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Tissue distribution and depuration of tributyltin for field-exposed Mytilus edulis
Marine Environmental
ELSEVlER
Research, Vol. 40, No. 4, pp. 409421, 1995 Copyright 01995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0141-1136/95 $9.50+0.00
0141-1136(94)00004-6
Tissue Distribution and Depuration of Tributyltin for Field-Exposed Mytilus edulis David S. Page,‘* Tamara M. Dassanayake”
& Edward S. Gilfillan’
“Bowdoin College Chemistry Department, Brunswick, Maine 04011, USA ‘Bowdoin College Marine Research Laboratory, Brunswick, Maine 04011, USA (Received 26 November
1994; revised version received 10 January 1995; accepted 23 January 1995)
ABSTRACT The disposition of tributyltin (TBT) within Mytilus edulis tissues and the depuration of TBT from various organs was determined for field populations of animals chronically exposed to environmental sources of TBT. Analysis of dissected organs demonstrated that TBT accumulated to the greatest extent in gill tissue, with TBT concentrations approximately twice that of whole-animal homogenates. A transplant study showed that the depuration of TBTfrom gill tissue and digestive gland tissue is a biphasic two-compartment process involving a rapid TBT loss process and a concurrent slower TBT depuration process. Whole-body and gonadal tissue depuration followed a slower monophasic depuration process. Depuration half ltfe values ranged from 2.2 -5.3 d for the fast depuration component and 28-69 dfor the slow component.
INTRODUCTION Tributyltin (TBT) is acutely toxic to many species of marine animals at very low water concentrations &g/liter-ng/liter range) (Maguire, 1987; Thain et al., 1987; Joshi & Gupta, 1990). While TBT antifoulants have seen widespread use in the past, their use has been restricted in many areas due to evidence of adverse effects on marine life (Waldock et al., 1987; Anon., 1994). Even though regulation has reduced the levels of environmental TBT (Waite et al., 1991; Anon., 1994), activities at localized sources such as shipyards, and commercial ship traffic remain as potential sources *To whom correspondence
should be addressed. 409
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of TBT. In addition, there is evidence that the resuspension of TBT-contaminated sediments in marinas and shipyards can serve as an on-going source of TBT in the water column and thus to biota (Harris et al., 1991; Kure & Depledge, 1994; Ritsema, 1994). In addition, TBT from contaminated sediment can be ingested directly by deposit-feeders (Langston & Burt, 1991). Consistent with the use of Myths edulis as a sentinel organism for the detection of marine pollutants in the Mussel Watch Program (Widdows & Donkin, 1992; O’Connor et al., 1994), data have been reported for TBT in mussels from various locations (e.g. Page & Widdows, 1991; Uhler et al., 1993). For a given pollutant exposure concentration, the steady-state concentration of a lipophilic pollutant such as TBT in the tissues of marine animals reflects the balance between the processes of absorption and depuration as described by various pharmacokinetic models (Barron et al., 1990). Myths edulis has been found to absorb TBT from seawater and TBT-exposed phytoplankton with bioconcentration factors inversely proportional to the TBT-source aqueous concentration (Laughlin et al., 1986; Zuolian & Jensen, 1989; Salazar & Salazar, in press). There is evidence that the rate of TBT absorption depends on the TBT-source aqueous concentration. For example, in a field transplant study Salazar & Salazar (in press) found that adult and juvenile Myths gulloprovinciulis reached a steady state between water and tissue TBT concentrations in less than 21 d of exposure at a seawater TBT concentration of about 70 ng/liter. For exposure to TBT at about 450 ng/liter in a transplant study, adult Myths gulloprovincialis required over 60 d to reach an apparent steady state between water and tissue TBT concentrations (Salazar et al., 1987). Absorption of 14C-labeled tributyltin oxide by various organs of Myths edulis was greatest in gill and viscera (Laughlin et al., 1986) indicating a partitioning process within the animals. There is a wide range of reported TBT depuration rates for Myths edulis. Laughlin et al. (1986) measured depuration half-life values of 1.94.1 d for the various tissues of laboratory-exposed mussels. Other workers found TBT (as total organic tin) to depurate from field-exposed mussels with a half-life of 40 d (Zuolian & Jensen, 1989). In a reciprocal transplant study with adult and juvenile Myths guZloproviniaZis(Salazar & Salazar, in press), TBT depuration was found to be essentially complete within 21 d (e.g. half-life < 21 d). The area of the present study, Maine (USA), has a low population density and a relatively unpolluted coastline. However, there are activities that do result in localized pollution threats. For TBT pollution in Maine, elevated levels of TBT have been measured in marine sediments associated with shipyards (Page, D., unpubl. data), and in mussel populations (Page
Tissue distribution and depuration of TBTfor
mussels
411
et al., 1990; Page & Widdows, 1991) associated with shipyards, commercial ship traffic, and wood lobster trap and fishing net treatment. The present study was undertaken to learn about the disposition and depuration of TBT in mussels from field locations in Maine identified as having elevated inputs of TBT.
METHODS Samples of the blue mussel, Myths Maine intertidal locations in 1989:
edulis, were collected from 2 coastal
Portland: The largest city in Maine, Portland is a major terminal for large oil tankers. There are also marinas and other commercial vessel activities. The sampling site was near an oil terminal facility. Boothbay Harbor: The Boothbay region is a major resort area with large and small pleasure craft, small shipyards, and commercial fishing facilities. The sampling site was near a lobster fisherman’s wharf and service facility. For the analyses of pooled samples of individual tissues dissected from a group of individual animals, 25-100 adult (5 cm) mussels were dissected immediately following collection in the field. All samples were frozen until analyzed. For the depuration study, a pool of 100 adult (5 cm) mussels was collected from the Boothbay Harbor site and transferred in a net container to a subtidal area at a location known to have no significant TBT inputs. The average water temperature was 13°C. Subsamples of 25 mussels were taken at 0, 7, 14 and 28 d depuration time. For each sampling time, 20 individuals were dissected into gills, gonads, and digestive glands. The dissected tissue samples were frozen until analyzed for alkyltins. In addition, 5 individuals were shucked and analyzed as a whole-animal homogenate for each time. The analytical method is described in detail elsewhere (Page, 1989). In summary, it consists of a wet extraction of a tripentyltin chloride (recovery standard) spiked homogenate with methylene chloride, followed by treatment with hexyl magnesium bromide to form the hexyl derivatives of any extractable organotin species present. The derivatized extract was cleaned up by liquid chromatography on silica gel, followed by addition of a second internal standard (butyltripentyltin) concentration, and quantification using capillary GC/FID. The analytical precision of the method was generally better than f 10% based on replicate field and laboratory samples.
D. S. Page et al.
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TABLE 1
Results of Analyses of Dissected Tissues from Mussels Collected in the Field: Portland July 1989 and Boothbay Harbor September 1989. Overall TBT and DBT Results are Given as pg/g Dry Tissue Weight. Data are Given as the Average of Duplicate Analyses f s Portland Tissue
Whole animal Gills Gonads Digestive gland Kidney Adductor muscle
Boothbay
pg TBTIg
pg DBTlg
2.35 3.93 2.09 1.03 0.74 0.36
0.34 0.62 0.41 0.40 0.32 0.12
zt i f f f zt
.43 .27 .05 .09 .03 .07
f i * * f f
.05 .26 .04 .oo .Ol .04
pg TBTlg
3.28 f .06 1.25 =t .06 1.64 * .Ol
pg DBTlg
1.93 f .02 1.20 f .05 1.23 f .08
RESULTS Tissue distribution Table 1 gives the results of TBT and DBT analyses of dissected tissues from mussels collected at the Portland Harbor site in July 1989, and at the Boothbay Harbor site in September 1989. The results are given as the average of duplicate analyses in pg/g on a dry tissue weight basis. Depuration Mussels from the Boothbay Harbor site were collected in October 1989 and allowed to depurate at an unpolluted site. The results are given in Table 2. The TBT and DBT concentrations are given as the average of duplicate analyses % the standard error, which is a composite of the analytical variability and the sampling variability. The TBT results are presented graphically in Fig. 1 together with the equations and the best-fit parameters for the one- and two-compartment depuration model regressions (Kaleidagraph Ver. 3.0.1, Abelbeck Software Inc.). Also shown in Fig. 1 are the best fit lines for the regressions and the r2 values for each regression reflecting the goodness of fit for each model. The two models used are as follows one-compartment (single exponential): (double exponential): [TBT] = [TBT] = [TBTlOe- kt two-compartment -klr + [TBT]02e-k2f where k values are rate constants (d-l); [TBTlole [TBTlo is the initial concentration of TBT in a given compartment; and t is the time of depuration in days.
Tissue distribution and akpuration of TBTfor
413
mussels
TABLE 2 Results of TBT and DBT Analyses of Dissected Tissues from Mussels Collected in the Field at the Boothbay Harbor Site Sampled in October 1989, and Allowed to Depurate at a Clean Location Over a 4 Week Period at an Average Temperature of 13°C. The TBT/ DBT Results are Given as pg/g Dry Tissue Weight. Data are Given as the Average of Duplicate Analyses f s Gill tissue
Whole animaI Days 0
7 14 28
pg TBTIg
pg DBTlg
0.82 0.78 0.76 0.62
0.99 0.78 0.84 0.79
% .05 ho.00 h.08 f .02
f .02 *O.OO zt.08 f .04
pg TBTIg
2.24 1.24 1.04 0.86
/lg TBTIg 1.21 zk.08
7 14 28
0.62 f .03 0.76 f .Ol 0.51 *.01
/lg DBTlg
1.19 0.91 1.08 0.80
1.52 1.48 0.99 0.93
+.22 %O.OO k.12 f .02
Digestive gland
Gonadal tissue Days 0
f .04 +O.OO h.12 zt .07
pg DBTlg
*.05 It.03 f .06 k.02
pg TBTlg
1.69 1.15 0.89 0.66
f.16 zt.01 *.04 l o.oo
/lg
DBTlg
1.40 0.90 0.99 0.72
k.19 * .05 f .06 *.04
Table 3 gives the depuration half-life values (days) calculated from the first order rate constant values given in Fig. 1 ‘using the equation: to.5 =0.693/k
DISCUSSION Tissue distribution
Whole-animal data are commonly used to relate biological effects to measured body burdens of pollutants (e.g. see Widdows et al., 1987; Donkin et al., 1989; Widdows & Donkin, 1989; De Kock & Kramer, 1994). Whole-animal pollutant concentrations may not accurately reflect the pollutant concentrations in individual target organs of the test animal. Laboratory uptake studies using radiolabelled TBT (Laughlin et al., 1986) showed that Mytilus edulis accumulated the TBT to the greatest extent in the gills. Analyses of dissected tissues of the clam, Mya arenaria, exposed to TBT in the laboratory (Kure & Depledge, 1994) found that gills and viscera had the highest proportion of TBT. These observations were confirmed for field-exposed populations of mussels in the present study where gill tissue TBT concentrations were roughly twice the whole-body values
0
0.5
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i
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0.5
1
1.5 E
0
1.5
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.
Gills
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8'
1.5
1.5
i
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0
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DAYS
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D. Whole Animal
20
B. Digestive Gland ,‘/,,,“/1”‘,1”“1’,‘/
Fig. 1. TBT depuration curves for: (a) Gill tissue; (b) digestive gland tissue; (c) gonadal tissue; (d) whole-animal homogenate. The error bars correspond to the standard error values given in Table 2. The TBT depuration data were fitted with a first order exponential (one-compartment) decay model and (for gills and digestive gland) with a double exponential (two-compartment) decay model. The regression results are given on the figure for each tissue.
1,
E 3'
2
2
E
2.5
2.5
Tissue distribution and depuration of TBTfor
415
mussels
TABLE 3 Half-Life Values (d) for TBT and DBT Depuration from Various Tissues in Mussels. For TBT Depuration from Gill and Digestive Gland Tissues, a Two-Compartment Model (Double Exponential) Gave the Best Fit to the Data. A Single Exponential Decay Model was Used for TBT Depuration from Whole-Animal Homogenate and Gonadal Tissue and for DBT Depuration from all Tissues. Half-Life Values Were Calculated from First-Order Rate Constants (Fig. 1) by the Formula to.5=0.693/k Tissue
Whole animal Gonad Digestive gland Gills
TBT rapid to.5 (d)
TBT slow to,5 id)
DBT to.5 (d)
5.3 2.2
69 28 58 53
115 58 35 36
for both the Portland mussels and the Boothbay mussels (Tables 1 and 2; time = 0). The elevated TBT concentrations in the gills and digestive gland are not likely due to the presence of particulate (paint chip) forms of TBT. Particulate matter in the gut would be purged by the animals within 24 h of depuration (Hawkins et al., 1990; De Kock & Kramer, 1994). The depuration kinetics (see below) indicate that TBT loss occurs over a longer time period than 24 h and is therefore not simply the expulsion of TBTcontaining particulate matter. Depuration During depuration; the Boothbay mussels lost TBT from gill, gonad, digestive gland and on a whole-animal basis at rates depending on the tissue. The data (Fig. 1) show that the overall apparent depuration rate is slowest for the whole-animal homogenate with a half life of 69 d which is over 3 times slower than the rate of TBT depuration (one compartment model) from the gills or digestive gland. Whole-animal depuration rates represent a composite of many contributing organ-specific depuration processes. As small contributors to the whole-animal dry weight, the contribution of individual organs to the whole-animal depuration kinetics is likely to be small. For example, the dry tissue weight of gills comprises ca. 3-5% (Zurburg et al., 1979) of the total dry weight of the mussel, depending on season and physiologic state. The slow whole-animal depuration rate could result from TBT being in a non-labile form in tissues representing a larger fraction of the total animal dry weight than the specific organs analyzed. Depuration-resistant ‘non-labile‘ TBT in shellfish tissue has been found in TBT depuration studies of a deposit-feeding clam (Langston & Burt, 1991) and for scallops (Davies et al., 1986).
416
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For the digestive gland and gill tissues (Fig. l), the TBT depuration kinetics exhibit an initial rapid component, followed by a much slower depuration rate reflecting a two-compartment depuration model similar to that observed for r4C-naphthalene depuration in individual tissues in Myths edulis (Widdows et al., 1983), for polycyclic aromatic hydrocarbons (PAH) depuration in whole oyster tissues (Stegeman & Teal, 1973; Page et al., 1987), for whole A4ytilu.s edulis tissue PAH depuration (Farrington, 1989), and for TBT depuration in fish (Tas, 1993). The preexponential factors in the regressions (Fig. 1) represent the initial concentration of TBT in each compartment. The results (Fig. 1) show that for the field population of mussels studied, the fast and slow depurating compartments of gill and digestive gland have approximately equal initial concentrations of TBT. Both gill and digestive gland tissue conduct processes that directly contact the aqueous environment. Therefore, the rapid depuration component in these 2 tissues could represent TBT loss from those parts of the gill and digestive gland structures most accessible to the water column. The slower depuration component could represent TBT loss from less water-accessible tissue structures. The depuration half-life values for DBT (Table 3) indicate that the rate of DBT loss is similar to the slower TBT depuration processes. This is contrary to the prediction that the more soluble substance (DBT) would be expected to partition into seawater more rapidly. The measured bioconcentration factors for DBT range from 100-500, approximately 2 orders of magnitude lower than for TBT, reflecting the greater water solubility of DBT (Widdows & Page, 1993). The loss of DBT from gills (Table 2) shows an initial steady state period (O-7 d) corresponding to the period of rapid TBT loss. These observations suggest that TBT depuration in mussels may not entirely be a simple phase transfer process, but a process in which DBT is an intermediate produced by the dealkylation of TBT (Lee, 1986; Lee, 1991) from labile and non-labile compartments within the gills and other tissues. Reported TBT depuration half-life values for Mytilus edulis range from 1.9 d (Laughlin et al., 1986) to over 40 d (Zuolian & Jensen, 1989). These previous studies analyzed their data using a single exponential decay model. The apparent differences in the reported results could be due to the depuration time period analyzed. The fast depuration half-life values of 2.2 d for gill tissue and 5.3 d for digestive gland tissue reported here agree well with the values of 2.3-3.2 d for gill tissue and 1.9-2.1 d for ‘viscera’ reported by Laughlin et al. (1986). Further analysis of the primary depuration data presented in Laughlin et al. (1986) shows a tailing at longer time values, yielding a half-life of approximately 50 d, corresponding closely to the 40 d depuration half-life observed by Zuolian & Jensen
Tissue distribution and depuration of TBTfor
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(1989), and the slow depurating compartment half-life of 53-58 d and whole-animal value of 69 d in the present study. Analysis of whole-animal depuration data in Salazar & Salazar (in press) for field-exposed adult and juvenile mussels transplanted from a seawater TBT concentration of ~70 ng/liter to one of ~3 ng/liter, yields a TBT depuration half-life of 67 d. This is similar to the half-life values for the rapid depuration process reported here. The differences in reported depuration rates for TBT most likely reflect differences in data analysis, and whether TBT loss from a labile or non-labile compartment was the dominant process. Mussels as bioindicators of TBT As discussed earlier (see Introduction), mussels are very useful bioindicators of pollution because the concentrations of pollutants measured in their tissues reflect time-averaged pollutant exposure levels. Field exposure differs from a laboratory or mesocosm constant exposure in that the water column concentrations of pollutants in the field can change rapidly due to environmental factors, as well as changes in rates of inputs (e.g. see Harris et al., 1991). This means that a field population of mussels is unlikely to be in a true equilibrium with respect to environmental pollutant exposure at any given time, but rather in a situation approximating a steady state with respect to absorption and depuration. The steady state tissue concentration of TBT in a population of mussels from a given location does not reflect a simple partitioning process between aqueous TBT species and a single ‘whole-animal’ compartment. It reflects the net result of a combination of partitioning processes involving different structures of the animal having different partitioning properties. While mussel tissues rapidly absorb TBT from the aqueous phase and from TBT-contaminated phytoplankton (Laughlin et al., 1986), the rate of loss of TBT as a result of changing exposure concentrations can be fast or slow depending on the tissue. For example, comparison of the September 1989 Boothbay tissue TBT distribution data (Table 1) with the October 1989 data (Table 2) from the same location shows that the gills had lost 32% of the TBT present over a period of one month reflecting probable seasonal decreases in TBT inputs. In contrast, gonadal tissue and digestive gland tissue showed little change in TBT concentration over the same period. The rate of TBT depuration from the rapidly-depurating compartment in gill tissue (Table 3) is 2.4 times faster than digestive gland tissue, which is consistent with the greater exposure of the gills to the aqueous phase. Gill tissue is a small fraction of the total whole-animal dry weight (Zurburg et al., 1979). If most of the TBT in those tissues comprising most of the whole-animal dry weight is in a non-labile form, then rapid changes in gill TBT
418
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concentrations due to depuration or changes in exposure levels would not be reflected in corresponding changes in whole-animal TBT concentrations (see Table 2). This means that mussels can appear to be in a steady state with respect to a given pollutant on a whole-animal basis, even though individual tissues may not be.
CONCLUSIONS The observed tissue distribution and disposition of butyltin species in mussels suggest that these factors should be considered in relating biological effects to chemical measurements for animals exposed to TBT in the field. If TBT depuration is a multi-compartment process, then the process of TBT uptake also involves the absorption and disposition of TBT among compartments within the mussel. Further investigation of the tissue-specific kinetics of TBT uptake and depuration could help to explain the apparent inverse TBT water concentration dependence of the whole-animal TBT bioconcentration factor in mussels (Laughlin et al., 1986; Salazar, 1989; Zuolian & Jensen, 1989). Biphasic kinetics appears to be a general phenomenon for the depuration of lipophilic pollutants from shellfish in cases where exposure times are sufficiently long to ensure that the pollutant has reached an approximate steady state with respect to all compartments within the animal (Widdows et al., 1983; Farrington, 1989; Widdows & Donkin, 1992). Kinetic data for pollutant depuration should therefore be analyzed by appropriate pharmacokinetic models (Barron et al., 1990) whenever possible. The wide range of depuration half-life values reported for TBT in Mytilus edulis reflects the likelihood that different studies have measured the depuration of TBT from different compartments within the animals.
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J. H., Pieters, H. & Zandee, D. I. (1979). The influence on energy metabolism in Mytilus eduiis (L.). II. Organ Phenomena in Marine Plants and Animals (E. Naylor & Pergamon Press, Oxford, pp. 293-300.
ELSEVlER
Research, Vol. 40, No. 4, pp. 409421, 1995 Copyright 01995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0141-1136/95 $9.50+0.00
0141-1136(94)00004-6
Tissue Distribution and Depuration of Tributyltin for Field-Exposed Mytilus edulis David S. Page,‘* Tamara M. Dassanayake”
& Edward S. Gilfillan’
“Bowdoin College Chemistry Department, Brunswick, Maine 04011, USA ‘Bowdoin College Marine Research Laboratory, Brunswick, Maine 04011, USA (Received 26 November
1994; revised version received 10 January 1995; accepted 23 January 1995)
ABSTRACT The disposition of tributyltin (TBT) within Mytilus edulis tissues and the depuration of TBT from various organs was determined for field populations of animals chronically exposed to environmental sources of TBT. Analysis of dissected organs demonstrated that TBT accumulated to the greatest extent in gill tissue, with TBT concentrations approximately twice that of whole-animal homogenates. A transplant study showed that the depuration of TBTfrom gill tissue and digestive gland tissue is a biphasic two-compartment process involving a rapid TBT loss process and a concurrent slower TBT depuration process. Whole-body and gonadal tissue depuration followed a slower monophasic depuration process. Depuration half ltfe values ranged from 2.2 -5.3 d for the fast depuration component and 28-69 dfor the slow component.
INTRODUCTION Tributyltin (TBT) is acutely toxic to many species of marine animals at very low water concentrations &g/liter-ng/liter range) (Maguire, 1987; Thain et al., 1987; Joshi & Gupta, 1990). While TBT antifoulants have seen widespread use in the past, their use has been restricted in many areas due to evidence of adverse effects on marine life (Waldock et al., 1987; Anon., 1994). Even though regulation has reduced the levels of environmental TBT (Waite et al., 1991; Anon., 1994), activities at localized sources such as shipyards, and commercial ship traffic remain as potential sources *To whom correspondence
should be addressed. 409
410
D. S. Page et al.
of TBT. In addition, there is evidence that the resuspension of TBT-contaminated sediments in marinas and shipyards can serve as an on-going source of TBT in the water column and thus to biota (Harris et al., 1991; Kure & Depledge, 1994; Ritsema, 1994). In addition, TBT from contaminated sediment can be ingested directly by deposit-feeders (Langston & Burt, 1991). Consistent with the use of Myths edulis as a sentinel organism for the detection of marine pollutants in the Mussel Watch Program (Widdows & Donkin, 1992; O’Connor et al., 1994), data have been reported for TBT in mussels from various locations (e.g. Page & Widdows, 1991; Uhler et al., 1993). For a given pollutant exposure concentration, the steady-state concentration of a lipophilic pollutant such as TBT in the tissues of marine animals reflects the balance between the processes of absorption and depuration as described by various pharmacokinetic models (Barron et al., 1990). Myths edulis has been found to absorb TBT from seawater and TBT-exposed phytoplankton with bioconcentration factors inversely proportional to the TBT-source aqueous concentration (Laughlin et al., 1986; Zuolian & Jensen, 1989; Salazar & Salazar, in press). There is evidence that the rate of TBT absorption depends on the TBT-source aqueous concentration. For example, in a field transplant study Salazar & Salazar (in press) found that adult and juvenile Myths gulloprovinciulis reached a steady state between water and tissue TBT concentrations in less than 21 d of exposure at a seawater TBT concentration of about 70 ng/liter. For exposure to TBT at about 450 ng/liter in a transplant study, adult Myths gulloprovincialis required over 60 d to reach an apparent steady state between water and tissue TBT concentrations (Salazar et al., 1987). Absorption of 14C-labeled tributyltin oxide by various organs of Myths edulis was greatest in gill and viscera (Laughlin et al., 1986) indicating a partitioning process within the animals. There is a wide range of reported TBT depuration rates for Myths edulis. Laughlin et al. (1986) measured depuration half-life values of 1.94.1 d for the various tissues of laboratory-exposed mussels. Other workers found TBT (as total organic tin) to depurate from field-exposed mussels with a half-life of 40 d (Zuolian & Jensen, 1989). In a reciprocal transplant study with adult and juvenile Myths guZloproviniaZis(Salazar & Salazar, in press), TBT depuration was found to be essentially complete within 21 d (e.g. half-life < 21 d). The area of the present study, Maine (USA), has a low population density and a relatively unpolluted coastline. However, there are activities that do result in localized pollution threats. For TBT pollution in Maine, elevated levels of TBT have been measured in marine sediments associated with shipyards (Page, D., unpubl. data), and in mussel populations (Page
Tissue distribution and depuration of TBTfor
mussels
411
et al., 1990; Page & Widdows, 1991) associated with shipyards, commercial ship traffic, and wood lobster trap and fishing net treatment. The present study was undertaken to learn about the disposition and depuration of TBT in mussels from field locations in Maine identified as having elevated inputs of TBT.
METHODS Samples of the blue mussel, Myths Maine intertidal locations in 1989:
edulis, were collected from 2 coastal
Portland: The largest city in Maine, Portland is a major terminal for large oil tankers. There are also marinas and other commercial vessel activities. The sampling site was near an oil terminal facility. Boothbay Harbor: The Boothbay region is a major resort area with large and small pleasure craft, small shipyards, and commercial fishing facilities. The sampling site was near a lobster fisherman’s wharf and service facility. For the analyses of pooled samples of individual tissues dissected from a group of individual animals, 25-100 adult (5 cm) mussels were dissected immediately following collection in the field. All samples were frozen until analyzed. For the depuration study, a pool of 100 adult (5 cm) mussels was collected from the Boothbay Harbor site and transferred in a net container to a subtidal area at a location known to have no significant TBT inputs. The average water temperature was 13°C. Subsamples of 25 mussels were taken at 0, 7, 14 and 28 d depuration time. For each sampling time, 20 individuals were dissected into gills, gonads, and digestive glands. The dissected tissue samples were frozen until analyzed for alkyltins. In addition, 5 individuals were shucked and analyzed as a whole-animal homogenate for each time. The analytical method is described in detail elsewhere (Page, 1989). In summary, it consists of a wet extraction of a tripentyltin chloride (recovery standard) spiked homogenate with methylene chloride, followed by treatment with hexyl magnesium bromide to form the hexyl derivatives of any extractable organotin species present. The derivatized extract was cleaned up by liquid chromatography on silica gel, followed by addition of a second internal standard (butyltripentyltin) concentration, and quantification using capillary GC/FID. The analytical precision of the method was generally better than f 10% based on replicate field and laboratory samples.
D. S. Page et al.
412
TABLE 1
Results of Analyses of Dissected Tissues from Mussels Collected in the Field: Portland July 1989 and Boothbay Harbor September 1989. Overall TBT and DBT Results are Given as pg/g Dry Tissue Weight. Data are Given as the Average of Duplicate Analyses f s Portland Tissue
Whole animal Gills Gonads Digestive gland Kidney Adductor muscle
Boothbay
pg TBTIg
pg DBTlg
2.35 3.93 2.09 1.03 0.74 0.36
0.34 0.62 0.41 0.40 0.32 0.12
zt i f f f zt
.43 .27 .05 .09 .03 .07
f i * * f f
.05 .26 .04 .oo .Ol .04
pg TBTlg
3.28 f .06 1.25 =t .06 1.64 * .Ol
pg DBTlg
1.93 f .02 1.20 f .05 1.23 f .08
RESULTS Tissue distribution Table 1 gives the results of TBT and DBT analyses of dissected tissues from mussels collected at the Portland Harbor site in July 1989, and at the Boothbay Harbor site in September 1989. The results are given as the average of duplicate analyses in pg/g on a dry tissue weight basis. Depuration Mussels from the Boothbay Harbor site were collected in October 1989 and allowed to depurate at an unpolluted site. The results are given in Table 2. The TBT and DBT concentrations are given as the average of duplicate analyses % the standard error, which is a composite of the analytical variability and the sampling variability. The TBT results are presented graphically in Fig. 1 together with the equations and the best-fit parameters for the one- and two-compartment depuration model regressions (Kaleidagraph Ver. 3.0.1, Abelbeck Software Inc.). Also shown in Fig. 1 are the best fit lines for the regressions and the r2 values for each regression reflecting the goodness of fit for each model. The two models used are as follows one-compartment (single exponential): (double exponential): [TBT] = [TBT] = [TBTlOe- kt two-compartment -klr + [TBT]02e-k2f where k values are rate constants (d-l); [TBTlole [TBTlo is the initial concentration of TBT in a given compartment; and t is the time of depuration in days.
Tissue distribution and akpuration of TBTfor
413
mussels
TABLE 2 Results of TBT and DBT Analyses of Dissected Tissues from Mussels Collected in the Field at the Boothbay Harbor Site Sampled in October 1989, and Allowed to Depurate at a Clean Location Over a 4 Week Period at an Average Temperature of 13°C. The TBT/ DBT Results are Given as pg/g Dry Tissue Weight. Data are Given as the Average of Duplicate Analyses f s Gill tissue
Whole animaI Days 0
7 14 28
pg TBTIg
pg DBTlg
0.82 0.78 0.76 0.62
0.99 0.78 0.84 0.79
% .05 ho.00 h.08 f .02
f .02 *O.OO zt.08 f .04
pg TBTIg
2.24 1.24 1.04 0.86
/lg TBTIg 1.21 zk.08
7 14 28
0.62 f .03 0.76 f .Ol 0.51 *.01
/lg DBTlg
1.19 0.91 1.08 0.80
1.52 1.48 0.99 0.93
+.22 %O.OO k.12 f .02
Digestive gland
Gonadal tissue Days 0
f .04 +O.OO h.12 zt .07
pg DBTlg
*.05 It.03 f .06 k.02
pg TBTlg
1.69 1.15 0.89 0.66
f.16 zt.01 *.04 l o.oo
/lg
DBTlg
1.40 0.90 0.99 0.72
k.19 * .05 f .06 *.04
Table 3 gives the depuration half-life values (days) calculated from the first order rate constant values given in Fig. 1 ‘using the equation: to.5 =0.693/k
DISCUSSION Tissue distribution
Whole-animal data are commonly used to relate biological effects to measured body burdens of pollutants (e.g. see Widdows et al., 1987; Donkin et al., 1989; Widdows & Donkin, 1989; De Kock & Kramer, 1994). Whole-animal pollutant concentrations may not accurately reflect the pollutant concentrations in individual target organs of the test animal. Laboratory uptake studies using radiolabelled TBT (Laughlin et al., 1986) showed that Mytilus edulis accumulated the TBT to the greatest extent in the gills. Analyses of dissected tissues of the clam, Mya arenaria, exposed to TBT in the laboratory (Kure & Depledge, 1994) found that gills and viscera had the highest proportion of TBT. These observations were confirmed for field-exposed populations of mussels in the present study where gill tissue TBT concentrations were roughly twice the whole-body values
0
0.5
0
5
~--‘,““1”“/‘T”l”‘r
10
DAYS
15 20
c. 25
Gonad 30
i
$
0
0.5
1
1.5 E
0
1.5
i0
2
.
Gills
2
A.
0.5
2.5
? =l
2.5
0
0.5
8'
1.5
1.5
i
/&l
I,“’
0
5
10
DAYS
15
25
D. Whole Animal
20
B. Digestive Gland ,‘/,,,“/1”‘,1”“1’,‘/
Fig. 1. TBT depuration curves for: (a) Gill tissue; (b) digestive gland tissue; (c) gonadal tissue; (d) whole-animal homogenate. The error bars correspond to the standard error values given in Table 2. The TBT depuration data were fitted with a first order exponential (one-compartment) decay model and (for gills and digestive gland) with a double exponential (two-compartment) decay model. The regression results are given on the figure for each tissue.
1,
E 3'
2
2
E
2.5
2.5
Tissue distribution and depuration of TBTfor
415
mussels
TABLE 3 Half-Life Values (d) for TBT and DBT Depuration from Various Tissues in Mussels. For TBT Depuration from Gill and Digestive Gland Tissues, a Two-Compartment Model (Double Exponential) Gave the Best Fit to the Data. A Single Exponential Decay Model was Used for TBT Depuration from Whole-Animal Homogenate and Gonadal Tissue and for DBT Depuration from all Tissues. Half-Life Values Were Calculated from First-Order Rate Constants (Fig. 1) by the Formula to.5=0.693/k Tissue
Whole animal Gonad Digestive gland Gills
TBT rapid to.5 (d)
TBT slow to,5 id)
DBT to.5 (d)
5.3 2.2
69 28 58 53
115 58 35 36
for both the Portland mussels and the Boothbay mussels (Tables 1 and 2; time = 0). The elevated TBT concentrations in the gills and digestive gland are not likely due to the presence of particulate (paint chip) forms of TBT. Particulate matter in the gut would be purged by the animals within 24 h of depuration (Hawkins et al., 1990; De Kock & Kramer, 1994). The depuration kinetics (see below) indicate that TBT loss occurs over a longer time period than 24 h and is therefore not simply the expulsion of TBTcontaining particulate matter. Depuration During depuration; the Boothbay mussels lost TBT from gill, gonad, digestive gland and on a whole-animal basis at rates depending on the tissue. The data (Fig. 1) show that the overall apparent depuration rate is slowest for the whole-animal homogenate with a half life of 69 d which is over 3 times slower than the rate of TBT depuration (one compartment model) from the gills or digestive gland. Whole-animal depuration rates represent a composite of many contributing organ-specific depuration processes. As small contributors to the whole-animal dry weight, the contribution of individual organs to the whole-animal depuration kinetics is likely to be small. For example, the dry tissue weight of gills comprises ca. 3-5% (Zurburg et al., 1979) of the total dry weight of the mussel, depending on season and physiologic state. The slow whole-animal depuration rate could result from TBT being in a non-labile form in tissues representing a larger fraction of the total animal dry weight than the specific organs analyzed. Depuration-resistant ‘non-labile‘ TBT in shellfish tissue has been found in TBT depuration studies of a deposit-feeding clam (Langston & Burt, 1991) and for scallops (Davies et al., 1986).
416
D. S. Page et al.
For the digestive gland and gill tissues (Fig. l), the TBT depuration kinetics exhibit an initial rapid component, followed by a much slower depuration rate reflecting a two-compartment depuration model similar to that observed for r4C-naphthalene depuration in individual tissues in Myths edulis (Widdows et al., 1983), for polycyclic aromatic hydrocarbons (PAH) depuration in whole oyster tissues (Stegeman & Teal, 1973; Page et al., 1987), for whole A4ytilu.s edulis tissue PAH depuration (Farrington, 1989), and for TBT depuration in fish (Tas, 1993). The preexponential factors in the regressions (Fig. 1) represent the initial concentration of TBT in each compartment. The results (Fig. 1) show that for the field population of mussels studied, the fast and slow depurating compartments of gill and digestive gland have approximately equal initial concentrations of TBT. Both gill and digestive gland tissue conduct processes that directly contact the aqueous environment. Therefore, the rapid depuration component in these 2 tissues could represent TBT loss from those parts of the gill and digestive gland structures most accessible to the water column. The slower depuration component could represent TBT loss from less water-accessible tissue structures. The depuration half-life values for DBT (Table 3) indicate that the rate of DBT loss is similar to the slower TBT depuration processes. This is contrary to the prediction that the more soluble substance (DBT) would be expected to partition into seawater more rapidly. The measured bioconcentration factors for DBT range from 100-500, approximately 2 orders of magnitude lower than for TBT, reflecting the greater water solubility of DBT (Widdows & Page, 1993). The loss of DBT from gills (Table 2) shows an initial steady state period (O-7 d) corresponding to the period of rapid TBT loss. These observations suggest that TBT depuration in mussels may not entirely be a simple phase transfer process, but a process in which DBT is an intermediate produced by the dealkylation of TBT (Lee, 1986; Lee, 1991) from labile and non-labile compartments within the gills and other tissues. Reported TBT depuration half-life values for Mytilus edulis range from 1.9 d (Laughlin et al., 1986) to over 40 d (Zuolian & Jensen, 1989). These previous studies analyzed their data using a single exponential decay model. The apparent differences in the reported results could be due to the depuration time period analyzed. The fast depuration half-life values of 2.2 d for gill tissue and 5.3 d for digestive gland tissue reported here agree well with the values of 2.3-3.2 d for gill tissue and 1.9-2.1 d for ‘viscera’ reported by Laughlin et al. (1986). Further analysis of the primary depuration data presented in Laughlin et al. (1986) shows a tailing at longer time values, yielding a half-life of approximately 50 d, corresponding closely to the 40 d depuration half-life observed by Zuolian & Jensen
Tissue distribution and depuration of TBTfor
mussels
417
(1989), and the slow depurating compartment half-life of 53-58 d and whole-animal value of 69 d in the present study. Analysis of whole-animal depuration data in Salazar & Salazar (in press) for field-exposed adult and juvenile mussels transplanted from a seawater TBT concentration of ~70 ng/liter to one of ~3 ng/liter, yields a TBT depuration half-life of 67 d. This is similar to the half-life values for the rapid depuration process reported here. The differences in reported depuration rates for TBT most likely reflect differences in data analysis, and whether TBT loss from a labile or non-labile compartment was the dominant process. Mussels as bioindicators of TBT As discussed earlier (see Introduction), mussels are very useful bioindicators of pollution because the concentrations of pollutants measured in their tissues reflect time-averaged pollutant exposure levels. Field exposure differs from a laboratory or mesocosm constant exposure in that the water column concentrations of pollutants in the field can change rapidly due to environmental factors, as well as changes in rates of inputs (e.g. see Harris et al., 1991). This means that a field population of mussels is unlikely to be in a true equilibrium with respect to environmental pollutant exposure at any given time, but rather in a situation approximating a steady state with respect to absorption and depuration. The steady state tissue concentration of TBT in a population of mussels from a given location does not reflect a simple partitioning process between aqueous TBT species and a single ‘whole-animal’ compartment. It reflects the net result of a combination of partitioning processes involving different structures of the animal having different partitioning properties. While mussel tissues rapidly absorb TBT from the aqueous phase and from TBT-contaminated phytoplankton (Laughlin et al., 1986), the rate of loss of TBT as a result of changing exposure concentrations can be fast or slow depending on the tissue. For example, comparison of the September 1989 Boothbay tissue TBT distribution data (Table 1) with the October 1989 data (Table 2) from the same location shows that the gills had lost 32% of the TBT present over a period of one month reflecting probable seasonal decreases in TBT inputs. In contrast, gonadal tissue and digestive gland tissue showed little change in TBT concentration over the same period. The rate of TBT depuration from the rapidly-depurating compartment in gill tissue (Table 3) is 2.4 times faster than digestive gland tissue, which is consistent with the greater exposure of the gills to the aqueous phase. Gill tissue is a small fraction of the total whole-animal dry weight (Zurburg et al., 1979). If most of the TBT in those tissues comprising most of the whole-animal dry weight is in a non-labile form, then rapid changes in gill TBT
418
D. S. Page et al.
concentrations due to depuration or changes in exposure levels would not be reflected in corresponding changes in whole-animal TBT concentrations (see Table 2). This means that mussels can appear to be in a steady state with respect to a given pollutant on a whole-animal basis, even though individual tissues may not be.
CONCLUSIONS The observed tissue distribution and disposition of butyltin species in mussels suggest that these factors should be considered in relating biological effects to chemical measurements for animals exposed to TBT in the field. If TBT depuration is a multi-compartment process, then the process of TBT uptake also involves the absorption and disposition of TBT among compartments within the mussel. Further investigation of the tissue-specific kinetics of TBT uptake and depuration could help to explain the apparent inverse TBT water concentration dependence of the whole-animal TBT bioconcentration factor in mussels (Laughlin et al., 1986; Salazar, 1989; Zuolian & Jensen, 1989). Biphasic kinetics appears to be a general phenomenon for the depuration of lipophilic pollutants from shellfish in cases where exposure times are sufficiently long to ensure that the pollutant has reached an approximate steady state with respect to all compartments within the animal (Widdows et al., 1983; Farrington, 1989; Widdows & Donkin, 1992). Kinetic data for pollutant depuration should therefore be analyzed by appropriate pharmacokinetic models (Barron et al., 1990) whenever possible. The wide range of depuration half-life values reported for TBT in Mytilus edulis reflects the likelihood that different studies have measured the depuration of TBT from different compartments within the animals.
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