., 2012) and in other species (e.g.
Zhang et al., 2007, in Bufo raddei). Such impairment in metamorpho-
sis may be due to growth retardation of the tadpoles as suggested
by Stebbins and Cohen (1995). Hirashima et al. (1993) report that
in red flour beetles larval-growth retardation is a likely cause of
delayed metamorphosis.
The abnormal morphological features in the tadpole tails
recorded in the present study (kyphosis and scoliosis) have been
documented by Peı́rez-Coll et al. (1988) and Herkovits et al. (1997)
in Bufo arenarum exposed to lead and Xenopus laevis to cadmium,
respectively. Hisaoka and List (1957) reported scoliosis in Rana syl-
vatica tadpoles and mentioned that it occurs primarily in the tail
and in the posterior tip of the urostyle of the frog. Thus, after meta-
morphosis this aberration would only be visible if the urostyle is
examined which would require sacrificing the frog. Some authors
have suggested that scoliosis in metal-exposed tadpoles may occur
due to a mutation (Hisaoka and List, 1957), and this may have been
a possibility in the present study where DNA damage was recorded.
An important finding of the current study, with regard to
sub-lethal impacts, is the capacity of Cr(VI) to alter several haema-
tological parameters in amphibians, where information is seriously
lacking. Short term exposure to chromium induced leucopenia,
i.e. the reduction in the number of white blood cells, suggesting
immuno-suppression. This has been demonstrated previously in
metal exposed fish (Wepener et al., 1992; Witeska et al., 2010).
Immuno-suppression from exposure to heavy metals is said to
occur due to the blockage of sites of active antibody molecules and
disruptions in normal metabolism, ionic balance, and the cellular
division of leucocytes (O’Neill, 1981). The Cr(VI) exposed toads also
suffered from neutropenia indicating effects essentially on non-
specific immunity, as also observed for chromium and lead exposed
fish in previous studies (Gill and Pant 1987; Witeska et al., 2010).
104 V.A.K. Fernando et al. / Aquatic Toxicology 177 (2016) 98–105
Decrease in neutrophil percentage leads to the reduction of phago-
cytic activity in organisms, impairing their capacity to deal with
pathogens in the natural environment.
Prolonged exposure to Cr(VI) also caused anaemic conditions
as evident by the marked decline of several other haematologi-
cal parameters and indices tested (RBC, Hb, PCV, MCV, MCH and
MCHC). Alarmingly, the LOAELs for the above parameters are
much lower than the environmentally relevant levels of 0.02 and
0.2 mg/ L. The decrease in erythrocytes may have been due to
haemolysis and the resulting haemorrhagic conditions in the kid-
ney, stomach and intestine, that are caused by the bioconcentration
of chromium as observed in fish (Trama and Benoit, 1960; Van der
Putte et al., 1981; Wepener et al., 1992). The production of ery-
throcytes in stressed animals initially increases to compensate for
the cells lost through haemolysis. As a consequence, there is an
increased proportion of small and immature red cells in the blood.
This fact is well portrayed by the decrease in the length and width
of erythrocytes and the MCV levels recorded in the present study.
It has been documented that chromium has the potential to retard
haemoglobin synthesis through the inhibition of steps such as iron
binding during biosynthesis (Tephly et al., 1978), due to struc-
tural alterations of the heme molecules and inhibitory effects on
the enzyme system (Johansson-Sjöbeck and Larsson, 1979). Previ-
ous studies have revealed a comparable decline in haematological
parameters of fish exposed to Cr(VI) (Gill and Pant, 1987; Wepener
et al., 1992; Vutukuru, 2005) while one study has revealed cor-
responding trends in the amphibian Bufo maculatus exposed to
cadmium (Ezemonye and Enuneku, 2011). The altered haemato-
logical parameters found in the present study are suggestive of
dose and duration dependency which has been previously shown
by Vutukuru (2005) for fish.
Besides haematotoxicity, one of the significant results generated
through the present study is the potential of chromium to induce
genotoxic effects in amphibians. We are unaware of previous
reports of chromium induced genotoxic effects on any amphibian
species. Cr(VI) induced considerable DNA damage in D. melanostic-
tus erythrocytes at low concentrations. Dose-dependency of DNA
damage observed in D. melanostictus confirms that genotoxicity in
this widely occurring species could be used as a monitoring end-
point for heavy metal pollution in Sri Lanka and in other Asian
countries. The MN test generated a Lowest Observable Adverse
Effect Level (LOAEL) of 0.2 mg/ L for short term exposure which
decreased to 0.002 mg/ L with long term exposure. Micronuclei can
originate from a chromosomal breakage as well as from a dysfunc-
tion in the mitotic spindle apparatus (Heddle, 1973). The MN test
detects chromosomal and/or genomic mutations (chromosomal
damage and/or alteration of mitotic spindles) after repair (Mouchet
et al., 2007) unlike the comet assay where DNA single/double strand
breaks and alkali-labile sites are detected only if repair has not
occurred (Kassie et al., 2000). This study is in agreement with
observations of Mouchet et al. (2007) where increased numbers
of micronuclei and DNA damage in terms of several comet assay
parameters were noted at trace levels of cadmium for tadpoles of
other species.
In this paper we demonstrate a wide range of lethal and sub-
lethal damage in D. melanostictus induced by exposure to Cr(VI),
which includes enhanced mortality, retarded growth and devel-
opment, and haematoxic and genotoxic effects. Haematotoxic and
genotoxic effects were observed at the levels of chromium recorded
in some urban freshwater water bodies in Sri Lanka, which were
around 0.2 mg/L. The sensitivity of D. melanostictus and the dose
dependent trends seen in many of its responses to the pollutant
indicate that this widely distributed toad is suitable for monitor-
ing heavy metal pollution in aquatic ecosystems. The evidence of
lethal and sub-lethal damage in D. melanostictus emphasizes the
need to control effluent discharge into water bodies, particularly
in developing countries where pre-treatment of industrial effluent
procedures are largely overlooked.
Acknowledgements
We acknowledge the financial assistance from the World
Bank (HETC/CMB/QIGW3/SCI) and the University of Colombo
(AP/3/2/2013/RG/Sc/06). We are grateful to the Department of
Zoology and Institute of Biochemistry, Molecular Biology and
Biotechnology of University of Colombo for providing the necessary
facilities for carrying out the research work.
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