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Changes in dietary bioaccumulation of tributyltin chloride (TBTCl) in red sea bream (Pagrus major) with the concentration in feed
Water Research 37 (2003) 1497–1504
Changes in dietary bioaccumulation of tributyltin chloride (TBTCl) in red sea bream (Pagrus major) with the concentration in feed Kumiko Ikeda*, Hisashi Yamada National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi Ohno, Saeki, Hiroshima 739-0452, Japan Received 16 July 2001; received in revised form 24 September 2002; accepted 4 October 2002
Abstract The effect of the concentration of tributyltin (TBT) in feed on the dietary bioaccumulation of tributyltin chloride (TBTCl) was studied in an 8-week uptake experiment and a 4-week elimination experiment using red sea bream (Pagrus major). The biomagnification factor (BMF) and the assimilation efficiency (AE) decreased from 0.30 to 0.15 and from 13% to 5.9%, respectively, as the TBT concentration in feed increased from 1.3 to 20 mg/g. The elimination rate constant (k2 ) was independent of the TBT concentration in the fish. Laboratory measurements of the BMF and AE of TBTCl underestimate actual field values if highly contaminated feed is used. Judging from the BMF and AE, the risk of the bioaccumulation of TBTCl through the food chain might be smaller than that of polychlorinated biphenyls. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bioaccumulation; TBTCl; Red sea bream; Biomagnification factor; Assimilation efficiency
1. Introduction Fish bioaccumulate contaminants through two main pathways: direct uptake from the water across the gills (bioconcentration) and dietary uptake (biomagnification). Information on dietary uptake is necessary for a better understanding of bioaccumulation through the food chain in aquatic ecosystems. The bioaccumulation of contaminants in fish is evaluated as the balance between uptake rate and elimination rate, called the bioaccumulation factor. The uptake and elimination rate constants of contaminants in fish depend on various biological or physicochemical conditions, such as fish species [1], size of experimental fish [2], and temperature of rearing water [3]. The concentrations of contaminants in the rearing *Corresponding author. Tel.: +81-829-55-0666; fax: +81829-54-1216. E-mail address: [email protected] (K. Ikeda).
water and feed also play an important role. The bioconcentration factor increases as the concentration of the contaminants in water decreases [4,5]. The biomagnification factor may also depend on the concentration of contaminants in feed, but this relationship is poorly understood. Two influences may affect the biomagnification factor: changes in the assimilation efficiency, which depends on the concentration of contaminants in feed, and changes in the elimination rate constant, which depends on the concentration in fish fed the feed. The concentrations of contaminants in fish possibly influence changes in the elimination rate constant through changes in the activity of xenobiotic-metabolizing enzymes such as cytochrome P-450. For instance, tributyltin (TBT) and polychlorinated biphenyls (PCBs) are metabolized through the cytochrome P-450 dependent monooxygenase system [6,7]. TBT also inhibits cytochrome P-450 activity [8–11]. On the other hand, PCBs induce cytochrome P-450 [12,13]. These facts
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indicate that the concentrations of contaminants in fish affect the elimination of the contaminants. Therefore, it is necessary to clarify how changes in the biomagnification factor and the uptake and elimination rate constants depend on the concentrations of contaminants in feed to understand the mechanism of bioaccumulation through the food chain. We studied how changes in the kinetic parameters that affect the bioaccumulation of tributyltin chloride (TBTCl) through dietary uptake—biomagnification factor, and uptake and elimination rate constants— depend on the concentration of TBT in feed. We used long-term uptake and elimination experiments with a marine fish, the red sea bream (Pagrus major). We also assessed the probability of TBTCl bioaccumulation through the food chain by comparing the kinetic parameters of TBTCl and PCBs, the major organic contaminants in marine environment.
Table 1 Experimental conditions for the bioaccumulation of tributyltin chloride and polychlorinated biphenyls by dietary uptake Contaminants Treatment Weight Concentration in feed of fisha (g) (mg/g wet wt) TBTC1
A B C D E
5.670.8 5.470.7 5.570.9 5.670.9 5.570.9
0.03570.001 1.370.1 2.470.2 1072 2071
PCBs
F G
5.770.4 5.770.4
o0.010 1.570.1
a
Weight of fish at beginning of the experiment.
2.4. Test system and rearing of fish 2. Materials and methods 2.1. Chemicals TBTCl (96% purity) was purchased from Aldrich Chemical Company (Milwaukee, WI, USA). PCB mixtures (Kanechlor-300, 400, 500, and 600; GC analytical standard) were purchased from GL Science Inc. (Tokyo, Japan). They were used without further purification. 2.2. Test fish Juvenile red sea bream (Pagrus major) were purchased from a public aquaculture center in Ibaraki. They were fed before the experiment with C-3000 formula (Kyowa Hakko, Tokyo, Japan). The body weights at the beginning of the experiment are shown in Table 1. All fish were already slightly contaminated by TBT at the beginning of the experiment, containing 9.370.6 ng/g body weight. No PCBs were detected. 2.3. Preparation of feed containing TBTCl or PCBs Feed was prepared by the method of Yamada et al. [14]. TBTCl or PCBs were dissolved in diethyl ether. The solution was gradually added to the feed and mixed in completely. Then the diethyl ether was evaporated overnight at room temperature. Five concentrations of TBTCl were prepared (treatments A–E; Table 1). The water content of the feed was 9.4–9.8%. Two concentrations of PCBs were also prepared (treatments F and G; Table 1). The water content of the feed was 8.3%.
Test fish were reared during an 8-week uptake experiment and a 4–6-week elimination experiment in the continuous flow-through system described by Yamada et al. [1], under various experimental conditions (Table 1). Seawater was filtered through activated carbon to remove P possible contaminants. The TBT and total PCB ( PCBs) concentrations in the filtered seawater were below P the detection limits (2 ng/L for TBT, 10 ng/L for PCBs). The temperature of the rearing water was maintained at 21.070.51C by a thermocontroller. The water was supplied to 50-L aquariums at the rate of 500 mL/min. Fish in the chemical treatments were fed on either TBTCl-treated or PCB-treated feed. Fish in the control treatments were fed on unmodified formula feed. The TBT concentration in the formula feed was 0.03570.001 mg/g, much lower than in the chemical treatments. The feeding rate was fixed on 0.027 (g feed/g fish/d) in all experiments. After 8 weeks of exposure, the fish were fed for a further 4 or 6 weeks on untreated formula feed to investigate the elimination of the TBTCl and PCBs. Air was introduced into the aquaria to maintain an adequate dissolved oxygen level in the water. The salinity of the seawater ranged from 31.5 to 33.0 psu. Under these conditions, the fish show no signs of tiredness or agitation during the experiment. During the uptake experiment, 3 fish were sampled in the 1st and 2nd weeks and every 2 weeks thereafter, and the concentrations of TBT and PCBs were analyzed. During the elimination experiment, the TBT concentrations in fish were determined every week. The PCB concentrations in fish were determined in the 1st, 2nd, 4th, and 6th weeks. The day before sampling fish were not fed on any feed.
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2.5. Chemical analysis
BMFd or BMFw ¼ ðCf 8 Cf 0 Þ=CF;
The concentration of TBT in fish was determined by the method described by Takami et al. [15]. A fish was homogenized with 1 N hydrochloric acid in a 1:1 (v/v) mixture of methanol and ethylacetate. TBT dissolved in the solvent was extracted with a 3:2 (v/v) mixture of ethylacetate and n-hexane. The extracted TBT was purified with both anion and cation ion-exchange resins. It was then converted to propyl derivatives with npropyl magnesium bromide. The propyl derivative of TBT was analyzed with a high-resolution gas chromatograph (HRGC; 15A; Shimadzu, Kyoto, Japan) and was detected by using a flame photometric detector (FPD) equipped with a tin-mode filter (610 nm). An Ultra-1 GC column (Hewlett-Packard, CA, USA) was used (length 25 m; internal diameter 0.32 mm; liquid phase: crosslinked methyl silicone gum; film thickness 0.25 mm). The column oven temperature was increased from 801C to 2501C, and both injector and detector temperatures were 2601C. The quantification of TBT was based on the known amount of tetrabutyltin as an internal standard. The average recovery of spiked TBT was 81% at 0.6 mg/ g. The coefficient of variation by this procedure was less than 5%, and the theoretical detection limit calculated from the least detectable amount of the instrument and the concentration ratio during preparation was 5.0 ng/g. The concentration of PCBs in the fish was determined by the alkaline digestion method of Wakimoto et al. [16] with minor modifications as described by Tanabe et al. [17,18]. A fish was digested in 1 N KOH–ethanol solution by refluxing for 1 h in a boiling water bath. Then PCBs were extracted into n-hexane. The hexane extracts were purified by the methods of Tanabe et al. [17] using concentrated H2SO4 and silica gel column chromatography. The PCBs were determined by HRGC (5890; Hewlett-Packard, CA, USA) equipped with a 63 Ni electron capture detector (ECD). A DB-1 GC column (J&W Scientific, CA, USA) was used (length 30 m; internal diameter 0.25 mm; film thickness 0.25 mm). The GC conditions were similar to those described by Tanabe et al. [18]. For the quantification of PCBs, an equivalent mixture of Kanechlor-300, 400, 500+600 was used as a standard. The sum of individually resolved P peaks was used to estimate the concentration of PCBs: The recoveries and detection limit calculated from standard deviation of the standard values were 94% and 10 ng/g wet wt, respectively.
where P BMFn is the BMF at the nth week; Cf n is the TBT or PCBs concentration in the fish at the nth week of theP uptake experiment (mg/g dry weight); Cf 0 is the TBT or PCBs concentration in the control fish; and CF is P the TBT or PCBs concentration in the feed. BMFd or BMFw is the BMF calculated on a dry- or wet-weight basis at the 8th week, when the TBT and PCB concentrations had reached equilibrium in all treatments. The elimination rate constant (k2 ) was calculated from the first-order model of the following equation:
2.6. Calculations of BMF, and uptake and elimination rate constants A BMF was calculated for the dietary uptake experiment by the following equation: BMFn ¼ ðCf n Cf 0 Þ=CF;
ð1Þ
CFLn ¼ CFL0 ek2 t ;
ð10 Þ
ð2Þ P
where CFLn is the TBT or PCBs concentration in the fish at nth week of the elimination experiment (mg/g wet weight); t is the period of the elimination experiment; k2 is Pthe elimination rate constant; and CFL0 is the TBT or PCBs concentration in the fish at the beginning of the elimination experiment. k2 is the observed elimination rate constant including growth dilution. The true elimination rate constant (ke ) was calculated from the following equation: k 2 ¼ ke þ G e ;
ð3Þ
where ke is the true elimination rate constant; and Ge is the growth rate constant. The uptake rate constant (k1 ) was estimated from the following equation, which is based on the work of Connell [19] and Yamada et al. [3]: BMFw ¼ k1 =k2 :
ð4Þ
The assimilation efficiency (AE) was calculated from the following equation: k1 CF k1 100 ¼ 100; ð5Þ 0:027CF 0:027 P where CF is the TBT or PCBs concentration in the feed (mg/g wet weight); and 0.027 is the feeding rate (g feed/g fish/d), described above. AE ¼
3. Results and discussion 3.1. Fish growth The growth of red sea bream is shown in Table 2. In all treatment groups, fish weights at the 8th week were higher than those at the beginning of the uptake experiment. The growth in treatment E (the highest TBT concentration in feed) was a little less than in treatment A (control treatment). The growth in treatment G (PCB treatment) was also a little less than in treatment F (control treatment for PCB exposure).
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Table 2 Growth of red sea bream during the experiments Ratio of fish weightb Treatment Weight of fisha (g) Uptake experiment (week)
TBTC1 A B C D E PCBs F G
Growth rate constant (Ge)c (/d)
Elimination experiment (week)
0
1
2
4
6
8
9
10
11
12
5.670.8 5.470.7 5.570.9 5.670.9 5.570.9
1.0 1.0 1.0 1.0 1.0
1.2 1.2 1.2 1.2 1.2
1.5 1.5 1.5 1.5 1.4
2.2 2.0 2.1 2.0 2.0
3.0 2.8 3.0 2.7 2.7
4.2 3.7 3.9 3.5 3.2
5.0 4.3 4.7 4.1 3.6
5.5 5.0 5.3 4.5 4.0
5.9 5.4 5.6 4.7 4.4
6.3 5.8 6.2 5.3 4.8
5.770.4 5.770.4
1.0 1.0
1.3 1.2
1.7 1.5
2.5 2.3
3.7 3.3
5.4 4.7
6.5 5.6
7.5 6.5
9.2 8.4
14 0.014 0.016 0.015 0.014 0.014 11 11
0.017 0.019
a
Weight of fish at the beginning of the experiment. Weight ratio (nth week)=weight at the nth week/weight at the beginning. c Growth rate constants were calculated with the data in the elimination experiment from the following equation: W ðtÞ ¼ W0 eGe t : b
The obvious differences between the chemical treatments and their controls were not shown in the growth rate constants (Ge ) during the elimination experiments. 3.2. TBT concentration in fish In the control treatment (treatment A), the TBT concentration in fish was low and remained fairly constant throughout the experiment (Fig. 1, panel B). It ranged from 6.5 to 14 ng/g (SD72.0). Changes in the TBT concentrations in fish during the uptake experiment are shown in Fig. 1. The concentrations in fish in treatments B–E increased remarkably in the uptake experiment: from 9.370.6 ng/g at the beginning of the experiment; to 6377, 110720, 300710, and 420770 ng/g at 1 week; to 110710, 170710, 5107120, and 7907130 ng/g at 6 weeks; to 130710, 190720, 510760, and 9107120 ng/g at 8 weeks. The bream accumulated the TBT by dietary uptake. The TBT concentration in the whole body was dependent on the TBT concentration in the feed; it was higher in fish fed with the feed containing a larger amount of TBTCl. In all treatments, the changes in the TBT concentrations from the 6th to the 8th weeks were small, which suggests that the TBT concentrations had reached equilibrium by the 6th week. Changes in the TBT concentrations during the elimination experiment are also shown in Fig. 1. The concentrations in fish decreased gradually with time, from 130 to 50 ng/g in treatment B, from 190 to 80 ng/g in treatment C, from 510 to 190 ng/g in treatment D, and from 910 to 260 ng/g in treatment E. The correlation coefficients of the regression analysis of Eq. (2) lie between –0.89 and –0.92 (n ¼ 15), and the correlations
are highly significant. The slope of each regression line is the observed elimination rate constant, k2 : 0.037/d for treatment B, 0.039/d for treatment C, 0.032/d for treatment D, and 0.040/d for treatment E (Table 4). The growth rate constant, Ge : 0.016/d for treatment B, 0.015/d for treatment C, 0.014/d for treatment D, and 0.014/d for treatment E (Table 2) was obtained. The true elimination rate constant, ke : 0.021/d for treatment B, 0.024/d for treatment C, 0.018/d for treatment D, and 0.026/d for treatment E was calculated from Eq. (3) with the values of k2 and Ge : The fact that these elimination rate constants are similar among the treatments suggests that the elimination rate constant of TBT from fish is independent of the TBT concentration in the fish. TBT is metabolized through the cytochrome P-450 dependent monooxygenase system in fish. It also inhibits cytochrome P-450 activity, yet the elimination rate constants did not change with the TBT concentrations in fish or feed. 3.3.
P
PCBs concentrations in fish
P In the control treatment (treatment F), the PCBs concentration in fish was below the detection limit (10 ng/g) throughout P the experiment. Changes in the PCBs concentration in fish during the uptake experiment are shown in Fig. 2. The concentration in fish (treatment G) increased from o10 to 760750 ng/g during the uptake experiment. PCBs were significantly bioaccumulated in fish by P dietary uptake. The change in the PCBs concentration from the 6thP to the 8th week was small, which suggests that the PCBs concentration had reached equilibrium by the 6th week.
K. Ikeda, H. Yamada / Water Research 37 (2003) 1497–1504
TBT concentration in fish (ng/g)
1000
100
C(2.4 g/g)
100 y=-0.039x+190 (r=0.90)
y=-0.037x+120 (r=0.91)
10
10 A(Control)
↓
uptake
1 0 10000 TBT concentration in fish (ng/g)
1000
B(1.3 g/g)
1501
2
4
6
elimination 8
10
12
1000
2
4
6
elimination
8
10
12
E(20 g/g)
1000
100
y=-0.032x+450 (r=0.92)
10
1
0 10000
D(10 g/g)
↓
uptake
1
100
y=-0.040x+820 (r=0.89)
10
↓
uptake 0
2
4
6
8
elimination 10
Experimental period (week)
12
1
↓
uptake 0
2
4
6
elimination
8
10
12
Experimental period (week)
Fig. 1. Changes in TBT concentrations in fish during the dietary uptake and elimination experiments.
P Changes in the PCBs concentration during the elimination experiment are also shown in Fig. 2. The concentration in fish decreased gradually with time, from 760 to 300 ng/g. The correlation between the P PCBs concentration and the elimination period (Eq. (2)) is highly significant (0.95, n ¼ 15). The observed elimination rate constant, k2 ; was 0.024/d (Table 5). The true elimination rate constant, ke : 0.005/d was calculated from Eq. (3) with the values of k2 and Ge (0.019/d, Table 2). These elimination rate constants were lower than that for TBT. The cytochrome P-450 pathway is induced by PCBs, yet PCBs were less easily eliminated than TBT in this study. 3.4. BMF The wet-weight BMFs of TBT at the 8th week (BMFw) are shown in Table 4. The dry-weight BMFs (BMFd) are shown in Table 3. The BMFds varied
significantly according to the TBT concentration in the feed, becoming smaller with increasing TBT concentration. Because the BMFds were all o1, TBT was not bioaccumulated in the fish at a higher concentration than in the feed. The dry-weight BMF (BMFd) of PCBs is shown in Table 3. Because the BMFd was >1, PCBs were bioaccumulated in the fish at a higher concentration than in the feed. 3.5. Calculated uptake rate constant ðk1 Þ and assimilation efficiency The k1 of TBTCl was calculated from Eq. (4) (Table 4). It became smaller with increasing TBT concentration in the feed. AEs of TBTCl were calculated from Eq. (5) with the values of k1 and feeding rate (Table 4). They also became smaller with increasing TBT concentration in
K. Ikeda, H. Yamada / Water Research 37 (2003) 1497–1504
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the feed. The BMF decreased with increasing TBT concentration in feed as a result of the decrease in k1 and AE with the increase in TBT concentration in feed. k2 was independent of the TBT concentration in feed. These results show that the bioaccumulation factor of
G(1.5 g/g)
ΣPCBs concentration in fish (ng/g)
1000
y=-0.024x+730 (r=0.95)
100
10
↓
uptake
elimination
TBTCl decreases in proportion to the increase in TBT concentrations in feed. Opperhuizen et al. [20] also reported that assimilation efficiencies of PCBs in fish decrease approximately from 50% to 25% with increasing PCBs concentration in feed after dietary exposure to five different concentrations ranging from 7.1 to 1400 mg/g. Hendriks et al. [21] reported that uptake rate constants, (near-)equilibrium accumulation and magnification ratios in aquatic species decreased with the reciprocal square root of the exposure concentration of transition metals. The BCF of TBTO ranged from 5000 to 9400 depending on the concentration in the rearing seawater and a larger BCF was obtained when the fish were reared in seawater containing a smaller amount of TBTO [1]. In general, contaminant concentrations in feed under experimental conditions will be higher than those in preys in the field. Hence, it is likely that laboratory measurements of BMF and AE will underestimate the actual values under field conditions. It is suggested that the experimental concentration of contaminant in feed should approach to the field condition as much as possible. The k1 of PCBs was calculated from Eq. (4) (Table 5) and is larger than those of TBTCl. The AE of PCBs was calculated from Eq. (5) (Table 5) and is also larger than those of TBTCl. These results suggest that PCBs in feed are more easily absorbed from the intestine than TBTCl. The AE of 56% in present study was higher than the range of 44% to 53% for guppies fed PCBs at 7.1– 150 mg/g of feed [20], but was smaller than the range of 67–93% for carp, determined directly from the difference in PCB concentrations between feed and feces [22].
1 0
2
4
6
8
10
12
14
Experimental period (week) P Fig. 2. Change in total PCB ( PCBs) concentrations in fish during the dietary uptake and elimination experiments.
3.6. Biomagnification of TBTCl and PCBs The biomagnification of TBTCl in treatment B was compared with that of PCBs in treatment G (Table 5). The concentrations in feed were similar.
Table 3 P Concentrations of TBT and PCBs in fish and feed, and biomagnification factors Contaminants
Treatment
(x) Concentration in feed (mg/g dry wt)
(y) Concentration in fisha (mg/g dry wt)
Biomagnification factorb (BMFd)
TBT
B C D E
1.470.1 2.770.2 1172 2371
0.4570.01 0.6670.10 1.870.3 3.370.4
0.3070.01 0.2370.04 0.1670.02 0.1570.02
1.670.1
2.770.2
1.770.1
P
PCBs
a
G P
Concentrations of TBT or PCBs in fish at the end of the uptake experiment (day 56). BMF calculated on a dry-weight basis. BMFd=(y(concentration in control fish [0.026 mg/g dry wt])/x). b
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Table 4 Kinetic parameters of the dietary bioaccumulation of TBTCl Treatment (x) (y) TBT concentration in TBT concentration BMFwa Elimination rate constant Uptake rate Assimilation efficiency feed (mg/g wet) in fish (mg/g wet) (k2 ) (/d) constant (k1 ) (/d) (AE)b (%) B C D E
1.370.1 2.470.2 1072 2071 a b
0.1370.01 0.1970.02 0.5170.06 0.9170.12
0.095 0.075 0.050 0.045
0.037 0.039 0.032 0.040
0.0035 0.0029 0.0016 0.0018
13 11 5.9 6.7
BMFw=(y(concentration in control fish [7.3 ng/g wet wt])/x)=k1 =k2 : k1 ð=dÞ CFðmg TBT=g feedÞ 100 ¼ k1 100=0:027: Assimilation efficiencyð%Þ ¼ CFðmg TBT=g feedÞ 0:027ðg feed=g fish=dÞ
Table 5 Comparison of kinetic parameters related to dietary TBTCl uptake with those of PCBs. Contaminants
Concentration in feed (mg/g wet wt)
Concentration in fisha (mg/g wet wt)
k1 (/d)
k2 (/d)
BMFd
Assimilation efficiency (%)
TBTC1 PCBs
1.370.1 1.570.1
0.1370.01 0.7670.05
0.0035 0.015
0.037 0.024
0.3070.01 1.770.1
13 56
a
TBT concentration in fish at the end of the uptake experiment.
The BMF of TBTCl (0.30) is a sixth of that of the PCBs (1.7). The k1 and AE of TBTCl (0.0035/d and 13%) are also smaller than those of the PCBs (0.015/d and 56%). The k2 and ke of TBT (0.037/d and 0.021/d) are larger than that those of the PCBs (0.024/d and 0.005/d). Judging from the BMF and AE, the risk of the bioaccumulation of TBTCl by dietary intake (through the food chain) might be smaller than that of PCBs. Judging from k1 ; k2 ; ke and AE, there seem to be two reasons why TBT is less easily bioaccumulated than PCBs: (1) TBTCl in feed is absorbed less easily than PCBs, and (2) TBT in fish is eliminated more easily than PCBs. 4. Conclusions 1. Red sea bream bioaccumulated TBTCl in feed by dietary uptake. This bioaccumulation depends on the TBT concentration in feed: TBT concentration in fish increased with increasing TBT concentration in feed. 2. The BMF and AE decreased from 0.30 to 0.15 and from 13% to 5.9%, respectively, as the TBT concentration in feed increased from 1.3 to 20 mg/g. The decrease in BMF was the result of the decrease in k1 and AE with the increase in TBT concentration in feed. k2 was independent of the TBT concentration in feed. 3. Laboratory measurements of the BMF and AE of TBTCl will underestimate actual field values if highly contaminated feed is used.
4. Judging from the BMF and AE, the risk of the bioaccumulation of TBTCl through the food chain might be smaller than that of PCBs.
Acknowledgements We are grateful to Dr. Jiro Koyama for his advice and critical reading of this paper.
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major) by two uptake pathways: dietary uptake and direct uptake from water. Environ Toxicol Chem 1994;13(9):1415–22. Takami K, Okumura T, Yamasaki H, Nakamoto M. Determination of triphenyltin and tributyltin compounds in fish and shellfish by capillary GC. Bunseki Kagaku 1988;37:449–55 (in Japanese). Wakimoto T, Tatsukawa R, Ogawa T. Analytical method of PCBs. J Environ Pollut Control 1971;7:517–22 (in Japanese). Tanabe S, Tatsukawa R, Phillips DJH. Mussels as bioindicators of PCB isomers and congeners in greenlipped mussels (Perna viridis) in Hong Kong waters. Environ Pollut 1987;47:41–62. Tanabe S, Sung JK, Choi DY, Baba N, Kiyota M, Yoshida K, Tatsukawa R. Persistent organochlorine residues in northern fur seal from the pacific coast of Japan since 1971. Environ Pollut 1994;85:305–14. Connell DW. Bioaccumulation behavior of persistent organic chemicals with aquatic organisms. Rev Environ Contam Toxicol 1988;101:117–54. Opperhuizen A, Schrap SM. Uptake efficiencies of two polychlorobiphenyls in fish after dietary exposure to five different concentrations. Chemosphere 1988;17(2): 253–62. Hendriks AJ, Heikens A. The power of size. 2. Rate constants and equilibrium ratios for accumulation of inorganic substances related to species weight. Environ Toxicol Chem 2001;20:1421–37. Tanabe S, Maruyama K, Tatsukawa R. Absorption efficiency and biological half-life of individual chlorobiphenyls in carp (Cyprinus carpio) orally exposed to Kanechlor products. Agric Biol Chem 1982;46(4):891–8.
Changes in dietary bioaccumulation of tributyltin chloride (TBTCl) in red sea bream (Pagrus major) with the concentration in feed Kumiko Ikeda*, Hisashi Yamada National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi Ohno, Saeki, Hiroshima 739-0452, Japan Received 16 July 2001; received in revised form 24 September 2002; accepted 4 October 2002
Abstract The effect of the concentration of tributyltin (TBT) in feed on the dietary bioaccumulation of tributyltin chloride (TBTCl) was studied in an 8-week uptake experiment and a 4-week elimination experiment using red sea bream (Pagrus major). The biomagnification factor (BMF) and the assimilation efficiency (AE) decreased from 0.30 to 0.15 and from 13% to 5.9%, respectively, as the TBT concentration in feed increased from 1.3 to 20 mg/g. The elimination rate constant (k2 ) was independent of the TBT concentration in the fish. Laboratory measurements of the BMF and AE of TBTCl underestimate actual field values if highly contaminated feed is used. Judging from the BMF and AE, the risk of the bioaccumulation of TBTCl through the food chain might be smaller than that of polychlorinated biphenyls. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bioaccumulation; TBTCl; Red sea bream; Biomagnification factor; Assimilation efficiency
1. Introduction Fish bioaccumulate contaminants through two main pathways: direct uptake from the water across the gills (bioconcentration) and dietary uptake (biomagnification). Information on dietary uptake is necessary for a better understanding of bioaccumulation through the food chain in aquatic ecosystems. The bioaccumulation of contaminants in fish is evaluated as the balance between uptake rate and elimination rate, called the bioaccumulation factor. The uptake and elimination rate constants of contaminants in fish depend on various biological or physicochemical conditions, such as fish species [1], size of experimental fish [2], and temperature of rearing water [3]. The concentrations of contaminants in the rearing *Corresponding author. Tel.: +81-829-55-0666; fax: +81829-54-1216. E-mail address: [email protected] (K. Ikeda).
water and feed also play an important role. The bioconcentration factor increases as the concentration of the contaminants in water decreases [4,5]. The biomagnification factor may also depend on the concentration of contaminants in feed, but this relationship is poorly understood. Two influences may affect the biomagnification factor: changes in the assimilation efficiency, which depends on the concentration of contaminants in feed, and changes in the elimination rate constant, which depends on the concentration in fish fed the feed. The concentrations of contaminants in fish possibly influence changes in the elimination rate constant through changes in the activity of xenobiotic-metabolizing enzymes such as cytochrome P-450. For instance, tributyltin (TBT) and polychlorinated biphenyls (PCBs) are metabolized through the cytochrome P-450 dependent monooxygenase system [6,7]. TBT also inhibits cytochrome P-450 activity [8–11]. On the other hand, PCBs induce cytochrome P-450 [12,13]. These facts
0043-1354/03/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 3 - 1 3 5 4 ( 0 2 ) 0 0 5 2 1 - 3
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indicate that the concentrations of contaminants in fish affect the elimination of the contaminants. Therefore, it is necessary to clarify how changes in the biomagnification factor and the uptake and elimination rate constants depend on the concentrations of contaminants in feed to understand the mechanism of bioaccumulation through the food chain. We studied how changes in the kinetic parameters that affect the bioaccumulation of tributyltin chloride (TBTCl) through dietary uptake—biomagnification factor, and uptake and elimination rate constants— depend on the concentration of TBT in feed. We used long-term uptake and elimination experiments with a marine fish, the red sea bream (Pagrus major). We also assessed the probability of TBTCl bioaccumulation through the food chain by comparing the kinetic parameters of TBTCl and PCBs, the major organic contaminants in marine environment.
Table 1 Experimental conditions for the bioaccumulation of tributyltin chloride and polychlorinated biphenyls by dietary uptake Contaminants Treatment Weight Concentration in feed of fisha (g) (mg/g wet wt) TBTC1
A B C D E
5.670.8 5.470.7 5.570.9 5.670.9 5.570.9
0.03570.001 1.370.1 2.470.2 1072 2071
PCBs
F G
5.770.4 5.770.4
o0.010 1.570.1
a
Weight of fish at beginning of the experiment.
2.4. Test system and rearing of fish 2. Materials and methods 2.1. Chemicals TBTCl (96% purity) was purchased from Aldrich Chemical Company (Milwaukee, WI, USA). PCB mixtures (Kanechlor-300, 400, 500, and 600; GC analytical standard) were purchased from GL Science Inc. (Tokyo, Japan). They were used without further purification. 2.2. Test fish Juvenile red sea bream (Pagrus major) were purchased from a public aquaculture center in Ibaraki. They were fed before the experiment with C-3000 formula (Kyowa Hakko, Tokyo, Japan). The body weights at the beginning of the experiment are shown in Table 1. All fish were already slightly contaminated by TBT at the beginning of the experiment, containing 9.370.6 ng/g body weight. No PCBs were detected. 2.3. Preparation of feed containing TBTCl or PCBs Feed was prepared by the method of Yamada et al. [14]. TBTCl or PCBs were dissolved in diethyl ether. The solution was gradually added to the feed and mixed in completely. Then the diethyl ether was evaporated overnight at room temperature. Five concentrations of TBTCl were prepared (treatments A–E; Table 1). The water content of the feed was 9.4–9.8%. Two concentrations of PCBs were also prepared (treatments F and G; Table 1). The water content of the feed was 8.3%.
Test fish were reared during an 8-week uptake experiment and a 4–6-week elimination experiment in the continuous flow-through system described by Yamada et al. [1], under various experimental conditions (Table 1). Seawater was filtered through activated carbon to remove P possible contaminants. The TBT and total PCB ( PCBs) concentrations in the filtered seawater were below P the detection limits (2 ng/L for TBT, 10 ng/L for PCBs). The temperature of the rearing water was maintained at 21.070.51C by a thermocontroller. The water was supplied to 50-L aquariums at the rate of 500 mL/min. Fish in the chemical treatments were fed on either TBTCl-treated or PCB-treated feed. Fish in the control treatments were fed on unmodified formula feed. The TBT concentration in the formula feed was 0.03570.001 mg/g, much lower than in the chemical treatments. The feeding rate was fixed on 0.027 (g feed/g fish/d) in all experiments. After 8 weeks of exposure, the fish were fed for a further 4 or 6 weeks on untreated formula feed to investigate the elimination of the TBTCl and PCBs. Air was introduced into the aquaria to maintain an adequate dissolved oxygen level in the water. The salinity of the seawater ranged from 31.5 to 33.0 psu. Under these conditions, the fish show no signs of tiredness or agitation during the experiment. During the uptake experiment, 3 fish were sampled in the 1st and 2nd weeks and every 2 weeks thereafter, and the concentrations of TBT and PCBs were analyzed. During the elimination experiment, the TBT concentrations in fish were determined every week. The PCB concentrations in fish were determined in the 1st, 2nd, 4th, and 6th weeks. The day before sampling fish were not fed on any feed.
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2.5. Chemical analysis
BMFd or BMFw ¼ ðCf 8 Cf 0 Þ=CF;
The concentration of TBT in fish was determined by the method described by Takami et al. [15]. A fish was homogenized with 1 N hydrochloric acid in a 1:1 (v/v) mixture of methanol and ethylacetate. TBT dissolved in the solvent was extracted with a 3:2 (v/v) mixture of ethylacetate and n-hexane. The extracted TBT was purified with both anion and cation ion-exchange resins. It was then converted to propyl derivatives with npropyl magnesium bromide. The propyl derivative of TBT was analyzed with a high-resolution gas chromatograph (HRGC; 15A; Shimadzu, Kyoto, Japan) and was detected by using a flame photometric detector (FPD) equipped with a tin-mode filter (610 nm). An Ultra-1 GC column (Hewlett-Packard, CA, USA) was used (length 25 m; internal diameter 0.32 mm; liquid phase: crosslinked methyl silicone gum; film thickness 0.25 mm). The column oven temperature was increased from 801C to 2501C, and both injector and detector temperatures were 2601C. The quantification of TBT was based on the known amount of tetrabutyltin as an internal standard. The average recovery of spiked TBT was 81% at 0.6 mg/ g. The coefficient of variation by this procedure was less than 5%, and the theoretical detection limit calculated from the least detectable amount of the instrument and the concentration ratio during preparation was 5.0 ng/g. The concentration of PCBs in the fish was determined by the alkaline digestion method of Wakimoto et al. [16] with minor modifications as described by Tanabe et al. [17,18]. A fish was digested in 1 N KOH–ethanol solution by refluxing for 1 h in a boiling water bath. Then PCBs were extracted into n-hexane. The hexane extracts were purified by the methods of Tanabe et al. [17] using concentrated H2SO4 and silica gel column chromatography. The PCBs were determined by HRGC (5890; Hewlett-Packard, CA, USA) equipped with a 63 Ni electron capture detector (ECD). A DB-1 GC column (J&W Scientific, CA, USA) was used (length 30 m; internal diameter 0.25 mm; film thickness 0.25 mm). The GC conditions were similar to those described by Tanabe et al. [18]. For the quantification of PCBs, an equivalent mixture of Kanechlor-300, 400, 500+600 was used as a standard. The sum of individually resolved P peaks was used to estimate the concentration of PCBs: The recoveries and detection limit calculated from standard deviation of the standard values were 94% and 10 ng/g wet wt, respectively.
where P BMFn is the BMF at the nth week; Cf n is the TBT or PCBs concentration in the fish at the nth week of theP uptake experiment (mg/g dry weight); Cf 0 is the TBT or PCBs concentration in the control fish; and CF is P the TBT or PCBs concentration in the feed. BMFd or BMFw is the BMF calculated on a dry- or wet-weight basis at the 8th week, when the TBT and PCB concentrations had reached equilibrium in all treatments. The elimination rate constant (k2 ) was calculated from the first-order model of the following equation:
2.6. Calculations of BMF, and uptake and elimination rate constants A BMF was calculated for the dietary uptake experiment by the following equation: BMFn ¼ ðCf n Cf 0 Þ=CF;
ð1Þ
CFLn ¼ CFL0 ek2 t ;
ð10 Þ
ð2Þ P
where CFLn is the TBT or PCBs concentration in the fish at nth week of the elimination experiment (mg/g wet weight); t is the period of the elimination experiment; k2 is Pthe elimination rate constant; and CFL0 is the TBT or PCBs concentration in the fish at the beginning of the elimination experiment. k2 is the observed elimination rate constant including growth dilution. The true elimination rate constant (ke ) was calculated from the following equation: k 2 ¼ ke þ G e ;
ð3Þ
where ke is the true elimination rate constant; and Ge is the growth rate constant. The uptake rate constant (k1 ) was estimated from the following equation, which is based on the work of Connell [19] and Yamada et al. [3]: BMFw ¼ k1 =k2 :
ð4Þ
The assimilation efficiency (AE) was calculated from the following equation: k1 CF k1 100 ¼ 100; ð5Þ 0:027CF 0:027 P where CF is the TBT or PCBs concentration in the feed (mg/g wet weight); and 0.027 is the feeding rate (g feed/g fish/d), described above. AE ¼
3. Results and discussion 3.1. Fish growth The growth of red sea bream is shown in Table 2. In all treatment groups, fish weights at the 8th week were higher than those at the beginning of the uptake experiment. The growth in treatment E (the highest TBT concentration in feed) was a little less than in treatment A (control treatment). The growth in treatment G (PCB treatment) was also a little less than in treatment F (control treatment for PCB exposure).
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Table 2 Growth of red sea bream during the experiments Ratio of fish weightb Treatment Weight of fisha (g) Uptake experiment (week)
TBTC1 A B C D E PCBs F G
Growth rate constant (Ge)c (/d)
Elimination experiment (week)
0
1
2
4
6
8
9
10
11
12
5.670.8 5.470.7 5.570.9 5.670.9 5.570.9
1.0 1.0 1.0 1.0 1.0
1.2 1.2 1.2 1.2 1.2
1.5 1.5 1.5 1.5 1.4
2.2 2.0 2.1 2.0 2.0
3.0 2.8 3.0 2.7 2.7
4.2 3.7 3.9 3.5 3.2
5.0 4.3 4.7 4.1 3.6
5.5 5.0 5.3 4.5 4.0
5.9 5.4 5.6 4.7 4.4
6.3 5.8 6.2 5.3 4.8
5.770.4 5.770.4
1.0 1.0
1.3 1.2
1.7 1.5
2.5 2.3
3.7 3.3
5.4 4.7
6.5 5.6
7.5 6.5
9.2 8.4
14 0.014 0.016 0.015 0.014 0.014 11 11
0.017 0.019
a
Weight of fish at the beginning of the experiment. Weight ratio (nth week)=weight at the nth week/weight at the beginning. c Growth rate constants were calculated with the data in the elimination experiment from the following equation: W ðtÞ ¼ W0 eGe t : b
The obvious differences between the chemical treatments and their controls were not shown in the growth rate constants (Ge ) during the elimination experiments. 3.2. TBT concentration in fish In the control treatment (treatment A), the TBT concentration in fish was low and remained fairly constant throughout the experiment (Fig. 1, panel B). It ranged from 6.5 to 14 ng/g (SD72.0). Changes in the TBT concentrations in fish during the uptake experiment are shown in Fig. 1. The concentrations in fish in treatments B–E increased remarkably in the uptake experiment: from 9.370.6 ng/g at the beginning of the experiment; to 6377, 110720, 300710, and 420770 ng/g at 1 week; to 110710, 170710, 5107120, and 7907130 ng/g at 6 weeks; to 130710, 190720, 510760, and 9107120 ng/g at 8 weeks. The bream accumulated the TBT by dietary uptake. The TBT concentration in the whole body was dependent on the TBT concentration in the feed; it was higher in fish fed with the feed containing a larger amount of TBTCl. In all treatments, the changes in the TBT concentrations from the 6th to the 8th weeks were small, which suggests that the TBT concentrations had reached equilibrium by the 6th week. Changes in the TBT concentrations during the elimination experiment are also shown in Fig. 1. The concentrations in fish decreased gradually with time, from 130 to 50 ng/g in treatment B, from 190 to 80 ng/g in treatment C, from 510 to 190 ng/g in treatment D, and from 910 to 260 ng/g in treatment E. The correlation coefficients of the regression analysis of Eq. (2) lie between –0.89 and –0.92 (n ¼ 15), and the correlations
are highly significant. The slope of each regression line is the observed elimination rate constant, k2 : 0.037/d for treatment B, 0.039/d for treatment C, 0.032/d for treatment D, and 0.040/d for treatment E (Table 4). The growth rate constant, Ge : 0.016/d for treatment B, 0.015/d for treatment C, 0.014/d for treatment D, and 0.014/d for treatment E (Table 2) was obtained. The true elimination rate constant, ke : 0.021/d for treatment B, 0.024/d for treatment C, 0.018/d for treatment D, and 0.026/d for treatment E was calculated from Eq. (3) with the values of k2 and Ge : The fact that these elimination rate constants are similar among the treatments suggests that the elimination rate constant of TBT from fish is independent of the TBT concentration in the fish. TBT is metabolized through the cytochrome P-450 dependent monooxygenase system in fish. It also inhibits cytochrome P-450 activity, yet the elimination rate constants did not change with the TBT concentrations in fish or feed. 3.3.
P
PCBs concentrations in fish
P In the control treatment (treatment F), the PCBs concentration in fish was below the detection limit (10 ng/g) throughout P the experiment. Changes in the PCBs concentration in fish during the uptake experiment are shown in Fig. 2. The concentration in fish (treatment G) increased from o10 to 760750 ng/g during the uptake experiment. PCBs were significantly bioaccumulated in fish by P dietary uptake. The change in the PCBs concentration from the 6thP to the 8th week was small, which suggests that the PCBs concentration had reached equilibrium by the 6th week.
K. Ikeda, H. Yamada / Water Research 37 (2003) 1497–1504
TBT concentration in fish (ng/g)
1000
100
C(2.4 g/g)
100 y=-0.039x+190 (r=0.90)
y=-0.037x+120 (r=0.91)
10
10 A(Control)
↓
uptake
1 0 10000 TBT concentration in fish (ng/g)
1000
B(1.3 g/g)
1501
2
4
6
elimination 8
10
12
1000
2
4
6
elimination
8
10
12
E(20 g/g)
1000
100
y=-0.032x+450 (r=0.92)
10
1
0 10000
D(10 g/g)
↓
uptake
1
100
y=-0.040x+820 (r=0.89)
10
↓
uptake 0
2
4
6
8
elimination 10
Experimental period (week)
12
1
↓
uptake 0
2
4
6
elimination
8
10
12
Experimental period (week)
Fig. 1. Changes in TBT concentrations in fish during the dietary uptake and elimination experiments.
P Changes in the PCBs concentration during the elimination experiment are also shown in Fig. 2. The concentration in fish decreased gradually with time, from 760 to 300 ng/g. The correlation between the P PCBs concentration and the elimination period (Eq. (2)) is highly significant (0.95, n ¼ 15). The observed elimination rate constant, k2 ; was 0.024/d (Table 5). The true elimination rate constant, ke : 0.005/d was calculated from Eq. (3) with the values of k2 and Ge (0.019/d, Table 2). These elimination rate constants were lower than that for TBT. The cytochrome P-450 pathway is induced by PCBs, yet PCBs were less easily eliminated than TBT in this study. 3.4. BMF The wet-weight BMFs of TBT at the 8th week (BMFw) are shown in Table 4. The dry-weight BMFs (BMFd) are shown in Table 3. The BMFds varied
significantly according to the TBT concentration in the feed, becoming smaller with increasing TBT concentration. Because the BMFds were all o1, TBT was not bioaccumulated in the fish at a higher concentration than in the feed. The dry-weight BMF (BMFd) of PCBs is shown in Table 3. Because the BMFd was >1, PCBs were bioaccumulated in the fish at a higher concentration than in the feed. 3.5. Calculated uptake rate constant ðk1 Þ and assimilation efficiency The k1 of TBTCl was calculated from Eq. (4) (Table 4). It became smaller with increasing TBT concentration in the feed. AEs of TBTCl were calculated from Eq. (5) with the values of k1 and feeding rate (Table 4). They also became smaller with increasing TBT concentration in
K. Ikeda, H. Yamada / Water Research 37 (2003) 1497–1504
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the feed. The BMF decreased with increasing TBT concentration in feed as a result of the decrease in k1 and AE with the increase in TBT concentration in feed. k2 was independent of the TBT concentration in feed. These results show that the bioaccumulation factor of
G(1.5 g/g)
ΣPCBs concentration in fish (ng/g)
1000
y=-0.024x+730 (r=0.95)
100
10
↓
uptake
elimination
TBTCl decreases in proportion to the increase in TBT concentrations in feed. Opperhuizen et al. [20] also reported that assimilation efficiencies of PCBs in fish decrease approximately from 50% to 25% with increasing PCBs concentration in feed after dietary exposure to five different concentrations ranging from 7.1 to 1400 mg/g. Hendriks et al. [21] reported that uptake rate constants, (near-)equilibrium accumulation and magnification ratios in aquatic species decreased with the reciprocal square root of the exposure concentration of transition metals. The BCF of TBTO ranged from 5000 to 9400 depending on the concentration in the rearing seawater and a larger BCF was obtained when the fish were reared in seawater containing a smaller amount of TBTO [1]. In general, contaminant concentrations in feed under experimental conditions will be higher than those in preys in the field. Hence, it is likely that laboratory measurements of BMF and AE will underestimate the actual values under field conditions. It is suggested that the experimental concentration of contaminant in feed should approach to the field condition as much as possible. The k1 of PCBs was calculated from Eq. (4) (Table 5) and is larger than those of TBTCl. The AE of PCBs was calculated from Eq. (5) (Table 5) and is also larger than those of TBTCl. These results suggest that PCBs in feed are more easily absorbed from the intestine than TBTCl. The AE of 56% in present study was higher than the range of 44% to 53% for guppies fed PCBs at 7.1– 150 mg/g of feed [20], but was smaller than the range of 67–93% for carp, determined directly from the difference in PCB concentrations between feed and feces [22].
1 0
2
4
6
8
10
12
14
Experimental period (week) P Fig. 2. Change in total PCB ( PCBs) concentrations in fish during the dietary uptake and elimination experiments.
3.6. Biomagnification of TBTCl and PCBs The biomagnification of TBTCl in treatment B was compared with that of PCBs in treatment G (Table 5). The concentrations in feed were similar.
Table 3 P Concentrations of TBT and PCBs in fish and feed, and biomagnification factors Contaminants
Treatment
(x) Concentration in feed (mg/g dry wt)
(y) Concentration in fisha (mg/g dry wt)
Biomagnification factorb (BMFd)
TBT
B C D E
1.470.1 2.770.2 1172 2371
0.4570.01 0.6670.10 1.870.3 3.370.4
0.3070.01 0.2370.04 0.1670.02 0.1570.02
1.670.1
2.770.2
1.770.1
P
PCBs
a
G P
Concentrations of TBT or PCBs in fish at the end of the uptake experiment (day 56). BMF calculated on a dry-weight basis. BMFd=(y(concentration in control fish [0.026 mg/g dry wt])/x). b
K. Ikeda, H. Yamada / Water Research 37 (2003) 1497–1504
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Table 4 Kinetic parameters of the dietary bioaccumulation of TBTCl Treatment (x) (y) TBT concentration in TBT concentration BMFwa Elimination rate constant Uptake rate Assimilation efficiency feed (mg/g wet) in fish (mg/g wet) (k2 ) (/d) constant (k1 ) (/d) (AE)b (%) B C D E
1.370.1 2.470.2 1072 2071 a b
0.1370.01 0.1970.02 0.5170.06 0.9170.12
0.095 0.075 0.050 0.045
0.037 0.039 0.032 0.040
0.0035 0.0029 0.0016 0.0018
13 11 5.9 6.7
BMFw=(y(concentration in control fish [7.3 ng/g wet wt])/x)=k1 =k2 : k1 ð=dÞ CFðmg TBT=g feedÞ 100 ¼ k1 100=0:027: Assimilation efficiencyð%Þ ¼ CFðmg TBT=g feedÞ 0:027ðg feed=g fish=dÞ
Table 5 Comparison of kinetic parameters related to dietary TBTCl uptake with those of PCBs. Contaminants
Concentration in feed (mg/g wet wt)
Concentration in fisha (mg/g wet wt)
k1 (/d)
k2 (/d)
BMFd
Assimilation efficiency (%)
TBTC1 PCBs
1.370.1 1.570.1
0.1370.01 0.7670.05
0.0035 0.015
0.037 0.024
0.3070.01 1.770.1
13 56
a
TBT concentration in fish at the end of the uptake experiment.
The BMF of TBTCl (0.30) is a sixth of that of the PCBs (1.7). The k1 and AE of TBTCl (0.0035/d and 13%) are also smaller than those of the PCBs (0.015/d and 56%). The k2 and ke of TBT (0.037/d and 0.021/d) are larger than that those of the PCBs (0.024/d and 0.005/d). Judging from the BMF and AE, the risk of the bioaccumulation of TBTCl by dietary intake (through the food chain) might be smaller than that of PCBs. Judging from k1 ; k2 ; ke and AE, there seem to be two reasons why TBT is less easily bioaccumulated than PCBs: (1) TBTCl in feed is absorbed less easily than PCBs, and (2) TBT in fish is eliminated more easily than PCBs. 4. Conclusions 1. Red sea bream bioaccumulated TBTCl in feed by dietary uptake. This bioaccumulation depends on the TBT concentration in feed: TBT concentration in fish increased with increasing TBT concentration in feed. 2. The BMF and AE decreased from 0.30 to 0.15 and from 13% to 5.9%, respectively, as the TBT concentration in feed increased from 1.3 to 20 mg/g. The decrease in BMF was the result of the decrease in k1 and AE with the increase in TBT concentration in feed. k2 was independent of the TBT concentration in feed. 3. Laboratory measurements of the BMF and AE of TBTCl will underestimate actual field values if highly contaminated feed is used.
4. Judging from the BMF and AE, the risk of the bioaccumulation of TBTCl through the food chain might be smaller than that of PCBs.
Acknowledgements We are grateful to Dr. Jiro Koyama for his advice and critical reading of this paper.
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