Acute and Subchronic Toxicity of Tributyltin Chloride (TBTCl) to the Marine Harpacticoid Copepod Tigriopus japonicus Mori

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Journal of Water and Environment Technology, Vol. 8, No.4, 2010


Address correspondence to Koichi Ara, Department of Marine Science and Resources, College of
Bioresource Sciences, Nihon University, E-mail: arakoich@brs.nihon-u.ac.jp
Received May 14, 2010, Accepted September 7, 2010.
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Acute and Subchronic Toxicity of Tributyltin Chloride
(TBTCl) to the Marine Harpacticoid Copepod Tigriopus
japonicus Mori


Koichi ARA*, Yusuke FUJITA*, Juro HIROMI*, Naoyuki UCHIDA*

*Department of Marine Science and Resources, College of Bioresource Sciences, Nihon
University, Kameino 1866, Fujisawa, Kanagawa 252-0880 Japan

ABSTRACT
Acute and subchronic toxicity experiments of tributyltin chloride (TBTCl) were conducted with
the marine harpacticoid copepod Tigriopus japonicus. The 48-hr LC
50
and highest non-lethal
concentration (NOLC) for adult females were 0.96 and 0.14 µg/L, respectively, whereas these
values for adult males were 0.58 and 0.07 µg/L, respectively. For the mean cumulative number
of nauplii produced per female, the 14-day highest no observed effect concentration (NOEC),
lowest observed effect concentration (LOEC) and EC
50
were 0.025, 0.05 and 0.055 µg/L,
respectively. The acute-subchronic ratio, i.e. the ratio of the 48-hr LC
50
for adult females to the
14-day highest NOEC, MATC (maximum acceptable toxicant concentration) and LOEC, was
38.5, 27.2 and 19.3, respectively. These results suggest that the concentrations of current ambient
TBT (tributyltin) compounds in Japanese coastal waters can be assumed as the safety range for
the survival, but are unlikely to cause a reduction in the number of nauplii produced per female
of T. japonicus. The high concentrations in seawaters, sediments and/or seawaters released from
sediments that have been observed in estuarine and coastal waters in Japan may lead to a
considerable reduction of survival and numbers of nauplii produced by females for T. japonicus.

Keywords: Tigriopus japonicus, toxicity tests, tributyltin chloride (TBTCl)


INTRODUCTION
Organotin compounds, especially tributyltin (TBT) and triphenyltin (TPT), dissolved
principally from organotin-based anti-fouling paint on ship-bottom and fishery
equipments established in estuarine and coastal waters, are one of the most hazardous
marine pollutants, and are biocidal to many aquatic organisms due to their high toxicity.
In addition, even extremely low concentration levels of these compounds can cause a
variety of serious abnormal symptoms for aquatic invertebrates and vertebrates, e.g.
impairments in morphogenesis, growth, maturity and reproduction, highly skewed sex
ratio toward females or males, and endocrine disruption (e.g. Bryan and Gibbs, 1991;
Koyama and Shimizu, 1992; Horiguchi and Shimizu, 1992; Fent, 1996).

Marine pollution by TBT and TPT has been occurring globally not only in estuarine and
costal waters, but also in offshore oceanic areas (e.g. Yamada, 1999; Antizar-Ladislao,
2008). Although their concentrations in many estuarine and coastal waters in Japan have
generally decreased in recent years after the regulation and prohibition of their use in
the 1990s, they have been detected, occasionally in high concentrations, e.g. maximum
TBT concentrations of 0.033-0.084 µg/L, 0.64-1.6 µg/g-dry weight and 0.025-0.78
µg/g-wet weight in seawaters, sea-bottom sediments and aquatic organisms in these
waters in the 1990s, respectively (Harino et al., 1997, 1998, 1999; Ministry of the
Environment, 2007).

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Many studies have been extensively performed to examine the acute and/or chronic
toxic effects of TBT to marine organisms such as commercially important fishes,
molluscs, shrimps and other crustaceans (e.g. Goodman et al., 1988; Kusk and Petersen,
1997; Lignot et al., 1998; Yamada, 1999; Hori et al., 2002; Ohji et al., 2002; Verslycke
et al., 2003; Kwok and Leung, 2005). Most of these studies have focused on the
evaluation of TBT concentration on acute and/or chronic toxicity to various marine
organisms. In general, acute toxicity studies have measured the LC
50
(the lethal
concentration to 50% of test organisms) at 24-96 hrs, whereas chronic toxicity studies
have examined mainly the reduction of growth and reproduction in test organisms
exposed to TBT. Although there is a huge amount of information available on acute
toxicity of TBT, information on chronic toxicity is still relatively scarce
(Antizar-Ladislao, 2008). Especially, there is yet little information on the toxic effects of
TBT compounds on small marine organisms such as copepods that are important prey
for various aquatic animals. For evaluating the detailed toxic effects of TBT that can be
applied to the environmental risk assessment, it is necessary to examine several indexes
such as the highest NOLC (no lethal concentration) in addition to the LC
50
in acute
toxicity tests, the highest NOEC (no observed effect concentration), LOEC (lowest
observed effect concentration), MATC (maximum acceptable toxicant concentration)
and EC
50
(median effect concentration) in chronic toxicity tests and the acute-chronic
ratio, which have not been simultaneously determined in most acute and chronic
toxicity studies. In the present study, the toxic effects of TBTCl (tributyltin chloride) to
the marine harpacticoid copepod Tigriopus japonicus Mori (T. japonicus) were
examined. TBTCl is one of the 13 TBT species that have been designated as Class 2
Specified Chemical Substances in 1990 under the “Law Concerning the Examination
and Regulation of Manufacture, etc. of Chemical Substances” in Japan. The toxicity
level of TBTCl may be as high as that of tributyltin oxide (TBTO) (e.g. Ohji et al.,
2002; Huang et al., 2006; Aono and Takeuchi, 2008), which has been designated a Class
1 Specified Chemical Substances under the same law. Tigriopus japonicus with body
length of approximately 1.0 and 0.9 mm for adult female and male, respectively (Ito,
1970; Koga, 1970), is widely distributed along the coast of Japan (Ki et al., 2009), and
can be an ideal marine model organisms for environmental studies such as ecotoxicity
testing (Raisuddin et al., 2007). In this study, the acute and subchronic toxicities of
TBTCl to T. japonicus were presented. The acute toxicity was expressed as the LC
50

and highest NOLC and the subchronic toxicity, which focused on the mean cumulative
number of nauplii produced per female during 14 days was expressed as the highest
NOEC, LOEC, MATC and EC
50
.


MATERIALS AND METHODS
Tigriopus japonicus was collected using a hand net (100 µm in mesh opening size) at
rocky tide pools located in Enoshima Island (Lat. 35º17’52”N, Long. 139º28’52”E),
Fujisawa, Kanagawa, Japan. These copepods were transferred into a bottle (volume: 5
L) containing approximately 4 L of ambient surface seawater and taken to the laboratory
within 1-2 hrs. Specimens were acclimated at least for 7 days under laboratory
condition (i.e. temperature: 24.0 ± 1.0ºC; salinity: 34; light:dark photoperiod of 12L:
12D) prior to experiment. The mono-cultured diatoms Skeletonema costatum or
Thalassiosira sp. and raphidophycean flagellate Heterosigma akashiwo were
sufficiently fed once a day prior to experiment. The seawater used for toxicity
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experiments was taken at the sea surface (1 m depth) in the neritic open area, 4.5 km off
Enoshima Island, Fujisawa, of Sagami Bay (Lat. 35º16’22.0”N, Long. 139º29’41.0”E;
local depth: 55 m).

The methods for preparing test solutions were based on Ohji et al. (2002), but an outline
is briefly given as follows. Seawater (salinity: 34 psu) filtered through a glass-fiber
filter (Whatman, GF/F) was used as control. Acetone solution of 0.05 mL/L, which was
made by adding 0.1 mL of acetone to 2 L of filtered seawater, was used as
acetone-control. The TBTCl solution was made by adding 2000 mg of tributyltin (IV)
chloride (TBTCl, Wako Pure Chemical Industries, Ltd., Japan) to 1 L of filtered
seawater with 0.05 mL/L acetone solution. The TBTCl solution of 500 µg/L was made
by adding 0.5 mL of 2000 mg/L TBTCl solution to 2 L of filtered seawater, and the
solution was stirred for 12 hrs by a magnetic stirrer. After stirring, the TBTCl solution
was transferred into lidded glass bottles and stored at 4ºC. In the present study, test
solutions of five TBTCl concentrations (0.1, 0.5, 1, 5 and 10 µg/L) and other five
concentrations (0.01, 0.025, 0.05, 0.075 and 0.1 µg/L) were prepared by dilution of
stock solution with 0.5% (v/v) acetone-filtered seawater solution. These condensed and
diluted solutions were made every week, and test solutions were used for 48 hrs or
renewed every 48 hrs as mentioned below. The TBTCl concentration of test solutions
utilized for experiments was considered as the nominal one, although it was not
determined in the present study, because Ohji et al. (2002) confirmed that the TBTCl
concentrations in test solutions were almost identical to the nominal ones and the
concentration levels remained the same even after 48 hrs.

The acute toxicity tests were conducted in conformity with the modified OECD Test
Guideline, i.e. the ecological effect testing method in the risk assessment program of the
Organization for Economic Cooperation and Development (OECD) (OECD, 1998). For
acute toxicity tests, five adult females or adult males of T. japonicus were introduced
into each glass bottle containing 50 mL of test solution. The experiment, with four
replicates of the control, acetone-control and test solutions (TBTCl concentration: 0.1,
0.5, 1, 5 and 10 µg/L), was run for 48 hrs in an incubator (temperature: 24.0 ± 1.0ºC;
light: dark photoperiod of 12L: 12D). The copepods were not fed during the experiment.
After the exposure, the degree of survival of the copepods in each bottle was checked
under a microscope.

The subchronic toxicity tests were conducted in conformity with the modified OECD
Test Guideline (OECD, 2008). The approximate threshold response concentrations (i.e.
highest NOLC) during the acute toxicity tests were selected as the highest test
concentration for subchronic toxicity tests. For subchronic toxicity tests, ten ovigerous
females of T. japonicus were individually introduced into each glass bottle containing
50 mL of test solution. For the control, acetone-control and test solutions (TBTCl
concentration: 0.01, 0.025, 0.05, 0.075 and 0.1 µg/L), triplicates were incubated for 14
days at the same condition as for acute toxicity tests. Test solutions were renewed every
48 hrs. Each time these female copepods were transferred into renewed test solutions,
the number of nauplii in each bottle was counted under a microscope.

Regression analysis was conducted to determine the relationships between the survival
rate of T. japonicus and TBTCl concentration and between the inhibition rate (the
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BA
0
20
40
60
80
100
0.01 0.1 1 10 100
TBTCl (µg/L)
Survival rate (%)
0
0
20
40
60
80
100
0.01 0.1 1 10 100
Survival rate (%)
TBTCl (µg/L)
0


Fig. 1 - Relationships between the survival rate (SR) of T. japonicus and TBTCl
concentration. Survival rate is expressed as mean (●) and SD (vertical bars).
A: adult females, SR = -25.7 × ln TBTCl + 49.0 (r
2
= 0.954, p < 0.05); B:
adult males, SR = -23.6 × ln TBTCl + 37.4 (r
2
= 0.998, p < 0.01)

proportion of inhibiting production of nauplii) and TBTCl concentration. These
relationships were converted to linearized equations and solved by the least-squares
method. For statistical comparison between the survival rates of adult females and
males, one-way ANOVA (Student’s t-test) was applied: a p-value of less than 0.05 was
considered statistically significant. The one-way ANOVA was applied also for statistical
comparison between the mean cumulative numbers of nauplii produced per female in
control and acetone-control and between the values in control and test solutions (for
each TBTCl concentration) in each experiment (every two days).


RESULTS
Acute toxicity in adult T. japonicus
All adult females and males of T. japonicus in the control and acetone-control solutions
were always alive during the experiment, whereas they died in test solutions with
TBTCl concentrations of 5 and 10 µg/L (Fig. 1). In addition, all adult females were
always alive in test solutions with TBTCl concentration of 0.1 µg/L. In test solutions
with TBTCl concentrations of 0.1, 0.5 and 1 µg/L, the survival rates of adult females
were significantly higher than those of adult males (ANOVA, p < 0.001 for each
concentration). In test solutions with TBTCl concentrations of 0.1-5 µg/L, the survival
rate of adult females and males decreased with increasing TBTCl concentration. There
were significant correlations between TBTCl concentration and survival rates of adult
females and males, respectively (Fig. 1). From the obtained regression equations of
TBTCl concentration-survival rate relationships, the 48-hr LC
50
for adult females and
males was calculated to be 0.96 and 0.58 µg/L, respectively, while the highest NOLC
for adult females and males was calculated to be 0.14 and 0.07 µg/L, respectively.

Subchronic toxicity in the number of nauplii
All ovigerous females in the control, acetone-control and test solutions were alive
during the 14-day experiments. The nauplii (N1) of T. japonicus appeared in the control,
acetone-control and test solutions on Day 2-4, and increased with experiment time,
especially in the control, acetone-control and test solutions at lower TBTCl
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concentrations (Fig. 2). All nauplii in the control, acetone-control and test solutions
were alive after hatching. The mean cumulative number (± SD) of nauplii produced per
female at the end of the experiment (i.e. on Day 14) was 32.2 ± 4.7 individuals/female
in control, 31.0 ± 4.2 individuals/female in acetone-control, and 31.2 ± 3.6, 28.2 ± 3.7,
17.7 ± 3.6, 6.7 ± 2.2 and 4.4 ± 1.2 individuals/female in test solutions with TBTCl
concentrations of 0.010, 0.025, 0.050, 0.075 and 0.1 µg/L, respectively. There was
statistically no significant difference between the mean cumulative numbers of nauplii
produced per female in the control, acetone-control and test solutions with TBTCl
concentrations of 0.01-0.025 µg/L throughout the experiment (Fig. 2). The mean
cumulative numbers of nauplii produced per female in the test solution with TBTCl
concentration of 0.05 µg/L were significantly lower than those in the control on Day 6
and 12-14, whereas the values in test solutions with TBTCl concentrations of
0.075-0.10 µg/L were significantly lower than those in the control on Day 4-14 (Fig. 2).
The highest NOEC and LOEC were 0.025 and 0.05 µg/L, respectively. The MATC (i.e.
geometric mean of NOEC and LOEC) was calculated to be 0.035 µg/L. The
acute-subchronic ratio, i.e. the ratio of the 48-hr LC
50
for adult females to the 14-day
highest NOEC, MATC and LOEC, was calculated to be 38.5, 27.2 and 19.3,
respectively.

The inhibition rate, i.e. the proportion of inhibiting production of nauplii during 14 days,
increased with increasing TBTCl concentration (Fig. 3). There was a significant
correlation between TBTCl concentration and inhibition rate. From the obtained
regression equations of TBTCl concentration-inhibition rate relationship, the 14-day
EC
50
was calculated to be 0.055 µg/L.



Fig. 2 - The cumulative number of nauplii produced per female of T. japonicus. The
number of nauplii is expressed as mean (●) and SD (vertical bars). Values with
asterisk (*) differed significantly from the values in control
0
10
20
30
40
02468101214
Experiment time (days)

Cumulative number of nauplii (inds./ female)

Control

Acetone control

TBTCl 0.01 µg/L

TBTCl 0.025 µg/L

TBTCl 0.05 µg/L

TBTCl 0.075 µg/L

TBTCl 0.1 µg/L

*
*
*
*
*
*

*
*
*
*

*
*
*
*

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0
20
40
60
80
100
0 0.04 0.08 0.12
TBTCl (µg/L)
Inhibition rate (%)


Fig. 3 - Relationship between the inhibition rate (IR) of T. japonicus and TBTCl
concentration. Inhibition rate is expressed as mean (●) and SD (vertical bars).
IR = 939×TBTCl - 2.04 (r
2
= 0.966, p < 0.0001)


DISCUSSION
In the present study, the organic solvent (i.e. acetone) used to facilitate the solubilization
of TBTCl into seawater was highly water-soluble and hard to enter the organism’s body
due to its low penetrability across biomembrane. In addition, the acetone concentration
in the acetone-control and test solutions was very low (approximately 0.05 mL/L). The
values of survival rate and cumulative number of nauplii in acetone-control were mostly
identical to those in control during the two experiments (Figs. 1 and 2). Thus, it was
assumed that the toxicity of acetone utilized in the present study would be very low and
negligible in comparison with that of TBTCl.

Acute toxicity tests of TBTCl have been conducted for several marine invertebrates
(Table 1). The 48-hr LC
50
values for T. japonicus obtained in the present study are
higher than that for Acartia tonsa (48-hr LC
50
: 0.24 and 0.47 µg/L (Kusk and Petersen,
1997)), and lower than those for all other marine invertebrates, especially the gammarid
amphipods Cerapus erae, Eohaustorioides sp. and Jassa slatteryi (48-hr LC
50
:
17.8-23.1 µg/L (Ohji et al., 2002)). This indicates that T. japonicus has higher toxic
sensitivity to TBTCl and can be a bioindicator for TBTCl pollution in environmental
waters as well as A. tonsa, rather than other tested organisms. For T. japonicus, the
48-hr LC
50
values of TBTCl to adult females and males obtained in the present study
are 6.5-fold and 3.9-fold higher than the 96-hr LC
50
values, respectively (0.15 µg/L
(Kwok and Leung, 2005)). This might be caused by the difference in the duration of the
experiment. Hori et al. (2002) showed that the LC
50
values of TBTCl to the marine
decapods Heptacarpus futilirostris and Marsupenaeus japonicus decline considerably
with lengthening exposure time (Table 1). Koyama and Shimizu (1992) reviewed the
time-dependent acute toxicity (i.e. 24-96-hr LC
50
) of TBTCl and TBTO to some marine
fishes. Similarly, Ara et al. (2004) stated the time-dependent acute toxicity (i.e. 24-96-hr
LC
50
, highest NOLC and lowest LC
100
) of Bunker C refined oil to the Japanese
littleneck clam Ruditapes philippinarum. Thus, the acute toxic effects of chemical
substances to marine organisms might be time-dependent, although the time-dependent
acute toxicity of TBTCl to T. japonicus was not evaluated in the present study.
Journal of Water and Environment Technology, Vol. 8, No.4, 2010

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Table 1 - Acute toxicity of TBTCl to marine invertebrates

Test animal
(life stage/body size)
Temp.
(
o
C)
Condition
(salinity)
T*
(hrs)
LC
50

(µg/L)
Reference
Copepoda
Acartia tonsa 17.5 ± 0.5 Brackish water (18) 48 0.47 Kusk and Petersen (1997)
A. tonsa 17.5 ± 0.5 Brackish water (28) 48 0.24 Kusk and Petersen (1997)
Tigriopus japonicus (adults) 25.0 ± 1.0 Seawater (34.5 ± 0.5) 96 0.15 Kwok and Leung (2005)
T. japonicus (adult females) 24.0 ± 1.0 Seawater (34) 48 0.96 This study
T. japonicus (adult males) 24.0 ± 1.0 Seawater (34) 48 0.58 This study

Mysidacea
Mysidopsis bahia (1 day-old) 25.0 ± 1.0 Brackish water (19.0-22.3) 96 1.1 Goodman et al. (1988)
M. bahia (3 day-old) 25.0 ± 1.0 Brackish water (19.0-22.3) 96 2.0 Goodman et al. (1988)
M. bahia (5 day-old) 25.0 ± 1.0 Brackish water (19.0-22.3) 96 2.2 Goodman et al. (1988)
Neomysis integer 15.0 Brackish water (5) 96 0.15 Verslycke et al. (2003)

Amphipoda: Caprellidae
Caprella danilevskii (5.7 ± 1.0 mm) 20.0 Seawater 48 5.9 Ohji et al. (2002)
C. equilibra (8.0 ± 2.3 mm) 20.0 Seawater 48 6.6 Ohji et al. (2002)
C. penantis R-type (5.5 ± 1.0 mm) 20.0 Seawater 48 1.2 Ohji et al. (2002)
C. subinermis (6.3 ± 1.4 mm) 20.0 Seawater 48 4.6 Ohji et al. (2002)
C. verrucosa (4.9 ± 1.1 mm) 20.0 Seawater 48 1.3 Ohji et al. (2002)

Amphipoda: Gammaridae
Cerapus erae (3.2 ± 0.5 mm) 20.0 Seawater 48 21.2 Ohji et al. (2002)
Eohaustorioides sp. (6.2 ± 0.7 mm) 20.0 Seawater 48 23.1 Ohji et al. (2002)
Jassa slatteryi (5.1 ± 0.7 mm) 20.0 Seawater 48 17.8 Ohji et al. (2002)

Decapoda
Heptacarpus futilirostris 25.0 Seawater (34) 24 7.8 Hori et al. (2002)
H. futilirostris 25.0 Seawater (34) 48 5.6 Hori et al. (2002)
H. futilirostris 25.0 Seawater (34) 72 4.4 Hori et al. (2002)
H. futilirostris 25.0 Seawater (34) 96 3.2 Hori
et al. (2002)
Marsupenaeus japonicus 25.0 Seawater (34) 24 85.5 Hori et al. (2002)
M. japonicus 25.0 Seawater (34) 48 5.2-53.5 Hori et al. (2002)
M. japonicus 25.0 Seawater (34) 72 4.8-42.8 Hori et al. (2002)
M. japonicus 25.0 Seawater (34) 96 3.0-42.8 Hori et al. (2002)
*T: exposure duration

On the basis of the LC
50
and highest NOLC values for T. japonicus obtained in the
present study, the toxic susceptibility of adult males to TBTCl was 1.6 to 2-fold higher
than adult females. This can be explained by the copepod susceptibility to external
stress that males are less tolerant to environmental stress than females (Davis, 1984).

The acute-subchronic ratio obtained for T. japonicus in the present study (19.3-38.5)
was similar to the acute-chronic ratio obtained for the marine copepod Eurytemora
affinis (15.2 to >25.0 (Hall et al., 1987, 1988; US EPA, 2003)). These values are within
the range of the 50-90th percentile for each of the three trophic levels (i.e. algae,
daphnids and fish) (Ahlers et al., 2006), although chemical substance, test organism (i.e.
species, life stage), endpoint for acute and chronic toxicity tests and obtained acute and
chronic values differed depending on the study. The present study showed that the
14-day EC
50
was 11 to 17-fold lower than the 48-hr LC
50
, and that the 14-day highest
NOEC was 2.8 to 5.5-fold lower than the 48-hr highest NOLC (Figs. 1 and 2). Egg,
larval and early life stages of marine organisms are generally more sensitive and much
less tolerant to environmental stress than adults and the later stages. The mean
cumulative numbers of nauplii produced per female in the control, acetone-control and
test solutions with TBTCl concentrations of 0.01-0.025 µg/L during 14 days were
similar to the mean brood size (15-35 eggs produced by a female copepod per brood) of
T. japonicus (Koga, 1970; Lee and Hu, 1981; Hagiwara et al., 1995; Takaku et al.,
2009). In fact, during the 14-day experiments, all ovigerous females of T. japonicus
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produced another brood, and nauplii hatched from these females two times, i.e. from the
first brood on Day 2-4 and from the second one on Day 10-14, in the control,
acetone-control and test solutions (Fig. 2). In these cases, the spawning interval would
be 2-4 days, which is similar to that of 1-6 days (mean: 2-3 days) at a temperature of
24ºC in laboratory culture experiments for T. japonicus (Takaku et al., 2009). In
addition, there was no significant difference between the numbers of nauplii hatched
from the first brood (previously produced before experiment) and second one (produced
during experiment) in the control, acetone-control and test solutions, respectively. This
implies that TBTCl would induce the failure of hatching success, but probably not the
decrease in brood size, although individual brood size and hatching success of T.
japonicus were not evaluated in the present study.

TBT compound (expressed as TBTO) concentrations in seawater, estuarine and coastal
waters in Japan have been 0.00044-0.00076 µg/L in 2005 (Ministry of the Environment,
2007), which are 770 to 2189-fold and 91 to 311-fold lower than the 48-hr LC
50
and
highest NOLC, respectively. These concentrations can be assumed as the “safety range”
for the survival of adult females and males of T. japonicus, because of the concentration
being 10 to 1000-fold lower than its LC
50
values (e.g. Howarth, 1989). These
concentrations are 72 to 125-fold and 33 to 57-fold lower than the 14-day EC
50
and
highest NOEC, respectively. This implies that these concentrations are unlikely to cause
a reduction in the number of nauplii of T. japonicus, because of the concentration being
lower than the safety (uncertainty) factor of 100. On the other hand, relatively high
concentrations (max. 0.027-0.043 µg/L) of TBT compound in seawater have been
observed in the innermost areas of estuaries and coastal waters, such as harbors, marinas,
fishery and trade ports, in Japan (Wang et al., 2004; Ohji et al., 2007; Onduka et al.,
2008; Suzuki et al., 2008). These concentrations are 14 to 36-fold, 1.6 to 5.1-fold, 1.3 to
2-fold and 0.6 to 0.9-fold higher than the 48-hr LC
50
, the highest NOLC, 14-day EC
50
,
and the highest NOEC, respectively. In these cases, T. japonicus are very much unlikely
to maintain their population, especially due to the reducing numbers of nauplii produced
by females. Similarly, relatively high TBT compound concentrations in sea-bottom
sediments have remained up to the present in estuarine and coastal waters in Japan, e.g.
0.000085-0.590 µg/g-dry-weight in 2005 (Ministry of the Environment, 2007) and
0.0004-0.0019 µg/g-dry-weight in 2009 (Japan Coast Guard, 2010). These high TBTCl
concentrations in sediments and/or seawaters released from sediments can considerably
reduce the survival, reproduction and hatching success of marine organisms (e.g. T.
japonicus) which inhabit in seawater-sediment environments, although the toxicity of
TBTCl in sediment to T. japonicus was not evaluated. The present study showed that
TBTCl concentration can be one of the important factors affecting the survival,
reproduction and population dynamics of T. japonicus in natural environments.
Consequently, for evaluating the toxic effects of TBTCl to T. japonicus, this suggests
the necessity of chronic toxicity tests using naupliar and copepodite stages in addition to
the acute and subchronic toxicity tests conducted in the present study.


CONCLUSIONS
Acute and subchronic toxicity of TBTCl to the marine harpacticoid copepod T.
japonicus was studied, and the following results were obtained.
(1) The 48 hr LC
50
for adult females and males was 0.96 and 0.58 µg/L, respectively.
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(2) The 48-hr highest NOLC for adult females and males was 0.14 and 0.07 µg/L,
respectively.
(3) The maximum acceptable concentration (i.e. highest NOEC) on the number of
nauplii produced per female during 14 days was 0.025 µg/L.
(4) The 14-day LOEC was 0.05 µg/L.
(5) The 14-day EC
50
was 0.055 µg/L.
(6) The acute-subchronic ratio, i.e. the ratio of the 48-hr LC
50
for adult females to the
14-day highest NOEC, MATC and LOEC, was 38.5, 27.2 and 19.3, respectively.


ACKNOWLEDGEMENTS
The authors would like to thank Mr. Kazuharu Yuasa, captain/owner of the fishery boat
“Genshun-maru”, for collecting seawater samples for experiments. Great appreciation is
also extended to Mr. Kazunori Hashiyama, Nihon University, for supplying the
mono-cultured diatoms and raphidophycean flagellate.


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