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Effect of mixtures of metals on the spotted Babylon snail (Babylonia areolata)
under different temperature conditions
Article  in  Toxicological and Environmental Chemistry · September 2013
DOI: 10.1080/02772248.2014.881077

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Effect of mixtures of metals on the
spotted Babylon snail (Babylonia
areolata) under different temperature
conditions
a

V.J. Vedamanikam & T. Hayimad

a

a

Institute of Oceanography, University Malaysia Terengganu,
Kuala Terengganu, Malaysia
Accepted author version posted online: 08 Jan 2014.Published
online: 31 Jan 2014.

To cite this article: V.J. Vedamanikam & T. Hayimad , Toxicological & Environmental Chemistry
(2014): Effect of mixtures of metals on the spotted Babylon snail (Babylonia areolata)
under different temperature conditions, Toxicological & Environmental Chemistry, DOI:
10.1080/02772248.2014.881077

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Toxicological & Environmental Chemistry, 2014
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Effect of mixtures of metals on the spotted Babylon snail (Babylonia
areolata) under different temperature conditions
V.J. Vedamanikam and T. Hayimad*
Institute of Oceanography, University Malaysia Terengganu, Kuala Terengganu, Malaysia

Downloaded by [Vikrant Vedamanikam] at 17:38 04 February 2014

(Received 19 November 2013; accepted 26 December 2013)
A study was conducted on the Babylon snail (Babylonia areolata) to examine the
effects of copper (Cu), cadmium (Cd), nickel (Ni), and zinc (Zn) on different life
stages of this gastropod. Metal toxicity significantly varied according to the life stage
of the snail. The different LC50 values obtained were 0.51, 5.49, 0.31, and 0.2 ppm for
Cu, Zn, Cd, and Ni for the larval stage and 4.98, 15.19, 0.91, and 1.21 ppm at the
juvenile stage and 8.54, 17.52, 1.14, and 1.44 ppm in the adult stage. Studies were
also conducted on the effects of dual metal concentrations and experiments were
repeated with temperature as a variable. Results demonstrated that metal toxicity
values were altered depending on the metals involved in the combination as well as
temperature under which the experiment was conducted.
Keywords: Babylonia areolata; toxicity; heavy metals; dual metal combinations; temperature variation

Introduction
In the natural environment organisms are subjected to a variety of pollutants sometimes
singularly and most to mixtures. Toxicity testing of heavy metals both acute and chronic
is well established. Various marine organisms such as fish, bivalve, shrimp, and gastropod
were examined and monitored to determine effects of the heavy metal contamination on
these organisms (Chan, 1995; Hashmi, Mustafa, and Tariq 2002; Neuberger-Cywiak,
Achituv, and Garcia 2003; Gammon, Turner, and Brown 2009). These studies were carried out predominantly with a single metal in each experiment. In reality, organisms in
nature undergo exposure to the effect of the metal mixtures which cannot be predicted
from the effects of individual toxicants (Utgikar et al. 2004). The toxic effects of metal
mixtures were studied in various organisms and different combination of metals
showed different adverse effects. The mixture of cobalt–cadmium, cadmium–zinc, cadmium–lead and copper–lead displayed antagonistic effects, while cobalt–copper and
zinc–lead showed synergistic effects in a bactericidal study (Fulladosa, Murat, and Villaescusa 2005). Data indicated that cadmium (Cd) became less toxic when combined
with other metals, and lead (Pb) seemed to be less toxic in the presence of Cd. Otituluju
(2002) reported that zinc (Zn) reduced the adverse effects of Cd and copper (Cu) in a
study conducted on the periwinkle, gastropod (Tympanotonus fuscatus). Synergistic
effects of Zn with other metals were reported in biting midge (Chironomus plumosus)

tested with different metal mixtures (Vedamanikam and Shazilli 2009). It was found that
the toxicity of Zn was increased when combined with chromium (Cr), nickel (Ni), mercury (Hg), and Cd (Vedamanikam and Shazilli 2009). This study reported further that
*Corresponding author. Email:
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V.J. Vedamanikam and T. Hayimad

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silver, Pb, Cu, and manganese reduced the toxicity of Zn (antagonistic effects). Thus, the
effects of metal mixtures need to be studied to better understand how an organism
responds to a variety of metals.
Methodology
To study the effects of different metals on Babylonia areolata, two definitive bioassays
were conducted. The first bioassay was a 96-hr median lethal concentration test to determine the 96-hr LC50 values for the different metals selected. The second bioassay again
consisted of a 96-hr LC50, but in this case, temperature was a variable and tests were conducted at different fixed temperature values. Four metal salts were selected for this investigation – zinc chloride, nickel (II) chloride, copper (II) chloride, and cadmium (II)
chloride. All metal salts were of analytical grade supplied by MERCK, Germany. Stock
solutions of 1000 mg/L were prepared for each metal.
A range-finding experiment was conducted to determine the appropriate concentrations of the four different metals for further toxicity studies on adult B. areolata. All tests
were conducted with three replicates and controls. Test containers with volumes of
600 ml were selected for execution of different bioassays. Each chamber was filled with
400 ml filtered sea water. To each test container, a single adult was added, until each
chamber had 10 adults, each adult randomly selected to prevent any bias. For each metal,
six test containers were allocated of which five were for metal concentrations and one a
control; each test had three replicates. Water quality parameters (temperature, salinity,
pH, and dissolved oxygen) were monitored throughout the 96-hr period. At termination
of each test, mortality data were compiled and 96-hr LC50 values calculated using the

trimmed Spearman–Karber toxicity program (Hamilton, Russo, and Thurston 1977). Animals were considered dead when all movement ceased and organisms exhibited nil
response to gentle stimulation. This was carried out by gently touching the organism with
a glass rod and observing the effect of stimulation on the organism. The concentrations
tested in for adult B. areolata for different metals are presented in Table 3. Concentrations
listed are the nominal values and measured values. The concentrations were measured
with a SpectrAA 220 Fast Sequential Atomic Absorption Spectrophotometer (AAS)
(manufactured by Varian - South Queensferry, Edinburgh, Scotland, UK). Prior to running the sample water, a calibration curve was obtained by running different concentrations of the various analytical standards (seven concentrations and a blank) through the
AAS. Further quality assurance was obtained with the use of the standard reference materials Cd, Cu, Ni, and Zn Standards for AAS (Fluka product codes 51994, 38996, 42242,
and 18827, respectively). The metal recovery percentage from the standard was 99.87%
for Cd, 98.81% for Cu, 99.24% for Ni, and 99.85% for Zn.
The effect of temperature on the 96-hr LC50 of the four different metals on B. areolata
was explored. The median lethal toxicity experiments were again conducted under conditions of fixed temperature. The temperatures selected were 10, 15, 20, 23, 25, 28, 30, 35,
and 40  C. The heavy metal concentration range for each metal tested remained the same
for the different temperatures. The concentrations used in each median lethal toxicity test
are provided in Table 1. Temperatures of 10, 15, and 20  C were maintained with the help
of a temperature control cabinet, while the higher temperatures were maintained with the
help of heating elements (Askol-IP68). Temperature fluctuations were observed to be
Æ0.5  C. For each temperature, a median lethal toxicity test was conducted for each metal.
The main part of this investigation was carried out in two phases. Phase 1 was to
obtain baseline data on effects of metal mixtures at a constant temperature of 25  C.


Toxicological & Environmental Chemistry

3

Table 1. Concentrations of heavy metals used in the 96-hr LC50 experiment.
Concentrations utilized in the bioassay (mg/L)
(measured concentrations are in parenthesis)

Metal

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Zinc
Nickel
Copper
Cadmium

0
0
0
0

10.0 (9.0Æ 0.5)
15.0 (14.9Æ0.1)
25.0 (23.9Æ0.5)
5.0 (4.9Æ0.1)

15.0 (14.8Æ0.1)
20.0 (19.5Æ0.3)
30.0 (28.9Æ0.5)
10.0 (9.0Æ0.2)

20.0 (18.9Æ0.3)
25.0 (24.1Æ0.5)
35.0 (34.9Æ0.02)
15.0 (14.9Æ0.1)

25.0 (26.1Æ0.5)

30.0 (29.8Æ0.1)
40.0 (40.1Æ0.02)
20.0 (21.1Æ0.4)

30.0 (29.8Æ0.1)
35.0 (34.9Æ0.02)
45.0 (44.9Æ 0.1)
25.0 (24.1Æ0.5)

Copper was used as the main metal and paired with Cd, Ni, and Zn. Variations in LC50
value would show if Cu was becoming more toxic or less toxic with the addition of the
second metal. The mixtures were tested against three life stages of the spotted Babylon
snail, the larva, juvenile, and adult, the concentrations of heavy metals used for each life
stage are found in Tables 2 and 3. Methodology used in the combination of metal followed Vedamanikam and Shazilli (2009) method PPMþPPM. In this method, actual test
concentrations are used in the mixtures rather than percentage of LC50 values. This allows
direct comparisons to be made as to whether a combination is more toxic than the individual metal. The basic set-up of the experiment is the same as the standard LC50 test with
the exception that a second metal is then added. The 96-hr LC50 values were calculated
based on individual metal concentrations and variation between the new value and the
original LC50values, i.e. the LC50 values obtained in the aquatic toxicity test versus the
LC50 values obtained from the mixture. All the tests were monitored at test initiation for
water quality – temperature, salinity, dissolved oxygen, and pH (Table 4).

Results and discussion
The toxicity tests of metal mixtures showed 96-hr LC50 values of each metal in every
stage of B. areolata life cycle varied with the individual heavy metal. With Cu case, 96hr LC50 value for the individual metal in larva, juvenile, and adult stage were 0.51, 4.98,
and 8.54 ppm, respectively. However, when paired with either Cd, Ni, or Zn, the LC50
Table 2. Nominal concentrations of metal used in the metal mixtures experiment for different life
stages of B. areolata.
Treatment
Life stage

Larval stage

Juvenile and adult stage

Copper þ cadmium
0.10
0.50
1.00
5.00
10.00
15.00
1.00
5.00
10.00
15.00
20.00
25.00

0.01
0.05
0.10
0.50
1.00
5.00
0.10
0.50
1.00
1.50
2.00
2.50

Copper þ nickel
0.10
0.50
1.00
5.00
10.00
15.00
1.00
5.00
10.00
15.00
20.00
25.00

0.01
0.05
0.10
0.50
1.00
5.00
0.50
1.00
1.50
2.00
2.50
3.00

Copper þ zinc
0.10

0.50
1.00
5.00
10.00
15.00
1.00
5.00
10.00
15.00
20.00
25.00

0.50
1.00
5.00
10.00
15.00
20.00
5.00
10.00
15.00
20.00
25.00
30.00


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V.J. Vedamanikam and T. Hayimad

Table 3. 96-hr LC50 values of B. areolata in all stages exposed to individual and mixtures of heavy
metals.
LC50 values relative to
single metal (ppm)

Type of metals

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Stage

Cd

Cu

Ni

Zn

95% lower
95% upper
confidence limit confidence limit

Larva

Single

Cu
Zn
Cd

Ni
Mixtures Cuþ Cd
Cuþ Ni
Cuþ Zn
Juvenile Single
Cu
Zn
Cd
Ni
Mixtures Cuþ Cd
Cuþ Ni
Cuþ Zn

–

0.51
–
0.31 –
–
–
0.05 0.50
–
1.01
–
0.70
4.98 –
15.19 –
0.91 –
1.21 –
0.41 4.11

–
6.46
–
7.13

–
–
–
0.20
–
0.10
–
–
–
–
–
–
1.22
–

–
5.49
–
–
–
–
2.12
–
–
–

–
–
–
12.84

0.36
4.03
0.19
0.11
0.03/0.35
0.53/0.05
0.43/1.34
3.34
13.44
0.71
1.07
0.23/2.30
4.16/1.08
5.29/11.06

0.71
7.50
0.53
0.34
0.07/0.71
1.95/0.20
1.12/3.36
7.42
17.16
1.17

1.37
0.73/7.33
10.05/1.21
9.60/14.91

Adult

Single

8.54
17.52
1.14
1.44
6.75
–
–

–
–
–
–
–
1.60
19.45

–
–
–
–
–

–
–

6.27
15.67
0.81
1.26
0.44/4.39
7.63/1.39
9.78/15.47

11.65
19.58
1.60
1.64
1.04/10.36
12.67/1.84
19.35/24.45

Cu
Zn
Cd
Ni
Mixtures Cuþ Cd
Cuþ Ni
Cuþ Zn

–
–
–

–
0.67
9.83
13.75

values were 0.5, 1.01, and 0.7 ppm in larva; 4.11, 6.46, and 7.13 ppm in juvenile; and
6.75, 9.83, and 13.75 ppm in adult stage. A similar trend was seen for other dual metal
combinations. Statistical analysis of data showed a significant difference in the LC50 values (f ¼ 0.002). The larval stage was also observed to be the most sensitive life stage of
the Babylon snail especially when exposed to dual combinations of Ni þ Cu (LC50
Table 4. Water quality parameters tested at 20 and 32  C for copper toxicity tests conducted on B.
areolata.
Condition

Temperature ( C)

Max
Min
Median
Standard deviation

20.05
20.00
20.025
0.035355

Max
Min
Median
Standard deviation

32.00
31.05
31.525
0.67175

Salinity (ppt)

Dissolved oxygen (ppm)

pH

20 C
36.61
32.50
34.74
1.34

8.23
5.35
6.23
0.88

8.25
7.88
8.05
0.10

32 C
36.66
32.93

34.91
1.19

9.20
3.98
5.53
2.01

7.87
6.66
7.51
0.36


5

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Toxicological & Environmental Chemistry

Figure 1. 96-hr LC50 values trend of metal mixture compared with normal temperature in larva
(A), juvenile (B), and adult (C) stages.

0.1 ppm) and Cd þ Cu (LC50 0.05 ppm). Adults were observed to be the least sensitive
(Table 3). When temperature was used as a variable it was observed that the 96-hr LC50 values varied, either increasing or decreasing depending upon the case (Figure 1). The results
showed that temperature played a role in either raising or lowering the toxicity of the metal


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6

V.J. Vedamanikam and T. Hayimad

mixtures. At the highest temperature tested (32  C), 96-hr LC50 values could not be calculated for several combinations as thermal toxicity came into effect.
Dual metal toxicity was been studied by different investigators. Otitoluju (2002)
examined the effects of Zn and Cu in the periwinkle juvenile, Tympanotonus fuscatus,
observing antagonistic effects of Cu in combination, a similar reaction as seen in this
experiment. Similar results were observed by others (Fulladosa, Murat, and Villaescusa
2005; Utgikar et al. 2004). The synergistic and antagonistic effects noted in this study
may be due to Cu enhancing the absorption of other metals or in the case of Zn decreasing
absorption of other metals as reported by Xu et al. (2011)
The present study demonstrated that non-essential heavy metals are more toxic to the
snail than essential metals in all experiments within this study. Nott and Langston (1993)
suggested that marine gastropod (Littoriana littorea) might detoxify essential metal such
as Zn via feces. Copper plays an important role in many enzyme systems of gastropods,
especially for hemocyanin loading. While Cd, which is the most toxic metal in the present
study, was found to disrupt respiration, feeding, and activities level of these organisms.
Cadmium is also known to induce a shift from aerobic to anaerobic metabolic pathway
(Moolman, Van Vuren, and Wepener, 2007). Data thus indicate that metal mixtures exert
adverse effects on the snail but due to the variability in responsiveness it is not possible to
utilize this model as a biomonitor for effects in other organisms.

Acknowledgments
The authors would like to acknowledge the Fundamental Research Grants System (FRGS 59180)
for funding this project.

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