The effect of salt composition on the salinity tolerance ...

The effect of salt composition on the salinity tolerance of mirror carp { Cyprinus carpio L.) during early ontogeny B. K. Karimov I), D. Keyser Z) Uzbekistan Academy ofSciences, Tashkent, Uzbekistan. 2J University ofHamburg, Hamburg, Germany. I)

Communicated by W Arntz Received: 24June 1997 Accepted: 21 July 1998 Abstract The effect of brackish water (BW), with the specific ionic composition of the water in the Aral Sea basin, and that of artificial standard seawater (SW) on sperm activity, egg fertilization, swelling and further development of the carp (Cyprinus carpio L.) until the stage of active feeding were studied. Spermatozoa are able to fertilize eggs up to 9 g/1 salinity. The fertilization rate is already extremely low at 6 g/1 salinity (13.4 % against 98 % in control) when the fertilization, swelling and further development take place in salinised water. However, the fertilization rate is nearly the same as in the control, even at 7 g/1 salinity, when the eggs are transferred to control water 5 minutes after fertilization. The swelling process is therefore regarded as the most sensitive stage in the salt tolerance of carp ontogeny. The most important result is the fact that BW with high Ca2 + and Mg2+ content is considerably less toxic than SW The present study confirms that mirror carp from Ahrensburg (Germany) can tolerate approx. 3 to 4 g/1 BW and SW without any visible changes during early ontogeny. Based on these results, possible adaptations to salinity in natural wild carp populations must be taken into consideration. Kurzfassung Salinitatstoleranz des Spiegelkarpfens ( Cyprinus carpio L.) wahrend der fri.ihen Ontogenese in Abhangigkeit von der Salzzusammensetzung des Wassers Untersucht wird die Wirkung von Brackwasser (BW) mit einer Salzzusammensetzung, die dem "des Aralseebeckens entspricht, und Standardseewasser (SW) auf Spermaaktivitat, Befruchtungsrate und weitere Entwicklung von Spiegelkarpfen (Cyprinus carpio L.) bis zu dem Stadium, in dem Nahrung aufgenommen wird. Spermien des Spiegelkarpfens sind moglicherweise bis zu einem Salzgehalt von 9 g/1 befruchtungsfahig. Die Befruchtungsrate ist schon bei 6 g/1 merklich geringer (13.4 % gegenliber 98 % in der Kontrolle), wenn die Befruchtung, Schwellung und weitere Entwicklung in Salzwasser erfolgt. Dennoch entspricht die Befruchtungsrate selbst bei einem Salzgehalt von 7 g/1 derjenigen der Kontrolle, wenn die Eier 5 Minuten nach der Befruchtung in Kontrollwasser iiberflihrt werden. Der Schwellungsprozd~ wird deshalb als entscheidend fur die Ontogenese angesehen. Das wichtigste Ergebnis dieser Untersuchung ist, daR BW mit hohem Ca2+und Mg2+ Gehalt erheblich weniger toxisch ist als SW Karpfen aus dem Untersuchungsgebiet

Arch. Fish. Mar. Res. 46(3), 1998, 225-239, 6 tables

B. K Karimov and D. Keyser

(Ahrensburg, Deutschland) tolerieren wahrend der fri.ihen Omogenese 3 bis 4 g/1 BW und SW ohne sichtbare Veranderungen. Bei der Anwendung dieser Ergebnisse milssen mogliche Adaptationen bei wildlebenden Karpfen-Populationen beri.icksichtigt werden.

Introduction Despite numerous publications on the subject, the response of aquatic organisms to various levels of ambient salt concentrations remains a subject of great importance. The most important and detailed reviews were published by Remane (1940, 1958), Privolnev (1964), Holliday (1969) and Khlebovich (1974). Many countries lack freshwater resources for aquaculture and brackish waters are used instead. Especially in arid zones, water resources are subject to increasing anthropogenic salinization. For example, during the last 20 to 30 years of 'planned agricultural development' in the Central Asian part of the former Soviet Union, a dramatic transformation of salt composition and rapid increase of mineralization of natural waters has occurred (Nikitin 1987, 1991; Kamilov et al. 1994; Karimov 1990). The cause was the huge extension of the irrigation system. The total volume of highly mineralized collector-drainage waters flowing into rivers and water bodies in Central Asia now amounts to 25 to 30 km3, which contains a total amount of various mineral salts estimated at about 70 to 80 million tonnes per year (Khabibullaev 1993). As a direct result, the mineralization in the middle and down stream section of the rivers increased from 0.5 to 0.6 g/1 to 1.2 to 2.5 g/1. Simultaneously the chemical composition of the waters changed from calcium hydrocarbonate to the sulphate-chloride-sodiummagnesra. By the 1980s the Aral Sea ecosystem had lost its importance as a fishery resource, mainly because of the absence of fish reproduction (Tleuov 1981). Due to the sharply reduced outflow from rivers into the lake a large number of Aral Sea fish species were denied the opportunity to migrate into the freshwater of the inflowing rivers in order to spawn. It is not surprising that in such conditions fish reproduction was impaired. Indeed even in Sarykamish and Arnasay, the two biggest lakes in the Aral depression, unsuccessful fish spawning was reported (Salikhov and Vundzettel 1986; Sanin et al. 1991). The water mineralization in these aquatic ecosystems varies between 5 and 15 g/1 (Kamilov et al. 1994). A similar increase in dissolved salts in the Veselov reservoir from 1933 to 1950 to 1000 mg CI-/1 caused the impairment of reproduction and the elimination of some cyprinid fishes (Syrovatski 195 5; Piskunova 1978). At the same time young cyprinid fishes such as wild carp ( Cyprinus carpio L.), bream (Abramis brama (L)) roach (Rutilus rutilus(L)) etc. were present in lakes with a salinity of about 6 to 9 g/1 (1122 to 1683 mg CI-/1). The spawning of carp was even higher in these lakes than in freshwater lakes. However, it is not known whether the shed eggs are capable of normal fertilization, development and hatching at such high levels of salinity. Young fish could have reached these lakes from a large number of freshwater collectors and irrigation canals, especially during the spawning period (Karimov and Razakov 1989).

226

Arch. Fish. Mar. Res. 46(3), 1998

Effect ofsalt composition on salinity tolerance

According to Nikitin (1987, 1991) and Kamilov et al. (1994) general mineralisation of the waters in the Aral Sea Basin is: • • • •

in rivers: in water reservoirs: in collectors: in lakes:

0.5-2.0 0.5-2.5 3-12 3-18

g/1 g/1 g/1 g/1.

Despite the fact that wild carp and its various domesticated forms is one of the main catches in natural waterbodies and is cultivated in fish farms, research on its reproduction has been very limited. The influence of salinity on freshwater fish, especially during their early ontogeny is of great importance. Large numbers of experimental data demonstrate that the salinity tolerance of fish is enhanced with the age of the fish. Eggs and larvae are the most sensitive stages in fish ontogeny (Evtjukhin 1933; Konovalov and Konovalova 1952; Karpevich 1960; Privolnev 1964; Khlebovich 1977; Karimov 1985a,b, 1995). Salinities of about 5 to 8 g/1 constitute the 'critical salinity range' (Khlebovich 1974) and represent an upper threshold for the fertilization and survival of the eggs of freshwater fish and a lower threshold for early ontogenetic stages of marine fish species. It is also the salinity range having the lowest species diversity in marginal brackish water (Remane 1934).

Materials and methods The experiments were carried out at the Ahrens burg Field Station of the Federal Research Centre for Fisheries, Germany, during spring 1996. 3 to 4 year-old adult mirror carp, weighing 2 to 4 kg and growing in big containers with a circulatory water supply were used. Sperm and eggs were obtained through hormonal induction with a carp pituitary powder suspension. Fertilization was performed by first adding the sperm to the eggs and then the water containing 4 g NaCl/1 and 3 g urea/1. There were several changes of water during the whole process. To ensure that the eggs were in direct contact with the sperm, there was constant stirring (up to 1.5 hours) until hardening of the egg's chorion occurred. The procedure was followed by a short dip into 0.5 g tannic acid per litre for 20 seconds to remove the sticky surface layer. This enabled the use of the eggs in the experiments without clustering (Woynarovich 1964). The fertilized eggs from each parent fish were incubated in glass dishes containing 120 to 130 ml of each test solution in 2 identical experiments. The total number of eggs in each experiment varied between 200 and 500. After routine chemical removal of the sticky layer the experimental dishes were immediately placed in a thermostatically controlled water bath, which stabilised the incubation temperature at between 19 and 23 °C. 1.5 to 2 hours after insemination the eggs were transferred to salt solutions in glass dishes and incubated under natural light conditions. The test salinities used in the experiments were divided into 3 types:


227


1) Fresh water (FW) from the underground drinking water supply of Hamburg city (tap water) with pH 8.3 to 8.4 and total mineralization of about 200 mg/1 (Table 1). This water was used in control experiments. 2) Highly mineralized (3, 6 and 9 g/1) artificial brackish water (BW) with the specific ionic composition of the Aral Sea basin (Table 2). The appropriate test salinities were prepared by dissolving reagent grade chemicals in tap water instead of distilled water to keep trace elements. 3) Artificial sea water (SW) at low concentrations: 3, 4,5 and 6 g/1; prepared by dissolving appropriate amounts of crystalline sea salt in distilled water. This was done to give the ionic composition shown in Table 2. To avoid any possible negative effects of trace elements, a special artificial sea water (SWS) was used in some experiments. It was prepared by dissolving various reagent grade salts in distilled water according to Schlieper (1968). During the first 24 hours the test solutions were changed 3 times. After that they were changed twice daily. The following observations were recorded:

-

motility of the spermatozoa, percentage fertilization, epiboly, egg diameter and solidity of egg membrane, embryo and larval mortality; developmental rate, number and character of morphological abnormalities, larval swimming behaviour, length and weight of the larvae.

The duration of sperm motility was measured at 21 to 22 °C incubation temperature (Belova 1981). Sperm from 4 different males was used. Two different phases of sperm motility were registered: Phase a: Phase b:

Time until approximately all spermatozoa ceased their forward movement. Time until approximately all spermatozoa ceased their oscillatory movement (Karimov 1985b).

Abbots formula (Finney 1971) was applied to distinguish the difference between the control and the experimental group, when more than 15 % mortality was also observed in the control:

where M

M

228

T

c

=

mortality in toxic solution (%) and

=

mortality in the control (%).


Effect ofsalt composition on salinity tolerance

Table 1: Chemical composition of tap water (average values for 1995). n.d. = not detected. Organic pollutants, e.g. pesticides, organochlorine compounds and polycyclic aromatic hydrocarbons were not detected. Source of data: Hamburger Wasserwerke 1996

Ions

Concentration Detection limit [mg/I]

Ions

Concentration Detection limit

[mg/1]

[mg/I]

[mg/1]

39.00

Aluminium

n.d.

0.01

Magnesium

3.00

Antimony

n.d.

0.001

Sodium

8.00

Arsenic

n.d.

0.001

Calcium

1

Potassium

1.10

0.5

Barium

n.d.

0.01

Iron

0.01

0.01

Lead

n.d.

0.001

Manganese

n.d.

0.01

Cadmium

n.d.

0.0005

Ammonium

0.03

0.03

0.002

Chromium

n.d.

Hydrocarbonate 126.30

Copper

n.d.

0.005

Chloride

Nickel

n.d.

0.002

9.00

Sulphate

11.0

Mercury

n.d.

0.0001

Nitrate

0.20

0.04

Selenium

n.d.

0.001

Nitrite

n.d.

0.01

Zinc

n.d.

0.01

Fluoride

0.09

0.01

Borate

0.02

0.01

Phosphate

n.d.

0.05

Silver

n.d.

0.01

15.00

0.1

Silicate

Table 2: Main ionic composition (in % of total salt content) and pH level of the various salinities used in the experiments. BW = artificial brackish water (Aral Sea type), SW = artificial brackish water (marine type with trace elements), SWS = artificial brackish water (marine type without trace elements) Ions

BW

SW

sws

Calcium

6.9

1.2

1.2

Magnesium

5.1

3.7

3.9

Sodium

18.7

30.7

30.6

Potassium

0.5

1.1

1.2

0.4

Hydrocarbonate

5.8

Chloride

18.7

55.3

58.5

Sulphate

44.3

7.74

4.1

pH


8.0-8.5

8.5-8.6

8.4-8.5

229


Table 3: The effect of various salinities on duration (in seconds) of sperm motility of carp. Phase a: forward movement, Phase b: only oscillatory movement

Male No.

Brackish water

Freshwater

Sea water

6 g/1

3 g/1 a

b

a

b

83

19

82

30

9 g/1 b

a

88

21

110

40

a

6 g/1 b

a

b

180

183

115

119

90

207

93

126

2

45

15

76

15

3

53

16

60

25

102

65

100

314

115

134

4

45

10

so

25

115

so

130

185

130

120

X±s

56± 18

15 ±4

67± 15

24±6

104 ± 12 44± 19 125 ±40 222±62 113 ± 15

125 ±7

Results Effect ofsalini-iy on sperm activi-iy and fertilization Within the investigated salinity ranges the duration of both phases of sperm motility (Table 3) was different. The spermatozoa fertilized eggs up to a salinity of 9 g/1 BW and 6 g/1 SW Up to a salinity of 6 g/1 BW, the time taken for all spermatozoa to cease their forward movement (phase a) was longer than the time taken until all spermatozoa ceased their oscillatory movement (phase b). However, between 6 g/1 SW and 9 g/1 BW the opposite picture was observed. Sperm motility was clearly prolonged in concentrations above 6 g/1 BW and in SW Immobile sperm at 6 g/1 BW or lower concentrations did not resume their activity by adding of fresh water. However, in 6 g/1 SW and 9 g/1 BW the addition of fresh water lead to the resumption of some spermatozoic oscillatory movements. The duration of sperm motility from different males varied sometimes more than 50 % in all tested salinities including freshwater. Since fertilization was performed in a solution of 4 g NaCl/1 and 3 g urea/1, this was accepted as a control. Therefore, eggs for incubation in freshwater and 3 g/1 salinity were always taken after the fertilization performed at 4 g/1 salinity. The rate of fertilization in this case was between 89 % and 98 %; only in one female it was as low as 76 %. The fertilization of eggs was studied in detail at higher salinities ( 6, 7 and 9 g/1 BW and SW). Less adhesiveness than in the control was observed when both fertilization and swelling were performed at these salinities; whereas the adhesiveness of eggs was similar to the control, when swelling was performed at 4 g NaCl/1 (Table 4). When fertilization and swelling took place in BW salt solutions, the chorion of the eggs already became hard after 45 to 50 min. The diameter of the eggs and the size of the

230


vJ

N

~ Clo

......

~

~

~

~ :'

:-.

~

~

~

~ ~

::i,.

BW BW BW

6 (6)

9 (9)

6 (6)

6 (6)

7 (7)

7 (4)

6

6

10

10

II

11

NaCl

NaCl

SW

NaCl

4 (4)

less less more

90 95*** 75***

less

less

130** 50

less

more

Adhesiveness of eggs during swelling

130**

90

Salinity Salt Duration range composition* of chorion during hardening fertilisation and swelling ( ) [g/1] [min]

6

Female No.

1.54 ± 0.11

1.52 ± 0.11

1.56 ± 0.07

1.54 ± 0.08

1.98 ± 0.08

[mm]

1.90 ± 0.09

1.63 ± 0.08

1.50 ± 0.09

1.63 ± 0.10

2.07± 0.05

[mm]

Diameter of eggs at end of start of swelling hatching

good

poor

poor 6SW

poor 6BW

poor 9BW

poor

good

Water hardening results

FW

FW

FW 6

FW 12

83

14

17

13

2

35

6BW FW 6

16

98

[%]

FW

FW

[g/1]

Fertilisation Salinity during the embryonic development

Table 4: Fertilisation of eggs in various salinities. * = 3 g/1 urea present in all salinities; ** = process of water hardening was in fact completed earlier; *** = data are means of 2 similar experiments. For definition of BW, FW, SW see Table 2.

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