Int. J. Mol. Sci. 2013, 14, 421-433; doi:10.3390/ijms14010421
OPEN ACCESSInternational Journal of

Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
ArticleEffects of Organoboron Antifoulants on Oyster and Sea Urchin
Embryo Development
Noritaka Tsunemasa 1,*, Ai Tsuboi 2 and Hideo Okamura 2
1

2

Environment Conservation Division, Environment Bureau, City of Hiroshima, 1-6-34 Naka-ku,
Hiroshima 730-8586, JapanLab Maritime Environment Management Graduate School of Maritime Science, Kobe University,
5-1-1 Fukaeminami, Higashinada-ku, Kobe 658-0022, Japan;
E-Mails: aixxxu_uxxx@yahoo.co.jp (A.T.); okamurah@matitime.kobe-u.ac.jp (H.O.)* Author to whom correspondence should be addressed; E-Mail: tunemasa-n@city.hiroshima.lg.jp;
Tel.: +81-82-504-2188; Fax: +81-82-504-2229.
Received: 9 October 2012; in revised form: 22 November 2012 / Accepted: 8 December 2012 /Published: 24 December 2012

Abstract: Prohibition of Ot (organotin) compounds was introduced in Japan in 1997 andworldwide from September 2008. This meant that the production of paints containing TBT
compounds was stopped and alternatives to the available Ot antifoulants had to be
developed. It has been claimed that the degradation by-products of these alternative
antifoulants were less toxic than those of Ot compounds. Since the introduction of thealternative antifoulants, the accumulation of these compounds has been reported in many
countries. However, the toxicity of these compounds was still largely unreported. In thisresearch, the toxicity of the alternative Ot antifoulants TPBP (triphenylborane pyridine)
and TPBOA (triphenylborane octadecylamine) and their degradation products on
Crassostea gigas and Hemicentrotus pulcherrimus were tested. The results showed that
toxic effects in Crassostea gigas was higher for each antifouling biocide than that in
Hemicentrotus pulcherrimus. Also, while the toxicity of the Organoboron antifoulants andthe Ots were the same, the former’s degradation products were much less harmful.
Keywords: bioassay; TPBP; TPBOA; alternative antifoulant; LC10; LC50

Int. J. Mol. Sci. 2013, 14422

1. Introduction
Organotin compounds (Ots), used for many years as antifouling biocides on ships, marine structures,and fishing nets, became a problem because of their toxicity and accumulation characteristics.
The movement toward the global regulation of these compounds began in October 2001 when theInternational Maritime Organisation (IMO) adopted the International Convention on the Control ofHarmful Antifouling Systems (AFS Convention), which prohibited the use of Ots as active ingredientsin antifouling agents for marine vessels. This convention came into effect on 17 September, 2008.
Currently, Irgarol 1051®, Diuron, Sea-nine 211®, copper pyrithione, and other organic booster
biocides are the major antifoulants used worldwide. In Tsunemasa and Okamura [1], a toxicity
evaluation method on fertilized oyster egg was developed, and the toxicity of organotin alternative
antifoulants (Irgarol 1051, Diuron and Sea-Nine 211) and organotin compounds (TBT and TPT) was
evaluated. From these results, it was clear that Sea-Nine 211 was as toxic as organotin compounds tofertilized oyster eggs.
Some organoboron antifoulants such as TPBP and TPBOA are only used in Japan and some other
Asian countries, but there is little data available on their impact, bioaccumulation, and environmental
toxicity. Only the research on Artemia salina and Skeletonema costatum [2] and that on
Anthocidaris crassispina [3] have looked at organoboron antifoulants.
The first biological examination of sea urchins was started by Kobayashi et al. [4], followed by
Loenning and Hagstroem [5], Dinnel et al. [6], Pagano et al. [7], and Beiras et al. [8]. The government
agencies Environment Canada [9], US EPA [10], ASTM [11] and APHA [12] conducted their own
research based on earlier test methods used on fertilized sea urchin eggs. In the research for this report,
the revised Kobayashi method [13] was used to determine the effects on embryonic development of
sea urchins.
Oysters and sea urchins were used in this research due to their high sensitivity to chemicalcompounds [14–16], as well as the fact that oysters and sea urchins can be found globally.
First, the rate of fertilization, development after fertilization, rate of deformity in the embryos, andnumber of underdeveloped embryos were measured. Then the effects of alternative Organoboron
antifoulants on Crassostea gigas and Hemicentrotus pulcherrimus embryos were evaluated. The
toxicity of Crassostea gigas and Hemicentrotus pulcherrimus were compared.
2. Results
The alternative Ot antifoulants (TPBP and TPBOA) and the degradation products from TPBP and
TPBOA (e.g., DPB, MPB, biphenyl, phenol, pyridine, benzene and boric acid), at concentrations
ranging from 0.1 to 1000 μg/L, were used in the toxicity test on the fertilized Crassostea gigas andHemicentrotus pulcherrimus eggs. The photograph of Crassostea gigas embryo development after
24 h at 25 °C is shown in Figure 1. Using these images, the toxicity of antifouling biocides wasevaluated by examining cell division at 2 h after fertilization and checking embryology (i.e., forD-shaped embryos) at 24 h after fertilization. The photograph of Hemicentrotus pulcherrimus embryo
development after 48 h at 20 °C is shown in Figure 2. Using these images, the toxicity of antifoulingbiocides was evaluated by examining cell division at 10 h after fertilization and checking embryology

Int. J. Mol. Sci. 2013, 14423(i.e., for two-armed echinopluteus embryos) at 48 h after fertilization. Controls after 24 h of
Crassostea gigas and controls after 48 h of Hemicentrotus pulcherrimus were normal. Controls after
24 h of Crassostea gigas and controls after 48 h of Hemicentrotus pulcherrimus are shown in
Figures 1G and 2G, respectively.
Figure 1. Effects of TPBP and TPBOA on Crassostea gigas embryo development after
24 h. (A) TPBP at 10 μg/L; (B) TPBP at 1 μg/L; (C) TPBP at 0.1 μg/L; (D) TPBOA at
10 μg/L; (E) TPBOA at 1 μg/L; (F) TPBOA at 0.1 μg/L; (G) Control.

Int. J. Mol. Sci. 2013, 14424

Figure 2. Effects of TPBP and TPBOA on Hemicentrotus pulcherrimus embryo
development after 48 h. (A) TPBP at 100 μg/L; (B) TPBP at 50 μg/L; (C) TPBP at
20 μg/L; (D) TPBOA at 100 μg/L; (E) TPBOA at 50 μg/L; (F) TPBOA at 20 μg/L;(G) Control.

2.1. Effects of Organoboron Antifoulants on Crassostea gigas Embryo
2.1.1. TPBP and TPBOAIn this report, the survival rate of fertilized eggs and the occurrence of deformity in D-shaped
embryos were investigated. Survival rate and deformity rates of Crassostea gigas embryo after aperiod of 24 h are shown in Figure 3. In the report by His et al. [17], four types of deformity wereInt. J. Mol. Sci. 2013, 14425

shown: convex hinge, indented shell margin, incomplete shell, and protruding mantle. In our
experiments, only protruding-mantle deformities were observed.
Figure 3. Survival and protruding-mantle deformity rates on Crassostea gigas embryo
after a period of 24 h.

In the case of TPBP, all of Crassostea gigas eggs in the 100 μg/L treatment died after 2 h, before
any cell division could take place. Approximately 25% of Crassostea gigas eggs in the 10 μg/L
treatment died before any cell division had occurred. Approximately 5% of the embryos whichsurvived showed signs of deformity or delayed development. Approximately 10% of Crassostea gigas
eggs in the 1 μg/L treatment died before any cell division. A small percentage of the embryos whichsurvived showed signs of deformity or delayed development.
Approximately 50% of Crassostea gigas eggs in the 10 μg/L treatment died after 24 h. Half of theeggs showed no signs of cell division, but in the case of the other half, cell division had occurred
before death. All of the surviving embryos, which became D-shaped embryos, developed
protruding-mantle deformity (Figure 1A). Approximately 85% of Crassostea gigas eggs in the 1 μg/L
treatment survived. Half of the embryos became D-shaped embryos but half showed signs of delayeddevelopment. Almost all of the D-shaped embryos developed protruding mantle deformity (Figure 1B).
Almost all of Crassostea gigas eggs in the 0.1 μg/L treatment survived. Approximately 70% ofembryos became a D-shaped embryo, while the other 30% of the embryos showed signs of delayeddevelopment. Most D-shaped embryos were normal, but slightly less than 10% developed
protruding-mantle deformity (Figure 1C).
In the TPBOA samples, approximately 85% of Crassostea gigas eggs in the 100 μg/L treatment
died after 2 h, before any cell division could take place. Slightly less than 20% of Crassostea gigas
eggs in the 10 μg/L treatment died before any cell division took place. A small percentage of theembryos which survived showed signs of deformity or delayed development. Slightly less than 10% of

Int. J. Mol. Sci. 2013, 14

426

Crassostea gigas eggs in the 1 μg/L treatment died before any cell division had taken place. A small
percentage of the embryos which survived showed signs of deformity or delayed development.
Slightly more than 30% of Crassostea gigas eggs in the 10 μg/L treatment died after 24 h. Most
eggs showed no signs of cell division, but in a few cases, cell division had occurred before death. Halfof the embryos became D-shaped embryos and half showed signs of delayed development. Almost allof the D-shaped embryos developed protruding-mantle deformity (Figure 1D). Slightly less than 10%
of Crassostea gigas eggs in the 1 μg/L treatment died. Approximately 10% of the embryos whichsurvived showed signs of delayed development, the other embryos became D-shaped embryos. Almost
all of the D-shaped embryos developed protruding-mantle deformity (Figure 1E). Most of the eggs in
the 0.1 μg/L treatment survived. All the surviving embryos became D-shaped embryos. Most D-shaped
embryos were normal with slightly less than 10% of the D-shaped embryos developed
protruding-mantle deformity (Figure 1F).
2.1.2. Degradation Products from TPBP and TPBOA
Crassostea gigas eggs had developed favorably after 2 and 24 h, and no evidence of any influenceon the development of the embryos was found, even at the maximum concentration of degradation
products (DPB, MPB, Biphenyl, Phenol, Pyridine, Benzene, Boric acid) studied (1000 μg/L).
2.1.3. LC10 and LC50 Values
The LC10 and LC50 values of each compound were calculated from the survival rate of the fertilized
Crassostea gigas eggs after the exposure times of 2 and 24 h using the Ecotox-Statics software
package. The results are listed in Table 1. It can be seen that the toxicity of the degradation products
was not influential on Crassostea gigas embryos at high concentrations (1000 μg/L). In the case of theother compounds, at 2 h the LC10 and LC50 values of TPBP and TPBOA were 1.1 (3.4) and2.7 (5.1) μg/L (nM), 7.5 (23) and 23 (44) μg/L (nM), respectively. At 24 h, the LC10 and LC50 values ofthese compounds were 0.58 (1.8) and 2.2 (4.2) μg/L (nM), 6.3 (20) and 10 (19) μg/L (nM), respectively.
Table 1. Lethal concentrations of antifouling compounds to oyster Crassostea gigas embryos.
2h

24 h

LC10

LC50

LC10

LC50

TPBP

1.1 (1.0–1.1)

7.5 (6.7–8.5)

0.58 (0.55–0.60)

6.3 (5.4–7.4)

TPBOA

2.7 (2.6–2.8)

23 (20–26)

2.2 (2.1–2.8)

10 (9.5–12)

DPB

>1000

>1000

>1000

>1000

MPB

>1000

>1000

>1000

>1000

Biphenyl

>1000

>1000

>1000

>1000

Phenol

>1000

>1000

>1000

>1000

Pyridine

>1000

>1000

>1000

>1000

Benzene

>1000

>1000

>1000

>1000

Boric acid

>1000

>1000

>1000

>1000

LC50: 50% lethal concentration (μg/L); LC10: 10% lethal concentration (μg/L); () 95% confidence interval.

Int. J. Mol. Sci. 2013, 14427

2.2. Effects of Organoboron Antifoulants on Hemicentrotus pulcherrimus Embryos
2.2.1. TPBP and TPBOAIn this report, the survival rate of fertilized eggs and the occurrence of deformity in
Hemicentrotus pulcherrimus embryos were investigated. Survival and deformity rates of the
Hemicentrotus puldherrimus embryo after a period of 48 h are shown in Figure 4. In the report by
His et al. [18], six types of deformity were shown: unequal length of postoral arms, twisted right oralrod, parts of right body rod missing, left body rod doubled, additional crossbarred body rod andapically “crossed” body rod. In our experiments, slight unequal length of postoral arms deformity
was observed. Deformity embryos are shown in Figure 5.
Figure 4. Survival and unequal length of postoral arms deformity rates on
Hemicentrotus pulcherrimus embryo after a period of 48 h.

Figure 5. Deformity embryo (A) Hemicentrotus pulcherrimus (B) Crassostea gigas.

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In the case of TPBP, all of Hemicentrotus puldherrimus eggs in the 100 μg/L treatment died after
10 h before any cell division could take place. Approximately 10% of Hemicentrotus puldherrimus
eggs in the 50 μg/L treatment died before any cell division had occurred. In the eggs which survived,
there were no signs of deformity or delayed development. Approximately 5% of
Hemicentrotus puldherrimus eggs in the 20 μg/L treatment died before any cell division. The surviving
embryos showed no signs of deformity or delayed development.
All of Hemicentrotus puldherrimus eggs in the 100 μg/L treatment died after 48 h before any cell
division could take place (Figure 2A). Almost all of Hemicentrotus puldherrimus eggs in the 50 μg/L
treatment died. Except for the eggs which died before any cell division took place, almost all of theembryos reached the blastula stage. A few of them developed signs of reaching the gastrula stage(Figure 2B). Almost all of Hemicentrotus puldherrimus eggs in the 20 μg/L treatment survived. All
surviving embryos became normal two-armed echinopluteus embryos (Figure 2C).
In the TPBOA samples, all of Hemicentrotus puldherrimus eggs in the 500 μg/L treatment died after
10 h before any cell division could take place. Approximately 10% of Hemicentrotus puldherrimus
eggs in the 200 μg/L treatment died before any cell division took place. In the embryos which survived,
there were no signs of deformity or delayed development. Approximately 5% of
Hemicentrotus puldherrimus eggs in the 100 μg/L treatment died. No cell division had taken place.
There were no signs of deformity or delayed development in the surviving embryos.
All of Hemicentrotus puldherrimus embryos in the 100 μg/L treatment died after 48 h. Almost all ofthem reached the gastrula stage (Figure 2D). Almost all of the embryos in the 50 μg/L treatment
survived. All surviving embryos became two-armed echinopluteus embryos. All of the echinopluteus
developed short arms (Figure 2E). Almost all of the embryos in the 20 μg/L treatment survived. All
surviving embryos became normal two-armed echinopluteus embryos (Figure 2F).
2.2.2. Degradation Products from TPBP and TPBOA
Hemicentrotus puldherrimus eggs had developed favorably after 10 and 48 h, and no evidence of
any influence on the development of the embryos was found, even at the maximum concentration of
degradation products (DPB, MPB) studied (1000 μg/L).
2.2.3. LC10 and LC50 Values
The LC10 and LC50 values of each compound were calculated from the survival rate of the fertilized
Hemicentrotus pulcherrimus eggs after exposure times of 10 and 48 h using the
Ecotox-Statics software package. The results are listed in Table 2. It can be seen that the toxicity of thedegradation products was not influential on Hemicentrotus pulcherrimus embryos at highconcentrations (1000 μg/L). In the case of the other compounds, at 10 h the LC10 and LC50 values of
TPBP and TPBOA were 22 (68) and 130 (250) μg/L (nM), 73 (230) and 290 (550) μg/L (nM),
respectively. At 48 h, the LC10 and LC50 values of these compounds were 6.4 (20) and 30 (57) μg/L(nM), 31 (96) and 73 (140) μg/L (nM), respectively.

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Table 2. Lethal concentrations of
Hemicentrotus pulcherrimus embryos.

antifouling

2h
TPBP
TPBOA
DPB
MPB

LC10
22 (21–23)
130 (120–130)
>1000
>1000

LC50
73 (67–80)
290 (270–320)
>1000
>1000

compoundstoseaurchin48 h
LC10
6.4 (5.7–6.6)30 (28–32)
>1000
>1000

LC50
31 (27–36)
73 (68–79)
>1000
>1000

LC50: 50% lethal concentration (μg/mL); LC10: 10% lethal concentration (μg/mL); () 95% confidence interval.

3. Discussion
At 24 h, the LC50 values of TBT, triphenyltin (TPT), Sea-Nine 211, Diuron and Irgarol 1051 in
Crassostea gigas embryos were 3.9, 3.7, 17, >1000, and >1000 μg/L, respectively [1]. TPBP, TBT and
TPT had almost the same toxicity in Crassostea gigas embryos. TPBOA and Sea-Nine 211 also had
similar levels of toxicity as one another in Crassostea gigas embryos.
The results of our laboratory tests showed that organoboron antifoulants are as toxic as Ots to theoysters and sea urchins. According to the results of this research and previous research [1], the effects
of antifouling biocides on oysters are shown as follows:
TBT = TPT = TPBP > TPBOA > Sea-Nine 211 >> Diuron = Irgarol 1051
Previous toxicity data of Ot alternative antifoulants on Hemicentrotus pulcherrimus could not befound, so the results of this research were compared with previously reported no observed-effectconcentration (NOEC) data on Zinc pyrithione, Chlorothalonil and Sea-Nine 211 from the sea urchin(Paracentrotus lividus). The values for Paracentrotus lividus were 11 nM (3.49 μg/L), 15 nM(3.98 μg/L) and 23 nM (6.49 μg/L), respectively [19]. Therefore, the toxicity of TPBP in the
Hemicentrotus pulcherrimus (in this case, the LC10 value at 48 h was used) was almost the same level
as the Paracentrotus lividus. However, that of TPBOA in the Hemicentrotus pulcherrimus was a little
lower than the Paracentrotus lividus.
According to the results of this research and previous researches [20], the effects of antifoulingbiocides on sea urchins are shown as follows:
TBT > TPBP = Sea-Nine 211 > TPBOA >> Irgarol 1051
The toxicity test in the Anthocidaris crassispina showed that the toxicity of TPBP and Sea-Nine 211
was high and that of Irgarol 1051 and Diuron was low [3].
These results showed almost the same pattern. However, the toxicity of each antifouling biocide
showed that the toxicity in oysters was higher than that in sea urchins. At first, it was thought that thistendency was due to the presence of a fertilization membrane in sea urchins which oysters do not have.
However, the fertilization membrane is less dense than the cell membrane, so it is difficult to believethat the fertilization membrane can prevent any materials passing through that the cell membrane could
not. Therefore, the difference of the toxicity in oysters and sea urchins could not be explained from thisstudy’s results.

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4. Experimental Section
4.1. Reagents and Materials
Triphenylborane pyridine (TPBP) and triphenylborane octadecylamine (TPBOA) were used as theantifoulants in this study. The chemical structures of TPBP and TPBOA are shown in Figure 6.
Diphenylborane hydroxide (DPB), phenylborane dihydroxide (MPB), biphenyl, pyridine, phenol,
benzene, and boric acid were used as the degradation products from TPBP or TPBOA [2,21]. The
TPBP, DPB, and MPB were donated by Hokko Chemical Industry (Tokyo, Japan). The TPBOA was
donated by Benny-Toyama (Osaka, Japan). The phenol, benzene, biphenyl (pesticide grade), andpyridine (spectroscopy grade) were purchased from Wako Pure Chemical Industries (Osaka, Japan).
The boric acid was obtained from Nakarai Chemical K.K. (Ibaraki, Japan). Dilute stock solutions(1000 mg/L) were prepared by dissolving the standard materials in dimethyl sulfoxide (DMSO). The
standard solutions (0.1, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100, 200, 500, 1000 μg/L) were formed bydiluting these solutions with artificial seawater which was prepared by diluting Daigo’s artificial
seawater SP purchased from Nihon Seiyaku Kogyo (Niigata, Japan). The dimethyl sulfoxide (forbiochemistry) and 10% formalin solution (for tissue fixation) were purchased from Wako Pure
Chemical Industries (Osaka, Japan). An alkaline formalin solution was prepared by further diluting
these solutions with the artificial seawater stock. The oysters were gathered from the breakwater in
Itsukaichi Nishi Ward, Hiroshima, Japan. Professor Kenji Torigoe, who is affiliated with the
Department of Education at Hiroshima University, identified the oysters used in the experiments. All
the oysters used were Crassostrea gigas. The sea urchins were purchased from Taguchi Educational
Laboratory. All the sea urchins used were Hemicentrotus pulcherrimus from the Miura Peninsula.
Figure 6. Chemical structures of triphenylborane compounds tested. (A) TPBP (B) TPBOA.

4.2. Equipment
An Olympus CK40 biological microscope with a magnification of 100× was used to photograph theoyster and sea urchin eggs, and the Motic Images Plus 2.2S image editing software package was used
to count the number of oyster and sea urchin eggs.
4.3. Oyster Toxicity Tests
After some initial trial and error tests, it was decided to use the procedure performed at the Fisheries
Experimental Station in Hiroshima Prefecture, which simulates the conditions in a nursery. The

Int. J. Mol. Sci. 2013, 14431

artificial seawater was bubbled for 1–2 h to oxygenate the solution. The oyster’s shell was cut openwith a scalpel, and the sex organs were removed. Then a slight incision was made in the sex organs anda sample collected; the sample was observed under the microscope; and the sex of the oyster wasdetermined. The scalpel used in the experiment was washed under running water each time a sample
was removed. The male oysters were placed in a laboratory dish and kept in a refrigerator. This cold
storage preservation stage was a new stage introduced in the experiments. Because of this, it waspossible to use these oysters in the experiments for >1 day. As in many cases, it was important to use
samples freely.
In the case of female oysters, beakers filled with artificial seawater were covered with a fine net; thesex organs were placed on top of the beaker; and then the sex organs were dissected. The eggs werecollected in the beakers and then washed in artificial seawater. The eggs were washed with artificial
seawater several times to separate the mature eggs from the immature eggs. Only the mature eggs that
settled at the bottom of the beaker were used, unlike the reports by His et al. [17,18], who used the
ASTM process to select the eggs [22]. A volume of 10 mL of standard solution was placed into a
six-hole microplate. The controls were adding DMSO to the artificial seawater at 0.1%. Three wellswith the same concentration were prepared. Approximately 200 mature eggs were added to each wellalong with a volume of 25 μL artificial seawater. The sperm, which was preserved in a refrigerator,
was diluted with artificial seawater 1000 times; a volume of 100 μL of artificial seawater was added toeach well; and then the well was used to fertilize the samples. This marked the beginning of the
toxicity tests. A constant temperature tank, maintained at 25 °C, was used for six-well microplates
during cultivation. Each well was observed under the microscope at 2 and again at 24 h. The
development stages of 200 oyster eggs or embryos (normal and abnormal) were identified andphotographed. After the experiments were concluded, the 10% lethal concentration (LC10) and the 50%
lethal concentration (LC50) values were calculated using the Ecotox-Statics software program
developed by the Japanese Society of Environmental Toxicology.
4.4. Sea Urchin Toxicity Tests
The sea urchin’s body fluid was drained after removing a part of their mouth. The gonad’s color wasthen confirmed. If the color was orange, it was identified as an ovary. If the color was light yellow or
white, it indicated a testis. And then, they were injected with 1 mL of 0.5 M KCL in a part of themouth. The male sea urchins were placed on their backs in a laboratory dish and kept in a refrigerator
and a high density of sperm was collected. In the case of female sea urchins, a volume of 100 mL of
the Erlenmeyer flask was filled with artificial seawater, the sea urchins were placed on their backs on
top of the flask. The eggs were collected in the flask and then washed in artificial seawater several
times to separate the mature eggs from immature eggs. A constant temperature tank, maintained at
20 °C, was used for six-well microplates during cultivation. Each well was observed under the
microscope at 10 and again at 48 h. The development stages of 200 sea urchin eggs or embryos(normal and abnormal) were identified and photographed. The other operations were performed thesame as the oysters toxicity test.

Int. J. Mol. Sci. 2013, 14432

5. Conclusions
The effect before degradation of TPBP on oyster and sea urchin eggs and embryos was high but its
degradation products showed no evidence of any influence.
24 h-LC50 of TPBP on oyster embryos had almost the same level of toxicity as that of TBT and TPT.
48 h-LC50 of TPBP on sea urchin embryos had almost the same level of toxicity as Sea-Nine 211
but was lower than TBT.
The effect of TPBOA on oyster and sea urchin eggs and embryos was lower than TPBP.
The toxicity of TPBP and TPBOA caused protruding-mantle deformity in the oyster embryos, andcaused unequal length of postoral arms deformity in the sea urchin embryos.
Acknowledgments
The TPBP, DPB, and MPB test compounds were kindly donated by the Hokko Chemicals Co. Ltd.,
Japan, and the TPBOA was provided by the Benny-Toyama Co. Ltd., Japan with much appreciation.
This work was partly supported by a Grant-in-Aid for Scientific Research (Monbukagakusyo
B20310019) and the Mitsui & Co., Ltd. Environmental Fund (2007). Kenji Torigoe, who is affiliatedwith Hiroshima University, identified the oysters used in the experiments. We wish to thank him forhis technical assistance.
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