The Influence of Organotin Alternative Antifoulants
on Oyster Embryology
11Tsunemasa N,2 Okamura H

Hiroshima City Institute of Public Health 2Graduate School of Maritime Science, Kobe Univ.

1.Introduction
Organotin(Ot) compounds that were used for many years as antifouling biocides have become a problem because of their
toxicity and accumulation characteristics. In September 2008, Ot compounds were prohibited from being used worldwide.
From 1997 in Japan, the production of paints that contain TBT compounds was prohibited, and so alternatives to Ot
antifoulants have been used since then. We have evaluated the toxicity of alternative Ot antifoulants (i.e.,Diuron, Irgarol 1051,and Sea-Nine211) and Ot compounds (TBT and TPT) using oysters that inhabit a large area of Hiroshima Bay.

2.Materials and Methods

3.Result and Discussion

2.1 Reagents and Materials
We used the alternative Ot antifoulants Irgarol 1051,Diuron and Sea-Nine211 for the toxicity tests, andwe used TBT and TPT to compare these alternative
Ot antifoulants with Ot compounds. We made dilutesolutions (1000mg/l) by dissolving the standard
materials in dimethyl sulfoxide (DMSO) and thenmade standard solutions (1.0-1000μg/l) by diluting
the dilute solutions with artificial seawater.

The condition of the oysters is shown in Fig.2.

ClN C8H17-n

SCH3

Sea-Nine 211
SNClClO
CH3NHCNCH3

CH3
H3C C HN
CH3NNIrgarol
NH CNCH2
CH2

DiuronFig.1Structers of the alternative Ot compounds

2.2 The oyster toxicity test
We placed a volume of 1ml of standard solution into a
24hole-microplate.We prepared three wells with thesame concentration.
↓We added a mature egg to each well, along with avolume of 25μl of artificial seawater.
↓We diluted the sperm, which was preserved in arefrigerator, with artificial seawater by1000 times,
added a volume of 25μl of artificial seawater to eachwell, and then used it to fertilized the sample.
↓We used a constant temperature tank, maintained at25℃, for the microplates during cultivation. After aperiod of 2h and 24 h, we observed each well underthe microscope and indentified the development
stages of 200 oyster eggs.

Table 1 Lethal effects of antifouling compounds on oyster embryos
2hLC10LC50TBT
2.7(2.6-2.9)
14(13-16)
TPT
4.1(3.9-4.3)
13(12-14)
Sea-Nine 211
4.3(4.0-4.5)27(24-30)
Diuron>1000>1000
Irgarol 1051
>1000
>1000
LC50 ： 50 % lethal concentration (µg/L)
LC10 ： 10 % lethal concentration (µg/L)

24h
LC10
LC50
0.69 (0.64-0.70)
3.3(2.9-3.7)
1.4(1.4-1.5)
2.5(2.4-2.7)
1.3(1.2-1.3)
13(11-15)
>1000
>1000
>1000
>1000
（ ）：95 % confidence interval
TBT(μg/L)

3.2 Influence on deformity
We have looked at thenumber of living and deadeggs after a fertilization
period of 24h.

Sea-Nine 211(μg/L)

controlcontrol

1

1

Test solution’s concentration

O3.1 LC10,LC50
We calculated the LC10 and LC50 values of eachcompound from the survival rate of the fertilized
oyster eggs after an exposure time of 2 and 24 h using
the data from the Ecotox-Statics software package.
The results are shown in Table1.

Test solution’s concentration

ClFig.2 Fertilized eggs after a period of 2 and 24 h.
(A):Oyster eggs with normal cell division(Sea-Nine211,1ppb,2h);(B):Oyster eggs that didn’t survive(TBT,100ppb,2h); (C):Oyster eggs
with no development(Sea-Nine211,100ppb,24h);(D)Normal D-shaped
embryos (control,24h); and (E) Deformed D-shaped embryos(TBT,5ppb,24h).

25102050

25102050

100

100
00%

2020%
deaｄ

40
60
40%60%
Ratio(%)

8080%

abnormalnormal

100
100%

00%

2020%
deaｄ

40
60
40%60%
Ratio(%)
abnormal

80
80%

100100%normalFig.3 The survival rate and deformity rate after a period of 24h.

Figure 3 shows the survival and deformity rates after adevelopment period of 24h.

4. Conclusions
We evaluated the toxicity of alternative Ot antifoulants
using the oyster’s embryology. The results showed thatthe toxicity of Diuron and Irgarol 1051 was very low,
and the toxicity of Sea-Nine 211 was almost the same asthat of TPT.