Eects of heavy metals on sea urchin embryo development.

Part 2. Interactive toxic eects of heavy metals

in synthetic mine euents
Naomasa Kobayashi
a,
*
, Hideo Okamura
b
a
Doshisha University, Karasuma-Imadegawa Kamikyo-ku, Kyoto 602-8580, Japan
b
Laboratory of Marine Environmental Management, Faculty of Maritime Sciences,
Kobe University, Fukaeminami 5-1-1, Higashinada, Kobe 658-0022, Japan
Received 19 May 2004; received in revised form 31 January 2005; accepted 22 February 2005
Abstract
Interactive toxic eects between heavy metals were investigated using a sea urchin (Anthocidaris crassispina) bioas-
say. An euent from an abandoned mine showed signicant inhibitory eects on embryo development as well as pro-
ducing specic malformations. The eects on the embryos were reproduced by synthetic polluted seawater consisting of
eight metals (manganese, lead, cadmium, nickel, zinc, chromium, iron, and copper) at the concentrations detected in the
mine euent. This indicated that the heavy metals were responsible for the eects observed. Five heavy metals were
ranked in decreasing order of toxicity as follows: Cu > Zn > Pb > Fe > Mn. Among these, zinc and manganese could
cause malformation of the embryos. From bioassay results using 27 combinations of heavy metals, 16 combinations
including zinc could produce specic malformations, such as radialized, exo-gastrulal, and spaceship Apollo-like gastr-
ulal embryos. Zinc was one of the elements responsible for causing malformations and its eects were intensied by the
presence of the other metals, such as manganese, lead, iron, and copper.
2005 Elsevier Ltd. All rights reserved.
Keywords: Bioassay; Disused mine; Malformation; Sublethal toxicity; Synthetic euent
1. Introduction
The toxicity of the polluted waters originating from a
disused lead mine was evaluated in our previous study
(Kobayashi and Okamura, 2004) using both sea urchin
bioassay and heavy metal analysis. The test waters, con-
taining high concentrations of eight heavy metals (man-
ganese, lead, cadmium, nickel, zinc, chromium, iron,
and copper), had inhibitory eects on the development
of sea urchin embryos at a 1/50 dilution of the raw
water. The test water induced various malformations,
such as radialized pluteus, exo-gastrula, and spaceship
Apollo-like embryos. It was speculated that zinc was
one of the metals causing malformations and that inter-
active eects occurred involving zinc and other metals.
Interactive eects between metals can result in synergis-
tic, additive or antagonistic actions and these eects
have been reported following bioassays using sea urchin
(Kobayashi and Fujinaga, 1976; Warnau et al., 1998;
0045-6535/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2005.02.071
*
Corresponding author. Address: 26 Kamitakano-Moroki-
cho, Sakyo-ku, Kyoto 606-0046, Japan. Fax: +81 75 791 7062.
E-mail addresses: kobayana@kyoto.zaq.ne.jp (N. Kobaya-
shi), okamurah@maritime.kobe-u.ac.jp (H. Okamura).
Chemosphere xxx (2005) xxxxxx
www.elsevier.com/locate/chemosphere
ARTICLE IN PRESS
Phillips et al., 2003), shrimp (Negilski et al., 1981), daph-
nia and trout (Enserink et al., 1991), the zebra mussel
(Kraak et al., 1994), and daphnia, fathead minnow
and freshwater mussel (Masnado et al., 1995).
The purposes of this study were (1) to determine
which metals in the mine euent were responsible for
the malformations, and (2) to evaluate the interactive ef-
fects between heavy metals detected in mine euent on
the development of sea urchin embryos.
2. Materials and methods
2.1. Test waters
Polluted water samples were collected around
Kanayama Cove, Shirahama, in Wakayama Prefecture,
Japan (Kobayashi and Okamura, 2004). Natural seawa-
ter collected near the Seto Marine Biological Laboratory
of Kyoto University has been successfully used as non-
contaminated seawater (control seawater) for sea urchin
bioassay. Dissolved concentrations of the eight heavy
metals in both water samples are shown in Table 1. Syn-
thetic polluted seawaters were prepared by adding heavy
metals into control seawater to reach the concentrations
measured in the left stream water (Table 1). Heavy metal
stock solutions dissolved in deionized water were pre-
pared using the following metal salts: MnCl
2
4H
2
O,
Pb(CH
3
COO)
2
3H
2
O, CdCl
2
, ZnCl
2
, K
2
Cr
2
O
7
, NiCl
2
,
FeCl
2
6H
2
O, and CuSO
4
5H
2
O. For sea urchin bioas-
says, the synthetic seawaters were successively diluted
with the control seawater at ratios of 1, 1/5, 1/10, 1/20,
1/50, and 1/100. Five metals (Mn, Pb, Zn, Fe, and Cu)
out of the eight detected in the left stream water were
the focus of experiments to assess interactive eects.
The following 27 heavy metal combinations were
prepared:
(A) one combination consisting of eight metals (Mn,
Pb, Cd, Zn, Cr, Ni, Fe, and Cu);
(B) one combination consisting of ve metals (Mn,
Pb, Zn, Fe, and Cu);
(C) ve combinations consisting of four of the ve
metals in B;
(D) 10 combinations consisting of three of the ve
metals;
(E) 10 combinations consisting of two of the ve
metals.
2.2. Bioassay
Anthocidaris crassispina (A. Agassiz), which has a
breeding season from May to September, was the sea
urchin used in the 20022003 experiments. In brief, eggs
and sperm were mixed and inseminated in each test
water at 2628 C. The method of sea urchin bioassay
was the same as in our previous studies (Kobayashi,
1990; Kobayashi and Okamura, 2004).
3. Results
3.1. Eects of synthetic polluted seawaters on embryo
development
The acute toxic eects of the polluted waters upon
the rst cleavage and pluteus formation are shown in
Table 2. Fertilization and further development of em-
bryos in the control seawater progressed normally. Ab-
sence of growth of the initial cell was observed in both
test waters at no dilution and at 1/5 dilution. Normal
development was achieved at 1/100 dilution for both
samples. The toxicity of the polluted water was generally
somewhat greater than that of the synthetic water. Both
samples caused various types of abnormalities to the em-
bryos. In order of increasing dilution, these were: retar-
dation of development, radialized pluteus, exo-gastrula,
spaceship Apollo-like embryos, remaining at the gas-
trula stage, and permanent blastula. The ultimate state
of developmental stages and abnormalities of both sam-
ples were quite similar (Table 2).
3.2. Eects of single metal on embryo development
It has been reported that zinc can cause malforma-
tions of developing embryos (Kobayashi and Okamura,
2004). In that study, manganese was also suspected to
cause malformations. Toxic eects of manganese on
sea urchin embryo development are listed in Table 3.
At the lowest concentration tested (870 lg/l), no inhibi-
tory eects were observed on the rst cleavage and plu-
teus formation. Retardation of the development was
observed at concentrations higher than 1700 lg/l. Exo-
gastrula and Apollo-like gastrula were observed at inter-
mediate concentrations (690028000 lg/l). The highest
concentration tested (55000 lg/l) completely inhibited
embryo development and induced permanent blastula
or gastrula. Thus, it was clearly shown that manganese
could cause malformations at high concentrations.
Table 1
Heavy metals detected in water samples
a
Test
samples
Dissolved concentration (lg/l)
Mn Pb Cd Zn Cr Ni Fe Cu
Natural
seawater
1.8 0.1 0.027 4.9 0.026 0.86 0.99 0.79
Left
stream
water
900 510 12 38 0.62 6.9 3700 8.4
a
Kobayashi and Okamura (2004).
2 N. Kobayashi, H. Okamura / Chemosphere xxx (2005) xxxxxx
ARTICLE IN PRESS
Toxic eects of a single metal on sea urchin embryo
development are summarized in Table 4 along with the
summary of two test waters in Table 2. The no observed
eect concentration (NOEC) of heavy metal, allowing
normal development, was 870 lg/l for manganese,
34 lg/l for lead, 7.2 lg/l for zinc, 210 lg/l for iron, and
3.8 lg/l for copper. Five metals are ranked in decreasing
order of toxicity according to the NOEC on embryo
development as follows: Cu > Zn > Pb > Fe > Mn.
3.3. Eects of heavy metal mixtures on embryo
development
The toxic eects of heavy metal mixtures on develop-
ment of sea urchin embryos are indicated in Table 5.
Each gure shows the dilution rate that caused eects
on pluteus formation only at the nal state reached in
the experiment (the ultimate state). A total of 27 com-
binations caused various types of malformations to the
embryos at various dilution rates. The results produced
by the eight-metal mixture shown in Table 2 are summa-
rized in Table 5 (A, number 1). Apart from this single
experiment, the focus was on the eects of the ve metals
(Mn, Pb, Zn, Fe, and Cu) among eight that were in high
concentration in the mine water.
3.3.1. Mixture of ve metals (B)
One combination (Table 5, number 2) of a ve-metal
mixture was examined. Stoppage of growth at one cell
was observed in a 1/5 dilution. Normal development
was achieved in a 1/100 dilution and abnormal develop-
ment was observed in a 1/50 dilution, the same as
observed in the eight-metal mixture. Since the abnormal-
ities observed for the eight-metal mixtures were repro-
duced in this combination, it was speculated that there
must be some key metals among these ve.
Table 2
Eects of test waters on embryo development of sea urchin Anthocidaris crassispina
Test sample Dilution 1st cleavage (60 min) Pluteus formation (48 h) Remarks on
the ultimate
state
a
Normal 1 cell Normal
(N)
Retard
(R)
Malform
(M)
Pre-pluteus
(P)
Control 1 98.2 0.3 1.8 0.3 98 0.3 1.3 0.5 0.7 0.5 N
Left stream water
b
1/100 96.3 0.5 3.7 0.5 96.3 0.8 2.2 0.2 1.4 2.4 0 N
1/50 88.8 1.5 11.2 1.5 50 3.6 21.8 2.1 27 3.0 1.2 2.5 R, RAD(EG)
1/20 30.3 2.5 69 2.7 0 0 100 0 R, EG(100)
1/10 13.8 3.0 85.5 3.3 0 0 77.8 5.5 22.2 5.5 R, AP, G
11/5 0 100 S
Synthetic polluted
seawater containing
eight heavy metals
1/100 98.5 0.3 1.5 0.3 95.8 1.0 0.8 0.2 1.7 0.2 1.7 0.3 N
1/50 88.2 0.3 11.8 0.3 61.5 5.3 26.3 4.1 7.0 0.5 5.2 1.2 R, RAD, EG
1/20 48.5 0.3 51.5 0.3 0 17.3 5.7 53.2 2.0 29.5 8.0 R, EG, AP
1/10 17.3 0.4 82.7 0.4 0 0 0 100 R, G
11/5 0 100 S
a
N: normal, R: retard, S: stoppage point, RAD: radialized pluteus, EG: exo-gastrula embryo, AP: Spaceship Apollo-like embryo, G:
gastrula, PB: permanent blastula.
b
Data on the sample collected at left stream (Kobayashi and Okamura, 2004).
Table 3
Eects of manganese on embryo development of sea urchin A.crassispina
Nominal
concentration
(lg/l)
1st cleavage (60 min) Pluteus formation (48 h) Remarks on
the ultimate
state
a
Normal 1 cell Normal Retard Malform Pre-pluteus
0 98.8 0.3 1.2 0.3 98.0 0.3 1.0 0.9 0.5 0.0 0.5 0.0 N
870 96.8 1.0 3.2 1.0 97.3 0.2 1.5 0.3 0.7 0.2 0.5 0.2 N
1700 97.3 0.3 2.7 0.3 91.1 4.1 6.7 4.4 1.5 0.5 0.7 0.5 R
3500 92.5 1.3 7.5 1.3 0 93.2 2.7 2.2 1.2 4.6 2.2 R
6900 87.2 4.5 12.8 4.5 0 64.5 2.7 30.5 2.9 5.0 3.7 R, AP, EG
14000 88.2 3.1 11.8 3.1 0 0 65.5 3.2 34.5 3.2 R, AP, EG
28000 86.0 3.0 14.0 3.0 0 0 32.8 3.2 67.2 3.2 R, AP
55000 71.5 4.2 28.5 4.2 0 0 0 100 R, PB, G
a
Same as shown in Table 2.
N. Kobayashi, H. Okamura / Chemosphere xxx (2005) xxxxxx 3
ARTICLE IN PRESS
3.3.2. Mixtures of four metals (C)
Five combinations (Table 5, numbers 37) of four-
metal mixtures were examined. Stoppage of growth at
one cell in a 1/5 dilution was observed for numbers 3
6. Normal development was achieved at a 1/100 dilution
for numbers 36 and abnormal development was ob-
served at a 1/50 dilution for numbers 36. Number 7
gave normal and abnormal development at 1/50 dilution
and 1/20 dilutions, respectively. The combinations are
ranked in decreasing order of malformations at the ulti-
mate state as follows: number 3 > 4 = 5 > 6. There were
no malformations observed in one combination (number
7) that contained no zinc.
3.3.3. Mixtures of three metals (D)
Ten combinations (Table 5, numbers 817) of three-
metal mixtures were examined. Complete stoppage of
growth at one cell was observed at a 1/5 dilution for
numbers 814. Normal development was achieved at 1/
1001/50 dilutions and abnormal development was ob-
served at 1/501/20 dilutions. The combinations
are ranked in decreasing order of malformations at
the ultimate state as follows: numbers 9 > 8 > 12 >
10 > 13 > 11. There were no malformations observed
in four combinations (numbers 1417) that contained
no zinc.
3.3.4. Mixture of two metals (E)
Ten combinations (Table 5, numbers 1827) of two-
metal mixture were examined. Complete stoppage of
growth at one cell was observed at a 1/5 dilution for
numbers 1821. Normal development was achieved at
1/1001/20 dilutions and abnormal development was ob-
served at 1/501/5 dilutions. The combinations are
ranked in decreasing order of malformations at the ulti-
mate state as follows: numbers 19 > 18 > 20 > 21. There
were no malformations observed in six combinations
(numbers 2227) that contained no zinc.
4. Discussion
The biological eects of the left stream water were
well reproduced by the synthetic seawater that included
eight heavy metals. This result suggests that the key
heavy metals that cause malformation of embryo devel-
opment were among the eight selected metals. It is obvi-
ous that the left stream water has severe toxic eects
because it contains heavy metals at concentrations
higher than the NOEC (Table 4). The concentrations
of these ve metals in a 1/50 dilution of left stream
water, in which abnormal eects were observed, were
much lower than the NOEC. Thus, no single metal
could cause the observed abnormal eects by itself and
interactive toxic eects between metals might have re-
sulted in a synergistic action in the real mine euent.
Three malformations (radialized, exo-gastrulal, and
spaceship Apollo-like gastrulal embryos) observed in left
stream water and synthetic polluted seawater were
reproduced by the presence of manganese or zinc. How-
ever, the manganese concentration (4590 lg/l) calcu-
lated from the dilution rate (1/201/10) to cause
malformations was much lower than the concentration
(690028000 lg/l) that causes malformations by itself.
This result was also true for zinc, but the dierence in
the concentrations was smaller. Retardation of develop-
ment was observed in most cases and stoppage of devel-
opment at the gastrula, blastula or earlier stages were
also found. The observed eects on embryo development
of the mine euent could not be explained simply by
heavy metal concentrations. Thus, interaction between
chemicals in the real euent may have caused inhibitory
eects on sea urchin embryo development.
The toxic eects of manganese (manganese chloride)
on sea urchin embryo development are indicated in
Table 3. Exo-gastrula and Apollo-like gastrula were
caused by manganese at relatively high concentrations.
These abnormalities were also reported by Lallier
Table 4
Eects of a single metal on sea urchin embryo development
Remarks on the
ultimate state
a
Single metal (nominal ion concentration in lg/l) Left stream water Synthetic polluted
seawater
Mn Pb Zn Fe Cu Dilution Dilution
Normal 870 34 7.2 210 3.8 1/100 1/100
Abnormal 1700 70 14 410 7.7 1/50 1/50
Radial 14 1/50 1/50
Exo-gastrula 690014000 2958 1/20 1/5020
Apollo-like 690028000 58 1/10 1/20
Retard 350028000 70250 1458 8201600 7.764 1/5010 1/5010
Gastrula 55000 250 1/10 1/10
Perm. blast 55000 250980 31130
Stoppage point 120480 3100 1/51 1/51
a
Same as shown in Table 2.
4 N. Kobayashi, H. Okamura / Chemosphere xxx (2005) xxxxxx
ARTICLE IN PRESS
Table 5
Eects of heavy metal combinations on sea urchin embryo development
Combination
a
no. A B C
1 2 3 4 5 6 7
Cd +
Cr +
Ni +
Mn + + + + + +
Pb + + + + + +
Zn + + + + + +
Fe + + + + + +
Cu + + + + + +
Normal 1/100 1/100 1/100 1/100 1/100 1/100 1/10050
Abnormal 1/50 1/50 1/50 1/50 1/50 1/50 1/20
Radial 1/50 1/50 1/5020 1/5020
Exo-gastrula 1/5020 1/20 1/20 1/20 1/20
Apollo-like 1/20 1/20 1/10 1/20 1/20 1/20
Retard 1/5010 1/5010 1/5010 1/5010 1/5010 1/5010 1/205
Gastrula 1/10 1/10 1/10 1/105
Perm. blast 1/10 1/10 1/10 1/10 1/10
Stoppage point 1/5 1/5 1/5 1/5 1/5 1/5
D
8 9 10 11 12 13 14 15 16 17
Mn + + + + + +
Pb + + + + + +
Zn + + + + + +
Fe + + + + + +
Cu + + + + + +
Normal 1/100 1/100 1/10050 1/100 1/10050 1/100 1/100 1/100 1/10050 1/10050
Abnormal 1/50 1/50 1/20 1/50 1/20 1/50 1/50 1/5020 1/20 1/20
Radial 1/20 1/20 1/20 1/20 1/20
Exo-gastrula 1/10 1/10 1/10 1/10 1/10 1/10
Apollo-like 1/10 1/10
Retard 1/2010 1/2010 1/2010 1/5010 1/2010 1/5010 1/2010 1/105 1/205 1/105
Gastrula
Perm. blast
Stoppage point 1/5 1/5 1/5 1/5 1/5 1/5 1/5
E
18 19 20 21 22 23 24 25 26 27
Mn + + + +
Pb + + + +
Zn + + + +
Fe + + + +
Cu + + + +
Normal 1/10050 1/100 1/10050 1/100 1/10050 1/10020 1/10050 1/10020 1/10020 1/10020
Abnormal 1/20 1/50 1/20 1/50 1/20 1/10 1/20 1/105 1/10 1/10
Radial 1/20 1/20 1/20 1/20
Exo-gastrula 1/10 1/10 1/10 1/10
Apollo-like 1/10 1/10 1/10
Retard 1/2010 1/5010 1/2010 1/2010 1/205 1/105 1/205 1/105 1/105 1/5
Gastrula 1/5 1/5 1/5
Perm. blast
Stoppage point 1/5 1/5 1/5 1/5
a
A: eight metals, B: ve metals, C: four metals, D: three metals, E: two metals.
N. Kobayashi, H. Okamura / Chemosphere xxx (2005) xxxxxx 5
ARTICLE IN PRESS
(1975), Kobayashi (1990), and Kobayashi and Okamura
(2004). Some papers reported that malformation of sea
urchin embryos could be induced by other heavy metals.
Kobayashi and Fujinaga (1976) reported the synergistic
inhibitory action of a combination of zinc and copper
upon sea urchin eggs. Phillips et al. (2003) reported the
toxicity of four-metal mixtures to larval sea urchins.
They used an index of 96-h EC50 on normally developed
larvae and reported that an antagonistic response oc-
curred in a combination of zinc and copper.
The toxic eects of metal mixtures on pluteus forma-
tion are shown for a total of 27 combinations (Table 5).
The dilution rate to achieve normal development was re-
corded as 1/1001/20 dilution. Stoppage of growth at
one cell was observed in 1/5 dilutions except for 10 com-
binations (numbers 7 for four-metal mixtures, numbers
1517 for three-metal mixtures, and numbers 2227 for
two-metal mixtures). Three abnormalities (radialized,
exo-gastrulal and spaceship Apollo-like gastrulal em-
bryos) were observed except for 11 combinations (num-
ber 7 for four-metal mixtures, numbers 1417 for three-
metal mixtures, and numbers 2227 for two-metal mix-
tures) that contained no zinc. In most cases of the 16
combinations where abnormalities were observed, zinc
concentration at 1/10 dilution was calculated as 3.8 lg/
l. This concentration is less than the NOEC of zinc
(7.2 lg/l). Thus, the current data indicate that abnormal-
ities were caused by low concentrations of zinc in com-
bination with other metals. It seems that these other
heavy metals (manganese, lead, iron, and copper) can
enhance the eect of zinc. Further experimentation is
needed to dene which metal is responsible for the ef-
fects, in terms of metal species and quantity.
5. Conclusions
Interactive toxic eects between heavy metals in mine
euent were investigated by sea urchin bioassay. Spe-
cic conclusions are noted as follows:
1. Toxic eects caused by mine euent were reproduced
by synthetic polluted seawater prepared by adding
eight heavy metals at the same concentrations as
detected in the euent.
2. Five heavy metals detected in mine euent were
ranked in decreasing order of toxicity as follows:
Cu > Zn > Pb > Fe > Mn. Only zinc or manganese
could cause specic malformation of embryos.
3. Bioassays with 27 combinations of heavy metals
clearly showed that zinc was the only element to
cause specic malformations and the eects were
intensied by the presence of the other metals such
as manganese, lead, iron, and copper.
Acknowledgments
The authors wish to express their gratitude to the
director and sta of the Seto Marine Biological Labora-
tory, Kyoto University, for the use of experimental facil-
ities. This work was partly supported by grants from
Salt Science Research Foundation and Kurita Water
and Environment Foundation.
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