Effects of saponin on survival, growth, molting and feeding of Penaeus japonicus juveniles

AqWCUltUtW

ELSEVIER

Aquaculture 144 ( 1996) 165-175

Effects of saponin on survival, growth, molting and feeding of Penaeus japonicus juveniles Jiann-Chu Chen

*, Kou-Wei

Chen, Jiann-Ming Chen

Deparhnenr of Aquaculture. National Taiwan Ocean University, Keelung, Taiwan 20224, ROC

Accepted 28 February 1996

Abstract The median lethal concentration (LC,,) of saponin on juvenile Penaeus japonicus was 20.82 mg 1.’ and 18.14 mg 1“ at 48 h and 96 h, respectively, in sea water of 34 practical salinity units. The mortality rate of juvenile P. juponicus exposed to 0 (control), 0.1, 0.5, 1 and 2 mg 1-l saponin after 60 days was 3.3%, 6.6%, lo%, 33.3% and 43.3%, respectively. After 36 days of exposure, the weight of shrimps exposed to 1 and 2 mg 1-r saponin was significantly lower than for those exposed to 0.1 and 0.5 mg 1-l saponin. The growth factor of shrimps exposed to 0.5 mg 1“ saponin was significantly lower than those exposed to 0.1 mg 1-l saponin and the control solution. Following exposure to saponin as low as 0.5 mg 1.‘, P. japonicus shortened the time to the first molt, and decreased its feeding, growth and molting frequency. The maximum acceptable toxicant concentration was 0.1 mg 1.’ saponin. Keywords:

Saponin; Survival; Growth; Molting; Feeding; Penaeus japonicus

1. Introduction Penaeus japonicus and Penaeus monodon are the most common penaeids currently being cultured commercially in Taiwan and other Pacific Rim countries (Chen, 1990). The mass mortality of P. monodon which occurred in 1986 (Lightner et al., 19871, the immunity of P. japonicus to monodon baculovirus (MBV) disease (Fukuda et al., 19881, and the capability for long-term shipping without water have attracted shrimp farmers to the intensive culture of P. japonicus since 1988.

l

Corresponding author. Tel.: 886-2-462-2192 Ext. 5205; fax: 886-2-463-3150.

00448486/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SOO44-8486(96)01301-4

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It is a common practice that prior to the stocking of penaeid shrimp, organic sludge on the bottom of the pond must be removed. This can be done through mechanical removal or through oxidation during sun-drying. After cleaning, the pond is then partially filled with 30-40 cm of water, and the predators, mostly fish, are removed by the application of tea seed cake at 2.510 mg 1.’ before the release of postlarvae (Chen, 1990). Tea seed cake, the residue of Camellia sp. seeds after oil extraction, contains 5.2-7.2% saponin (Minsalan and Chiu, 1986). Saponin is a glucoside that may destroy erythrocytes and is highly toxic to fish (Minsalan and Chiu, 1986; Boyd, 1990). The utilization of tea seed cake is very effective for the eradication of predatory fish in shrimp ponds (Tang, 1961; Terazaki et al., 1980). The recommended level of crude saponin for use in eradicating undesirable fish is 1.1 mg 1.’ (Terazaki et al., 1980). Minsalan and Chiu (1986) reported that application of tea seed cake at 15 mg 1.’ to ponds was adequate to kill fish within 6 h. The lethal concentrations of saponin to several species of finfish and penaeid shrimp have been provided (Terazaki et al., 1980; Minsalan and Chiu, 1986). It was reported that finfish are more susceptible to saponin than shrimp. Since tea seed cake is widely used in shrimp ponds, the toxicity of saponin to penaeid shrimp is of primary concern to shrimp farmers. This paper provides information on the effects of different concentrations of saponin on survival, growth, molting and feeding in P. juponicus juveniles.

2. Materials

and methods

Three experiments with saponin were conducted. The first was a study of the acute toxicity of saponin, the second evaluated chronic effects on growth and molting, and the third, the effects on feeding. 2.1. Animals and water P. japonicus juveniles obtained from a private nursery located in Iilan, Taiwan were shipped to the laboratory and acclimated for one week before bioassay testing. The average wet weight of shrimps used was 1.36 f 0.22 g and 0.31 + 0.41 g for the first and second experiments, respectively. For the third experiment, two sizes of shrimp weighing 1.71-1.84 g and 8.23-8.36 g were used. Sea water (34 practical salinity units (p.s.u.)> pumped from the Keelung coast adjacent to the University was filtered through sand and gravel filters by air-lift pumping.

2.2. Test solution Saponin test solutions were prepared by dissolving 9.009 g of saponin (Product No. S-7900, Sigma Chemical Company, USA, from Quillaja bark, containing 11.l% sapogenin) in 1000 ml of distilled water to prepare 1000 mg 1-l saponin stock solution and then diluted with sea water to make 0, 15, 17.5, 20, 22.5,25, 27.5, 30 mg 1-l saponin, 0,

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0.1, 0.5, 1, 5 mg 1-l saponin, and 0, 0.1, 0.5, 1, 2, 5, 10 mg 1-l saponin as test solutions for the first, second and third experiment, respectively. 2.3. Effect of saponin on lethal effect The short-term median lethal concentration (LC,,) toxicity tests were carried out according to the method described by Buikema et al. (1982) and the American Public Health Association et al. (1985). Shrimps were taken from the holding tanks and transferred to each test solution in triplicate tanks. Bioassay experiments to establish tolerance limits were conducted in 8-l polyethylene tanks containing 2 1 of test solution. Each tank contained eight shrimps, and water was aerated continuously by an air stone with a blower. Each test solution was renewed daily, in accordance with the static renewal method for toxicity tests (Buikema et al., 1982; American Public Health Association et al., 1985). During the experiment, the shrimps were fed a commercial diet (39% protein) designed for P. monodon by Tairoun Products Co. Ltd. (Taipei, Taiwan) twice a day (09:OO and 21:00 h) at 5% of body weight per day. Water temperature was maintained at 27 f l”C, dissolved oxygen was 6.02 f 0.4 mg l-t, and the pH ranged from 8.10 to 8.25. Observations were usually made at 24 h intervals up to the 96th hour. Death was assumed when shrimps were immobile and showed no response when touched with a glass rod. The concentration response of test organisms was determined for LC,, of saponin with a computer program (Trevors and Lusty, 1985). 2.4. Effect of saponin on growth Shrimps were taken from the holding tank, and were individually housed in a cylindrical cage (10 cm in diameter and 30 cm in height) made of plastic screens (2 mm X 3 mm). Each tank contained 20 1 of test solution, and ten cages, and was aerated with an air stone. There were triplicates for each test solution with a total number of 30 juveniles (ten per replicate) for each test solution. The shrimps were also fed the commercial diet mentioned above. Bioassay tests were conducted using the static renewal method with test solutions renewed daily. Dead juveniles and uneaten feed were removed every day in the afternoon (14:00-15:00 h) when the water was renewed. Exuviae (molted exoskeletons) were also removed daily when they were found. Observations were made every morning at 11:OOh, and the number of surviving animals was recorded every 12 days. During the experiment, the water temperature, pH level and dissolved oxygen averaged 26.5 f 0.6”C, 8.05 f 0.2, and 6.1 f 0.4 mg l-‘, respectively. Ten shrimps in one replicate of 0.1 mg 1-l and one of 0.5 mg 1-l saponin solutions died on the 37th and on the 15th day, respectively, due to aeration failure. The wet weight was measured at IO-day intervals for 60 days. Each individual shrimp was netted, placed on a gauze to remove excess water and then weighed. The relationship between wet weight and time elapsed was calculated according to the

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Table 1 Number of Penaeus japonicus juveniles exposed to different concentrations 60 days during the first and second experiment

of saponin (mg I- ‘) for 96 h and

(a) Fist experiment Saponin

Time elapsed (h)

(mg I-‘)

0

12

24

48

72

96

15 17.5 20 22.5 25 21.5 30

24 24 24 24 24 24 24

24 24 24 24 24 24 20

24 24 24 24 23 18 0

22 20 18 15 13 10 0

22 20 10 8 0 0 0

20 18 7 4 0 0 0

(b) Second experiment Saponin

Time elapsed (days)

(mg 1-l)

0

12

24

36

48

60

Control 0.1 a 0.5 a 1 2

30 30 30 30 30

30 30 30 30 30

30 29 19 27 28

29 28 19 27 27

29 18 19 21 20

29 18 17 20 17

a Ten shrimps in one of three replicates died due to aeration failure on the 37th and 15th day for the 0.1 and 0.5 mg I- ’ saponin treatment, respectively.

equation of Ricker (1975): W = AeET, where W is wet weight (g), T is time elapsed (days) and B is a growth factor. 2.5. Effect of saponin on feeding In order to compare how the feeding of shrimp was affected by the concentration and exposure time to saponin, two tests were conducted. Shrimps (1.71-l .84 g) that had been exposed to 0 (control), 0.1, 0.5, 1 and 2 mg 1-l saponin for 70 days (in the second experiment) were used for the first test. Shrimps (8.23-8.36 g) that had been exposed to 0, 0.5, 1, 5 and 10 mg 1-l for half an hour were used for the second test. The feeding tests were conducted in 8-l polyethylene tanks containing 2 1 of test solution. Each test solution was conducted in five replicates with one shrimp in each replicate. The shrimps were fed individually with the same feed as above. After 1.5 h of feeding, unconsumed feed was pipetted and placed on filter paper and dried to a constant weight. The feeding rate was expressed as ( A-B)/C X 100, where A was the amount of feed applied (g), B was amount of feed (g) remaining after 1.5 h feeding, and C was the wet weight of a shrimp (g). 2.4. Statistical analysis All data were subjected to a one-way analysis of variance (Steel and Torrie, 1980). If a significant difference was indicated at the 0.05 level, then Duncan’s multiple range test was used to identify any significant differences between treatments (Duncan, 1955).

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3. Results 3.1.

Eflect

of

saponin on lethal effect

The survival of shrimps exposed to each test solution is given in Table 1. All shrimps exposed to 30 mg l- ’ saponin died after 24 h, and all shrimps exposed to 25 and 27.5 mg 1-l saponin died after 72 h. However, no mortality occurred among the shrimps exposed to 15, 17.5, 20 and 22.5 mg 1-l saponin after 24 h. The LC,, values of saponin and their 95% confidence limits at different time exposures are presented in Fig. 1. The 24, 48, 72 and 96 h LC,, of saponin on P. japonicus juveniles was 27.08, 20.83, 18.91 and 18.14 mg l-r, respectively. 3.2. EfSect of saponin on growth The survival of shrimps exposed to different concentrations of saponin at different times is shown in Table 1. The mortality of shrimps exposed to 1 and 2 mg I- ’ saponin after 60 days was 33.3% and 43.3%, respectively. The effect of saponin on growth is shown in Fig. 2, in which a photograph of representative shrimps reared at 0 (control), 0.1, 0.5, 1, 2 mg I- ’ saponin after 60 days is shown. The growth in wet weight of shrimps exposed to each test solution is shown in Table 2. One-way analysis of variance indicated that the weight of shrimps exposed to 1 mg I-’ saponin was significantly lower (P < 0.05) than those exposed to 0 (control), 0.1, and 0.5 mg l- ’ saponin after 36 days. However, no significant growth was observed among any treatment after 12 days. The maximum acceptable toxicant concentration (MATC) was 0.5 and 0.1 mg I- ’ saponin after 36 and 60 days of exposure, respectively.

15

I

I

12

24

/ 36

48

60

72

84

96

108

Time elapsed (h) Fig. 1. The LC,, values and their 95% confidence after different time periods.

limits of saponin (mg I- ’ ) on Pemeus japonicus juveniles

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0.1 mg/l

Control

Fig. 2. Effects of different concentrations exposure.

144 (1996) 165-175

0.5 mg/l

1 mg/l

2 mg/l

of saponin on growth of Penaeus japonicus juveniles

after 60 days

The relationship between weight and time elapsed for the P. juponicus juveniles exposed to each test solution is shown in Table 3. Statistical analysis indicated that no significant difference in growth factor (B) was observed among shrimps exposed to 0.5

Table 2 Mean (SE) weight (g) of Penaeus japonicus days

juveniles

exposed

to different concentrations

of saponin for 60

Saponin

Time elapsed (days)

(mgl-‘)

0

12

24

36

48

60

Control

0.31a (0.01) 0.31a (0.01) 0.31a (0.01) 0.31a (0.01) 0.31a (0.01)

0.47a (0.01) 0.46a (0.02) O&a (0.02) 0.44a (0.01)

0.72a (0.02) 0.73a to.021 0.67ab (0.03) 0.64b (0.02) 0.62b (0.02)

1.02a (0.03) 1.OSa (0.03) 0.97a (0.03) 0.85b (0.03) 0.85b (0.03)

1.47a (0.04)

1.92a (0.05) 1.91a (0.03) 1.71b (0.06) I .48c (0.06)

0.1 0.5 1 2

Values in the same column followed by different

letters are significantly

1.47a (0.04) 1.37a (0.05) 1.16b (0.04) 1.12b (0.05) different (P < 0.05).

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171

Table 3 Relationship between wet weight of Penaelrs japonicus juveniles and time elapsed after shrimps were exposed to different concentrations of saponin for 60 days. W = AeST, where W is wet weight (g) and T is time elapsed (days) Saponin

A

B

R2

n

0.3268 0.3257 0.3255 0.3125 0.3259

0.0308a 0.0307a 0.0283b 0.0267bc 0.0252~

0.9845 0.9861 0.9855 0.9883 0.9839

29 18 17 20 17

(mg I-‘) Control 0.1 0.5 1 2

Values in the same column coefficient of determination.

followed

by different

letters are significantly

different

(P < 0.05).

R* is the

and 1 mg I-’ saponin, and among those exposed to 1 and 2 mg 1-l saponin. However, a significant difference of growth factor was found among those exposed to 0.5 mg 1-l saponin and 0.1 mg I- ’ saponin. The MATC for P. japonicus juveniles was 0.1 mg I-’ saponin as determined from the growth factor for the shrimps exposed to each test solution after 60 days. Following exposure to increased concentration of saponin, P. juponicus shortened the time to the first molt. The time interval to the first molt of shrimps exposed to the control solution, and 0.1, 0.5, 1 and 2 mg 1-l saponin was 5.72, 5.44, 4.06, 3.85, and 3.53 days, respectively (Fig. 3). P. juponicus following exposure to saponin at 0.5 mg 1-l significantly decreased its molting frequency. In the control solution, one molted four times, 13 molted five times, 12 molted six times. and three molted seven times. In

z zf&2)

-1

(0.45)

c

3.85 (0.49)

1

2

7

(0:40)

1

1

L

Control

Saponin concentration

(mg/l)

Fig. 3. Effects of different concentrations of saponin (mg I- ’ ) on the time to first molting (days) of Penaeus japonicus. Values arc mean (SE). Bars with the same letter are not significantly different (P > 0.05).

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Table 4 Number of Penaeus japonicus juveniles molting, different concentrations of saoonin for 60 davs

I44 (1996) 165-175

and the mean molting

Saponin

No. of shrimp for molting frequency

(mg I-‘>

4

5

6

7

Control 0.1 0.5 1 2

1 0

13 7 12 9 9

12 11 4 6 2

3 0 0 0 0

1 5 6

Values in the same column followed by different

frequency

of shrimps

exposed

to

Mean molting frequency

5.59a 5.61a 5.18b 5.05b 4.771,

letters are significantly

different (P < 0.05).

the 2 mg 1-l saponin solution, six molted four times, nine molted five times, and two molted six times (Table 4). The mean molting frequency was 5.59, 5.61, 5.18, 5.05 and 4.77 for the shrimps exposed to the control solution, and 0.1, 0.5, 1 and 2 mg 1-l saponin, respectively. The MATC was 0.1 mg 1-l saponin as determined from the mean molting frequency of P. juponicus when exposed to each test solution for 60 days.

3.3. EfSect of saponin on feeding The feeding test indicated that the amount of feed the shrimps consumed declined with increasing saponin concentration. For the juveniles (1.71-1.84 g) exposed to the control solution, and O-1,0.5, 1 and 5 mg 1-l saponin after 70 days, the feeding rate was 2.40%, 2.28%, 1.51%, 1.14%, and 0.71%, respectively (Table 5). For the adolescents (8.23 - 8.36 g) exposed to the control solution, and 0.5, 1, 5, and 10 mg 1-l saponin after 30 mitt, the feeding rate was 2.03%, 0.98%, 0.42%, 0.14% and 0.04%, respectively (Table 6). The feeding rate of shrimps exposed to 0.5 mg I-’ saponin was significantly

Table 5 Effect of saponin on feeding rate (%) for Penaeus japonicus juveniles, concentrations of saponin for 70 days at 34 p.s.u.

after they had been exposed to different

Saponin

Weight

Feeding rate (%)

(mg I-‘)

(g)

First

Second

Third

Mean

Control

1.944 (0.093) 1.915 (0.061) 1.847 (0.056) 1.737 (0.060) 1.713 (0.063)

2.495a (0.055) 2.434a (0.249) 1.496b (0.268) 0.912c (0.057) 0.538~ (0.089)

2.304a (0.218) 2.199ab (0.092)

2.410a (0.455) 2.205ab (0.376) 1.289bc (0.339) 1.14Obc (0.256) 0.862~ (0.248)

2.403a

0.1 0.5

1 2

Values in the same cohmm followed by different

1.736bc (0.122) 1.376c (0.148) 0.73Od (0.081) letters are significantly

2.279a

I .507b 1.143b 0.71Oc

different (P < 0.05).

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144 (19961 165-175

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Table 6 Effect of saponin on feeding rate (%) for Penaeus japonicus juveniles, after they had been exposed to different concentrations of saponin for 30 min at 34 p.s.u. Saponin

Weight

Feeding rate (%)

(mg 1-l)

(g)

First

Second

Mean

Control

8.298 (0.190) 8.350 (0.218) 8.229 (0.228) 8.242 (0.141) 8.240 (0.179)

1.956a (0.108) 1.009b (0.108) 0.414.c (0.064) 0.149d (0.030) 0.048e (0.005)

2.111a (0.157) 0.958b (0.081) 0.419c (0.058) 0.126d (0.013) 0.037e (0.007)

2.034a

0.5

1 5 10

0.984b 0.417c 0.138d 0.043e

Values in the same column followed by different letters are significantly different (P < 0.05).

lower than those exposed to the control solution and 0.1 mg I-’ saponin. The MATC was 0.1 mg 1-l saponin as determined from the effect of saponin on feeding of P. japonicus juveniles and adolescents.

4. Discussion Tilapia, Oreochromis mossambicus and shrimp Exopalaemon orientis are very common competitors, found in a great number of shrimp ponds in Taiwan. They compete with cultured penaeids for food and oxygen. Ten pounders (Elopsawaiensis), gobies (Acentrognbius caninus), sea bass (Serranids) and teraponids are common predatory fishes, and prey directly on shrimps (Chen, 1990). Therefore, shrimp farmers have customarily used tea seed cake as a toxin to kill undesirable fish in shrimp ponds. Terazaki et al. (1980) compared the toxicities of saponin on several teleosts at 15 p.s.u., and found that the median lethal time (LT,,) of saponin at 1.1 mg 1-l was 0.7 h, 2.5 h, 5.0 h, 6.0 h and 6.5 h for Eleutheronema tetradactylum, Mugil trade, Scatophagus argus, 0. mossambicus and Mystus sp., respectively. They also reported that the LTso of saponin at 40 mg 1-l was 0.5 h for 0. mossambicus and 30 h for P. merguiensis. 0. mossambicus is much more susceptible to saponin than P. merguiensis. Minsalan and Chiu (1986) also reported that finfish Glossogobicus giurus is more sensitive to saponin than P. monodon and Metapenaeus ensis. The present study indicated that no P. japonicus juvenile died following exposure to saponin at 22.5 mg 1-l for 24 h. Fish catching with the aid of piscicidal plants is an ancient practice. A number of researchers have studied the reactions of fish to the poisons of these plants. Saponin, a water soluble glucoside, acts chiefly by lowering the surface tension between water and the gills of fish, thus preventing the uptake of oxygen by the fish and leading to slow death by oxygen deprivation (Lamba, 1970). Saponin is considered to cause swelling in the lamella and interlamellar epithelia of gills and affect respiratory epithelium of fish (Roy et al., 1986; Roy and Munshi, 1989).

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In a field trial, Minsalan and Chiu (1986) observed that P. monodon juveniles did not feed, when tea seed cake at 15 mg 1-l was applied, and they appeared to return to normal feeding after the concentration was reduced to 3 mg I-’ (equivalent to 0.15 mg I-’ saponin). The present study indicated that saponin at 0.5 mg 1-l affected the feeding, growth and molting frequency of P. japonicus juveniles. Roy and Munshi (1989) reported that the perch Anabas testudineus, following 24 h exposure to 2-5 mg 1-l saponin increased its oxygen uptake by 20% and increased its number of erythrocyte, amount of hemoglobin and hematocrit. Roy et al. (1990) documented that saponin could damage the respiratory epithelium of Heteropneustes fossilis. Similar reactions may also occur in hemocyanin containing penaeids. Further research is needed to study the hemocyanin level in the hemolymph, oxygen uptake and oxygen binding capability of penaeid under saponin stress. Molting of decapod crustaceans is affected by the extrinsic factors like temperature, salinity, light intensity and pollutants and by the intrinsic factors like nutritional state and eyestalk ablation, and has been reviewed by Kleinholz (1985). The present study indicated that the time before the first molting decreased with increased saponin concentration. Mean molting frequency decreased significantly for the shrimps following 60 days exposure to saponin at 0.5 mg 1-l and higher. It is known that external factors like light and temperature, which stimulate the central nerve system and cause the secretion of hormones, would affect the molting cycle of Crustacea (Wassenberg and Hill, 1984). However, the shrimps in the present study were tested in a controlled light/dark (L:D 12:12) and temperature (25.5 k 0.7”C) environment. It would be interesting to study the mechanism of why shrimps subjected to saponin reduce the time for the first molting, and decrease the molting frequency after long-term exposure. Based on the 96-h LC,, (18.14 mg 1.’ saponin) and an empirical application factor of 0.1 (Sprague, 1971), the ‘safety level’ was 1.81 mg 1.’ saponin on P. juponicus. The threshold concentration that produces statistically significant deleterious effects is commonly expressed as the MATC (Wickins, 1976). The MATC obtained from the present study was 0.1 mg 1-l saponin for feeding, growth factor and molting frequency. The present study reveals that P. japonicus exposed to a concentration of saponin much lower than that of the ‘safe value’ may exhibit different degrees of chronic response knowledge of which would be useful in pond management during shrimp farming.

Acknowledgements We are very grateful for the grant supported by the Council of Agriculture of the Republic of China. We would also like to thank Y.H. Chen for his assistance in creating graphs.

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