4(1): 20-27 (2010)
DOI: 10.3153/jfscom.2010003
Journal of FisheriesSciences.com
E-ISSN 1307-234X
© 2010 www.fisheriessciences.com
RESEARCH ARTICLE
ARAŞTIRMA MAKALESİ
BIOACCUMULATION OF COBALT IN
MOSQUITOFISH (Ga m bu sia a ffin is
BAIRD&GIRARS, 1853) AT DIFFERENT FLOW
RATES AND CONCENTRATIONS
Utku Güner∗
Trakya University, Faculty of Arts and Sciences, Department of Biology. 22030 Edirne, Turkey
Abstract:
The aim of the present study was to determine Co accumulation pattern in bodies of
mosquitofish (Gambusia affinis Baird & Girard, 1853) at differing flow rates and
concentrations following a 180 hours of exposure period. Co values in each experimental group
were determined by flame atomic absorption spectrophotometer (FAAS). We found no
differences in the mean accumulated Co level between experimental groups.
Keywords:
∗
Correspondence to:
Cobalt, accumulation, flow rate, Gambusia affinis
Utku GÜNER, Trakya University, Faculty of Arts and Sciences, Department of Biology. 22030
Edirne -TURKEY
Tel: (+90 284) 235 28 26 / 1194 Fax: (+90 284) 235 40 10
E-mail: uguner@trakya.edu.tr or utku_guner@hotmail.com
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Introduction
The pollution of aquatic ecosystems by heavy
metals is an important environmental problem
(Rayms-Keller et al. 1998), as heavy metals
constitute some of the most hazardous substances
that can bioaccumulate (Tarifeno-Silva et al.
1982). Bioaccumulation is a process in which a
chemical pollutant enters into the body of an
organism and is not excreted, but rather collected
in the organism’s tissues (Zweig et al. 1999).
Although some of these metals (e.g., Cu, Zn, Co)
are essential trace elements for living organisms,
they become toxic at higher concentrations. Some
of these heavy metals were used as micronutrient
such as cobalt (Co).
Co is of relatively low abundance in the
Earth's crust and in natural waters, from which it
is precipitated as the highly insoluble Cobalt
sulphite CoS. Although the average level of Co in
soils is 8 ppm, there are soils with as little as 0.1
ppm and others with as much as 70 ppm. In the
marine environment Co is needed by blue-green
algae (cyanobacteria) and other nitrogen fixing
organisms (Watanabe et al.1997).
Co is an essential metal as an integral
component of cyanocobalamin (vitamin B12)
constituting nearly 4.5% of its molecular weight
which is indispensable for animal growth.. Most
animals need Co for vitamin B12 synthesis by
intestinal microflora. Members of these intestinal
flora have also been isolated from the intestinal
tracts of fish (Kashiwada et al., 1970). Co, as a
part of vitamin B12 is associated with nitrogen
assimilation and synthesis of haemoglobin and
muscle protein. It also influences certain
enzymes, binds to insulin (Cunningham et al.
1955) and also reduces plasma glucose levels
(Roginski and Mertz, 1977).
Previous studies reported results on Co
accumulation and their toxicity effect in aquatic
organisms (Nolan etal 1992). Co acute to chronic
ratios determined for Daphnia were 111 and 1163
in soft water and hard water, respectively
(Diamond et al 1992) indicating large differences
between acute and chronic effects thresholds. Co
toxicity data also exist for several fish species
goldfish (Carassius auratus), fathead minnow
(Pimephales promelas), zebrafish (Brachydanio
rerio), and banded gouramis (Colisa fasciatus)
(Marra et al.1998). Baudin and Fritsch (1989)
examined the related contribution of food and
water in the accumulation of cobalt in fish. Carp
fed Co-contaminated snails were found to
accumulate Co only slightly. Marra et al (1992)
reported that a clear dose-response relationship
between Co concentration and mortality,
although rainbow trout did not start dying in
concentrations that eventually produced 100%
mortality until after the first 2 days.
Fish accumulated Co-cobalamine 21 times
more rapidly from seawater than CoCl2 and
retained ingested Co-cobalamine 20 times more
efficiently (100 %) than ingested CoCl2 (5 %).
Two thirds of the ingested Co-cobalamine was
retained in the fish with a retenhon half-time of 8
d. The remaining one third of the organic form
was retained with a half-time of 54 d, a value
which was not significantly different from that of
CoCl2 (47 d) (Nolan et al, 1992). On the other
hand, very high doses of Co (0.1-5 g Co kg-1)
were toxic to rainbow trout, resulting in
haemorrhages in the digestive tract and
alterations in white blood cells (Watanabe et
al.1997). Co deprivation reduced the intestinal
synthesis of vitamin B12 in catfish (Limsuwan
and Lovell, 1981).
The mosquitofish Gambusia affinis (Baird
and Girard, 1853) is a member of family
Poeciliidae (Poeciliids), subfamily Poeciliinae
and inhabits standing to slow-flowing water;
most common in vegetated ponds and lakes,
backwaters and quiet pools of streams. G. affinis
feeds on zooplankton, small insects and detritus
and is used as live food for carnivorous aquarium
fishes (Page and Burr, 1991). G. affinis is
commonly present in the pond and streams near
agricultural areas and also it is easy to maintain
this species in laboratory (Öztürk and İkiz, 2003,
Güner, 2009).
Since G. affinis is widespread, used for biological control of mosquitoes and also is an easy
organism to be maintained in laboratory, we used
this organism as the experimental animal and
focused in this present study on mainly
bioaccumulation of different consantrations
(4000, 2000, 1000 ppm) of Co with different
flow rates (0.5-1-2 ml//h) in animals with
different exposure times (1, 12, 24, 36, 48, 60,
72, 84, 96, 108, 132 and 180 h).
Materials and Methods
Mosquito fish, Gambusia affinis (Order:
Cyprinodontiformes, Family: Poeciliidae), were
obtained from Güllapoğlu Lake Edirne and
transferred in our controlled laboratory and kept
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in 100 l continuous aerated glass aquarium tanks
for 2 weeks before experimentation in flowing
dechlorinated (active carbon filtered) and aerated
Edirne city tap water [Gülapoğlu Lake water:
Ca+2 40 mg/l, pH 8.0, 14°C]. The temperature,
oxygen content and pH of the aquarium water
were monitored daily.
Animals were fed
approximately 3% body weight per day (as three
1% meals per day) with a commercial fish bait
(Pınar Fish Bait; Zn 70 mg/kg, Mg 25 mg/kg, Mn
25 mg/kg, Fe 2 mg/kg, Co 0.2mg/kg, wet
weight).
After a two weeks accustimation period in
holding tanks (maintained at 21 oC, 12:12 h
light:dark regime), 300 fishes were randomly
transferred to three (50x50x100 cm, 100 l) glass
aquarium exposure tanks which operated with
static systems with continuous aeration (three
experiemental groups). Prior to the experiments,
each aquaria were continuously mixed by motor
(see Figure 1).
CoSO4 salts were used for preparation of three
different stock solutions (4000, 2000, 1000 ppm
Co). Fishes were exposed for different times (1,
12, 24, 36, 48, 60, 72, 84, 96, 108, 132 and 180
h) in different stock solutions (4000, 2000, 1000
ppm Co) with different flow rates (0,5-1-2 ml//h)
(see fig 1).
First experimental group was exposed to 4000
ppm Co at a flow rate of 0.5 ml/h, second
experimental group was exposed to 2000 ppm Co
at a flow rate of 1 ml/h and third experimental
group was exposed to 1000 ppm Co at a flow rate
of 2 ml/h. But, at all sampling period, all
aquariums were equal Co concentrations (see fig
2). At the end of experiment time (180 h) all of
aquarium Co concentrations were 7.2 ppm.
Figure1. Experimental design (dosimeter, motor and air pomp).
8.000
7.000
Co (ppm)
6.000
5.000
4.000
3.000
2.000
1.000
0.000
0
20
40
60
4000 ppm 0.5 ml/h
80
100
120
hour
2000 ppm 1 ml/h
140
160
180
200
1000 ppm 2ml/h
Figure 2. Sampling times and final doses of three experimental groups.
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The total lengths of all fishes from each
sampling time were measured using digital
callipers in order to obtain an average length for
that group (see table 1). The total sample was
then placed into a 5 ml close polypropylene tube
and placed into shaker for 24 hours at 30°C. All
fishes had 1 ml of nitric acid and 1 ml percloric
acids added and were left overnight on a cold
digest (Güner 2007, 2008). The levels of Co, in
digests were determined using flame atomic
absorption (Unicom Model 929 AA) . The
standards used to make calibration cue were 1, 3
and 5 mg\1. The SNK test was used to compare
Co concentrations among replicates, Co
treatments and time. As metal accumulation in
replicate aquaria was not significantly different
from each other, samples were pooled and a twoway Anova analysis was performed followed by
a SNK test as a post hoc test. Groups were
considered to be significantly different from each
other if p< 0.05. All analyses were performed
using an SPSS version 10.0 and MS Excel
program.
Results and Discussion
In an aquatic environment with Co inside,
exposure of fish to this metal in water results in
50 % of the metal being associated with the skin
and external organs, 30 % with the kidneys, and
the remainder being evenly distributed
throughout the body, whereas as a result of an
uptake within food, only 30 % becomes
associated with the external organs, and the
remainder is uniformly present in other tissues
(Coughtrey & Thorne 1983). Koyanagi et al.
(1980) reported the major Co-accumulating
organs in flounder Karejus bicoloratus as being
the intestine (8 %), bone (9), liver (12 %), muscle
(12%), skin (15 %), (l7 %), and head (20 %).
Suzuki et al. (1979) reported that 75 % of Co in
yellowtail Seriola quinqueradiata was present in
the blood, viscera, and muscle. In this study, to
avoid different accumulation in tissues, all body
Co accumulation was determined.
The natural concentrations of Co in fishes are
very low and Co accumulation in fishes was not
observed in areas where Co concentration in
water was close to the background values
(Moiseenko and Kudryavtseva 1990). During our
experimental period Co accumulated in G. affinis
body.
Total body length and weight of all fish were
measured, the average of groups, the largest and
the smallest value was shown taple 1. Partial
correlation coefficients was found between Co
and total wieght as -0,5218 (P<0.001).
In all experimental groups, accumulation of
Co increased depending on time and with increasing Co water level (Table 2).
Figures 3-5 show comparison
exposure times and body Co levels.
between
Accumulation pattern of 4000 ppm Co group
with 0.5 ml/h flow rate indicated that there
existed a step between 24 and 84 hours (see fig
3).
Figure 3. Accumulation of Co in 4000 ppm and 0.5 ml/h flow rate in G.affinis
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0,5 ml/h
4000 ppm Co
1 ml/h
2000 ppm Co
2 ml/h
1000 ppm
Table 1. Some described statics of weighs of fish in experimental groups.
Dose
Time Minimum Maximum
Std. Dev.
Mean
0,0800
0,6100
0,2012
0,2133
1
0,0900
0,2800
0,0796
0,2140
12
0,1000
0,2000
0,0406
0,1400
24
0,0380
0,5300
0,1929
0,2336
36
0,0900
0,6000
0,2090
0,2360
48
0,1100
0,6000
0,2056
0,2420
60
0,0700
0,2300
0,0669
0,1480
84
0,0900
0,1500
0,0217
0,1180
132
0,0900
0,2400
0,0677
0,1360
180
0,1300
0,5500
0,1702
0,2620
1
0,1200
0,2700
0,0611
0,1760
12
0,0800
0,2000
0,0476
0,1180
24
0,1100
0,3400
0,0920
0,2080
36
0,1300
0,3000
0,0669
0,1980
48
0,0700
0,1200
0,0207
0,0940
60
0,0900
0,2900
0,0815
0,1500
84
0,0700
0,2100
0,0526
0,1280
132
0,1200
0,1500
0,0141
0,1300
180
0,0600
0,2200
0,0740
0,1480
1
0,0900
0,2300
0,0505
0,1500
12
0,0800
0,1700
0,0356
0,1120
24
0,0700
0,2000
0,0592
0,1550
36
0,1000
0,1700
0,0237
0,1300
48
0,1300
0,3100
0,0716
0,1860
60
0,1100
0,5100
0,1877
0,2720
84
0,1000
0,2000
0,0391
0,1540
132
0,0800
0,1400
0,0235
0,1200
180
Fig 4. Accumulation of Co at 2000 ppm and 1 ml/h flow rate in G.affinis
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Figure 5. Accumulation of Co at 1000 ppm with 2 ml/h flow rate in G.affinis
0.5 ml/h
4000 ppm Co
1 ml/h
2000 ppm Co
2 ml/h
1000 ppm
Table 2. Co analysis results of experimental groups.
Dose Time
Mean Std. Error Minimum
Maximum
of Mean
4.882
2.918
37.144
1 17.183
9.202
17.636
66.056
12 30.638
4.184
31.410
52.318
24 43.084
36.264
12.798
216.658
36 79.488
13.430
15.472
88.482
48 55.121
15.374
17.889
106.664
60 65.999
16.296
56.578
144.757
84 89.697
8.300
78.520
130.889
132 104.597
19.870
54.500
147.217
180 110.229
4.215
5.660
29.392
1 16.771
2.891
12.404
29.950
12 22.197
9.726
33.888
88.556
24 68.383
8.906
21.421
68.127
36 44.441
12.142
26.112
95.911
48 60.510
11.979
69.025
137.129
60 108.494
16.352
29.310
119.878
84 78.808
21.506
47.090
176.100
132 105.170
7.795
75.547
118.563
180 91.377
9.597
17.132
64.467
1 32.624
4.505
19.465
45.589
12 28.761
10.455
44.676
108.644
24 75.606
23.140
40.558
139.679
36 71.121
7.969
45.832
102.485
48 74.352
8.702
34.784
85.254
60 65.586
17.558
20.471
110.609
84 62.706
10.551
59.743
120.985
132 83.788
10.784
91.657
147.900
180 107.631
SNK
A
B
C
D
C
C
D
E
E
A
A
B
C
B
D
E
D
F
A
b
c
c
c
d
d
d
e
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(Student-Newman-Keuls (SNK) test P<0.001)
Very high doses of Co (0.1-5 g Co kg-1) were
toxic to rainbow trout, resulting in hemorrhages
in the digestive tract and alterations in white
blood cells (Watanabe et al.,1997). In this study,
at the end of experimental period (180 hours), no
mortality was observed in all groups.
the end of experiment period. The accumulation of Co in the fish body investigated increased depending on dose and time. However, accumulation of heavy metals at different
flow rate more detailed in other heavy metals
should be investigated.
Generally, exposure of fish to cobalt in water
results in 50 % of the metal being associated with
the skin and external organs (Nolan et al, 1992).
Co is essential as an integral component of vitamin B12 which is indispensable for fish growth
(Miyazaki et al., 2001). Co was reported to accumulate in many tissues such as liver, gonads,
and digestive duct (Nolan et al., 1992). Different
fish tissues have got different Co accumulation
capacities. In this study total Co accumulation in
the body tissues were analyzed.
Coughtrey, P.J., Thorne, M.C., (1983).
Radionuclide distribution and transport in
terrestrial and aquatic ecosystems: a cntical
review of data. Vol. 2. A. A. Balkema,
Rotterdam, p. 191-217.
The present results showed that there was no
difference in the mean Co accumulation levels
between some experiment periods; i.e. 0.5 ml/h
flow rate 4000 ppm Co concentration group between 24-48 and also 60-132 hours (see table 2).
Cunningham, L.W., Fischer, R.L. and Vestling,
C.S., (1955). The binding of zinc and cobalt
by insulin, Journal of the American
Chemical
Society,
77:
5703-5707.
doi:10.1021/ja01626a072
Marra et al (1992) reported that the temporal
pattern of Co toxicity was different than Cu. Cuinduced lethality occurred as early as the first 24
h of exposure, with most mortality occurring
within the initial 72 h. In contrast, Co-induced
lethality did not occur prior to 72 h of exposure,
with the majority of toxicity observed between 72
and 192 h.
Diamond, J.M., Winchester, E.L., Muckler, D.G.,
Rasnake, W.J., Fanelli, J.K., Gruber, D.,
(1992). Toxicity of cobalt to freshwater
indicator species as a function of water
hardness, Aquatic Toxicology, 22: 163–180.
In this study, when three flow rates and Co
concentrations groups were compared there were
some similarities and differences between
groups. First, because of accumulation pattern
much uniform, 4000 ppm Co concentration group
at 0.5 ml/h flow rate, accumulation showed
different pattern. Second, the first two
experiments period except, at first period of 1
ml/h flow rate of 2000 ppm Co concentration
group, accumulation was showed a continuous
increased. Co accumulation pattern was not much
All of groups showed time depend accumulation
pattern (see table 2). Because of the same final
accumulation level at end of experiment period
(180 hour) all of the groups showed similar patterns.
Conclusions
In conclusion it appeared that three flow
rates applied and Co concentrations did not
affect Co accumulation in mosquito fish at
26
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