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Comparison of absorption rates of halogenated phenols across fish gills in fresh and marine water
Marine Environmental Research 28 (1989) 275-278
Comparison of Absorption Rates of Halogenated Phenols Across Fish Gills in Fresh and Marine Water Peter P~irt Department of Zoophysiology, Uppsala University, Box 560, S-751 22 Uppsala, Sweden
ABSTRACT The absorption rates of five halogenated phenols across the gill epithelium of freshwater- and seawater-adapted rainbow trout was measured. At pH6, when the compounds mainly are present in the non-ionized form, the absorption rates in fresh water were higher than in seawater. At p H 9 no differences in absorption rates between the two water qualities were observed for those phenols present in ionizedJbrm. It is concluded that adaptation to seawater to some extent reduces the permeability of the gills for the nonionized forms of the compounds.
The importance of water quality in modifying the absorption rates of heavy metals in fish is well-recognized. However, the impact of water quality on the absorption of organic compounds has not received much attention. The aim of the present investigation was to test whether there is a difference in absorption rates of halogenated phenols across the gill epithelium of freshwater- and seawater-adapted fish. The background is an observation of Tulp et al.~ showing a more efficient absorption of 2,2',4,5,5'-pentachlorobiphenyl (PCB) in freshwater- than in seawater-adapted salmon (Salmo salar). Phenols are weak acids. The degree of ionization is determined by the ambient pH and the pK a of the compound. Fish gills are approximately 1000 times more permeable for the non-ionized form than for the ionized form 2 (Part & Saarikoski, in preparation). The absorption and toxicity of phenols in fish is therefore pH-dependent. 3'4 However, the absorption rate at a pH > pK a is significantly higher than that predicted on the basis of the concentration and permeability of the non-ionized form. 2 It indicates that 275
Marine Environ. Res. 0141-1136/90/$03.50 .~ 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
276
P e t e r Piirt
the ionized form contributes to the absorption rate. Local acidification of the gill surface, s unstirred layers impeding the diffusion rate of the nonionized form 2 or the formation of ion pairs between the phenate ion and cations in the water 6 have all been suggested as responsible for these deviations. Ion-pair formation would be expected to be more pronounced in the ion-rich seawater than in fresh water. Thus, the pH-dependence of the absorption of weak acids would be expected to be less obvious in salt water than in fresh water. A perfused gill preparation from rainbow trout (Salmo gairdneri) was used. v This method allows direct measurement of absorption rates across the gill epithelium. Experiments were performed at pH 6 and p H 9 in fresh water (Na + 0"06, Cl- 0"02, Ca 2 + 0"2 mmol/litre; conductivity 6(~70/~S) and in artificial seawater (2"5% salinity). The originally freshwater fish were acclimated for 6 weeks to seawater prior to the perfusions. A mixture containing 0"5-5/tmol/litre of the following compounds was used: 4bromophenol (4-BrP) (pK~ 9.2), 2,4-dichlorophenol (2,4-DCP) (pK~ 7.85), 2,4-dibromophenol (2,4-DBrP) (pK~ 7.8), 2,4,5-trlchlorophenol (2,4,5-TCP) (pK~ 7-05) and 2,4,6-trichlorophenol (2,4,6-TCP) (pK,, 6.2). They were analysed by gas chromatographyfl The non-ionized form of the phenols dominates at pH 6. Absorption rates at this pH were higher in freshwater- than in seawater-adapted trout gills (Fig. 1). This observation confirms the conclusion of Tulp et al. 1 of a more efficient absorption of non-ionized hydrophobic compounds in freshwaterthan in seawater-adapted fish gills. At pH 9 no difference in absorption rates between freshwater- and ~j
Fresh wateP
.5 Sea watem
~f
4-8pP
2,4-DBPP 2,4-OCP
2,4,5-TCP
2, 4,6-7CP
Fig. I. The absorption rate constants (K min 1) at p H 6 of halogenated phenols in pcrfused gills from fresh- and seawater-adapted rainbow' trout. Values are mean ± S D , nrr~h,,~,~r = 15, n,ea~,~,r 16: *p < 0"05, Mann-Whitney U-test.
Absorption rates of phenols across fish gills
T
277
L
~resh watep
]
•
Sea water
F~ ~redi~te~
01
11_ 4-BrP
2, 4-OBrP
2, 4-DCP
2, 4, 5-TCP
2, 4, 6 TCP
Fig. 2. The observed and predicted absorption rate constants (K min 1) at p r i g of halogenated phenols in perfused gills from fresh- and seawater-adapted rainbow trout. The predicted constants are calculated from the permeability and relative concentration of the non-ionized form of the phenols, assuming that the gills only are permeable for the nonionized form (pH-partition hypothesis). Values are mean + SD, t/i,re~h,~aler 15, n,~,,~,~, = 16: * p < 0"05, M a n n - W h i t n e y U-test. =
seawater-adapted gills was observed except for 4-BrP which had a higher absorption rate in fresh water (Fig. 2). At this pH, 4-BrP is still mainly nonionized and it confirms the previous observation of a salinity-induced reduction of gill permeability for the non-ionized form of the compounds. The other phenols are mainly present as phenate ions. The absorption rates in both water qualities were markedly higher than expected on the basis of the concentration of the non-ionized form (Fig. 2). Since there was no difference in absorption rates between fresh and seawater, the results do not support the idea of ion-pair formation as being responsible for the higher than expected absorption at pH > pK a. As a conclusion, the present investigation shows that the gill epithelium of freshwater-adapted rainbow trout is more permeable for the non-ionized form of halogenated phenols than the epithelium of seawater-adapted trout. This difference may, however, be of minor importance with respect to accumulation of phenols in marine fish. The pH of the water appears to be the most important water quality parameter in modifying the absorption rates of these compounds. Seawater has, by comparison, generally a higher pH (pH around 8) than most natural fresh waters. Since the pK, of most halogenated phenols of environmental concern are lower than 8 they will be mainly ionized in seawater and are thus less available than in fresh water. The net result will be a lower absorption of phenols in the marine than in the limnic environment.
278
Peter Piirt ACKNOWLEDGEMENTS
This research was funded by the National Protection Board.
Swedish Environmental
REFERENCES 1. Tulp, M. Th. M., Haya, K., Carson, W. G., Zitko, V. & Hutzinger, O., Chemosphere, 4 (1979) 243-9. 2. Saarikoski, J., Lindstr6m, R., Tyynel~i, M. & Viluksela, M., Ecotoxicol. Environ. Safety, 11 (1986) 158-73. 3. Saarikoski, J. & Viluksela, M., Arch. Environm. Contam. Toxicol., 10 (1981) 747-53. 4. Saarikoski, J. & Viluksela, M., Exotoxicol. Environ. Safety, 6 (1982) 501 12. 5. Lloyd, R. & Herbert, D. V. M., Ann. Appl. Biol., 48 (1960) 399404. 6. Esser, H. O. & Moser, P., Ecotoxicol. Environ. Safety, 6 (1982) 131-48. 7. P~irt, P., Saarikoski, J., Tuurala, H. & Havaste, K., Aquatic Toxicol. (submitted).
Comparison of Absorption Rates of Halogenated Phenols Across Fish Gills in Fresh and Marine Water Peter P~irt Department of Zoophysiology, Uppsala University, Box 560, S-751 22 Uppsala, Sweden
ABSTRACT The absorption rates of five halogenated phenols across the gill epithelium of freshwater- and seawater-adapted rainbow trout was measured. At pH6, when the compounds mainly are present in the non-ionized form, the absorption rates in fresh water were higher than in seawater. At p H 9 no differences in absorption rates between the two water qualities were observed for those phenols present in ionizedJbrm. It is concluded that adaptation to seawater to some extent reduces the permeability of the gills for the nonionized forms of the compounds.
The importance of water quality in modifying the absorption rates of heavy metals in fish is well-recognized. However, the impact of water quality on the absorption of organic compounds has not received much attention. The aim of the present investigation was to test whether there is a difference in absorption rates of halogenated phenols across the gill epithelium of freshwater- and seawater-adapted fish. The background is an observation of Tulp et al.~ showing a more efficient absorption of 2,2',4,5,5'-pentachlorobiphenyl (PCB) in freshwater- than in seawater-adapted salmon (Salmo salar). Phenols are weak acids. The degree of ionization is determined by the ambient pH and the pK a of the compound. Fish gills are approximately 1000 times more permeable for the non-ionized form than for the ionized form 2 (Part & Saarikoski, in preparation). The absorption and toxicity of phenols in fish is therefore pH-dependent. 3'4 However, the absorption rate at a pH > pK a is significantly higher than that predicted on the basis of the concentration and permeability of the non-ionized form. 2 It indicates that 275
Marine Environ. Res. 0141-1136/90/$03.50 .~ 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
276
P e t e r Piirt
the ionized form contributes to the absorption rate. Local acidification of the gill surface, s unstirred layers impeding the diffusion rate of the nonionized form 2 or the formation of ion pairs between the phenate ion and cations in the water 6 have all been suggested as responsible for these deviations. Ion-pair formation would be expected to be more pronounced in the ion-rich seawater than in fresh water. Thus, the pH-dependence of the absorption of weak acids would be expected to be less obvious in salt water than in fresh water. A perfused gill preparation from rainbow trout (Salmo gairdneri) was used. v This method allows direct measurement of absorption rates across the gill epithelium. Experiments were performed at pH 6 and p H 9 in fresh water (Na + 0"06, Cl- 0"02, Ca 2 + 0"2 mmol/litre; conductivity 6(~70/~S) and in artificial seawater (2"5% salinity). The originally freshwater fish were acclimated for 6 weeks to seawater prior to the perfusions. A mixture containing 0"5-5/tmol/litre of the following compounds was used: 4bromophenol (4-BrP) (pK~ 9.2), 2,4-dichlorophenol (2,4-DCP) (pK~ 7.85), 2,4-dibromophenol (2,4-DBrP) (pK~ 7.8), 2,4,5-trlchlorophenol (2,4,5-TCP) (pK~ 7-05) and 2,4,6-trichlorophenol (2,4,6-TCP) (pK,, 6.2). They were analysed by gas chromatographyfl The non-ionized form of the phenols dominates at pH 6. Absorption rates at this pH were higher in freshwater- than in seawater-adapted trout gills (Fig. 1). This observation confirms the conclusion of Tulp et al. 1 of a more efficient absorption of non-ionized hydrophobic compounds in freshwaterthan in seawater-adapted fish gills. At pH 9 no difference in absorption rates between freshwater- and ~j
Fresh wateP
.5 Sea watem
~f
4-8pP
2,4-DBPP 2,4-OCP
2,4,5-TCP
2, 4,6-7CP
Fig. I. The absorption rate constants (K min 1) at p H 6 of halogenated phenols in pcrfused gills from fresh- and seawater-adapted rainbow' trout. Values are mean ± S D , nrr~h,,~,~r = 15, n,ea~,~,r 16: *p < 0"05, Mann-Whitney U-test.
Absorption rates of phenols across fish gills
T
277
L
~resh watep
]
•
Sea water
F~ ~redi~te~
01
11_ 4-BrP
2, 4-OBrP
2, 4-DCP
2, 4, 5-TCP
2, 4, 6 TCP
Fig. 2. The observed and predicted absorption rate constants (K min 1) at p r i g of halogenated phenols in perfused gills from fresh- and seawater-adapted rainbow trout. The predicted constants are calculated from the permeability and relative concentration of the non-ionized form of the phenols, assuming that the gills only are permeable for the nonionized form (pH-partition hypothesis). Values are mean + SD, t/i,re~h,~aler 15, n,~,,~,~, = 16: * p < 0"05, M a n n - W h i t n e y U-test. =
seawater-adapted gills was observed except for 4-BrP which had a higher absorption rate in fresh water (Fig. 2). At this pH, 4-BrP is still mainly nonionized and it confirms the previous observation of a salinity-induced reduction of gill permeability for the non-ionized form of the compounds. The other phenols are mainly present as phenate ions. The absorption rates in both water qualities were markedly higher than expected on the basis of the concentration of the non-ionized form (Fig. 2). Since there was no difference in absorption rates between fresh and seawater, the results do not support the idea of ion-pair formation as being responsible for the higher than expected absorption at pH > pK a. As a conclusion, the present investigation shows that the gill epithelium of freshwater-adapted rainbow trout is more permeable for the non-ionized form of halogenated phenols than the epithelium of seawater-adapted trout. This difference may, however, be of minor importance with respect to accumulation of phenols in marine fish. The pH of the water appears to be the most important water quality parameter in modifying the absorption rates of these compounds. Seawater has, by comparison, generally a higher pH (pH around 8) than most natural fresh waters. Since the pK, of most halogenated phenols of environmental concern are lower than 8 they will be mainly ionized in seawater and are thus less available than in fresh water. The net result will be a lower absorption of phenols in the marine than in the limnic environment.
278
Peter Piirt ACKNOWLEDGEMENTS
This research was funded by the National Protection Board.
Swedish Environmental
REFERENCES 1. Tulp, M. Th. M., Haya, K., Carson, W. G., Zitko, V. & Hutzinger, O., Chemosphere, 4 (1979) 243-9. 2. Saarikoski, J., Lindstr6m, R., Tyynel~i, M. & Viluksela, M., Ecotoxicol. Environ. Safety, 11 (1986) 158-73. 3. Saarikoski, J. & Viluksela, M., Arch. Environm. Contam. Toxicol., 10 (1981) 747-53. 4. Saarikoski, J. & Viluksela, M., Exotoxicol. Environ. Safety, 6 (1982) 501 12. 5. Lloyd, R. & Herbert, D. V. M., Ann. Appl. Biol., 48 (1960) 399404. 6. Esser, H. O. & Moser, P., Ecotoxicol. Environ. Safety, 6 (1982) 131-48. 7. P~irt, P., Saarikoski, J., Tuurala, H. & Havaste, K., Aquatic Toxicol. (submitted).