EFFECTS OF SALINITY ON THE DURATION AND COURSE OF EMBRYOGENESIS IN SEA TROUT (SALMO TRUTTA L.)

Małgorzata Bonisławska
Department of Aquatic Sozology, West Pomeranian University of Technology in Szczecin, Szczecin, Poland

ABSTRACT

Effects of salinity (0.5–0.7; 1; 2; 3‰) on the duration and course of embryogenesis in sea trout were studied using a system consisting of a microscope coupled with a digital camera, computer screen, and video recorder. The observation system used made it possible to analyze the videotaped images and frames, and to measure the eggs (diameters) and newly hatched larvae (body length and yolk sac volume). The control embryos took the longest to hatch. Depending on the water salinity, the newly hatched larvae showed significant differences: the longest and largest larvae hatched from the control eggs and those incubated at the lowest salinity. The larvae incubated at 2‰ showed the highest percentage of malformed individuals. The salinity of 3‰ proved too high for the sea trout embryos; they failed to hatch and died while still in the egg. Low salinity (up to 2‰) did not affect the duration and course of embryogenesis in the sea trout.

Key words: fertilization, embryonic, development, salinity, sea trout (Salmo trutta L.).

INTRODUCTION

Aquatic habitats supporting numerous organisms including fish differ widely in terms of quality and properties of the water (i.a., temperature, pH, dissolved oxygen content, nutrient content, salinity, content of suspended particulates, and anthropogenic pollutants). Each of those factors, alone or in combination with others, may, and do, affect the distribution, reproduction, and embryonic development of various fish taxa.

It is not surprising, then, that effects of salinity on fertilization and course of embryonic development of numerous fish species, both marine and freshwater, have been at the focus of attention of numerous authors worldwide. As early as in 1930, Bogucki [3] studied permeability of sea trout egg membranes to different substances, including salts (KCl, NaCl). Russian scientists have been particularly successful in their studies on salinity effects on the course of embryogenesis in fish. In the 1940s, Olifan [20], who studied salinity effects on embryonic development of carp (Cyprinus carpio L.), bream (Abramis brama (L.)), and Caspian roach (Rutilus rutilus caspicus J.), found each species' embryonic and larval development to be adapted to a particular salinity range; she also demonstrated stage-specific salinity sensitivity. Vernidub [29] dealt with effects of Ringer fluid-based salt solutions (12, 15, 16, 18‰) on different developmental stages of percids: perch (Perca fluviatilis L.), bream, zander (Stizostedion lucioperca (L.)), and ruffe (Gymnocephalus cernua (L.)). She found the strongest solutions to exert the strongest effects on the developing eggs during gastrulation and at the stage of embryo formation and growth, resulting in size differences between larvae hatched from eggs incubated at different salinities. Pietrova [23] used Ringer fluid-based salt solutions (5.5; 11; 16.5; 22; 27.5; 33‰) to incubate eggs of smelt (Osmerus eperlanus (L.)) and whitefish (Coregonus lavaretus (L.)). The smelt embryos were observed to develop regularly at the salinity of up to 5.5‰; the whitefish embryos were more resistant, as some larvae hatched from eggs incubated at 11‰. Rykova [25] found the spawning ground salinity to be decisive for reproduction and to affect spawning by reducing fertility and egg diameter in grass carp.

Vetemaa and Saat [30] studied effects of salinity (1–11‰) on the developing eggs of ruffe from freshwater reservoirs and brackish water of a Baltic bay. They found larvae of the freshwater ruffe to develop regularly when the eggs were incubated at 1–6‰. The embryos developing from eggs of the Baltic bay ruffe were more salinity-tolerant, as normal individuals hatched from eggs incubated at 7, 8, and 9‰ and their maximum body length was always attained at a salinity higher than that applied to the eggs of the freshwater ruffe.

Swanson [27] carried out a very interesting study involving milkfish (Chanos chanos), a species providing an evolutional link between clupeiforms and cypriniforms and occurring in open ocean and marine water (Indian Ocean, Pacific, Red Sea). The species spawns in clear water in the vicinity of coral reefs and the newly hatched larvae migrate to estuaries and bays to feed. They are able to live both in fresh and saline (even up to 40‰) water, are high-temperature (up to 40°C) tolerant and die when the temperature drops below 15°C [34]. The eggs were experimentally incubated in saline water (15, 20, 35, 50, 55‰). Observations made by the author unequivocally pointed to the salinity of 35‰ as optimal for the embryonic development of the species [27].

Fashina-Bombata and Busari [9] followed embryonic development of the catfish (Heterobranchus longifilis) in eggs incubated at salinities of 0 to 15‰. The eggs were capable of hatching within the salinity range of 0–7.5‰, but the hatching rate was observed to decrease with increasing salinity, from 71–75% (at 1.5 and 3‰) to 41% at 7.5‰ [9].

In Poland, studies on effects of saline (3‰) water of the Vistula Lagoon and water of higher salinities (c. 3.7; 5.6; and 11‰) on sperm mobility, fertilization, and embryonic development of bream were carried out by Dziekońska [7,8]. The sperm in her study, although motile at a salinity higher than 3‰ (5.6‰), failed to fertilize the egg cells [8].

Effects of increased or much reduced salinity on the embryonic development were studied also in marine fish. The numerous studies dealing with the problem confirmed the adverse effect of both a too low and too high salinity on embryogenesis [15,19,22,28,33].

Salinity is a result of accumulation of dissolved salts in fresh water. The salinity spectrum of inland waters is very wide. Soil irrigation and river damming are anthropogenic activities which may produce increased salinity of surface waters [1]. The major sources of inland water salinity in Poland include saline mine water discharges (55%), industrial sewage (23%), and others (22%) [16]. A serious problem is posed by increased salinity (increased concentration of chlorides in water) in winter and early spring as a result of salt being used for removal of frozen snow and ice from the roads.

The solutions used in this study encompass the brackish water range of 0.5–5‰ (mixohaline – mixooligohaline) [21]. As indicated by the evidence described above, technological developments worldwide result in increasing salinity of inland waters (including streams and brooks serving as spawning grounds for the sea trout). Therefore, this study was aimed at presenting effects of salinity on fertilization and embryonic development as well as on the survival rate and size of the newly hatched larvae of this valuable salmonid species.

MATERIAL AND METHODS

The study was carried out during two seasons: November-March of 2005/2006 and 2006/2007.

Egg fertilization and incubation
Reproductive materials (eggs: a mix of eggs from 5 females; sperm: a mix of sperm from 8 males) were obtained from adult sea trout caught in the River Rega (NW Poland, near Trzebiatów). The eggs were fertilized dry at an isothermal laboratory. The fertilized eggs, placed in fresh and saline water, were left to swell for about 1 hour, whereupon they were distributed between plastic containers placed in tanks filled with water of different salinity. To ensure optimal incubation conditions, the containers were kept in an isothermal room and were equipped in aerators and filters.

In the 2005/2006 season, the following experimental treatments were applied:
IA: eggs fertilized and incubated in saline water (0.5–0.7‰);
IB: eggs fertilized and incubated in saline water (1‰);
IC: eggs fertilized and incubated in saline water (2‰);
IIA: eggs fertilized in tap water and incubated in saline water (0.5–0.7‰);
IIB: eggs fertilized in tap water and incubated in saline water (1‰);
IIC: eggs fertilized in tap water and incubated in saline water (2‰);
K: control, eggs fertilized and incubated in tap water (0.2–0.3‰).

In the 2006/2007 season, the experiment was repeated using the following treatments:
I B: eggs fertilized and incubated in saline water (1‰);
IC: eggs fertilized and incubated in saline water (2‰);
K: control, eggs fertilized and incubated in tap water (0.2–0.3‰);
in addition, the scope of the experiment was broadened by introducing new experimental treatments: ID: eggs fertilized and incubated in saline water (3‰); R: eggs fertilized and incubated in riverine water (River Wiśniówka, NW Poland near Goleniów) (0.3–0.4‰). Solutions with salinities of 0.5–0.7; 1; 2; 3‰ were prepared by dissolving appropriate amounts of sea salt in distilled water.

Embryogenesis
An observation set (a microscope, a digital camera, a computer monitor, a video recorder, a personal computer) was used to follow the embryonic development of live eggs; the equipment made it possible to videotape the observations and to analyse the video images [32].

The duration of embryonic development was expressed in degree-days (°D); the rate of embryogenesis was determined by following the consecutive stages (as shown by about 60% of the individuals observed): blastopore closure; the appearance of pigment in the eyes ('eyeing'); hatching (as of the first hatched larvae); 50% hatching; and termination of hatching.

The per cent egg fertilization rate was calculated on 100 eggs from each sample at the blastopore closure stage. The survival rate, expressed as the percentage of hatched larvae, was determined on termination of hatching, based on a group of fertilized eggs picked out at the blastopore closure stage.

Egg measurements
Images of swollen eggs (20 individuals each in K, IA, IB, and IC treatments of the 2005/2006 season) were videotaped. Two egg diameters were measured (± 0.001 mm) on the images with the aid of the MultiScan v. 13.01 software (Computer Scanning System, Ltd. Poland) , and the measurements were averaged. The egg volume was subsequently calculated from the sphere volume formula:

where: r, sphere radius (mm)

Measurements of larvae
The total body length (longitudo totalis – l.t.) as well as the length (l) and height (h) of the yolk sac were measured (± 0.001 mm) in 1-d-old larvae (about 20–25 individuals per treatment). The data obtained were used to calculate the yolk sac volume from the elongated ellipsoid volume formula [2,11]:

where: l, length of the yolk sac (mm), h, height of the yolk sac (mm).

The larvae were weighed (to 0.001g) on a WXD 200/2000 electronic balance.
Images of the embryos developing in the eggs and those of the newly hatched larvae were measured precisely with the aid of the Multi Scan Base v. 13.01 software.

Physical and chemical parameters of water
In each experimental treatment, the basic physical and chemical water quality parameters were determined several times (Table 1).

Statistical treatment
The data supplied by the experiment were subjected to statistical treatment carried out using the Statistica 7.1 PL software (StatSoft Poland). The following tests were applied:

– one-way analysis of variance (ANOVA, p<0.01) for length and volume of the yolk sac in the larvae newly hatched from eggs incubated in different salinities;
– Tukey's T test (p<0.01) to compare mean dimensions of eggs and larvae [26].

RESULTS

As shown by the water quality parameters for the two experimental seasons (Table 1), none of the chemical or physical property of the water (of those analysed) constituted an additional factor which could potentially affect the developing sea trout embryos and distort results of the experiment.

2005/2006 season
Sea trout egg size
As shown by the results of Tukey's T test, there were no significant differences (p>0.01) in egg diameter and volume between sea trout eggs fertilized and incubated in freshwater and those fertilized and incubated in saline water (Table 2).

Table 2. Size parameters ( ± SD) of sea trout (Salmo trutta L.) eggs in the control (K) and treatments IA, IB, IC; 2005/2006 season

Embryonic development
The study showed water salinities of 0.5–0.7; 1; and 2‰, applied both in the treatments in which the eggs were fertilized and incubated in saline water (IA, IB, IC) and in those involving fertilization in tap water and incubation in saline water (IIA, IIB, IIC), to only shorten the embryonic development duration by 1–2 days, compared to the time taken by embryogenesis in the control (K) (Table 3).

The embryogenesis stages identified as the blastopore closure and intensive eye pigmentation were reached in all the treatments at the same value of degree-days (Table 3).

The first to hatch (at 357°D) were the larvae in IB, IC, IIB, and IIC. The duration (days) of hatching itself was at its shortest in the control (10 days), the remaining treatments showing the process to be extended from 13 (IIB) to as many as 17 days (IA). The highest fertilization and survival rates were recorded in the control (Table 3).

Characteristics of the newly hatched larvae
The highest weight on hatching was typical of the larvae from the control eggs (K) (Table 4), while the weights of larvae hatched from the eggs fertilized and incubated in saline water (IA, IB, IC) and those hatched from the eggs fertilized in tap water and incubated in saline water (IIA, IIB, IIC) showed no significant differences (Tukey's T test, p<0.01) (Table 4).

Table 4. Characteristics ( ± SD) of sea trout larvae hatched from eggs subjected to various treatments; 2005/2006 season

Significant differences were apparent in the length of the larvae. The longest and largest larvae were those hatched in the control (16.80 mm) (Fig. 1a) and in IIB (16.39 mm) (Table 4). The lengths in those treatments were significantly different (Tukey's T test, p<0.01) from those involving the larvae hatched from eggs fertilized and incubated at 2‰ (treatment IC). Those larvae were the shortest and showed large yolk sacs (Fig. 1b). The IC larvae showed the highest rate of malformations (Fig. 2a, b) (Table 4).

2006/2007 season
Embryonic development
Similarly to the 2005/2006 season, low salinities (1 and 2‰) did not significantly affect the duration of sea trout embryonic development in treatments IA and IB, compared to the control (K) and to the treatment involving eggs fertilized and incubated in riverine water (R) (Table 5). Although the embryogenesis stages identified (blastopore closure, eye pigment appearance) were attained by all the embryos at the same time, the hatching began at the earliest (at 359°D) in treatment IIC.

In the season discussed, the hatching process was more extended in time; it was the shortest in the treatment involving eggs fertilized and incubated in riverine water (R), the most extended hatching being observed in treatment IIB (eggs fertilized in incubated at 1‰) (Table 5).

In the additional treatment, involving egg fertilization and incubation at 3‰, the embryos began to die out as of about 300 °D.

The rates of egg fertilization and embryo survival were at their highest in the control (Table 5).

Characteristics of the newly hatched larvae
Similarly to the preceding season, the heaviest and longest individuals carrying the smallest yolk sacs were obtained from the eggs fertilized and incubated in tap water (K) and at 1‰ (IA) (Table 6); they were significantly (Tukey's T test, p<0.01) different from the larvae hatched from eggs fertilized and incubated in the riverine water (R) (14.92 mm) which carried large yolk sacs (Table 6).

Table 6. Characteristics ( ± SD)of sea trout larvae hatched from eggs subjected to various treatments; 2006/2007 season

The highest rate of deformations was recorded in the newly hatched larvae originating from the eggs fertilized and incubated at 2‰ (IC) (Table 6).

DISCUSSION

The salinities used in this study showed concentration-dependent effects on different stages of the sea trout embryonic development.

Egg fertilization took place in all the treatments, even at the highest salinity (3‰) (Table 4), although the eggs subjected to the highest salinity did not hatch. Such results confirm the findings reported by Ivlev [14] and Dziekońska [8] that it is not the sperm that – subjected to elevated salinity – is unable to fertilize eggs or performs poorly. Sperm activity does not decrease; on the contrary, when tested at 11‰ salinity, bream sperm proved to be still viable, while eggs became unfertilizable [14].

Perivitelline space formation
Inhibition of the perivitelline space formation under the influence of increased salinity was reported by Bogucki [3], and later on by Zotin [35,36]. They explained the inhibition by invoking inhibitory effects of salt solution on the secretion, by cortical alveoles, of hydrophilous colloids which, after egg activation, are responsible for water absorption by the egg and thus for the perivitelline space formation [35,36]. The content of cortical alveoli is secreted into the perivitelline slit; then, the water absorbed (by osmosis) from the outside of the egg forms a highly hydrophilous perivitelline fluid [3,5]. A subsequent study published in 1981 [25] confirmed the effect of salinity on the perivitelline space formation. Under normal conditions of embryonic development, fertilized eggs of grass and silver carps show large perivitelline space (about 98% of the total egg volume) [18]. The study demonstrated reduction of the perivitelline space with increasing salt concentration; slowing down of the response involving the "luminous circle" formation during cortical alveole break-down was observed: while in fresh water the circle appeared 1–2 minutes after egg contact with water, it took 7 minutes to form at elevated salinity, and even then the circle was not complete [25].

Similarly to pike eggs [4], the size of the sea trout eggs was not affected by salinity. Contrary to expectations, there were no volume differences between eggs fertilized and incubated in saline water (IA, IB, IC in the 2005/2006 season) and in tap water (K) (Table 2). It may be then concluded that the size of the perivitelline space, which accounts for an average of about 20% of the sea trout egg volume, did not change. The lack of differences can be explained by the fact that salmonid eggs have egg membrane which is very thick (50 µm) and resistant to pressure [35] as a result of specific adaptations to embryonic development taking place in eggs buried under a gravel mound. It has to be borne in mind that the egg membrane is not so much a barrier as it is a "filter" for mass exchange between the egg content and the external medium [31].

As described by Domurat in his 1956 publication on embryonic development (starting from the receiving mound appearance) of sea trout, pike, and roach in water and anhydrous medium (paraffin oil) [6], the water absorbed during egg swelling (perivitelline space formation) was sufficient for the embryonic development to proceed normally throughout. However, the sea trout and pike eggs kept in oil did not hatch [6].

This study showed the sea trout eggs fertilized in tap water and, following the perivitelline space formation, transferred to the salinity of 2‰ (IIC in the 2005/2006 season) to have higher fertilization and survival rates, compared to the eggs fertilized and incubated at 2‰ (IC) (no such pronounced differences were observed in IA and IB as well as IIA and IIB) (Tables 3, 5). This is evidence that tap (fresh) water provides a more favourable environment for developing embryos at the initial stages of embryogenesis (perivitelline space formation). The perivitelline space is the immediate surroundings of the developing embryo, the fluids it contains – as demonstrated earlier by Domurat [6] – being capable of adversely affecting the emerging embryo. The effect is visible as size differences between the newly hatched larvae.

Size of the larvae
Data obtained in the 2005/2006 season show the larvae hatched from eggs fertilized in fresh (tap) water (K) and transferred to saline water (IIA, IIB, IIC) were significantly longer, their yolk sac being significantly shorter, than those fertilized and incubated in saline water (IA, IB, IC) (Table 4). It should be, however, pointed out that the control individuals (K) were best adapted to independent life, as indicated by their size parameters (Fig. 1a,b, Table 4). A similar pattern was observed in cyprinid eggs which had been initially incubated in fresh water and transferred to high-salinity solutions (11 and 16‰) at the final stage of gastrulation: despite progressing embryonic development, the embryo was unable to attain the normal length and was shorter than the control embryo [29].

The lower salinities used in the experiment (0.5–0.7; 1; 2‰) resulted only in earlier (by 2–3 days) hatching than in the control, which can be explained by an attempt, on the part of the embryos, to change the unfavourable environment. The results obtained confirmed earlier findings of Vernidub [29] that prolonged exposure to low-salinity solutions exerts no adverse effect on morphogenesis of spring spawning fish, but interfers with water regime in the egg. A higher salinity resulted in various damages which disturbed the embryonic development, including a change in oxygen demand of the embryo and the breathing process itself [27,29]. Vernidub [29] found salt to penetrate inside the egg and into the embryo, which resulted in a rapid change in the embryo's physiology during the initial period of exposure. A 3–4 hour exposure to 1% (10‰) salinity reduced pH of the embryo to 7.0 and increased its respiration rate to 120–200%. The highly elevated salinity reduced pH of the embryo (to below 7.0) and reduced the respiration rate. Similarly, Swanson [27] found oxygen consumption (µlO₂ x individual h^-1) of the milk fish (Chanos chanos) embryos toward the end of their development, both at the highest (50–55‰) and the lowest (15–20‰) salinity, to be lower than that in the embryos developing at 35‰. Consequently, the newly hatched larvae differed in their size parameters: those larvae developing at the salinity optimum (35‰) were the longest, their yolk sacs being the smallest [27]. A similar pattern was revealed in this study: the largest and longest larvae hatched from the eggs incubated under optimal conditions, in tap water (K) (Fig. 1a) and at the very low salinity (IA, IB, IIA, IIB) (Tables 3 and 5). The highest salinity (3‰, ID) proved lethal for the developing embryos, and hatching did not occur. The salinity of 2‰ did not stop hatching, but reduced the size of the newly hatched larvae (Fig. 1b), confirming earlier findings from different fish species [9,10,20,24,29].

Deformations of the larvae
The larval deformation rate, particularly high in the treatment involving embryogenic development at 2‰, does not deviate, however, from results reported by Oven [22] (eggs of bluntsnouted mullet) and Rykova [24] (eggs of silver carp). They found the embryos, developing in eggs incubated at elevated salinity, and the newly hatched larvae to show various deformations: yolk "leakage", body curvature, yolk sac swelling, poor pigmentation of body and eyes, and even arrested jaw development [22,24].

Data obtained for the sample containing eggs fertilized and incubated in the River Wiśniówka water showed the newly hatched larvae, the shortest and carrying the largest yolk sac, to have been somewhat less advanced in their development (Table 5). This could have been related to the Wiśniówka water quality which, in contrast to the tap (treated) water, had a different composition, containing more organic compounds, microorganisms, and – primarily – suspended solids. These, by adhering to the eggs, could have interfered with oxygen uptake by the egg, and thus caused differences in size parameters of the newly hatched larvae.

Differences in the duration of embryogenesis between the control and experimental treatments could have resulted also from effects of Na⁺ and Cl^- ions on the hatching enzyme activity. In their study on chorionase activity in coregonids kept at different NaCl concentrations, Luberda et al. [17] found that the enzyme's activity was weakened to 30% only at very high (55.1‰) NaCl concentration. In this study on the sea trout eggs incubated in low-salinity water, no weakening of the hatching enzyme activity was observed; on the contrary, hatching occurred earlier in the experimental treatment rather than in the control.

In addition to the structural effects described above (size differences among the newly hatched larvae), embryonic development in saline water must have involved certain physiological changes. Due to poor transparency of the sea trout egg membrane, it was difficult to examine the embryos developing in the eggs. The relevant literature contains reports of physiological changes taking place in fish embryos developing at different salinities, e.g., reduced salinity slowed down the heartbeat rate in embryos and larvae of herring [12,28] and of the Atlantic salmon at 2.5–24‰ [13].

CONCLUSIONS

Compared to other fish species (e.g., cyprinids), the developing eggs of sea trout (Salmo trutta L.) seem to be more sensitive to water salinity. Although the salinity below 2‰ had no effect on the duration and course of embryonic development, fertilization and incubation of eggs at 2‰ induced unfavourable changes in size parameters of the newly hatched larvae and increased the rate of malformations, whereas salinity of 3‰ proved lethal.

Anthropogenic salination of rivers and streams supporting sea trout spawning grounds may, and does, pose a serious threat to sea trout populations.

ACKNOWLEDEGMENTS

The author wishes to thank Professor A. Winnicki for his invaluable suggestions offered during the study. Thanks are also due to Professor K. Formicki and all the staff of the Department of Fish Anatomy and Embryology for access to the isothermal laboratory.

REFERENCES

Accepted for print: 9.10.2009

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