Malformations of the endangered Chinese sturgeon,
Acipenser sinensis, and its causal agent
Jianying Hua,1, Zhaobin Zhanga, Qiwei Weib, Huajun Zhena, Yanbin Zhaoa, Hui Penga, Yi Wana, John P. Giesyc,d,e,
Luoxin Lib, and Bo Zhangf
aCollege of Urban and Environmental Sciences, Peking University, Beijing 100871, China; bKey Laboratory of Freshwater Biodiversity Conservation and
Utilization, Yangtze River Fisheries Research Institute, Chinese Academy of Fisheries Science, Ministry of Agriculture of China, Jingzhou, Hubei 434000,
China; cDepartment of Veterinary Biomedical Sciences and Toxicology Center, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK, Canada
S7N 5B3; dDepartment of Zoology, Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824; eDepartment of Biology and
Chemistry, Research Centre for Coastal Pollution and Conservation, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, SAR, China;
and fKey Laboratory of Cell Proliferation and Differentiation, Center of Developmental Biology and Genetics, College of Life Sciences, Peking University,
Beijing 100871, China
Edited by Derek Muir, Environment Canada, Burlington, ON, Canada, and accepted by the Editorial Board April 8, 2009 (received for review
September 20, 2008)
teratogenesis 兩 fish 兩 triphenyltin 兩 Yangtze River
H
uman activities have contributed to extinctions of species,
and can be a contributing factor to decreases in populations.
In particular, some pesticides can adversely affect endangered
species (1–3). Sturgeons belong to one of the most ancient
groups of the Osteichthyes. Because of their desirability as food,
their long-life, and changes in their habitats, populations of
sturgeon have declined globally. All extant sturgeon species are
listed as ‘‘protected’’ under the Convention on the International
Trade of Endangered Species. Among the 25 extant sturgeon
species, the Chinese sturgeon (Acipenser sinensis) is an anadromous fish that has survived at the edge of extinction, and is listed
among the first class of protected animals in China (4).
The Chinese sturgeon inhabits the East China and Yellow
Seas, and spawns in the Yangtze River. Loss of critical spawning
habitat because of construction of the Three-Gorges Dam and
Gezhouba Dam on the Yangtze River is thought to have
contributed to a steep population decline (4, 5). To save this
endangered species, in the 1980s, the Chinese government began
an artificial propagation program. However, this program has
not resulted in the recovery of the Chinese sturgeon population.
Also, the female:male sex ratio has changed from 0.79 in
1981–1993 (5) to 5.9 in 2003–2004 (6), the motility of sperm has
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0809434106
decreased (7), and intersex has been observed (5). These observations have indicated that synthetic chemicals may be having
adverse effects that could contribute to the population declines
observed for this endangered species. Chinese sturgeon are
exposed to relatively great concentrations of synthetic compounds, including musk fragrances and organochlorines, which
possibly affect the fertilization and, therefore, affect populations
(8). However, until now, there has been no direct evidence that
exposure to synthetic compounds was related to adverse effects on
the Chinese sturgeon population. Thus, it has been difficult to make
appropriate management policies for the protection of Chinese
sturgeon.
Both triphenyltin (TPT) and tributyltin (TBT) have been used
extensively in paints to prevent fouling of ship hulls and fishnets.
In addition to the fact that TPT concentrations measured in
marine fish were unexpectedly greater than those of TBT
because of the trophic magnification of TPT (9, 10), TPT
continues to be used as a contact fungicide to treat crops in
China. TPT acetate and TPT hydroxide are registered for use in
China especially as molluscicides to eliminate the golden apple
snail (Pomacea canaliculata) in paddy fields where it has seriously threatened aquatic crops. Based on a questionnaire among
the pesticide companies that registered TPT pesticides, ⬇200
tons of TPT pesticides are manufactured in China. Although all
of the TPT usage in agriculture in Taiwan was completely
prohibited in 1999, 27% of the surveyed farmers are still using
TPT acetate illegally after the ban (11).
Ocular and morphological malformations have been observed
in embryos and larvae of European minnows (Phoxinus phoxinus) and zebrafish (Danio rerio) after in ovo exposure of TPT
(12, 13) and in the offspring of medaka (Oryzias latipes) maternally exposed to TBT and TPT (14, 15). Also, TBT and TPT can
inhibit reproduction (14, 15). Therefore, in the present study, the
following questions were addressed. (i) Is TPT accumulated by
Chinese sturgeon and then transferred to the eggs? (ii) Can the
malformation be observed in wild Chinese sturgeon population?
(iii) Can the malformations observed in larvae and fry of wild
Chinese sturgeon be caused by TPT under controlled laboratory
conditions? Nanoinjection techniques were used to accurately
determine the effects of known concentrations including environmentally relevant concentrations of TPT on both Chinese
sturgeon and Siberian sturgeon eggs.
Author contributions: J.H., Q.W., Y.W., and J.P.G. designed research; Z.Z., H.Z., Y.Z., H.P.,
and L.L. performed research; B.Z. contributed new reagents/tools; and J.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. D.M. is a guest editor invited by the Editorial Board.
1To
whom correspondence should be addressed. E-mail: hujy@urban.pku.edu.cn.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0809434106/DCSupplemental.
PNAS 兩 June 9, 2009 兩 vol. 106 兩 no. 23 兩 9339 –9344
ENVIRONMENTAL
SCIENCES
The anadromous Chinese sturgeon (Acipenser sinensis) is endangered and listed among the first class of protected animals in China.
The possible causes for the decline of this species are the effects of
synthetic chemicals, and loss of critical habitat. Chinese sturgeon in
the Yangtze River have accumulated triphenyltin (TPT) to 31–128
ng/g wet weigh (ww) in liver, which is greater than the concentrations of tributyltin (<1.0 ng/g ww). Maternal transfer of TPT has
resulted in concentrations of 25.5 ⴞ 13.0 ng/g ww in eggs of wild
Chinese sturgeon, which poses a significant risk to the larvae
naturally fertilized or hatched in the Yangtze River. The incidence
of deformities in fry was 7.5%, with 1.2% of individuals exhibiting
ocular abnormal development, and 6.3% exhibited skeletal/morphological deformations. The incidences of both ocular and skeletal/morphological deformations were directly proportional to the
TPT concentration in the eggs of both the Chinese sturgeon and the
Siberian sturgeon (Acipenser baerii) in controlled laboratory studies. The rates of deformities in the controlled studies were consistent with the rates caused at the similar concentrations in eggs
collected from the field. Thus, TPT is the causal agent to induce the
malformation of larvae of Chinese sturgeon. The incidence of
deformed larvae of Chinese sturgeon is an indicator of overall
population-level effects of TPT on Chinese sturgeon, because TPT
at environmentally relevant concentrations can result in significantly decrease both quality and quantity of eggs and spawning
frequency of fish.
Table 1. Concentrations of BTs and PTs in different tissues (ng/g ww) of the Chinese sturgeon
Tissue
Lipid, %
Liver, n ⫽ 8
12.2 ⫾ 7.4
Heart, n ⫽ 7
4.2 ⫾ 1.7
Muscle, n ⫽ 8
1.9 ⫾ 1.2
Gill, n ⫽ 6
2.4 ⫾ 0.6
Roe, n ⫽ 15
33.7 ⫾ 9.8
Gonad, n ⫽ 6
3.6 ⫾ 1.7
Adipose, n ⫽ 5
66 ⫾ 18
Intestine, n ⫽ 7
2.8 ⫾ 1.6
Stomach, n ⫽ 5
1.3 ⫾ 0.4
Pancreas, n ⫽ 2
6.8
Kidney, n ⫽ 1
Gallbladder, n ⫽ 1
Spleen, n ⫽ 1
31.5
23.0
ND
Value
MBT
DBT
TBT
⬍1.0
11.8
9.1
Min
⬍1.0
257
1,115
Max
—
Mean ⫾ SD 293 ⫾ 366 72.1 ⫾ 81.0
⬍1.0
7.6
5.2
Min
3.8
15.9
12.5
Max
Mean ⫾ SD 9.3 ⫾ 3.0 10.8 ⫾ 2.9 1.4 ⫾ 1.5
⬍1.0
2.2
1.5
Min
4.3
7.5
8.3
Max
4.6 ⫾ 1.7 1.3 ⫾ 1.4
Mean ⫾ SD 3.7 ⫾ 2.3
⬍1.0
3.1
12.3
Min
⬍1.0
14.6
23.8
Max
—
7.7 ⫾ 3.9
Mean ⫾ SD 18.2 ⫾ 4.7
⬍1.0
2.2
⬍1.5
Min
⬍1.0
10.8
10.8
Max
—
4.0 ⫾ 2.0
Mean ⫾ SD 5.9 ⫾ 3.7
⬍1.0
4.0
3.4
Min
⬍1.0
14.0
20.0
Max
—
8.9 ⫾ 4.2
Mean ⫾ SD 8.8 ⫾ 6.7
⬍1.0
⬍1.0
⬍1.5
Min
⬍1.0
5.3
3.6
Max
—
1.9 ⫾ 2.1
Mean ⫾ SD 1.3 ⫾ 1.3
⬍1.0
2.8
⬍1.5
Min
⬍1.0
9.7
20.1
Max
—
6.3 ⫾ 2.3
Mean ⫾ SD 12.4 ⫾ 6.1
⬍1.0
4.3
⬍1.5
Min
⬍1.0
8.5
4.4
Max
—
6.5 ⫾ 1.8
Mean ⫾ SD 2.3 ⫾ 1.3
Min
2.6
6.4
⬍1.0
Max
10.2
8.3
⬍1.0
—
33.5
36.9
4.0
—
9.0
5.7
⬍1.0
—
11.2
13.6
⬍1.0
BTs
MPT
DPT
5.8
⬍2.0
20.9
324
104
1,373
365 ⫾ 447 33.3 ⫾ 30.9 66.3 ⫾ 105
1.9
⬍2.0
14.6
6.4
4.4
28.6
3.6 ⫾ 1.7
1.5 ⫾ 1.3
21.4 ⫾ 5.7
0.7
⬍2.0
4.1
3.3
⬍2.0
20.2
1.8 ⫾ 0.8
—
9.6 ⫾ 4.8
⬍1.0
⬍2.0
18.0
8.2
⬍2.0
37.7
2.4 ⫾ 3.2
—
25.9 ⫾ 7.5
⬍1.0
⬍2.0
3.4
2.4
⬍2.0
15.8
1.3 ⫾ 0.8
—
9.9 ⫾ 4.0
⬍1.0
⬍2.0
8.2
2.4
⬍2.0
34.0
1.0 ⫾ 0.7
—
17.7 ⫾ 10.5
⬍1.0
⬍2.0
⬍3.5
2.5
⬍2.0
6.2
0.9 ⫾ 0.9
—
3.2 ⫾ 2.7
⬍1.0
⬍2.0
3.5
1.5
⬍2.0
26.7
0.6 ⫾ 0.4
—
18.7 ⫾ 8.0
⬍1.0
⬍2.0
6.1
⬍1.0
⬍2.0
11.3
—
—
8.9 ⫾ 2.4
9.0
⬍2.0
⬍1.0
18.5
⬍2.0
5.1
74.4
12.5
40.0
14.7
4.4
7.1
24.8
3.4
2.1
TPT
PTs
30.8
128
68.0 ⫾ 31.2
28.3
73.5
53.0 ⫾ 15.8
17.7
56.7
38.2 ⫾ 14.9
7.6
42.6
25.5 ⫾ 13.0
7.8
53.5
25.6 ⫾ 13.0
7.1
32.6
16.6 ⫾ 9.3
⬍1.0
37.3
15.8 ⫾ 17.7
6.1
16.3
11.5 ⫾ 4.4
5.5
15.1
10.1 ⫾ 4.0
22.3
22.6
70.0
11.0
40.4
37.6
468
168 ⫾ 134
31.2
79.6
58.1 ⫾ 16.9
18.4
58.8
40.0 ⫾ 15.4
8.1
45.4
27.9 ⫾ 14.3
9.1
55.6
26.9 ⫾ 13.4
7.9
35.0
17.6 ⫾ 9.8
⬍4.0
39.9
16.7 ⫾ 18.3
6.6
17.6
12.1 ⫾ 4.5
5.5
15.1
10.1 ⫾ 4.0
23.1
27.4
122
22.4
46.0
ND, not determined.
Results and Discussion
Concentrations of Organotins in Chinese Sturgeon Tissues. Concen-
trations of both TPT and TBT and their metabolites were
measured in tissues of wild Chinese sturgeon (Table 1). TBT was
detected in 2 heart samples [n ⫽ 7, ⬍ limit of quantification
(LOQ)-3.8 ng/g wet weight (ww)], 3 muscle samples (n ⫽ 8,
⬍LOQ-4.3 ng/g ww), and 1 kidney sample (4.0 ng/g ww), whereas
dibutyltin (DBT) and monobutyltin (MBT) were detected in all
tissues, but were more prevalent in liver (n ⫽ 8, 11.8–257 ng of
DBT/g ww, 9.1–1115 ng of MBT/g ww) and kidney (n ⫽ 1, 36.9
ng of DBT/g ww, 33.5 ng of MBT/g ww). Although TPT was
found in all of the tissues except for 2 adipose samples (n ⫽ 5,
⬍LOQ-37.3 ng/g ww), diphenyltin (DPT) and monophenyltin
(MPT) occurred with the greatest prevalence and concentrations in liver (5.8–324 ng of DPT/g ww, ⬍LOQ-104 ng of MPT/g
ww), kidney (n ⫽ 1, 40.0 ng of DPT/g ww, 12.5 ng of MPT/g ww),
and gallbladder (n ⫽ 1, 7.1 ng of DPT/g ww, 4.4 ng of MPT/g
ww). The greatest concentration of TPT (n ⫽ 8, 30.8–128 ng/g
ww) was found in liver. This concentration was greater than those
in fishes from Inner Danish waters (16), and comparable with
those in marine fishes from the Japanese market (17), but less
than those from the Mediterranean (10), Taiwan (15), Netherlands (18), and Japan (19). Although concentrations of total BTs
in liver (365 ⫾ 447 ng/g ww) were greater than those of PTs
(168 ⫾ 134 ng/g ww), concentrations of TPT (68.0 ⫾ 31.2 ng/g
ww) were greater than those of TBT (⬍1.0 ng/g ww). This
observation suggests different toxico-kinetic behaviors of TPT
and TBT in Chinese sturgeon. Because the Chinese sturgeon is
an anadromous fish and lives most of its life in the deep ocean,
9340 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0809434106
where concentrations of organotins are less than in coastal areas,
these results suggest that Chinese sturgeon have a greater
capacity to accumulate TPT relative to TBT than do other fishes.
The distributions of BTs and PTs among tissues are described
in SI Materials and Methods. Organotins did not accumulate in
sturgeon in proportion to lipid content as do many neutral
organochlorine compounds. This phenomenon may be because
of the close affinity of trialkyltin compounds with some amino
acids, peptides, and proteins (20). As shown by the tissue
distributions of BTs and PTs (Fig. S1), TPT was transferred from
female Chinese sturgeon to their eggs, in which concentration
ranged from 7.8 to 53.5 ng of TPT/g ww and there was an
age-related accumulation (Fig. 1), whereas TBT was not detected in eggs. Maternal transfer was described by the ratio of the
concentration of TPT in eggs to that in the liver of females (21).
The value for this ratio was 0.34 ⫾ 0.11, which was comparable
with those of organochlorines such as hexachlorobenzene
(HCB; 0.61) and total 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDTs including o,p⬘-DDD, p,p⬘-DDD, o,p⬘-DDT, p,p⬘DDT, o,p⬘-DDE, and p,p⬘-DDE, 0.27) in the same Chinese
sturgeon (8).
Malformation of Wild Chinese Sturgeon Larvae. To determine
whether malformations in Chinese sturgeon occurred in the
Yangtze River, ⬇2- to 3-day-old larvae of Chinese sturgeon were
captured on 26, 27, 28, 30 November, and 1 December 2007 from
the spawning area below Gezhouba Dam, which is located 38 km
downstream from the Three-Gorges Dam in Yangtze River. The
larvae or embryos of Chinese sturgeon were cultured in conHu et al.
Fig. 1. Relationships between age of adult female Chinese sturgeon and
concentration (ng/g ww) of TPT (䊐) and DBT (Œ) in their eggs. Chinese
sturgeon were captured from the spawning location,Yichang of Yangtze
River, and died during the artificial propagation in each year between 2003
and 2006. TPT: log [TPT] ⫽ 0.0294 ⫻ age ⫹ 0.7127, r2 ⫽ 0.1496, P ⫽ 0.154. DBT:
log [DBT] ⫽ ⫺0.0295 ⫻ age ⫹ 1.2142, r2 ⫽ 0.3250, P ⫽ 0.026.
In Ovo Exposure of Sturgeon by Nanoinjection. Eighty Chinese
sturgeon eggs for each exposure group were exposed to TPT in
ovo via nanoinjection. The background concentration of TPT in
Chinese sturgeon eggs was 19.3 ng of TPT/g ww, and for other
organotins, were 8.7 ng of MBT/g ww, 1.6 ng of DBT/g ww, ⬍1.0
ng of TBT/g ww, ⬍2.0 ng of MPT/g ww, and 1.1 ng of DPT/g ww,
respectively. Injection of the eggs with control (triolein), 30 or
150 ng/g ww TPT during the period just after fertilization until
mid to lategastrula formation resulted in 4.0% (2 of 50), 11.1%
(5 of 45), and 22.6% (7 of 31) skeletal/morphological deformations, and 0% (0 of 50), 4.4% (2 of 45), and 9.7% (3 of 31) ocular
deformations, respectively. Skeletal/morphological deformation
were significantly related to concentration of TPT (␹2 ⫽ 4.244,
1° of freedom, P ⫽ 0.039). Even thought a dose-dependant
response was also observed for ocular deformation, because of
the limited sample size, no statistically significant (␹2 ⫽ 1. 917,
1° of freedom, P ⫽ 0.166) relationship with TPT concentration
was observed.
Because the Chinese sturgeon is endangered, it was possible
to obtain only a few eggs from only few adults. Therefore, a
hybrid study was conducted, in which larger numbers of eggs of
Siberian sturgeon (Acipenser baerii) were used as a surrogate
species to obtain more robust statistics by nanoinjection studies
of all of the organontim compounds. Siberian sturgeon eggs were
propagated and exposed to TPT in ovo via nanoinjection at
concentrations of 0 (control, triolein), 27, 136, or 681 ng of
TPT/g ww. The background concentration of TPT in Siberian
Hu et al.
Fig. 2. Malformations of 18 d posthatch wild Chinese sturgeon (A. sinensis)
larvae. (A) Abnormal ocular development (left to right, normal larva, single
eye larva, and no eye larva). (B) Skeletal/morphological deformation (Upper,
normal larva; Lower, curved larva).
sturgeon eggs was ⬍1.0 ng/g ww. Both ocular and morphological
malformations were observed (Fig. 3A and B), and their frequencies were directly proportional to exposure concentration
(Table 2). At concentrations that were similar to those observed
in wild Chinese sturgeon from the Yangtze River (27 ng of
TPT/g ww), malformation incidences for ocular and morphological deformation were 2.7 (17/625) and 4.2% (26/625), respectively. The association between malformation incidences
and exposure TPT concentration was statistically significant (for
morphological malformation: ␹2 ⫽ 15.85, 1° of freedom, P ⫽
0.000; for abnormal ocular malformation: ␹2 ⫽ 16.85, 1° of
freedom, P ⫽ 0.0).
To further clarify the potential for organotin compounds to
cause malformations, in ovo exposure to other organotins (MBT,
DBT, and DPT) detected in the eggs of Chinese sturgeon were
investigated individually, by injecting them into Siberian sturgeon eggs at concentrations of 0.0 (control; triolein), 30, 150, or
750 ng/g ww. In all of the in ovo exposure groups, no ocular
deformation was obser ved, and the rates of skeletal/
morphological deformation for MBT and DBT were 1.17%–
1.72%, but no dose-dependant response was observed for both
organotins. The rate of skeletal/morphological deformation for
DPT was 1.03%–2.03%, and dose-dependant response was
observed, but no statistically relationship was obtained (Table
S1). The skeletal/morphological deformation rates in blank and
injection control groups were 0.70 and 0.92%, respectively. This
result indicates organotin compounds other than TPT that were
detected in eggs of Chinese sturgeon would not cause the
observed malformations of wild Chinese sturgeon.
TPT has been observed to inhibit osteoclast differentiation
through a retinoic acid receptor-dependent signaling pathway
(22). Because TPT can bind to the retinoid X receptor (RXR)
with even greater affinity than the endogenous ligand, 9-cis
retinoic acid (23), and RXR␣ null mice exhibited an ocular
PNAS 兩 June 9, 2009 兩 vol. 106 兩 no. 23 兩 9341
ENVIRONMENTAL
SCIENCES
trolled facilities at the Chinese Sturgeon Hatchery, Jingzhou,
Hubei, until ⬇18 d posthatch, and then were inspected for
deformities. The incidence of skeletal/morphological deformations was 6.3% (65/1039), and 1.2% (12/1039) had no eyes or only
1 eye. At the same time, 2 adult female and 2 adult male Chinese
sturgeon were captured from the Yangtze River for artificial
propagation. Spawning was induced, and mature eggs were
artificially fertilized. A subsample of eggs was retained for
subsequent quantification of organotins. Of the larvae artificially
propagated from the 2 wild Chinese sturgeon, 3.9% (40/1075) of
juveniles 18 d posthatch exhibited skeletal/morphological deformations, whereas 1.7% (18/1075) had only 1 eye or no eyes (Fig.
2 A and B). Alternatively, TPT concentrations in the eggs of the
2 wild Chinese sturgeon were 20.0 and 23.7 ng of TPT/g ww,
whereas the concentrations of TBT were both under detection
limit (⬍1.0 ng/g ww). A single metabolite of TBT, MBT, was
detected at concentrations of 3.0 and 4.0 ng of MBT/g ww.
Fig. 4.
Dose-response curves of triphenyltin chloride (TPT), diphenyltin
dichloride (DPT), monophenyltin trichloride (MPT), tributyltin chloride (TBT),
dibutyltin dichloride (DBT), and monobutyltin trichloride (MBT). A natural
RXR ligand, 9-cis-retinoic acid (RA), was used as positive control.
Fig. 3. Malformations of Siberian sturgeon (A. baerii) larvae exposed to TPT
via nanoinjection of eggs. (A) Abnormal ocular development (left to right,
normal larva from control, single eye larva, and no eye larva). (B) Skeletal/
morphological deformation (Upper, normal larva from control; Lower, curved
larva with no eyes).
abnormality (24), it could modulate this receptor in a way that
could lead to the observed deformities. Therefore, in addition to
the in ovo studies, the relative potencies of the 6 organotins to
interact with the RXR were determined by use of a 2-hybrid
yeast assay system with RXR␣. The relative potencies of DPT,
MPT, TBT, DBT, and MBT relative to TPT were estimated to
be 2.8 ⫻ 10⫺3, 9.4 ⫻ 10⫺4, 0.48, 8 ⫻ 10⫺5, and ⬍2 ⫻ 10⫺6,
respectively (Fig. 4; Table S2). This result suggests that none of
the organotins except TPT were sufficiently potent to cause
deformities at the concentrations that were detected in the eggs
of Chinese sturgeon.
Together, these multiple lines of evidence were consistent with
the hypothesis that TPT was the likely cause of the malformations observed in larvae of wild Chinese sturgeon, although other
contaminants may be present that could produce similar effects.
Polychlorinated biphenyls (PCBs) are widespread in the environment, and a relatively great concentration of PCBs (5810 ng/g
ww) has been detected in the eggs of Shovelnose sturgeon from
the Mississippi River (25). In Chinese sturgeon eggs, the mean
total concentration of PCBs was 95.1 ng/g ww (16.8–229 ng/g ww)
(Fig. S2 and Table S3). When eggs of Siberian Sturgeon were
injected with 100 or 300 ng/g ww Aroclor 1254 (a commercially
manufactured PCB product), 1.13 (9 of 798 larvae) and 1.33%
(7 of 526 larvae) had skeletal/morphological deformities, respectively, and no ocular deformations were observed (Table S1).
In this study, we observed malformations in wild Chinese
sturgeon, and then correlated the response with the putative
causative agent, TPT. We isolated the putative causative agent
and introduced it into eggs of both Chinese and Siberian
sturgeon, and were able to reproduce the same deformities at the
similar proportions for similar doses in the laboratory; these
rates were similar to those observed in embryos spawned in the
wild. Thus, we have completed Kock’s postulates, and concluded
that TPT was the most likely cause of the observed deformities.
The deformities observed in Chinese sturgeon in the Yangtze
River are a measurable indicator of the adverse effects of TPT,
but the rates of deformities observed in the larvae of Chinese
sturgeon alone would be unlikely to have severe adverse effects
on the population. In our study, it was observed that exposure of
medaka to TPT at environmentally relevant concentrations
could also inhibit reproduction (15). In particular, the quality
and quantity of eggs and spawning frequency were significantly
decreased (Table S4), because TPT inhibited the induction of
vitellogenin, which is essential material for vitellogenesis, oocyte
maturation, and yolk biosynthesis in fish (15). As an overall
result, exposure to TPT at environmentally relevant concentrations can reduce the capacity to produce offsprings. The signal
inducing deformations by TPT exposure are much weaker than
that on decrease of capacity to produce viable offspring. When
rates of ocular and skeletal/morphological deformities reached
1.2 and 6.3, the capacities to produce viable offspring would be
reduced by 58.4% and 75.9%, respectively. Even though the
effects of TPT on the capacity to produce viable offspring are
unknowable under field conditions, because the same types of
Table 2. Malformations of Siberian sturgeon (A. baerii) larvae developed from eggs exposed via nanoinjection to TPT
No.
Exposure, ng
TPT/g ww
Blank
Control
27
136
681
Frequency, %
Eggs
injected
Hatched
larvae
Survived
larvae*
Abnormal
skeletal larvae
Abnormal
ocular larvae
Abnormal
skeletal larvae
Abnormal
ocular larvae
5,287
1,048
1,090
1,190
1,044
3,431
660
717
701
450
3,226
606
625
600
316
19
4
26
37
42
0
0
17
26
24
0.59
0.66
4.16
6.17
13.3
0
0
2.72
4.33
7.59
*Eighteen days posthatch larvae.
9342 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0809434106
Hu et al.
Materials and Methods
Eggs of Chinese Sturgeon for Artificial Fertilization. Two female and 2 male
Chinese sturgeon were captured from the Yichang region of the Yangtze
River in 2007. The female individuals were both 24 years old with body weights
(BWs) of 228 and 242 kg, and body lengths 336 and 332 cm, respectively. The
2 male individuals were 63.5 and 103.5 kg, and 210 and 260 cm, respectively.
After injecting luteinizing hormone releasing hormone A2 (LHR-A2) (Ningbo
second hormone factory) of 9 ␮g/kg BW for female, and 4.5 ␮g/kg BW for
male, fish spawned in the following 16 h at 18 °C. Egg and sperm were
collected, and the eggs were artificially fertilized by the female with BW 228
kg of to the male with BW 63.5 kg, and the 242.2-kg female was spawned with
the 103.5-kg male. The fertilized eggs were cultured in active carbon-treated
water until ⬇18 d old, and then were inspected for malformation. Also, 2 samples
of wild Chinese sturgeon eggs were collected before propagation for analyzing
to determine concentrations of TPT. Concentrations of TPT in eggs of each fish
were determined in triplicate and expressed as the mean ⫾ SD (n ⫽ 3).
Sample Collection. The Chinese sturgeon is a typical anadromous fish that
lives in the sea and returns to spawn in rivers, primarily the Yangtze River.
As a valuable, ancient fish species, the Chinese sturgeon is protected by the
Chinese government (4), and the capture for these individuals was done
under a permit that authorized collection strictly for scientific purposes.
During the 1980s, the population of Chinese sturgeon declined rapidly (5).
Since then, artificial propagation has begun to save this endangered
species, and 8 to 10 Chinese sturgeon were allowed to be captured for this
study. After propagation, surviving sturgeon are released back into the
Yangtze River. However, some do not survive. In this study, the eggs and
other tissues were collected for quantification of TPT before propagation,
and the other organs and tissues came from 17 sturgeon that had died
during the artificial propagation between 2003 and 2006. After collection,
samples of tissues were frozen immediately at ⫺20 °C and kept at that
temperature until analysis. The ages of fish were determined by counting
growth layers in the cleithrum, as described (5, 6). The details of the
samples analyzed in this study are shown in Table S5.
Nanoinjection. Because there were few eggs of Chinese sturgeon available to
conduct statistically robust studies, under controlled laboratory conditions
where rates of deformities could be accurately measured, Siberian sturgeon
eggs propagated from adults in artificial culture were exposed to TPT and
other organotins in ovo via nanoinjection. Eggs of Siberian sturgeon were
obtained from the Yangtze River Fisheries Research Institute. Detailed information on the procedure for nanoinjection is given in a previous article (26).
Briefly, aluminosilicate capillary tubes (1.0-mm outer diameter and 0.58internal diameter; Sutter Instrument) were used to make injection needles
with 5- to 10-␮m internal-diameter tips. Approximately 7 nL (⬍0.1% of egg
volume; egg weight, 16 mg ⫾ 0.2 mg/egg) of trioline (control) or TPT stock
solution was injected directly into the fertilized egg within 8 h using a
picoinjector (PLI-90; Harvard Apparatus) and Stereomicroscope (Zeiss Stemi
2000; Diagnostic Instruments). Exposure concentrations of TPT were 0 (con-
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River (Shanghai Scientific and Technical Publishers, Shanghai, China), p 83.
Hu et al.
trol, trioline), 27, 136, or 681 ng/g ww. After injection, the eggs were incubated in flow-through containers (30 ⫻ 30 ⫻ 16 cm), suspended in stainless
steel barrels (120-cm i.d. ⫻ 80-cm height), supplied with activated-carbon
treated water renewed daily at 16 °C–18 °C, and dead embryos were removed
daily and hatched larvae number were recorded. Finally, ⬇18 d posthatch,
larvae were inspected for malformation. No obvious effect was observed in
mortality and development in control (trioline) compared with blank (uninjected groups).
The methods used for in ovo exposure of Siberian sturgeon to other
organotins and PCBs were the same as described above. Injected concentrations of DBT, MBT, DPT were 0 (control, trioline), 30, 150, or 750 ng/g ww, and
those of PCBs (as Aroclor 1254, a commercial PCBs product) were 100 and 300
ng/g ww. Details of the methods used for nanoinjection of Chinese sturgeon
eggs are provided in SI Materials and Methods.
Quantification of TPT and Its Related Chemicals. The method used to quantify
the 6 organotins was based on a previous article (9) with some modifications,
and a detailed method description is provided in SI Materials and Methods.
The fortification experiments were conducted by adding a comparable
amount of the 6 deuterium-labeled surrogate analogues with the typical
concentrations in Chinese sturgeon samples, which ranged from 10 to 50 ng/g
ww. Recoveries of the 6 deuterium-labeled surrogates were calculated by
response relative to that of the internal standard TeBT-d36. Recoveries were
56 ⫾ 7% for MBT-d9, 113 ⫾ 3% for DBT-d18, 115 ⫾ 5% for TBT-d27, 112 ⫾ 4%
for DPT-d10, and 109 ⫾ 3% for TPT-d15, but the recovery of MPT-d5 was limited
to 47% (n ⫽ 6). Limits of quantification for MBT, DBT, TBT, MPT, DPT, and TPT
at S/n ⫽ 3 were 1.5, 1.0, 1.0, 2.0, 1.0, and 1.0 ng/g ww, respectively.
Analysis for PCBs. A detailed method description is provided in SI Materials and
Methods.
Pathological Examination and Statistics. An external inspection of larvae was
observed under a microscope with an ocular micrometer each day. The malformations were sorted into skeletal/morphological deformation and ocular
deformation. The skeletal/morphological deformation included shortening of
the notochord, deletions of pinna, and curvature of body or tail, and the
ocular deformation was those with no eyes and only 1 eye. Most of larvae with
skeletal/morphological deformation lost normal swimming behavior. A ␹2 test
was used to test the differences in morphological malformation or abnormal
ocular malformation between the control and the TPT exposure group, and
difference was considered significant if P ⬍ 0.05. The statistical analyses were
performed with SPSS 15.0.
ACKNOWLEDGMENTS. We thank Hao Du, Hui Zhang, and Xihua Chen
(Yangtze River Fisheries Research Institute, Beijing, China) for capturing wild
larvae of Chinese sturgeon and Hongbo Yang, Chong Huang, Jian Jiao, Fujun
Ma, Lini Hao, Jianxian Sun, and Wanfeng Wang (Peking University, Peking,
China) for preparing nanoinjection experiment. This work was supported by
National Natural Science Foundation of China Grants 40632009 and 20777002;
National Basic Research Program of China Grant 2007CB407304; Fund of
Three-Gorges Project Corporation for Ecological and Environmental Compensation Grant 071490; Canada Research Chair Program and Chair Professorship
at the Department of Biology and Chemistry and Research Centre for Coastal
Pollution and Conservation, City University of Hong Kong (J.P.G.); and the
National Science and Engineering Research Council of Canada Discovery Grant
Project 6807.
8. Wan Y, et al. (2007) Levels, tissue distribution, and age-related accumulation of
synthetic musk fragrances in Chinese sturgeon (Acipenser sinensis): Comparison to
organochlorines. Environ Sci Technol 41:424 – 430.
9. Hu JY, et al. (2006) Trophic magnification of triphenyltin in a marine food web of Bohai
Bay, North China: Comparison to tributyltin. Environ Sci Technol 40:3142–3147.
10. Borghi V, Porte C (2002) Organotin pollution in deep-sea fish from the Northwestern
Mediterranean. Environ Sci Technol 36:4224 – 4228.
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90:S8 –S15.
12. Fent K, Meier W (1994) Effects of triphenyltin on fish early life stages. Arch Environ
Contam Toxicol 27:224.
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success, and liver ultrastructure of early life stages of zebrafish (Danio rerio). Ecotox
Environ Safety 44:25–39.
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medaka, orzias latipes, to mixtures of tributyltin and polychlorinated biphenyls.
Environ Toxicol Chem/SETAC 24:591–596.
15. Zhang ZB, Hu JY, Zhen HJ, Wu XQ, Huang C (2008) Reproductive inhibition and
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PNAS 兩 June 9, 2009 兩 vol. 106 兩 no. 23 兩 9343
ENVIRONMENTAL
SCIENCES
deformities were observed in both Chinese sturgeon and medaka
exposed to the same concentrations of TPT, suggesting that the
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fitness of the Chinese sturgeon population in the Yangtze River.
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9344 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0809434106
22. Yonezawa T, et al. (2007) Tributyltin and triphenyltin inhibit osteoclast differentiation
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Hu et al.
Supporting Information
Hu et al. 10.1073/pnas.0809434106
SI Materials and Methods
Chemicals and Standards for Analysis of TPT and Its Ralted Chemicals.
Monobutyltin trichloride (MBT, 97%), monophenyltin trichloride (MPT, 98%), and tropolone (98%) were purchased from
ACROS ORGANICS. Diphenyltin dichloride (DPT, 96%) was
obtained from Aldrich. Dibutyltin dichloride (DBT, 97%), tributyltin chloride (TBT, 95%), triphenyltin chloride (TPT, 95%),
and sodium tetraethylborate (NaBEt4, 98%) were purchased
from Wako. Deuterated organotins, MBT-d9, DBT-d18, TBTd27, tetrabutyltin-d36 (TeBT-d36), MPT-d5, DPT-d10, and TPTd15 were obtained from Hayashi Pure Chemicals. Dichloromethane, methanol, and hexane were HPLC grade obtained from
Fisher Scientific, and tetrahydrofuran was HPLC grade obtained
from DIKMA. Diethyl ether was HPLC grade and purchased
from Siyou Chemicals. Acetic acid, hydrochloric acid, and sodium acetate were AR grade. Anhydrous sodium sulfate and
sodium chloride were heated at 450 °C for 6 h before usage.
Florisil columns (1 g) were obtained from Waters. Water was
obtained by a compact ultrapure water system (Easypure UV).
Fresh NaBEt4 solution of 5% (wt/vol) was prepared with tetrahydrofuran every month. An acetate buffer was made from
acetic acid and sodium acetate solution. All of the solutions were
stored at 4 °C in the dark.
Quantification of Organotins. One to 3 g (dependent on the lipid
content) of different tissues of the Chinese sturgeon were
homogenated and 100 ␮L of surrogate standard solution containing 1 mg/L of MBT-d9, DBT-d18, TBT-d27, MPT-d5, DPTd10, and TPT-d15 was added to each. The mixture was first
extracted with 25 ml of 1 M HCl-methanol/ethyl acetate (1/1) by
shaking for 10 min. Then, the samples were centrifuged for 10
min, and the supernatant was transferred to a separation funnel
containing 100 mL of saturated NaCl solution. The extraction
procedure was repeated again. Analytes were then extracted
with 30 mL of ethyl acetate/hexane (3:2) solution twice by
mechanical shaking for 5 min each time. Then, 100 mL hexane
was added to the combined organic layer, and the mixture was
left to stand still for 30 min. After the mixture had been passed
through a layer of anhydrous sodium sulfate to remove moisture,
it was concentrated by use of a rotary evaporator. Then the
concentrate was mixed with 10 mL acetate buffer solution and
200 ␮L ethylborate reagent to derivatize target organotins. The
derivatized samples were combined with 40 mL of 1 M KOHethanol solution to decompose any fat for 1 h. After the
saponification, 40 mL pure water was added to the solution, and
then extracted with 20 mL hexane by mechanical shaking for 10
min twice. The combined hexane extract was first concentrated
and passed through a florisil cartridge column (conditioned with
10 mL hexane) covered with a layer of anhydrous sodium sulfate
and then eluted with 7 mL of hexane/diethyl ether (9:1). After
TeBT-d36 was added as the internal standard, the final solution
was concentrated to 0.3 mL for GC/MS analysis.
All equipment was rinsed with acetone and hexane to avoid
sample contamination, and a laboratory blank was also included
during the process of analysis. To automatically correct the losses
of analytes during extraction or sample preparation, and to
compensate for variations in instrument response from injection
to injection, quantification of the 6 organotins was conducted out
by use of relative response factors of the analyte with their
internal standards of deuterium-labeled surrogate analogues. In
this study, the 6 deuterium-labeled surrogates were used to
correct for extraction efficiencies during sample preparation and
Hu et al. www.pnas.org/cgi/content/short/0809434106
the signal variation of the GC-MS from one injection to another,
and the internal standard TeBT-d36 was used to calculate the
recoveries of the 6 deuterium-labeled surrogates. The calibration
standard was prepared every day according to the following
procedure. A 100 ␮L solution of organotin standards (1 mg/L)
and their surrogates (1 mg/L) were added in 50 mL of acetate
buffer solution (pH ⫽ 5.0). Then, 200 ␮L of ethylborate reagent
and 2 mL hexane were combined with the mixture and shaken
mechanically for both derivatization and extraction for 5 min.
After the organic layer was removed, another 2 mL hexane was
added to extract the analyte again. The organic layer was
combined with the former one and concentrated to 0.3 mL under
gentle nitrogen gas.
Quantification of PCBs. The mixture of standards of PCBs containing 142 PCBs (PCB 1, 2, 3, 4/10, 5/8, 6, 12/13, 14, 15, 16/32,
17, 18, 19, 20/33, 22, 24/27, 25, 26, 28/31, 29, 34, 35, 37, 40,
41/64/71, 42/59, 44, 45, 46, 47/48/75, 49, 51, 52/73, 53, 54, 56/60,
63, 66, 67, 69, 74, 70, 77, 81, 82, 83, 84/92, 85, 91, 93/95, 97, 99,
100/101, 103, 104, 105, 107, 110, 114, 87/90/115, 117, 118, 119,
122, 123, 124, 128, 129, 130, 131, 132/153, 134, 135/144, 136, 137,
138/163/164, 141, 146, 147, 149, 151, 154, 156, 157, 158, 165, 167,
170/190, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 183, 185,
187, 189, 191, 193, 194, 195, 196/203, 197, 199, 200, 201, 202, 205,
206, 207, 208, and 209; ordered by International Union of Pure
and Applied Chemistry number) was purchased from AccuStandard. Aroclor 1254 and 1242 were purchased from Sino-Japan
Friendship Centre for Environmental Protection. Hexane, dichloromethane and acetone were pesticide grade purchased
from Fisher Scientific, and sodium sulfate and silica gel (100–200
mesh size) from Beijing Chemical Reagent Company.
Analysis for PCBs. The analytical method used to quantify the
PCBs was based on a previous article (1) with some modifications; 2 g of freeze-dried eggs of the Chinese sturgeon were
homogenized, and 200 ␮L of surrogate standard solution containing PCB 30, PCB 121, and PCB 198 were added to the each
sample. The mixtures were transferred to 250 mL roundbottomed flasks and Soxhlet extraction were carried out for 24 h
with 250 mL hexane/dichloromethane (1:3 vol/vol) added by 2 g
of Na2SO4. After being concentrated to ⬇1 mL by use of a rotary
evaporator, the residue was sequentially passed through silica gel
packed glass columns (impregnated with sulfuric acid) covered
with 1 cm Na2SO4 layers. The columns were then eluted by 15
mL hexane and 10 mL dichloromethane sequentially. The elution was transferred to 4-mL bottles, and concentrated to dryness
under gentle nitrogen followed by 200 ␮L hexane addition for
GC-MS analysis.
All equipment was rinsed with acetone and hexane to avoid
sample contamination, and a laboratory blank was also included
during the process of analysis. To automatically correct the losses
of analytes during extraction or sample preparation, and to
compensate for variations in instrument response from injection
to injection, quantification of PCB congeners was conducted out
by use of relative response factors of the analyte with the internal
standards. In this study, the PCB 30, PCB 121, and PCB 198 were
used to correct for extraction efficiencies during sample preparation and the signal variation of the GC-MS from one injection
to another. The calibration standard was prepared every day
according to the following procedure.
The fortification experiments were conducted by adding a
comparable amount of PCB congeners with the typical concen1 of 10
trations in Chinese sturgeon samples, which ranged from 10 to
50 ng/g wet weigh (ww). Recoveries of PCBs were in the range
of 50–124%. The limit of quantification (LOQ) for different
PCB congeners ranged from 0.02 to 1.21 ng/g ww.
Instrumental Conditions. The analysis for the 6 derivatized or-
ganotins was carried out on a GC-MS coupled with a HewlettPackard 5890 GC and a Hewlett-Packard 5971 MS. An HP-5MS
capillary column (60 m 0.25 mm i.d. with a film thickness of 0.25
␮m) was used for organotin analysis. The injector temperature
was maintained at 270 °C. The temperature program increased
from 60 (2 min) to 130 °C at a rate of 20 °C/min (26 min), and
then to 280 °C at 20 °C/min (7 min). The injection volume was 1
␮L, and the splitless mode was used. The mass spectrometer was
operated in the electron impact ionization mode with an ionizing
energy of 70 eV, and the data were acquired with selected ion
monitoring mode (40 ms dwell time). The fragment ions were
selected according to the most abundant ions in each oligomer.
The concentrations of organotins are expressed as cationic
species.
Instrumental analysis for PCBs was performed by GC-EI-MS
(Shimadzu QP 2010 plus).
A DB-5MS capillary column (30 m ⫻ 0.25 mm ⫻ 0.1 ␮m film
thickness; J&W Scientific) was selected for PCBs analysis. The
injector and ion source temperature was held at 280 and 320 °C,
respectively. The temperature program increased from 80 (4
min) to 160 °C at 20 °C/min, then to 230 °C at 2 °C/min. and then
to 295 °C (5 min) at a rate of 30 °C/min. The injection volume was
1 ␮L, and the splitless mode was used. The MS was operated in
the electron impact ionization mode with an ionizing energy of
70 eV, and the data were acquired with selected ion monitoring
mode. The fragment ions were selected according to the most
abundant ions in each PCB.
Nanoinjection of Eggs of Chinese Sturgeon. A limited number of
eggs were collected from 1 wild-caught chinese sturgeon during
the reproductive season (last 10 days of November 2008). One
anadromous female (length 330 cm) and 1 anadromous male
Chinese sturgeon (length 265 cm) were captured from Yichang
of Yangtze River in 2008. After injecting luteinizing hormone
releasing hormone A2 (LHR-A2; Ningbo second hormone
factory) of 9 ␮g/kg body weight (BW) for female and 4.5 ␮g/kg
body weight for male, fish spawned in the following 16 h at 18 °C.
Egg and sperm were collected, and the eggs were artificially
fertilized, and then the procedure for nanoinjection was carried
out according to the method as described in the main text for
Siberian sturgeon. Eighty eggs were injected with each treatment. Exposure concentrations of TPT were 0 (control, trioline),
30 or 150 ng/g ww with a final injection volume of 20 nL of
trioline (vehicle control) or TPT stock solution per egg. The
embryos and larvae were maintained in active carbon-treated
water until ⬇12 d posthatch, and then were inspected for
deformities.
Yeast Assay for RAR-mediated Activity. The yeast 2-hybrid transactivation assay, which has been described in ref. 2 was applied
to evaluate the RAR-mediated activity of samples. Yeast cells
were preincubated at 30 °C for 16 h in 5 mL medium (6.7 g/L
Difco yeast nitrogen base without amino acids, 0.2% glucose, 300
mg/L L-isoleucine, 1500 mg/L L-valine, 200 mg/L L-adenine
hemisulfate salt, 200 mg/L L-arginine HCl, 200 mg/L L-histidine
HCl monohydrate, 300 mg/L L-lysine HCl, 200 mg/L Lmethionine, 500 mg/L L-phenylalanine, 200 mg/L L-threonine,
300 mg/L L-tyrosine, 200 mg/L L-uracil; Sigma); 50 ␮L of
overnight culture and 2.5 ␮L of DMSO solution diluted to the
desired concentrations were then added to 200 ␮L of fresh
medium (2% galactose) in a microtube (Axygen Scientific),
respectively. After yeasts were cultured for 4 h at 30 °C, 150 ␮L
Hu et al. www.pnas.org/cgi/content/short/0809434106
of the above culture was fractionated, and its absorbance at 595
nm was detected. The residual culture (100 ␮L) was centrifuged
at 4 °C (15,000 ⫻ g) for 5 min, and the collected cells were
resuspended in 200 ␮L of Z buffer (0.1 M sodium phosphate, pH
7.0/10 mM KCl/1 mM MgSO4) containing 1 mg/mL Zymolyase
20T (Seikagaku), and incubated for 20 min at 30 °C. The
enzymatic reaction was started by the addition of 40 ␮L of 4
mg/mL 2-nitrophenyl-␤-D-galactopyranoside (ONPG; Tokyo
Kasei), and incubated for 20 min at 30 °C. Then the enzymatic
reaction was stopped by adding 1 M Na2CO3 (100 ␮L). After the
above solution was centrifuged, 150-␮L aliquots were placed into
96-wells of a microplate. Absorbances at 415 and 570 nm were
read on a microplate reader (Bio RAD 550) to estimate the
RAR-mediated activity, and the ␤-galactosidase activity (U) was
calculated according to the following equation: U ⫽ 1,000 ⫻
([OD415] ⫺ [1.75 ⫻ OD570]/([t]⫻[v]⫻[OD595]), where t represents the reaction time (min); v is the volume of the culture used
in the assay (mL); OD595 is the cell density at the start of the
assay; OD415 is the absorbance by O-nitrophenol at the end of the
reaction, and OD570 is the light scattering at the end of the
reaction. In this assay, all-transRA was used as positive control,
and the molar concentration for each organotin that produces
50% (EC50) of the maximum response of corresponding RAR
agonistic activity was calculated by the Prism 4 for Windows
program (GraphPad).
Tissue Distribution of Oragnotins in Chinese Sturgeon. The tissue
distributions of BTs and PTs are given in Fig. S1. Because of the
limited number of samples, kidney, spleen, and gallbladder are
not included in the graph. The greatest concentration of BTs was
measured in liver tissue (mean: 365 ng/g ww, ranging from 21 to
1,373 ng/g ww), followed by 1 kidney sample (74.4 ng/g ww). The
BTs residues in other organs were 1 or 2 orders of magnitude less
than those in liver. This profile of BTs distribution in tissues was
similar to that of finless porpoise (3) and Dall’s porpoise (4) from
Japanese coastal waters. Concentrations of PTs decreased in the
order: liver (mean: 168 ng/g ww, in the range of 38–468 ng/g
ww)⬎kidney (122 ng/g ww)⬎heart (mean: 58 ng/g ww, in the
range of 31–80 ng/g ww)⬎spleen (46 ng/g ww)⬎muscle (mean:
40 ng/g ww, in the range of 18–59 ng/g ww)⬎gill, roe, pancreas,
gonad, gallbladder, adipose, intestine and stomach (range: 1.0–
55.6 ng/g ww), which was in accordance with that of the Dall’s
porpoise collected in the northern North Pacific Ocean (5). Both
BTs and PTs distributions revealed that liver and kidney had an
important role in the burden of organotins in Chinese sturgeon,
which is different from those of organochlorine compounds and
musk fragrances in Chinese sturgeon (6). The lipid content in
organs is often associated with the accumulation of many organic
chemicals; however, there was no correlation between lipid
contents of different tissues and the PTs or BTs concentrations,
suggesting that organotins did not accumulate in organisms in a
lipid-specific way as traditional lipophilic compounds do. The
relatively great accumulation of BTs and PTs in the liver and
kidney of Chinese sturgeon may be because of the close affinity
of trialkyltin compounds with some amino acids, peptides and
proteins (7).
Concentrations of PCBs in the Eggs of Chinese Sturgeon. Of 142
target PCBs, PCB 28/31, 52/73, 54, 47/48/75, 74, 56/60, 66, 93/95,
84/92, 100/101, 99, 87/90/115, 85, 118, 107, 105, 151, 149, 146, 165,
132/153, 138/163/164, 178, 187, 183, 177, 171, 180, and 170/190
were detected in the eggs of Chinese sturgeon. Of these PCBs
detected, PCB 99, 132/153, and 138/163/164 were detected in all
of the 14 egg samples, and PCB 85, 100/101, 118 and 28/31 were
detected in 13, 12, 11 among 14 samples, respectively (Table S2).
The mean concentration of total PCB congeners was 95.1 ⫾ 78.2
ng/g ww (from 16.8 to 229 ng/g ww). Among 39 PCBs including
coeluting congeners detected in the eggs of Chinese sturgeon,
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PCB 132/153, 99, and 138/163/164 were predominant which
accounted for 20.1 ⫾ 5.4%, 15.2 ⫾ 4.4% and 13.4 ⫾ 8.1% of total
PCBs. PCB 85, 118 were almost at the same level, which
accounted for 7.8 ⫾ 4.9%, and 6.0 ⫾ 4.6% of the total PCBs
concentration, respectively. The profile of PCBs in eggs of
Chinese sturgeon is relatively similar to that of Aroclor 1254
compared with other commercial manufactured Aroclors such as
Aroclor 1242, 1248, and 1260 (8) (Fig. S2). Compositions of
Aroclor 1254 and 1242 which are similar to the 2 main PCB
industrial products (i.e., PCB-3 and PCB-5) made in China, and
the result indicated that 81% PCB congeners detected in samples
were included in Aroclor 1254. Therefore, to investigate the
potential toxicity of PCBs, sturgeon embryos were exposed to
Aroclor 1254 under laboratory conditions.
1. Gudrun B, et al. (1995) Uptake of PCBs in fish in a contaminated river system: Bioconcentration factors measured in the field. Environ Sci Technol 29:2010.
2. Kostyniak PJ, et al. (2005) Formulation and characterization of an experimental PCB
mixture designed to mimic human exposure from contaminated fish. Toxicol Sci
88:400 – 411.
3. Iwata H, Tanabe S, Mizuno T, Tatsukawa R (1995) High accumulation of toxic butyltins
in marine mammals from Japanese coastal waters. Environ Sci Technol 29:2959 –2962.
4. Yang J, Miyazaki N (2006) Transplacental transfer of butyltins to fetus of Dall’s porpoise
(Phocoenoides dalli). Chemosphere 63:716 –721.
5. Yang J, Harino H, Miyazaki N (2007) Transplacental transfer of phenyltins from a
pregnant Dall’s porpoise (Phocoenoides dalli) to her fetus. Chemosphere 67:244 –249.
6. Wan Y, et al. (2007)) Levels, tissue distribution, and age-related accumulation of
synthetic musk fragrances in Chinese sturgeon (Acipenser sinensis): Comparison to
organochlorines. Environ Sci Technol 41:424 – 430.
7. David AG, Smith PJ (1980) Recent advances in organotin chemistry. Adv Inorg Chem
Radiochem 23:1–77.
8. Nishikawa J, et al. (1999) New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator. Toxicol Appl
Pharmacol 154:76 – 83.
9. Zhang ZB, Hu JY, Zhen HJ, Wu XQ, Huang C (2008) Reproductive inhibition and
transgenerational Toxicity of Triphenyltin on Medaka (Oryzias latipes) at environmentally relevant levels. Environ Sci Technol 42:8133– 8139.
Hu et al. www.pnas.org/cgi/content/short/0809434106
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Fig. S1. Concentrations of BTs (right cluster) and PTs (left cluster) in different tissues of Chinese sturgeon. Data are presented in box-and-whisker plots; 50%
of the cases have values within the boxes, and the edges of the box mark the 25th and 75th percentiles. a, PTs; b, BTs.
Hu et al. www.pnas.org/cgi/content/short/0809434106
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Fig. S2.
Comparison of PCB profiles in the eggs of Chinese sturgeon with commercially manufactured Aroclor 1254 and 1242.
Hu et al. www.pnas.org/cgi/content/short/0809434106
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Table S1. Malformations of Siberian sturgeon (Acipenser baerii) larvae developed from eggs exposed via nanoinjection to Arcolor
1254, DBT, MBT, and DPT
No.
Type
Blank
Control
Arcolor 1254
—
—
DBT
—
—
MBT
—
—
DPT
—
Treatment
Eggs
injected
Hatched
larvae*
Survived
larvae*
Abnormal
skeletal larvae
Abnormal
ocular larvae
Frequency of abnormal
skeletal larvae, %
Untreated
triolein
100 ng/g ww
300 ng/g ww
30 ng/g ww
150 ng/g ww
750 ng/g ww
30 ng/g ww
150 ng/g ww
750 ng/g ww
30 ng/g ww
150 ng/g ww
750 ng/g ww
—
666
1,220
814
663
569
521
876
643
819
559
658
452
—
472
842
564
462
397
199
623
452
576
402
441
323
1000
436
798
526
426
349
168
569
414
506
387
410
296
7
4
9
7
5
6
2
6
3
5
4
6
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0.70
0.92
1.13
1.33
1.17
1.72
1.19
1.05
0.72
0.99
1.03
1.46
2.03
*Eighteen days posthatch larvae.
Hu et al. www.pnas.org/cgi/content/short/0809434106
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Table S2. Half maximal EC50 and relative potency factors (RePs) of 6 organotins based on RXR
reporter gene expression
TPT
TBT
DPT
MPT
DBT
MBT
9cis-RA
Hu et al. www.pnas.org/cgi/content/short/0809434106
EC50
ReP
9.6 ⫻
2.0 ⫻ 10⫺8
3.4 ⫻ 10⫺6
1.0 ⫻ 10⫺5
1.2 ⫻ 10⫺4
⬎5.0 ⫻ 10⫺3
7.8 ⫻ 10⫺8
1
0.47648
0.00283
0.00094
0.00008
⬍2 ⫻ 10⫺6
—
10⫺9
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Table S3. Concentrations of PCBs in eggs (ng/g ww) of Chinese sturgeon
Sample code
PCBs
28/31
52/73
54
47
74
56/60
66
93/95
92/84
100/101
99
90
85
118
107
105
151
149
146
165
153/132
138/163/164
178
187
183
177
171
180
170/190
TOTAL
A0410
A0406
A0408
A0412
A0414
A0500
A0439
A0449
A0444
A0403
A0440
A0452
A0447
A0438
Mean
SD
1.7
ND
1.3
0.5
1.7
ND
1.4
0.6
ND
0.4
2.8
ND
ND
0.8
ND
ND
ND
ND
ND
ND
4.4
1.3
ND
ND
ND
ND
ND
ND
ND
16.8
ND
ND
ND
ND
1.0
0.3
ND
ND
ND
0.4
2.1
ND
1.2
1.3
ND
ND
ND
ND
ND
2.1
4.3
2.0
ND
1.2
ND
ND
ND
1.9
ND
17.7
ND
ND
0.7
ND
1.9
ND
ND
ND
ND
2.6
6.3
0.4
5.6
5.0
ND
ND
ND
ND
16.8
ND
9.5
17.3
ND
ND
ND
ND
ND
ND
ND
66
2.0
1.4
ND
0.6
7.7
2.4
4.5
1.5
ND
4.4
25.6
ND
19.3
15.7
ND
ND
ND
ND
8.7
ND
41.4
47.7
ND
12.3
1.4
ND
ND
3.8
ND
200
ND
ND
ND
ND
2.6
1.8
ND
ND
ND
1.0
6.5
ND
5.4
2.6
ND
ND
ND
ND
ND
ND
10.8
12.6
ND
7.0
ND
ND
ND
ND
ND
50.4
5.9
2.9
ND
0.4
8.7
ND
5.7
2.1
6.6
6.7
34.3
1.4
7.6
ND
2.3
ND
7.2
ND
16
ND
48.6
20.4
2.9
13.4
3.5
1.9
1.5
23
6.4
229
0.6
0.6
ND
0.2
ND
ND
ND
0.6
ND
1.5
4.8
ND
5.0
1.8
ND
ND
ND
ND
ND
ND
6.1
6.8
ND
ND
ND
ND
ND
ND
ND
27.9
1.2
ND
1.6
ND
2.2
0.7
ND
ND
ND
ND
6.0
0.5
2.4
1.4
ND
ND
ND
ND
ND
2.6
6.5
2.3
ND
1.7
ND
ND
ND
1.4
ND
30.4
4.3
ND
ND
ND
4.3
ND
ND
1.5
4.8
3.0
18.6
0.7
5.4
29.5
2.2
5.5
3.2
2.6
8.9
25.7
12.3
1.9
6.9
2.2
1.2
12.8
3.2
ND
ND
161
1.3
0.7
ND
0.3
ND
1.4
ND
0.6
ND
1.1
8
ND
3.6
2.1
ND
ND
0.6
ND
1.1
3.7
7.4
3.1
ND
ND
ND
ND
ND
ND
ND
35.1
3.7
1.7
ND
1.0
5.8
3.4
4.4
2.1
2.1
2.7
23.9
2.3
7.3
15.5
2.0
ND
4.2
2.8
15.0
ND
27.7
12.5
2.2
12.6
3.0
1.0
ND
17.4
5.3
182
3.0
ND
ND
ND
ND
ND
ND
ND
ND
1.3
7.0
0.6
3.6
ND
ND
ND
ND
ND
2.7
ND
6.9
2.6
ND
1.8
ND
ND
ND
ND
ND
29.4
5.2
ND
ND
ND
6.6
ND
3.7
ND
ND
4.6
30.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
53.0
21.2
2.4
ND
3.0
19.2
ND
24.7
4.5
178
ND
ND
0.5
0.4
4.0
0.6
1.7
ND
ND
ND
11.8
1.0
7.2
5.7
ND
ND
ND
ND
ND
6.6
21.5
10.5
2
7.8
2.6
ND
ND
14.3
9.5
108
2.1
0.5
0.3
0.2
3.3
0.7
1.5
0.6
1.0
2.1
13.4
0.5
5.3
5.8
0.5
0.4
1.1
0.4
4.9
2.9
18.6
11.6
1.2
4.3
1.0
2.5
0.3
6.2
1.8
95.1
2.0
0.9
0.5
0.3
2.8
1.0
2.0
0.8
2.0
1.9
11
0.7
4.6
8.3
0.9
1.4
2.1
0.9
6.5
6.6
17
12
1.9
5.0
1.3
5.7
0.9
9.0
3.1
75
ND, less than limit of quantification.
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Table S4. Rates of deformities in larvae of F1 generation and the capacity to produce viable offspring of medaka after TPT-Cl
exposure
Exposure
groups, ng/L
0 (Control)
1.6
8
40
200
1,000
Accumulated TPT levels
in female, ng/g ww
Rate of ocular
deformities, %
Spawning
frequency, %*
Spawned egg no.
per female per day
Egg protein,
␮g/egg
No. of viable offspring
produced by 1 female pre day
Not detected
6.52 ⫾ 0.56
28.9 ⫾ 5.73
141 ⫾ 9.18
720 ⫾ 113
4919 ⫾ 571
0
0
0.95 ⫾ 0.78
1.97 ⫾ 1.31
3.48 ⫾ 1.62
6.72 ⫾ 1.66
86.9 ⫾ 8.13
83.33 ⫾ 6.80
65.47 ⫾ 10.12
55.95 ⫾ 9.27
40.47 ⫾ 11.21
42.86 ⫾ 13.11
24.25 ⫾ 3.97
21.15 ⫾ 4.08
18.43 ⫾ 3.29
15.88 ⫾ 3.06
15.23 ⫾ 2.93
13.85 ⫾ 2.94
164 ⫾ 5.44
155 ⫾ 5.12
152 ⫾ 2.62
146 ⫾ 4.78
136 ⫾ 3.7
133 ⫾ 3.40
19.5 ⫾ 1.88
15.2 ⫾ 3.42
9.37 ⫾ 1.07
6.15 ⫾ 1.21
4.14 ⫾ 1.47
3.67 ⫾ 0.96
Data are presented as means ⫾ standard deviation (9).
*Spawning frequency is the fraction of spawning females per day.
Hu et al. www.pnas.org/cgi/content/short/0809434106
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Table S5. Details of Chinese sturgeon samples used in this study
Length, cm
Sample code
A0466
A0406
A0410
A0412
A0414
A0408
A0447
A0445
A0403
A0444
A0452
A0449
A0500
A0439
A0438
A0440
A0441
Date of collection
Tissue
Age, year
Wt, kg
2003
2004
2004
2004
2004
2004
2005
2005
2005
2005
2005
2005
2005
2006
2006
2006
2006
L, M, H, Go, St, I, A, Gi, K
R, L, M, H, Go, St, P
R, L, M, H, Go, St, I, A, Gb
R, L, M, H, Go, St, I, Gi, P
R, L, M, I, A, Gi
R
R, L, M, H, Go, I, Gi
L, M, H, Go, I, A, Gi
R
R
R
R
R
R, L, M, H, Go, St, I, A, Gi, Sp
R
R
R
24
18
17
24
25
22
19
18
24
23
23
22
22
21
27
17
24
254
174
140
230
263
230
192
187
260
224
207
252
227
223
334
176
240
Total
length
Body
length
339
297
288
334
337
312
303
285
338
320
322
327
317
312
343
290
340
285
245
246
287
285
258
247
237
280
270
282
275
261
262
290
250
300
L, liver; M, muscle; H, heart; Go, gonad; St, stomach; I, intestines; A, adipose; Gi, gill; P, pancreas; K, kidney; Gb, gallbladder; R, roe; Sp, spleen. All sturgeons
were female. Eggs and other tissues were collected from 17 sturgeon that were captured from the spawning location at Yichang on the Yangtze River, and died
during the artificial propagation in each year between 2003 and 2006.
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