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Shell thickening in Anodonta cygnea by TBTO treatments
Camp. Biochem. Physiol. Printed in Great Britain
Vol. 92C.
SHELL
No. I, pp, 77-80,
1989 0
THT~KENIN~ IN A~~~~~TA TBTO TREATMENTS
0306~4492/89 53.00 + 0.00 1989 Pergamon Press plc
CYGNEA
BY
J. MACHADO*, J. COIMBRA* and C. Slit *Departamento de Fisiologia, Instituto de CiCncias Biomidicas Abel Salazar, Largo do Prof. Abel Salatar, 2,400O Porto, Portugal and $Departamento de Engenharia Electrot&cnica. Faculdade de Engenharia, Universidade do Porto, Porto, Portugal (Received 4 January 1988) Abstract-l.
Low level TBTO (bis(tributyltin)oxide) treatments (0.2 pg/l) caused, in uivo, a secretion of a calcified organic matrix over the inner face of the shell of Anodonfa cygnet after eight days. 2. Electrophysiological studies carried out on the outer mantle epithelium (OME) showed that the short-circuit current was strongly inhibited by TBTO from the shell side. On the contrary, the effect is very slow from the haemolymph side. 3. The TBTO effect on the proton and/or bicarbonate transport mechanisms across the OME can be a strong factor in the shell thickening
in the experiments is composed of CaCl, (I mM), MgClz (0.5 mMf. KC1 (6.75 mM). NaCl (8.75 mM) and NaHCO, (10mMi. Adequate convection df the solutions in both half-chambers was achieved by bubbling carbogen (9.5%0,+ S%CO,) through them. The epithelium was short-circuited by means of a simple electronic voltageclamp system. The transepithelial conductance was mea. sured bv disnlacing membrane notential bv 10 mV for 15 s and recording current pulse. After a control period, small TBTO amounts were added separately to the haemolymph and shell sides reaching a final concentration of about lO-s M.
Organotin compounds, including bis(tributyltin)oxide (TBTO) and tributyltin fluoride (TBTF), are
presently used as a biocide component in marine antifouling paints (Zuckerman et al., 1978; Alzieu et al., 1980, 1982). Tributyltin are present in water and sediments where their release from antifouling paints, found usually on small boats and recreational craft, is the main source (Alzieu et al., 1980, 1982; Maguire et ai., 1982; Maguire, 1984). The increasing annual usage of organotin compound raises the possibility of environmental pollution (Maguire et al., 1984). The presence of high concentration of fine particles in the water may have been responsible for some shell malformation (Key et al., 1976; Alzieu, 1981; Alzieu et al., 1982; Waldock and Thain, 1983, 1986). In this work we report the effects of TBTO on the shell thickening by their action on the ion transport mechanisms across the OME of Anodontu cygnea.
RESULTS All animals treated in oivo with TBTO present a strong pellicle resulting from secretion of organic matrix over the inner shell surface. Calcareous deposits over organic matrix is observed by SEM (Figs 1 and 2). The pellicle is without any apparent texture. The calcareous crystals do not present any typical shape and form isolated groups distributed with regularity over the pellicle. Only one of the control animals presented a fragile pellicle, and without any calcareous deposits. Through electrophysiological studies a rapid fall in the short-circuit current of 81% (hO.02) was observed when TBTO was added to the shell side (Fig. 3). Minor decreases in current were also obtained when lowest amounts of TBTO were used. The transepithelial conductance decreased by 36% (kO.02) in the first condition (Fig. 3). A very slow effect occurred when TBTO was added to the haemolymph side, decreasing the current by 6% ( f 0.03) after 30min (Fig. 3). In this case, the transepithelial conductance is practically unchangeable (Fig. 3).
MATERIAL AND METHODS Fresh water clams (Anodonra cygnet) were collected from lagoon of Mira in the North of Portugal, and kept in aerated and dechlorinated water for up to two weeks. Animals were considered healthy if they strongly closed their valves when disturbed. One group of 8 animals was treated in experimental tanks (10 1) where 80 pl of TBTO stock solution had been added. The estimated final concentration of TBTO, about 0.2 @l/l, was calculated from specific gravity of stock solution and from solubility in water. Another group of 8 animals was kept in control situation. The water was always aerated in the two grouus. After eight davs of TBTO treatments the inner face of the shells was observed. Recent pellicles were removed from the shell and observed bv a JEOL JSM-3X scanning electron microscope (SEM). The calcium carbonate was identified on the fragments formed over the pellicie by dispersive energy microanalysis (DES). For an electrophysiological study the OME was carefully dissected in situ and pieces of 1.2cm diameter from the central area were mounted in Ussing type chambers. According to Coimbra er nl. (1988) the control solution used
DISCUSSION
Bioaccumulation experiments with bis(tributyltin)oxide (TBTO) by marine mussels, Mytilus edulis, indicated that an uptake occurred rapidly when it was either dissolved in water (OSg/l) or associated with 77
J. MACHADO et al.
78
Fig. I. Organic matrix (OM) secreted over the shell inner face of an animal in TBTO treatment: a group of calcareous fragments (CF) formed on the organic matrix; smooth (*) and rugous (-) faces of the calcareous fragments x 9000. Fig. 2. A magnification
of calcareous
fragments. Porous fragments matrix (OM) x 50.000.
phytoplankton Isochrysis gQlbQnQ (Laughlin et al., 1986). They showed that there is a clear accumulation in some tissues (viscera, gill, mantle and muscle). This product or similar compounds, when accumulated in the tissues, can induce shell deformity which is characterized by extreme thickening of the valves (Galtsoff, 1969; Key et al., 1976; Alzieu et al., 1980;
(P) and compact
fragments
(C): organic
Alzieu, 198 1; Alzieu et al., 1982; Waldock and Thain, 1983, 1986). ’ Our observations of the shell of A. cygnea, in low level TBTO treatments (0.2pg/l), showed a strong organic matrix secretion (pellicle) over the entire inner face of the shell. This pellicle already presents some calcareous deposits. All animals in such condi-
Tributyltin
(A)
oxide
79
(B)
Il.04 5 2 0.8--
-- 0.8
2z
d .z 0.6.. I5
-- 0.6
5 z s
e _ 0.4--
-- 0.4
8 s
-E al u’ :: 0.2-0.0
. 0
. 8
.
. . . . , 16 24 30
Time
(min)
1%
-- 0.2
2
0.0
5 0
H (71
b
Con&l
D
TBTO
Fig. 3. Effect of TBTO observed separately from both haemolymph and shell sides of OME. (A) The inhibitory effect from haemolymph side (A) and from shell side (0) was studied on seven and twelve
preparations respectively. The values plotted are the average of the ratios between the values measured at time I and those at time zero. (B) The control and experimental values of conductance plotted at the top of each bar are the average of ratios. H: inhibitor added to the haemolymph side of seven preparations (7). S: inhibitor
added
to shell side of twelve preparations
tion show identical characteristics which indicate that this biocide can be a strong thickening inductor of the valves. On the other hand, our electrophysiological studies proved that TBTO has a rapid inhibitory effect on the current when added to the shell side. This effect of TBTO showed that this barrier is strongly electrogenie. According to previous results (Machado et al., 1988a) there is a clear 1: 1 extrusion of H + across the apical membrane (OME) towards the shell and of HCO, across the basal membrane towards the haemolymph. Therefore, the lowering of the proton extrusion to the shell side, by TBTO, can be an important factor to induce CaCO, deposition causing the shell thickening when the animals are in TBTO treatment. Simultaneously, the expected decrease of bicarbonate extrusion to the haemolymph side will support the increase of HCOq and Ca + + from microspherules (Machado et al.. 1988a) which diffuse across the outer mantle epithelium (OME), which is very permeable to these ions (Coimbra ef al., 1988). Also. during the natural cycles of the shell calcification, high levels of calcium deposition from spring and low levels from autumn (Zandee et al., 1980) occur with low and high currents respectively across the OME (Coimbra et nl.. 1988). In relation to the inhibitory effect on the conductance we can point out some decrease of the ion diffusional mechanisms. The gradual fall in the current when TBTO is added to the haemolymph side indicates a slow dissipation of the electrochemical gradients. This slow effect can be due either to a direct action in the complex transport mechanisms (Coimbra et al., 1988) located in the haemolymph side or more likely to a delayed action in the H + “pump” in the opposite side which involves the crossing of the epithelium. It is not impossible that TBTO is a specific inhibitor for this electrogenic “pump”. Our data strongly suggest that the CaCO, deposits in the shell of A. cygnea by TBTO in c~ivoresult from an inhibition of the proton transport towards the
(12).
shell and from a greater mobilization the haemolymph reserves.
of calcium from
Acknowledgements--We thank Prof. Arala Chaves of Department of Immunology of the ICBAS for the critical reading of the manuscript. This work was supported by a JNICT grant (Junta National de InvestigaCZo Cientifica e Tecnolbgica).
REFERENCES
Alzieu C. (1981) Evaluation des risques dus a I’emploi des peintures antisalissures dans les zones conchylicoles. Anomalies de calcification. Theme Surveillance continue Mecanisme d’action des polluants. Addendum du rapport due 15 juin 1981. Institut Scientifique et Technique des Peches Maritimes. Nates le I5 decembre 1981. Alzieu C.. Hera1 M., Thibaud Y., Dardignac M. J. and Feuillet M. (1982) Influence des peintures antisalissures a base d’organostaniques sur la calcification de la coquille d’huitre Crassostrea gigas. Rev. Trae. Inst. Pech. Marit. 45, 101-l 16. Alzieu C., Thibaud Y., Hera1 M. and Boutier B. (1980) Evaluation des risques dus a I’emploi des peintures antisalissures dans les zones conchylicoles. Rec. Trau. Insf. Pech. Marif. 44, 301-349.
Coimbra J., Machado J., Fernandes P. L., Ferreira H. G. and Ferreira K. G. (1988) Electrophysiology of the mantle of Anodonta cygnea. J. exp. Biol. (in press). Galtsoff P. S. (1969) Anomalies and malformations in the shells of Crassosrrea cirginiea. Natn. Cancer Inst. Monogr. 31, 575-380. Key D., Nunny R. S., Davidson P. E. and Leonard M. A. (1976) Abnormal shell growth in the Pacific oyster Crassosirea gigas. Some preliminary results from experiments undertaken in 1975. ICES Paper CM 1976/K: I I (mimeograph). International Council for the Exploration of the sea,- Copenhagen. Laughlin R. B.. French W. and Guard H. E. (1986) A&umulation of Bis(tributyltin)oxide by the Marink Mussel MyGlus edulis. Envir. Sri. Technol. 20, 884-890. Machado J., Coimbra J., SB C. and Cardoso I. (1988a) Shell thickening in Anodonm cygnea by induced acidosis. Camp. Biochem. Physiol. (in press).
80
J. MACHADO et al.
Machado J., Ferreira K. G. and Ferreira H. G. (1988b) The acid-base balance in the outer mantle epithelium of Anodonta cygnea (in preparation). Maguire R. J., Chau Y. K., Bengert G. A., Hale E. J., Wong P. T. S. and Kramar 0. (1982) Occurrence of organotin compounds in Ontario lakes and rivers. Envir. SC;. Technol. 16, 698-702. Maeuire R. J.. Wone P. T. S. and Rhamev J. S. (1984) ,&umulation and metabolism of tri-n-buiylin cation by a green alga, Ankisrrodesmus falcafus. Can. J. Fish. Aquat. Sci. 41, 537-540. Thain J. E. and Waldock M. J. (1986) The impact of tributyltin (TBT) antifouling paints on molluscan fisheries. War. Sci. Technol. 18. 193-202.
Waldock M. J. and Thain J. E. (1983) Shell thickening in Crassosfrea gigas: organotin antifouling or sediment induced? Mar. Pollur. Bull. 14, 411415. Zandee D. I., Kluytmans J. H., Zurburg W. and Pieter H. (1980) Seasonal variations in biochemical composition of Myfilus edulis with reference to energy metabolism and gametogenesis. Netherlands Journal of Sea Research 14(l), l-29. Zuckerman J. J., Reisdorf R. P., Ellis III H. V. and Wilkinson R. R. (1978) Organotins in biology and the environment. In Organomefals and Organome~alloia?, Occurrence and Fate in the Environment (Edited by Brinckman F. E. and Bellama J. M.), pp. 388-422. ACS Symp. Ser. 82. American Chemical Society, Washington, DC.
Vol. 92C.
SHELL
No. I, pp, 77-80,
1989 0
THT~KENIN~ IN A~~~~~TA TBTO TREATMENTS
0306~4492/89 53.00 + 0.00 1989 Pergamon Press plc
CYGNEA
BY
J. MACHADO*, J. COIMBRA* and C. Slit *Departamento de Fisiologia, Instituto de CiCncias Biomidicas Abel Salazar, Largo do Prof. Abel Salatar, 2,400O Porto, Portugal and $Departamento de Engenharia Electrot&cnica. Faculdade de Engenharia, Universidade do Porto, Porto, Portugal (Received 4 January 1988) Abstract-l.
Low level TBTO (bis(tributyltin)oxide) treatments (0.2 pg/l) caused, in uivo, a secretion of a calcified organic matrix over the inner face of the shell of Anodonfa cygnet after eight days. 2. Electrophysiological studies carried out on the outer mantle epithelium (OME) showed that the short-circuit current was strongly inhibited by TBTO from the shell side. On the contrary, the effect is very slow from the haemolymph side. 3. The TBTO effect on the proton and/or bicarbonate transport mechanisms across the OME can be a strong factor in the shell thickening
in the experiments is composed of CaCl, (I mM), MgClz (0.5 mMf. KC1 (6.75 mM). NaCl (8.75 mM) and NaHCO, (10mMi. Adequate convection df the solutions in both half-chambers was achieved by bubbling carbogen (9.5%0,+ S%CO,) through them. The epithelium was short-circuited by means of a simple electronic voltageclamp system. The transepithelial conductance was mea. sured bv disnlacing membrane notential bv 10 mV for 15 s and recording current pulse. After a control period, small TBTO amounts were added separately to the haemolymph and shell sides reaching a final concentration of about lO-s M.
Organotin compounds, including bis(tributyltin)oxide (TBTO) and tributyltin fluoride (TBTF), are
presently used as a biocide component in marine antifouling paints (Zuckerman et al., 1978; Alzieu et al., 1980, 1982). Tributyltin are present in water and sediments where their release from antifouling paints, found usually on small boats and recreational craft, is the main source (Alzieu et al., 1980, 1982; Maguire et ai., 1982; Maguire, 1984). The increasing annual usage of organotin compound raises the possibility of environmental pollution (Maguire et al., 1984). The presence of high concentration of fine particles in the water may have been responsible for some shell malformation (Key et al., 1976; Alzieu, 1981; Alzieu et al., 1982; Waldock and Thain, 1983, 1986). In this work we report the effects of TBTO on the shell thickening by their action on the ion transport mechanisms across the OME of Anodontu cygnea.
RESULTS All animals treated in oivo with TBTO present a strong pellicle resulting from secretion of organic matrix over the inner shell surface. Calcareous deposits over organic matrix is observed by SEM (Figs 1 and 2). The pellicle is without any apparent texture. The calcareous crystals do not present any typical shape and form isolated groups distributed with regularity over the pellicle. Only one of the control animals presented a fragile pellicle, and without any calcareous deposits. Through electrophysiological studies a rapid fall in the short-circuit current of 81% (hO.02) was observed when TBTO was added to the shell side (Fig. 3). Minor decreases in current were also obtained when lowest amounts of TBTO were used. The transepithelial conductance decreased by 36% (kO.02) in the first condition (Fig. 3). A very slow effect occurred when TBTO was added to the haemolymph side, decreasing the current by 6% ( f 0.03) after 30min (Fig. 3). In this case, the transepithelial conductance is practically unchangeable (Fig. 3).
MATERIAL AND METHODS Fresh water clams (Anodonra cygnet) were collected from lagoon of Mira in the North of Portugal, and kept in aerated and dechlorinated water for up to two weeks. Animals were considered healthy if they strongly closed their valves when disturbed. One group of 8 animals was treated in experimental tanks (10 1) where 80 pl of TBTO stock solution had been added. The estimated final concentration of TBTO, about 0.2 @l/l, was calculated from specific gravity of stock solution and from solubility in water. Another group of 8 animals was kept in control situation. The water was always aerated in the two grouus. After eight davs of TBTO treatments the inner face of the shells was observed. Recent pellicles were removed from the shell and observed bv a JEOL JSM-3X scanning electron microscope (SEM). The calcium carbonate was identified on the fragments formed over the pellicie by dispersive energy microanalysis (DES). For an electrophysiological study the OME was carefully dissected in situ and pieces of 1.2cm diameter from the central area were mounted in Ussing type chambers. According to Coimbra er nl. (1988) the control solution used
DISCUSSION
Bioaccumulation experiments with bis(tributyltin)oxide (TBTO) by marine mussels, Mytilus edulis, indicated that an uptake occurred rapidly when it was either dissolved in water (OSg/l) or associated with 77
J. MACHADO et al.
78
Fig. I. Organic matrix (OM) secreted over the shell inner face of an animal in TBTO treatment: a group of calcareous fragments (CF) formed on the organic matrix; smooth (*) and rugous (-) faces of the calcareous fragments x 9000. Fig. 2. A magnification
of calcareous
fragments. Porous fragments matrix (OM) x 50.000.
phytoplankton Isochrysis gQlbQnQ (Laughlin et al., 1986). They showed that there is a clear accumulation in some tissues (viscera, gill, mantle and muscle). This product or similar compounds, when accumulated in the tissues, can induce shell deformity which is characterized by extreme thickening of the valves (Galtsoff, 1969; Key et al., 1976; Alzieu et al., 1980;
(P) and compact
fragments
(C): organic
Alzieu, 198 1; Alzieu et al., 1982; Waldock and Thain, 1983, 1986). ’ Our observations of the shell of A. cygnea, in low level TBTO treatments (0.2pg/l), showed a strong organic matrix secretion (pellicle) over the entire inner face of the shell. This pellicle already presents some calcareous deposits. All animals in such condi-
Tributyltin
(A)
oxide
79
(B)
Il.04 5 2 0.8--
-- 0.8
2z
d .z 0.6.. I5
-- 0.6
5 z s
e _ 0.4--
-- 0.4
8 s
-E al u’ :: 0.2-0.0
. 0
. 8
.
. . . . , 16 24 30
Time
(min)
1%
-- 0.2
2
0.0
5 0
H (71
b
Con&l
D
TBTO
Fig. 3. Effect of TBTO observed separately from both haemolymph and shell sides of OME. (A) The inhibitory effect from haemolymph side (A) and from shell side (0) was studied on seven and twelve
preparations respectively. The values plotted are the average of the ratios between the values measured at time I and those at time zero. (B) The control and experimental values of conductance plotted at the top of each bar are the average of ratios. H: inhibitor added to the haemolymph side of seven preparations (7). S: inhibitor
added
to shell side of twelve preparations
tion show identical characteristics which indicate that this biocide can be a strong thickening inductor of the valves. On the other hand, our electrophysiological studies proved that TBTO has a rapid inhibitory effect on the current when added to the shell side. This effect of TBTO showed that this barrier is strongly electrogenie. According to previous results (Machado et al., 1988a) there is a clear 1: 1 extrusion of H + across the apical membrane (OME) towards the shell and of HCO, across the basal membrane towards the haemolymph. Therefore, the lowering of the proton extrusion to the shell side, by TBTO, can be an important factor to induce CaCO, deposition causing the shell thickening when the animals are in TBTO treatment. Simultaneously, the expected decrease of bicarbonate extrusion to the haemolymph side will support the increase of HCOq and Ca + + from microspherules (Machado et al.. 1988a) which diffuse across the outer mantle epithelium (OME), which is very permeable to these ions (Coimbra ef al., 1988). Also. during the natural cycles of the shell calcification, high levels of calcium deposition from spring and low levels from autumn (Zandee et al., 1980) occur with low and high currents respectively across the OME (Coimbra et nl.. 1988). In relation to the inhibitory effect on the conductance we can point out some decrease of the ion diffusional mechanisms. The gradual fall in the current when TBTO is added to the haemolymph side indicates a slow dissipation of the electrochemical gradients. This slow effect can be due either to a direct action in the complex transport mechanisms (Coimbra et al., 1988) located in the haemolymph side or more likely to a delayed action in the H + “pump” in the opposite side which involves the crossing of the epithelium. It is not impossible that TBTO is a specific inhibitor for this electrogenic “pump”. Our data strongly suggest that the CaCO, deposits in the shell of A. cygnea by TBTO in c~ivoresult from an inhibition of the proton transport towards the
(12).
shell and from a greater mobilization the haemolymph reserves.
of calcium from
Acknowledgements--We thank Prof. Arala Chaves of Department of Immunology of the ICBAS for the critical reading of the manuscript. This work was supported by a JNICT grant (Junta National de InvestigaCZo Cientifica e Tecnolbgica).
REFERENCES
Alzieu C. (1981) Evaluation des risques dus a I’emploi des peintures antisalissures dans les zones conchylicoles. Anomalies de calcification. Theme Surveillance continue Mecanisme d’action des polluants. Addendum du rapport due 15 juin 1981. Institut Scientifique et Technique des Peches Maritimes. Nates le I5 decembre 1981. Alzieu C.. Hera1 M., Thibaud Y., Dardignac M. J. and Feuillet M. (1982) Influence des peintures antisalissures a base d’organostaniques sur la calcification de la coquille d’huitre Crassostrea gigas. Rev. Trae. Inst. Pech. Marit. 45, 101-l 16. Alzieu C., Thibaud Y., Hera1 M. and Boutier B. (1980) Evaluation des risques dus a I’emploi des peintures antisalissures dans les zones conchylicoles. Rec. Trau. Insf. Pech. Marif. 44, 301-349.
Coimbra J., Machado J., Fernandes P. L., Ferreira H. G. and Ferreira K. G. (1988) Electrophysiology of the mantle of Anodonta cygnea. J. exp. Biol. (in press). Galtsoff P. S. (1969) Anomalies and malformations in the shells of Crassosrrea cirginiea. Natn. Cancer Inst. Monogr. 31, 575-380. Key D., Nunny R. S., Davidson P. E. and Leonard M. A. (1976) Abnormal shell growth in the Pacific oyster Crassosirea gigas. Some preliminary results from experiments undertaken in 1975. ICES Paper CM 1976/K: I I (mimeograph). International Council for the Exploration of the sea,- Copenhagen. Laughlin R. B.. French W. and Guard H. E. (1986) A&umulation of Bis(tributyltin)oxide by the Marink Mussel MyGlus edulis. Envir. Sri. Technol. 20, 884-890. Machado J., Coimbra J., SB C. and Cardoso I. (1988a) Shell thickening in Anodonm cygnea by induced acidosis. Camp. Biochem. Physiol. (in press).
80
J. MACHADO et al.
Machado J., Ferreira K. G. and Ferreira H. G. (1988b) The acid-base balance in the outer mantle epithelium of Anodonta cygnea (in preparation). Maguire R. J., Chau Y. K., Bengert G. A., Hale E. J., Wong P. T. S. and Kramar 0. (1982) Occurrence of organotin compounds in Ontario lakes and rivers. Envir. SC;. Technol. 16, 698-702. Maeuire R. J.. Wone P. T. S. and Rhamev J. S. (1984) ,&umulation and metabolism of tri-n-buiylin cation by a green alga, Ankisrrodesmus falcafus. Can. J. Fish. Aquat. Sci. 41, 537-540. Thain J. E. and Waldock M. J. (1986) The impact of tributyltin (TBT) antifouling paints on molluscan fisheries. War. Sci. Technol. 18. 193-202.
Waldock M. J. and Thain J. E. (1983) Shell thickening in Crassosfrea gigas: organotin antifouling or sediment induced? Mar. Pollur. Bull. 14, 411415. Zandee D. I., Kluytmans J. H., Zurburg W. and Pieter H. (1980) Seasonal variations in biochemical composition of Myfilus edulis with reference to energy metabolism and gametogenesis. Netherlands Journal of Sea Research 14(l), l-29. Zuckerman J. J., Reisdorf R. P., Ellis III H. V. and Wilkinson R. R. (1978) Organotins in biology and the environment. In Organomefals and Organome~alloia?, Occurrence and Fate in the Environment (Edited by Brinckman F. E. and Bellama J. M.), pp. 388-422. ACS Symp. Ser. 82. American Chemical Society, Washington, DC.