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Sensitivity of fish eggs to acid stress
Water Research Vol. 14, pp. 1679 to 1681 Pergamon Press Lid 1980. Printed in Great Britain
SENSITIVITY OF FISH EGGS TO ACID STRESS RAYMOND M. LEE and SHELBY D. GERKING Department of Zoology, Arizona State University, Tempe, AZ 85281, U.S.A.
(Received August 1979) Abstract---Craig & Baksi (Water Res. 11, 621-626, 1977) may have exaggerated the effect of acid stress on oogenesis by comparing the low viability of eggs laid and incubated at pH 4.5 and 5.0 with the higher survival of eggs laid at pH 6.7 and transferred after 12 h to pH 4.5 and 5.0. On the basis of our findings, a lower survival would have been observed in the latter group if the eggs had been transferred immediately to the acid conditions. The egg changes its character rapidly after being laid. Permeability decreases and the chorion hardens during the first few hours after release, allowing the egg to become more resistant with time. The results of two experiments are reported to demonstrate this effect.
Pollution of aquatic habitats resulting from acid pre- transfer was compared with that of eggs which were cipitation and mine drainage is a widespread and laid at the control pH and then transferred to the test escalating environmental problem. It has been estab- conditions after a 12-h delay (delayed transfer). In this fished that acidification has a detrimental effect upon experiment, neither the female nor the eggs she was fish reproduction (Mount, 1973; Bcamish, 1976; carrying were subjected to acid conditions. Menondez, 1976; Lee & Gerking, In press~ Craig & In the first test, eggs undergoing the 1-h transfer Baksi (1977), using the flagfish, Jordanella floridae, hatched with a greater frequency than did those eggs report that acid stressed fish produce very few eggs at subjected to continuous exposure (Table 1). Hatching pH 4.5 and 5.0. However, about 86% of eggs laid by percentages of eggs with the 1-h transfer were 57.0, fish at a control pH of 6.7 and subsequently trans- 44.0, 21.2 and 15.4 at pH 8.3, 7.0, 6.5 and 6.0, respectferred to these reduced pH levels hatch successfully, ively. Of those eggs with continuous exposure, the From these results, they conclude that the maturation hatching percentages were 50.6, 32.0, 8.7 and 3.3% for of the eggs at pH 6.7 contributed to the success of the same pH levels. No significant difference was hatching and, therefore, that oogenesis is more sensi- demonstrated between the controls at pH 8.3 (57.0 tive to acidity than is the egg after it is laid. and 50.6%), but significant differences occurred at pH Using the desert pupfish, Cyprinodon n. nevadensis, levels 7.0 (Z2 = 4.73, P < 0.05), 6.5 (X2 = 8.50, P < for similar reproductive studies, we also found that 0.005) and 6.0 (Z2 = 7.37, P < 0.01). Eggs experiencfish suffered significant declines in egg production at ing continuous acid exposure during maturation and reduced pH levels. Our results differed, however, from incubation are, therefore, at a definite disadvantage. those of Craig and Baksi, in as much as egg viability The experiment does not rule out either of two alterwas very low in acid conditions, native interpretations, however. Exposure of the adult Differences in the test procedures probably contri- females to acid conditions may affect oogenesis suffibuted to the conflicting result~ Craig and Baksi u ~ l ciently to alter the eggs' ability to hatch. The other several fish in each breeding tank at the control pH possibility is that immediate contact with acid con6.7 and checked for the presence of eggs daily. The ditions is in itself sufficient cause to reduce hatching eggs were removed from the tank and placed into the success. test pH solution within 24 h after being laid. In our The results of the second experiment showed that experiments pairs of pupfish are placed separately in 201. aquaria at a control pH 8.3. Each pair is allowed Table I. Percentage hatching success of eggs laid by the a 30-rain spawning period after which the eggs are desert pupfish, Cyprinodon n. nevadensis, when subjected to immediate or delayed acid exposure removed from a spawning mop, inspected for abnormalities and placed in nylon baskets immersed in the Transferred test solution within I h after being laid. Not transferred after 1 h Two tests were conducted to ascertain whether egg pH Number Hatching Number Hatching viability was related to the time of transfer from nor8.3 510 50.6 100 57.0 mal to acidic conditions. In the first test, the hatching 7.0 278 32.0* 100 44.0 success of eggs which underwent oogenesis, were laid 6.5 196 8.71" 95 21.2 and incubated at the reduced pH levels (continuous 6.0 92 3.3~ 75 1.5.4 exposure) was compared with that of eggs which were 5.5 36 0.0 65 0.0 laid at the control pH (8.3) and transferred within 1 h * Chi-square test significant at P < 0.05. to the test pH (1-h transfer). In the second test, the I" Chi-square test significant at P < 0.01. hatching success of eggs which underwent the l-h ~:Chi-square test significant at P < 0.005. 1679
1680
RAYMONDM. LEE and SHELBYD. GERKING
Table 2. Percentage hatching success of eggs laid by the desert pupfish, Cyprinodon n. nevadensis, when subjected to delayed acid exposure
pH 6.5 6.0 5.5
Transferred after l h Adv. devel. and/or Number hatching 92 63 55
32.3t 21.1 12.5'
Transferred after 12 h Adv. devel, and/or Number hatching 85 46 63
55.9 32.3 30.0
* Chi-square test significant at P < 0.05. t Chi-square test significant at P < 0.01.
eggs subjected to the delayed transfer hatched more frequently than did those eggs which underwent the l-h transfer (Table 2). The percentages of eggs with the delayed transfer that either hatched successfully or reached an advanced stage of development were 55.9, 32.3 and 30.0 and pH levels 6.5, 6.0 and 5.5, respectively. When the eggs underwent the l-h transfer, 32.3, 21.1 and 12.5~ of the eggs either hatched or developed to an advanced stage at the corresponding pH levels. The differences between the two groups are significant at two of the three pH levels (pH 6.5, Z2 = 6.88, P < 0.01 and pH 5.5, Xz = 3.94, P < 0.05). Johansson & Milbrink (1976) found similar results with perch, Perca fluviatilis, eggs at their lower pH levels. At pH 5.6, 5.2 and 4.7, a greater percentage of eggs hatched if they remained at the control pH (7.7) for 24 h prior to being placed into the test solutions, than if the eggs were placed immediately after fertilization into the test solution. Evidently changes are occurring during the first hours after fertilization that significantly affect survival. The process of chorion hardening might explain why delayed transfer results in higher egg viability. Only a few of many references are cited to show that the chorion is at first freely permeable to water and various molecules and later is resistant to osmosis and diffusion. During the first few hours after fertilization, the developing eggs undergo several changes. After closing of the micropyle, the chorion separates from the cortex producing the perivitelline space. Kao et al. (19541 noted that the permeability of the Fundulus plasma membrane decreased rapidly within the first hour after fertilization. Prescott (19551 used D 2 0 to show that, during this period soon after egg laying, only the perivitelline space is penetrable by water, Zotin (1965) confirmed this by placing salmonid eggs in hypertonic solutions and observing that water loss occurred only from the perivitelline space and not from the embryo. Wedemeyer (19681 used 6SZn to show ion uptake in developing eggs. The chorion of the treated eggs contained 71% of the 6SZn taken up by the egg, the perivitelline fluid contained 26%, the yolk 2%, and the embryo only 1~/~ Cykowska & Winnicki (1972) reported that egg membrane strength increased 7-fold in the first 12h after fertilization and
had increased 15 times after 24 h. After hardening, the chorion and the plasma membrane form a resistant barrier to protect the developing embryo from the external medium. The results of the present experiments, combined with these earlier findings, indicate that eggs are suseeptible to acid exposure immediately after fertilization until the membranes have hardened. This explains the very low hatching percentages of eggs laid, fertilized and incubated in low pH waters and the high hatching percentages of eggs laid in neutral pH waters and transferred after a period of time to low pH levels. Although we agree with Craig and Baksi that oogenesis may be affected by acid exposure, we believe that the effect they observed was accentuated by delayed transfer to low pH solutions. The best evidence for an oogenesis effect arose from their observations of inhibited maturation of the oocyte and abnormalities in the eggs, both of which we have confirmed in the pupfish. We concluded in earlier studies that oogenesis was the most sensitive stage in the life cycle to temperature (Shrode & Gerking, 1977; Gerking et al., 1979). If oogenesis does prove to be sensitive to a variety of stresses, then tolerance tests on eggs laid under unstressed conditions may indicate resistances greater than those the same eggs would experience if they had matured in the body of a female subjected to that stress (see Kellogg & Latiman, 1979; Daye & Garside, 1979). As far as acid exposure is concerned, future investigators should bear in mind that in recently acidified lakes and streams both the female and the eggs she produces are exposed to the same degree of acidity.
REFERENCES Beamish R. J. (1976) Acidification of lakes in Canada by acid precipitation and the resulting effects on fishes. War. Air Soil Pollut. 6, 501-514. Craig G. R. & Baksi W. F. (19771 The effects of depressed pH on flagfish production, growth and survival. Water Res. 11, 621-626. Cykowska C. & Winnicki A. (19721 Embryonic development of the Baltic sea trout [Salmo trutta) in buffer solutions. Acta Ichth. Pisca 2, 3--12. Daye P. G. & Garside E. T. (19791 Lower lethal levels of pH for embryos and alevins of Atlantic salmon. Salmo salar L. Can. J. Zool. 57, 1713-1718. Johansson N. & Milbrink G. (19761 Some effects of acidified water on the early development of roach (Rutilus rutilus L.) and perch (Perca [luviatilis L.) Water Resour. Bull. 12, 39-48. Gerking S. D., Lee R. M. & Shrode J. B. 11979) Effects of generation-long temperature acclimation on reproductive performance of the desert pupfish, Cyprinodon n. nevadensis. Physiol. Zool. 52 (2), 113-121. Kao C. Y., Chambers R. & Chambers E. L. (1954) Internal hydrostatic pressure of the Fundulus egg. II. Permeability of the chorion. J. cell. comp. Physiol. 44, 447-461. Kellog R. L. & Latiman D. L. (19781 Effect of acute and chronic thermal exposures on the eggs of three Hudson River anadromous fishes. Eneroy and Environmental Stress in Aquatic Ecosystems (Edited by Thorp J. H. &
Sensitivity of fish eggs to acid stress Gibbons J. W.) pp. 714-725. U. S. Department of Energy. Lee R. M. & Gerking S. D. (1980) Survival and reproductive performance of the desert pupfish, Cyprinodon n. nevadensis, in acid waters. In press, Menendez R. (1976) Chronic effects of reduced pH on brook trout (Salvelinusfontinalis). J. Fish. Res, Bd. Can. 33, 118-123. Mount D. I. (1973) Chronic effect of low pH on fathead minnow survival, growth and reproduction. Water Res. 7, 987-993.
1681
Pre~r,ott D. M. (1955) Effect of activation on the water perme,ability of salmon eggs. J. cell. comp. Physiol. 45, 1-12. Shrode J. B. & Gerking S, D. (1977) Effects of constant and fluctuating temperatures in reproductive performance of a desert pupfish, Cyprinodon n. nevadensis. Physiol. Zool. 50, 1-10. Wedemeyer G. (1968) Uptake and distribution of Zn 65 in the coho salmon egg (Oncorhynchus kisutch). Comp. Biochem. Physiol. 26, 271-279. Zotin A. I. (1965) The uptake and movement of water in embryos. Syrap. Soc. exp. Biol. 19, 365-384.
SENSITIVITY OF FISH EGGS TO ACID STRESS RAYMOND M. LEE and SHELBY D. GERKING Department of Zoology, Arizona State University, Tempe, AZ 85281, U.S.A.
(Received August 1979) Abstract---Craig & Baksi (Water Res. 11, 621-626, 1977) may have exaggerated the effect of acid stress on oogenesis by comparing the low viability of eggs laid and incubated at pH 4.5 and 5.0 with the higher survival of eggs laid at pH 6.7 and transferred after 12 h to pH 4.5 and 5.0. On the basis of our findings, a lower survival would have been observed in the latter group if the eggs had been transferred immediately to the acid conditions. The egg changes its character rapidly after being laid. Permeability decreases and the chorion hardens during the first few hours after release, allowing the egg to become more resistant with time. The results of two experiments are reported to demonstrate this effect.
Pollution of aquatic habitats resulting from acid pre- transfer was compared with that of eggs which were cipitation and mine drainage is a widespread and laid at the control pH and then transferred to the test escalating environmental problem. It has been estab- conditions after a 12-h delay (delayed transfer). In this fished that acidification has a detrimental effect upon experiment, neither the female nor the eggs she was fish reproduction (Mount, 1973; Bcamish, 1976; carrying were subjected to acid conditions. Menondez, 1976; Lee & Gerking, In press~ Craig & In the first test, eggs undergoing the 1-h transfer Baksi (1977), using the flagfish, Jordanella floridae, hatched with a greater frequency than did those eggs report that acid stressed fish produce very few eggs at subjected to continuous exposure (Table 1). Hatching pH 4.5 and 5.0. However, about 86% of eggs laid by percentages of eggs with the 1-h transfer were 57.0, fish at a control pH of 6.7 and subsequently trans- 44.0, 21.2 and 15.4 at pH 8.3, 7.0, 6.5 and 6.0, respectferred to these reduced pH levels hatch successfully, ively. Of those eggs with continuous exposure, the From these results, they conclude that the maturation hatching percentages were 50.6, 32.0, 8.7 and 3.3% for of the eggs at pH 6.7 contributed to the success of the same pH levels. No significant difference was hatching and, therefore, that oogenesis is more sensi- demonstrated between the controls at pH 8.3 (57.0 tive to acidity than is the egg after it is laid. and 50.6%), but significant differences occurred at pH Using the desert pupfish, Cyprinodon n. nevadensis, levels 7.0 (Z2 = 4.73, P < 0.05), 6.5 (X2 = 8.50, P < for similar reproductive studies, we also found that 0.005) and 6.0 (Z2 = 7.37, P < 0.01). Eggs experiencfish suffered significant declines in egg production at ing continuous acid exposure during maturation and reduced pH levels. Our results differed, however, from incubation are, therefore, at a definite disadvantage. those of Craig and Baksi, in as much as egg viability The experiment does not rule out either of two alterwas very low in acid conditions, native interpretations, however. Exposure of the adult Differences in the test procedures probably contri- females to acid conditions may affect oogenesis suffibuted to the conflicting result~ Craig and Baksi u ~ l ciently to alter the eggs' ability to hatch. The other several fish in each breeding tank at the control pH possibility is that immediate contact with acid con6.7 and checked for the presence of eggs daily. The ditions is in itself sufficient cause to reduce hatching eggs were removed from the tank and placed into the success. test pH solution within 24 h after being laid. In our The results of the second experiment showed that experiments pairs of pupfish are placed separately in 201. aquaria at a control pH 8.3. Each pair is allowed Table I. Percentage hatching success of eggs laid by the a 30-rain spawning period after which the eggs are desert pupfish, Cyprinodon n. nevadensis, when subjected to immediate or delayed acid exposure removed from a spawning mop, inspected for abnormalities and placed in nylon baskets immersed in the Transferred test solution within I h after being laid. Not transferred after 1 h Two tests were conducted to ascertain whether egg pH Number Hatching Number Hatching viability was related to the time of transfer from nor8.3 510 50.6 100 57.0 mal to acidic conditions. In the first test, the hatching 7.0 278 32.0* 100 44.0 success of eggs which underwent oogenesis, were laid 6.5 196 8.71" 95 21.2 and incubated at the reduced pH levels (continuous 6.0 92 3.3~ 75 1.5.4 exposure) was compared with that of eggs which were 5.5 36 0.0 65 0.0 laid at the control pH (8.3) and transferred within 1 h * Chi-square test significant at P < 0.05. to the test pH (1-h transfer). In the second test, the I" Chi-square test significant at P < 0.01. hatching success of eggs which underwent the l-h ~:Chi-square test significant at P < 0.005. 1679
1680
RAYMONDM. LEE and SHELBYD. GERKING
Table 2. Percentage hatching success of eggs laid by the desert pupfish, Cyprinodon n. nevadensis, when subjected to delayed acid exposure
pH 6.5 6.0 5.5
Transferred after l h Adv. devel. and/or Number hatching 92 63 55
32.3t 21.1 12.5'
Transferred after 12 h Adv. devel, and/or Number hatching 85 46 63
55.9 32.3 30.0
* Chi-square test significant at P < 0.05. t Chi-square test significant at P < 0.01.
eggs subjected to the delayed transfer hatched more frequently than did those eggs which underwent the l-h transfer (Table 2). The percentages of eggs with the delayed transfer that either hatched successfully or reached an advanced stage of development were 55.9, 32.3 and 30.0 and pH levels 6.5, 6.0 and 5.5, respectively. When the eggs underwent the l-h transfer, 32.3, 21.1 and 12.5~ of the eggs either hatched or developed to an advanced stage at the corresponding pH levels. The differences between the two groups are significant at two of the three pH levels (pH 6.5, Z2 = 6.88, P < 0.01 and pH 5.5, Xz = 3.94, P < 0.05). Johansson & Milbrink (1976) found similar results with perch, Perca fluviatilis, eggs at their lower pH levels. At pH 5.6, 5.2 and 4.7, a greater percentage of eggs hatched if they remained at the control pH (7.7) for 24 h prior to being placed into the test solutions, than if the eggs were placed immediately after fertilization into the test solution. Evidently changes are occurring during the first hours after fertilization that significantly affect survival. The process of chorion hardening might explain why delayed transfer results in higher egg viability. Only a few of many references are cited to show that the chorion is at first freely permeable to water and various molecules and later is resistant to osmosis and diffusion. During the first few hours after fertilization, the developing eggs undergo several changes. After closing of the micropyle, the chorion separates from the cortex producing the perivitelline space. Kao et al. (19541 noted that the permeability of the Fundulus plasma membrane decreased rapidly within the first hour after fertilization. Prescott (19551 used D 2 0 to show that, during this period soon after egg laying, only the perivitelline space is penetrable by water, Zotin (1965) confirmed this by placing salmonid eggs in hypertonic solutions and observing that water loss occurred only from the perivitelline space and not from the embryo. Wedemeyer (19681 used 6SZn to show ion uptake in developing eggs. The chorion of the treated eggs contained 71% of the 6SZn taken up by the egg, the perivitelline fluid contained 26%, the yolk 2%, and the embryo only 1~/~ Cykowska & Winnicki (1972) reported that egg membrane strength increased 7-fold in the first 12h after fertilization and
had increased 15 times after 24 h. After hardening, the chorion and the plasma membrane form a resistant barrier to protect the developing embryo from the external medium. The results of the present experiments, combined with these earlier findings, indicate that eggs are suseeptible to acid exposure immediately after fertilization until the membranes have hardened. This explains the very low hatching percentages of eggs laid, fertilized and incubated in low pH waters and the high hatching percentages of eggs laid in neutral pH waters and transferred after a period of time to low pH levels. Although we agree with Craig and Baksi that oogenesis may be affected by acid exposure, we believe that the effect they observed was accentuated by delayed transfer to low pH solutions. The best evidence for an oogenesis effect arose from their observations of inhibited maturation of the oocyte and abnormalities in the eggs, both of which we have confirmed in the pupfish. We concluded in earlier studies that oogenesis was the most sensitive stage in the life cycle to temperature (Shrode & Gerking, 1977; Gerking et al., 1979). If oogenesis does prove to be sensitive to a variety of stresses, then tolerance tests on eggs laid under unstressed conditions may indicate resistances greater than those the same eggs would experience if they had matured in the body of a female subjected to that stress (see Kellogg & Latiman, 1979; Daye & Garside, 1979). As far as acid exposure is concerned, future investigators should bear in mind that in recently acidified lakes and streams both the female and the eggs she produces are exposed to the same degree of acidity.
REFERENCES Beamish R. J. (1976) Acidification of lakes in Canada by acid precipitation and the resulting effects on fishes. War. Air Soil Pollut. 6, 501-514. Craig G. R. & Baksi W. F. (19771 The effects of depressed pH on flagfish production, growth and survival. Water Res. 11, 621-626. Cykowska C. & Winnicki A. (19721 Embryonic development of the Baltic sea trout [Salmo trutta) in buffer solutions. Acta Ichth. Pisca 2, 3--12. Daye P. G. & Garside E. T. (19791 Lower lethal levels of pH for embryos and alevins of Atlantic salmon. Salmo salar L. Can. J. Zool. 57, 1713-1718. Johansson N. & Milbrink G. (19761 Some effects of acidified water on the early development of roach (Rutilus rutilus L.) and perch (Perca [luviatilis L.) Water Resour. Bull. 12, 39-48. Gerking S. D., Lee R. M. & Shrode J. B. 11979) Effects of generation-long temperature acclimation on reproductive performance of the desert pupfish, Cyprinodon n. nevadensis. Physiol. Zool. 52 (2), 113-121. Kao C. Y., Chambers R. & Chambers E. L. (1954) Internal hydrostatic pressure of the Fundulus egg. II. Permeability of the chorion. J. cell. comp. Physiol. 44, 447-461. Kellog R. L. & Latiman D. L. (19781 Effect of acute and chronic thermal exposures on the eggs of three Hudson River anadromous fishes. Eneroy and Environmental Stress in Aquatic Ecosystems (Edited by Thorp J. H. &
Sensitivity of fish eggs to acid stress Gibbons J. W.) pp. 714-725. U. S. Department of Energy. Lee R. M. & Gerking S. D. (1980) Survival and reproductive performance of the desert pupfish, Cyprinodon n. nevadensis, in acid waters. In press, Menendez R. (1976) Chronic effects of reduced pH on brook trout (Salvelinusfontinalis). J. Fish. Res, Bd. Can. 33, 118-123. Mount D. I. (1973) Chronic effect of low pH on fathead minnow survival, growth and reproduction. Water Res. 7, 987-993.
1681
Pre~r,ott D. M. (1955) Effect of activation on the water perme,ability of salmon eggs. J. cell. comp. Physiol. 45, 1-12. Shrode J. B. & Gerking S, D. (1977) Effects of constant and fluctuating temperatures in reproductive performance of a desert pupfish, Cyprinodon n. nevadensis. Physiol. Zool. 50, 1-10. Wedemeyer G. (1968) Uptake and distribution of Zn 65 in the coho salmon egg (Oncorhynchus kisutch). Comp. Biochem. Physiol. 26, 271-279. Zotin A. I. (1965) The uptake and movement of water in embryos. Syrap. Soc. exp. Biol. 19, 365-384.