VOL. 17, NO. 6 WATER RESOURCES BULLETIN

AMERICAN WATER RESOURCES ASSOCIATION DECEMBER 1981

PERIVITELLINE pH OF SALMONIDE EGGS IN RELATION TO AMBIENT pH'

Niklas Johansson, Per Runn, and Morgan Sohtell'

ABSTRACT: Eggs from salmon and brown trout were reared at 6 pH levels in the pH range 4.1-7.5. By micropuncture, the pH of the peri- vitelline fluid (PVF) was measured at different stages of development with an antimony microelectrode system. The pH of the PVF falls with decreased pH of the ambient media, although no point of equili- brium was found at the lower pH levels. When eggs were reared at pH levels 4.1-4.7, the pH of the PVF in salmon eggs was 0.4 units above the ambient media, while that in trout eggs was 1.0 pH units above. De- creased pH of the PVF during the later, actively ion accumulation, em- bryonic stages will subject the embryos to ion regulating stresses similar to those registered for adult fishes. (KEY TERMS: acid rain; acidification; fish; Salmonidae egg develop ment; perivitelline fluid; pH.)

INTRODUCTION The increasing acidification of lakes and watersheds at-

tributed to precipitation of airborne acidifying substances, primarily oxides of sulphur from combustion of sulphur con- taining fossil fuels and nitrogen oxides from high temperature processes, is causing drastic changes in the fish fauna in parts of Europe and on the North American continent. In Scandi- navia the most severely affected areas are fresh waters with low buffering capacity on the west coast of Sweden and in southern and southwest Norway. Through prevailing wind direction these areas are largely subjected to airmasses transported from regions of high industrial activity in western Europe. When these airmasses reach the coastal areas of Sweden and Norway precipitation occurs through orographic effects and exposes these areas to an intensive precipitation. On the Swedish west coast the influence of local industrial activities such as petro- chemical industries increases the severity of the acidification situation.

In Norway the acidification has led to the extinction of salmon and trout populations in a number of rivers resulting in great economical losses. On the Swedish west coast the sal- mon and trout populations are severely threatened and the perch-pike-roach community of thousands of lakes have been severely distorted or wiped out. This process is now spreading in an increasing number of regions in Sweden (Almer, et d , 1974).

The effect on the fish fauna has largely been attributed to a direct toxic effect of the increased hydrogen activity; recent studies indicate that fishes are affected in different life stages by acidification of their environment (Beamish and Harvey, 1972; Beamish, 1974; Eddy, 1976; Jensen and Snekvik, 1972; Johansson and Milbrink, 1976; Leivestad, etal., 1976; Mount, 1973; Packer and Dunson, 1972). A number of studies have emphasized the specific problems associated with embryonic development, e.g., the viability of embryos during the late stages of development and the interaction between ambient pH and hatching (Carrick, 1979; Johansson, et al.. 1977; Runn, et al., 1977; Trojnar, 1977).

Before hatching the embryo is surrounded by the chorion. The space between the chorion and the vitelline membrane, the perivitellinar space, is formed by the inflow of ambient water during the first minutes after the eggs have been shed into water (Nakano, 1969). This inflow is caused by the re- lease into the forming perivitallinar space of a colloid which is impermeable through the chorion. Due to the hydrostatic pressure built up by the osmotic gradient the eggs thereby be- come turgid. The colloid together with the water now present forms the perivitellinar fluid (PVF).

From fertilization to hatching, the embryo is dependent on the composition of the PVF, as it is the primary environment of the embryo and all transports to and from the surrounding water must take place through the PVF. Although permeable to both water and small ions, including small molecules, the chorion is impermeable to large molecules. The PVF would therefore, after an infinite time, reach the same ionic composi- tion as the surrounding water, if it were not for the ion binding properties of the large molecules present that might modify the total ion content of the PVF, as has been shown for sodium (Rudy and Potts, 1969). The composition of the PVF also is affected by compounds excreted and taken up by the embryo.

Although the effects of a decreased environmental pH on adult fish and survival of eggs and fry are fairly well estab- lished no data have been reported on the effect of pH on the PVF constituting the embryonic environment. The objectives of this study were to determine:

'Paper No. 80127 of the Wuter Resources Bulletin. Discussions are open until August 1, 1982. Respectively, BS, Graduate Student, Department of Zoophysiology, Uppsala University, Box 560, $75 1 22 Uppsala, Sweden; BS, Graduate Student

Department of Zoology, Uppsala University, Box 561, 5751 22 Uppsala, Sweden; and PhD, Assistant Professor, Department of Physiology and Medical Biophysics, Uppsala University, Box 572, 5751 23 Uppsala, Sweden.

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Johansson, RUM, and Sohtell

1) whether an increased H' activity in the rearing water

2) whether there are any differences between species and/ wil l give a decreased pH in the PVF, and

or the developmental stages studied regarding PVF pH.

previous study with the same species under similar conditions (Johansson, et al., 1977).

TABLE 2. Mortality of Salmon and Trout Eggs Reared at Six pH Levels in the pH Range 7.24.1, From Johansson, et al., 1977.

MATERIALS AND METHODS

Roe of salmon (Salmo salm L.) and brown trout ( S a l m mtfu L.) were fertilized on November 2, 1976, in water from the River Dddven (pH 6.5) at the fish rearing station at &v- karleby (one female and two males used for both species). After about 15 minutes, the fertilized eggs were divided into groups and transferred to the rearing solutions described be- low and transported to the Department of Zoophysiology , Uppsala (transport time was about one hour). The eggs were reared in six identical recirculating systems, similar to the ones described by Runn, et al (1977). The total volume of the rearing water in each system was 80 liters.

The rearing solutions were made by mixing one part of tap water with 10 parts of distilled water, giving the following concentrations of: Ca2', 10.1; K', 0.5; Mg2', 1.0; Na', 1.5; C1-, 2.5; and NOT, 1.1 mg 1-l. These solutions were ad- justed to the desired pH values with diluted H2SO4 (analytical grade) and aerated for at least 24 hours before use. The speci- fic conductance (recorded at 25OC with Radiometer CDM 2 d) and pH (recorded at the actual rearing temperature with Radiometer PHM 29r) are listed in Table 1. The pH in each solution was recorded at least every second day and, if neces- sary, adjusted to the desired pH with diluted H2SO4. Forty percent of the rearing solution in each apparatus was changed approximately every tenth day. The temperature in the rear- ing solution was t4.OoC on the first day of the experiment, and was decreased gradually so that it reached t1.6OC on day 40. The temperature then was kept constant until day 90 when it was raised again to reach t5.OoC on the 160th day of rearing.

TABLE 1. pH (maximum and minimum) and Conductance of the Rearing Solutions.

solution Highest pH Lowest pH Conductance No. pH Adjusted To Recorded Recorded (25OC) pS

1 -- 7.7 6.9 70 2 5.8 6.1 5.7 70 3 5.0 5.2 5.0 95 4 4.7 4.9 4.7 107 5 4.4 4.6 4.4 120 6 4.1 4.3 4.1 148

Mortality, Percent, from the Eyed stage to Hatching

PH Salmon Brown Trout

7.2 3 2 5.8 1 2 5.0 1 2 4.8 4 2 4.4 16 10 4.1 81 29

Measurements of the pH of the perivitelline fluid (PVF) were performed with an antimony electrode system designed for micropuncture in a manner similar to that first described by Vieira and Malnic (1968). The manufacture of the elec- trodes is described by Karlmark (1 973). In our investigation, the antimony electrodes had short tips and were angularly ground to a diameter of about 5 pm (Figure 1 b). The silver- silverchloride reference electrode had a liquid junction of 2 percent agar in 1 M KC1 , ending in a tip of about 6 p (Fig- ure la). The two electrodes were connected to a mV meter (PHM 26, Radiometer, Copenhagen).

epoxyresin cabk

I

pol yethene b connect ion antimony

Figure 1 (a). Silversilverchloride Reference Electrode (tip of glass capillary, filled with 2 percent agar in 1 M KCl, angularly ground to a tip diametez of 6 pm); (b) Anti- mony pH-Electrode (tip of glass capillary, filled with anti- mony, angularly ground to a tip diameter of 5 pm).

Hatching occurred for both salmon and trout around the 135th day of rearing. Due to the limited number of eggs (- 250) reared at each pH level from each of the species, al- most all were used during the study. Consequently, it was im- possible to obtain data concerning mortality during the rearing period. The mortality data in Table 2 are taken from a

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The electrode system was calibrated in citric acid phosphate buffers prior to and after each pH measurement. The buffers were placed in tubes and mounted in the specimen stage (Fig- ure 2b). The electrode system was standardized further against a bulb glass electrode (CK 2321C, Radiometer) in the measur- ing chamber fluid to avoid an additional electrode potential due to an oxygen sensitivity of the antimony electrode (Ives and Janz, 1961). The antimony electrode has been found to give a lower pH when measuring in bicarbonate containing

WATER RESOURCES BULLETIN

Perivitelline pH of Mmonide Eggs in Relation to Ambient pH

biological tissue (Karlmark and Sohtell, 1973) and, recently, this value has been found to be 0.09 pH units lower than the actual pH (Sohtell, 1979). All pH values given in this investi- gation have been corrected accordingly.

thermistor a

- 1 r v stirrer

water .I#." ........ n .... C

mantle

drain valve

0

4

b

I I

iffer tube

Figure 2(a). Cross Section of Measuring Chamber With Water Mantle and Specimen Stage (the dotted line shows water level in the center container; the total diameter of the measuring chamber is 15 cm). (b) Side and top view of specimen stage (in the side view, a fixed egg is shown in cross section; the size of the upper surface of the spedmen stage is 4.5 x 4.5 cm).

MEASURING PROCEDURE The fertilized eggs from salmon and trout were placed on

specimen stages in a thermostated measuring chamber filled with the same solution as that in which the eggs were reared (Figure 2a). Four eggs were fNed with plasticine to each specimen stage (Figure 2b). In a preparatory investigation, lasting for three days, no harmful effects on the eggs due to the method of fixation could be observed. The viability of the embryos at the early recordings was difficult to determine. Embryos showing any signs of diverging appearance were al- ways excluded. Occasionally, eggs were opened after the re- cordings to confirm their viability.

The temperature, when mounting the eggs, was always the same as in the rearing solution and the recordings were per- formed at +4.0°C. The low temperature was achieved by us- ing a cooling compressor regulated with a thermistor probe placed in the bath surrounding the eggs.

The electrodes were mounted separately onto two micro- manipulators and the puncture of the object was observed through a binocular microscope. When puncturing the chorion, it was almost impossible to introduce both electrodes into the

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perivitelline space without injuring the vitelline membrane. Consequently, the reference electrode was always placed out- side and close to the chorion. In a preparatory investigation the potential difference over the chorion was measured, but was found not to differ significantly from zero. This means that no additive potential, but only a hydrogen ion activity potential, occurred with the antimony electrode in the peri- vitelline space.

Day 85 was the last day of successful measurement of the PVF pH. Attempts were made to accomplish measurements after day 85, but these were unsuccessful because the em- bryos broke the electrode tips when they moved.

RESULTS On each day of measurement three (occasionally two or

four) measurements were performed on eggs from the same group. The pH values recorded are given (range and arithmetic mean of hydrogen ion activity converted to pH) in Figure 3.

For the eggs reared in the pH range 5.84.1 , except for pH 4.4, the initial pH recorded on day 1 is significantly higher and close to fertilization pH, 6.5, than the results obtained during the later part of development. The time delay in reach- ing a stable relation between ambient media and the perivitel- linar fluid, PVF, during the initial part of development shows that this stabilization is not instantaneous but requires at least one to two days. In the pH range 4.1-5.0, the Ht activity of the PVF never equilibrates fully with the ambient water. As shown in Figure 4, where the mean values of the last record- ing, approximately day 80, are plotted against rearing pH, the PVF shows a lower H+ activity compared to that of the am- bient media, most pronounced at lower pH levels. The situa- tion at pH 7.5 was an increase in pH to a level close to or slightly below that of the ambient media. Furthermore, there was a difference between the two species studied as illus- trated in Figure 4. The slopes of the lines for salmon and trout are 0.83 and 0.75, respectively, and in the pH range 4.1-4.7, the mean deviation of pH of the PVF above the ambient media was - 0.4 for salmon and - 1.0 for trout (as calculated from the actual individual measurements).

The ranges given in Figure 3 are considerable in some cases, especially for trout. The variation between individual eggs is assumed to be mainly biological, as there is little doubt of the reliability of the measuring technique, Conclusions drawn here are not based on single mean values or ranges but on trends or comparisons between mean values or groups of indi- vidual values.

It should be emphasized that as the experiment continued, embryos that could not withstand the rearing conditions died and were discarded (Table 2). Accordingly eggs that were sampled for recordings at the end of the experimental period were taken out of groups that were more or less selected to withstand acidification. The deviating values recorded at pH 4.4 between days 40 and 50 for trout are, however, hard to explain.

No continuous measurement of the pH change in the PVF of eggs exposed to decreased pH has been successful. However,

WATER RESOURCES BULLETIN

Johansson, Runn, and Sohtell

pH P V F

4 , pH 7.5 P

7.0

'V F El pH 5.8

pH P V F DH 5.0 pH P V F pH 4.7

DAYS . t

20 40 W 80

Figure 3. The pH Values of the Perivitelline Fluid (PVF) Measured on Three or Four Different Occasions During the Development. Mean values (open circle = trout, and filled circle = salmon) are connected for clarity. The vertical bars show range at each measurement and the horizontal broken line indicates the pH of the rearing solution. The first measurements were performed within 16 hours of fertilization for all group.

997 WATER RESOURCES BULLETIN

Perivitelline pH of Salmonide Eggs in Relation to Ambient pH

measurements on eggs exposed for 12 hours at various de- creased pH levels during the later part of development yield a pH close to that obtained on eggs reared at the corresponding pH through development. This is in contrast to the initial measurements performed on the eggs, approximately 12 hours after fertilization in which the pH of the PVF, except for eggs reared at pH 4.4, was considerably above the values obtained during the later part of development (see above).

pH PVF

pH REARING WATER c-

5.0 6.0 TO

Figure 4. Mean Values of the Last Recordings (approximately, day 80). The pH of the perivitellinar fluid (PVF) is plotted against the pH' of the rearing water. The broken line represents a one-to-one relationship.

DI SCUSSI ON The present study shows that unhatched embryos of salmon

and trout, in an acidified environment, are exposed to a pH close to or slightly above that 0.f the environment. Moreover, differences in the pH of the PVF between the species studied were measured, when the eggs were reared under identical con- ditions.

The decreased Ht activity in the PVF as compared to the ambient media might be caused by a number of different factors, the relative contributions of which are today undocu- mented. The excretion and uptake of substances diffusible through the chorion by the embryo causes a flux of these sub- stances through the chorion. Thus, the permeability of the chorion for different substances affects the composition of the PVF, e.g., excretion products formed (such as HC05, N H a would under conditions of low permeability influence the Ht activity of the PVF. A further factor that might modulate the Ht activity is the relative volume of the PVF in each case.

The ion composition of the PVF has been shown to deviate from that of the surrounding media regarding sodium. At low ambient concentrations of sodium the content of this ion in the PVF is increased by a factor of ten (Rudy and Potts, 1969). ns has been attributed to the ion binding properties of the colloid in the PVF. The colloid might also affect the Ht acti- vity and induce the deviations of pH reported here between the PVF and the ambient media.

It is not suggested that the more pronounced reduction in Ht activity of the PVF recorded for trout compared to salmon at low pH values is to be mediated by some mechanism absent in salmon. The discrepancy between salmon and trout is to be regarded as a result of qualitative and/or quantitatitve dif- ferences of the factors discussed above.

One of the major effects of a decreased environmental pH on adult fishes is an increased stress on the ion regulating mechanisms, resulting in a reduction in the net accumulation of ions, or at low pH levels even in a net loss of ions (Packer and Dunson, 1972; McWilliams and Potts, 1978). The result, when rearing egg and yolk sac stages of some species, includ- ing the two studied here, is a relation between decreased pH and increased mortality. This was most significant during the period around hatching and during the stages of yolk resorb- tion (Johansson, et al., 1977; Leivestad, et al., 1976). Sodium exchange and accumulation in early salmon embryos have been shown to be insignificant but increase rapidly during the later stages of development, and from the eyed stage to hatch- ing, total Na content is increased by 20 percent (Rudy and Potts, 1969).

As our measurements show, even prior to hatching, embryos reared at low pH levels are subjected to a pH close to or slightly above that of the ambient media. The onset of active ion accumulation in the later stages of egg development will cause an increased stress on the ion regulating mechanisms. Thus, the decreased pH of the PVF in acid ambient water may ac- count for the increased mortality observed during this period (Johansson, et al., 1977; Leivestad, er d , 1976). Except for interaction with the hatching process (see below), i t is sug- gested that this increased mortality is mediated by similar mechanisms as those described earlier for adult fish. The ob- servation that salmon embryos before hatching are exposed to a lower pH than trout embryos when their eggs are simul- taneously subjected to decreased environmental pH, may be a factor promoting the higher degree of mortality measured in eggs of salmon reared at pH < 4.4 compared to those of trout (Table 2).

The higher hydrogen activity of the ambient media com- pared to the PVF at low pH levels will, at the perforation of the chorion at hatching, increase the osmoregulatory stress on the fry. This is in accordance with previous results where an increased mortality is reported at hatching and the post hatch- ing stages (Johansson, er al., 1977).

The measurements performed on eggs exposed to a de- creased pH for 12 hours shows that even short episodes of low pH might effect developing embryos although enclosed by the chorion. Such a situation can arise during spring snow melt where massive amounts of melt water with a low pH flows out during a short period.

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Johansson, RUM, and Sohtell

Apart from being a major factor affecting the ion regula- tion properties of the embryo, the decreased pH of the PVF also might be one of the explanations for the impairment of the hatchmg process of salmonid eggs in acid water described earlier, since the activity of the hatching enzymes depends on the actual pH of the PVF (Hagenmaier, 1974; Johansson,etul., 1977; Yamagami, 1973).

CONCLUSIONS Our result show that salmon and brown trout embryos

developing in an acidified environment are subjected to pH stresses even prior to hatching. The hydrogen ion activity of the PVF does not at low pH levels equilibrate with the ambient water but shows a slightly lower activity, 0.4-1.0, pH units. Accordingly, the perforation of the chorion at hatching will result in an increased stress at hatching and during the post hatching stages. These findings are in agreement with the in- creased mortality obtained during these stages in egg fry sur- vival tests. The rapid adjustment of the PVF pH (<12 h) to changes of the ambient water leads to that even short episodes of low pH, such as during the spring snow melt, can effect developing embryos.

Our results also indicates that when selecting species and strains suitable for introduction in acidified areas the pro- perties of the PVF constituting the microenvironment of the developing embryo is of considerable interest.

ACKNOWLEDGMENTS

We wish to express our appreciation to MIS. Manta Karlsson for her technical assistance, to MIS. Kerstin Ahlfors for preparing the illus- trations and to Professor Jan Erik Kihlstrb'm and Dr. Jan Gfberg for valuable criticism of the manuscript. This work was supported by the National Swedish Environment Protection Board (Contract No. 7-1 281 76 and 7-1 28/77).

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Beamish, R. J. and H. Harvey, 1972. Acidification of the La Cloche Mountains Lakes, Ontario, and Resulting Fish Mortalities. J. Fish. Res. Bd. Can. 29:1131-1143.

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Hagenmaier, H. E., 1974. The Hatching Process of Fish Embryos. IV. The Enzymological Properties of a Highly Purified Enzyme (chorionase) From the Hatching Fluid of the Rainbow Trout, Salrno gairdneri. J. Comp. Biochem. Physiol. 49 B:313-324.

Ives, D. J. C. and G. J. Janz, 1961. Reference Electrodes. Academic Press, New York, New York, pp. 336-351.

Jensen, K. W. and E. Snekvik, 1972. Low pH-Levels Wipe Out Salmon and Trout Populations in Southernmost Norway. Ambio 1(6):223- 225.

Johansson, N. and C. Milbrink, 1976. Some Effects of Acidified Water on the Early Development of Roach, Rutilus rutilus, and Perch, Perca fluviatilis. Water Resources Bulletin 12(1):3948.

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Sohtell, M., 1979. C02 Along the Proximal Tubules in the Rat Kidney. Acta Physiol. Scand. 105:146-155.

Trojnar, J. R., 1977. Egg Hatchability and Tolerance of Brook Trout (Salvelinus fontinalis) Fry at Low pH. J. Fish Res. Bd. Can. 34: 574-579.

Vieira, F. L. and G. Malnic, 1968. Hydrogen Ion Secretion by Rat Renal Cortical Tubules as Studied by an Antimony Electrode. Am. J. Physiol. 214 (4):7 10-7 18.

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