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Acute toxicity of ammonia on

Litopenaeus vannamei Boone
juveniles at different salinity levels
Jiann-chu Chen

Journal of Experimental Marine Biology and Ecology

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 Journal of Experimental Marine Biology and Ecology
259 Ž2001. 109–119
www.elsevier.nlrlocaterjembe

Acute toxicity of ammonia on Litopenaeus

Õannamei Boone juveniles at different
salinity levels
Yong-Chin Lin, Jiann-Chu Chen )
Department of Aquaculture, National Taiwan Ocean UniÕersity, Keelung, Taiwan, 202,
People’s Republic of China
Received 25 October 2000; received in revised form 13 January 2001; accepted 15 February 2001

Abstract

Litopenaeus Õannamei juveniles Žtotal length 22 " 2.4 mm. were exposed to different concen-
trations of ammonia-N Žun-ionized plus ionized ammonia as nitrogen., using the static renewal
method at different salinity levels of 15‰, 25‰ and 35‰ at pH 8.05 and 238C. The 24, 48, 72,
96 h LC50 values of ammonia-N on L. Õannamei juveniles were 59.72, 40.58, 32.15, 24.39 mg
ly1 at 15‰; 66.38, 48.83, 43.17, 35.4 mg ly1 at 25‰; 68.75, 53.84, 44.93, 39.54 mg ly1 at 35‰,
respectively. The 24, 48, 72, 96 h LC50 values of NH 3-N Žun-ionized ammonia as nitrogen. were
2.95, 2.00, 1.59, 1.20 mg ly1 at 15‰; 2.93, 2.16, 1.91, 1.57 mg ly1 at 25‰; 2.78, 2.18, 1.82,
1.60 mg ly1 at 35‰, respectively. As the salinity decreased from 35‰ to 15‰, susceptibility of
ammonia-N increased by 115%, 132%, 140% and 162% after 24, 48, 72 and 96 h exposure,
respectively. The Asafety levelB for rearing L. Õannamei juveniles was estimated to be 2.44, 3.55,
3.95 mg ly1 for ammonia-N and 0.12, 0.16, 0.16 mg ly1 for NH 3-N in 15‰, 25‰ and 35‰,
respectively. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Ammonia; Litopenaeus Õannamei; Salinity; Toxicity

1. Introduction

White shrimp Litopenaeus Õannamei ŽBoone, 1931., which is distributed throughout

the Pacific coast from Gulf of California to northern Peru is the major species of penaeid
shrimp in the east hemisphere and contribute 30% of farmed production of penaeid
shrimps in the world ŽFarfante and Kensley, 1997.. This species inhabits brackish water

)
Corresponding author. Tel.: q886-2-2462-2192 ext. 5205; fax: q886-2-2462-0295.
E-mail address: Jcchen@mail.ntou.edu.tw ŽJ.-C. Chen..

0022-0981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 0 9 8 1 Ž 0 1 . 0 0 2 2 7 - 1
110 Y.-C. Lin, J.-C. Chen r J. Exp. Mar. Biol. Ecol. 259 (2001) 109–119

of 1–2‰ and saline water of 40‰ ŽMenz and Blake, 1980.. L. Õannamei exhibits
hyper-osmotic regulation in low salinity levels, and exhibits hypo-osmotic regulation at
high salinity levels with an isomotic point of 718 mosM kgy1 , which is equivalent to
25‰ ŽCastille and Lawrence, 1981.. The juveniles have their best survival between
temperatures of 208C and 308C and salinities above 20‰ ŽPonce-Palafox et al., 1997..
The optimal temperature for better growth is 28–308C ŽPonce-Palafox et al., 1997.. Bray
et al. Ž1994. reported that 15–25‰ are considered ideal for optimum salinity for L.
Õannamei. However, Ponce-Palafox et al. Ž1997. reported that the survival and growth
coincide best at around 33–40‰.
L. Õannamei has been introduced to Taiwan since June 1985 and their spontaneous
spawning, propagation and larval rearing have been conducted successfully for several
generations cultured ŽLin et al., 1990.. Seasonal ranges of water temperature in shrimp
farms of Taiwan vary from 168C to 308C. For more than 10 years, the commercial
shrimp farming, mainly from local species tiger shrimp Penaeus monodon and kuruma
shrimp P. japonicus, shrunken due to disease outbreak associated with bacteria, like
Vibrio harÕeyi and V. alginolyticus, and virus, like monodon baculoviros virus ŽMBV.
and white spot syndrome virus ŽWSSV. ŽLo and Kou, 1998.. Therefore, L. Õannamei
has been receiving attention and about 2000 ha of ponds have been devoted to the
culture of this species in Taiwan ŽYu and Song, 2000..
Culture of penaeid shrimps has been intensified due to limitation and availability of
ponds. In an intensive culture system, ammonia is the commonest toxicant resulting
from excretion of cultured animals and mineralization of organic detritus like uncon-
sumed feed and feces. It has been reported that concentration of ammonia-N Žun-ionized
plus ionized ammonia as nitrogen. increased directly with culture period and might
reach as high as 46 mg ly1 in intensive grout-out ponds ŽChen et al., 1988.. Accumula-
tion of ammonia in pond water may deteriorate water quality, reduce growth, increase
oxygen consumption and ammonia-N excretion, alter concentrations of hemolymph
protein and free amino acid levels, and even cause high mortality ŽWickins, 1976; Chen
and Lin, 1992; Chen et al., 1994.. Elevated ammonia in water has also been reported to
increase the susceptibility of rainbow trout Oncorhynchus mykiss to pathogen ŽHurvitz
et al., 1997..
The lethal effects of ammonia on the juveniles of penaeid shrimps have been
provided for P. chinensis ŽChen and Lin, 1992., P. monodon ŽChen and Lei, 1990., P.
paulensis ŽOstrensky and Wasielesky, 1995., P. penicillatus ŽChen and Lin, 1991., P.
semisulcatus ŽWajsbrot et al., 1990., and Metapenaeus ensis ŽNan and Chen, 1991..
Reported 96-h LC50 varied, 23.70–45.58 mg ly1 for ammonia-N and 0.87–2.47 mg ly1
for NH 3-N Žun-ionized ammonia as nitrogen. ŽChen and Lei, 1990; Nan and Chen,
1991; Wajsbrot et al., 1990; Chen and Lin, 1992.. However, little information is
available on the lethal effect of ammonia at different salinity levels for penaeid shrimps
ŽChen and Lin, 1991, 1992.. The purpose of the present study is to estimate ammonia
toxicity of L. Õannamei juveniles at salinity levels of 15‰, 25‰ and 35‰.

2. Materials and methods

L. Õannamei postlarvae were shipped from a shrimp farm in Pingtung, and cultured in
the laboratory of National Taiwan Ocean University for 3 months prior to experimenta-
 Y.-C. Lin, J.-C. Chen r J. Exp. Mar. Biol. Ecol. 259 (2001) 109–119 111

tion. The salinity of water for culturing was 25‰. In the period of stocking, the shrimp
was fed a diet Žprotein no less than 39%. prepared by Tairoun Feedstuff ŽTaipei,
Taiwan.. The shrimps were divided randomly into three groups in the same salinity, and
then adjusted 2‰ each day to reach three different salinity levels of 15‰, 25‰ and
35‰.
Seawater Ž35‰. pumped form the Keelung coast adjacent to the university was
adjusted to 35‰, 25‰ and 15‰, with municipal water dechlorinated with sodium
thiosulfate, and then filtered through a gravel and sand bed by air-lifting and aerating for
3 days before use. Stock solution of ammonia-N at 10 000 mg ly1 was prepared with
ammonium chloride in the same way as that reported by Chen and Lei Ž1990., and then
diluted to the desired concentrations of ammonia-N with 35‰, 25‰ and 15‰ seawater.
The nominal concentration of ammonia-N for test solution ranged from 20 to 100 mg
ly1 at 10-mg ly1 interval and 125 and 150 mg ly1 . Experimental concentrations of
ammonia-N were 20, 30, 40, 50, 60, 70, 80, 90, 100, 125 and 150 mg ly1 . The actual
concentrations of ammonia-N in test solution were measured using the method described
by Solorzano Ž1969.. The concentration of NH 3-N Žun-ionized ammonia as nitrogen.
was calculated according to the equations of Whitfield Ž1978. based on pH 8.05,
temperature 238C, and salinity level of 15‰, 25‰ and 35‰. The concentration of
NH 3-N contributed 4.93%, 4.42% and 4.05% of ammonia-N concentration at 15‰,
25‰ and 35‰, respectively ŽBower and Bidwell, 1978..
Short-term LC50 Žmedian lethal concentration. toxicity tests were carried out accord-
ing to the methods described by American Public Health Association et al. Ž1985..
Shrimps were sampled randomly from the holding tanks and transferred to test solutions
and control solutions Žwithout added ammonia.. Bioassay experiments to establish
tolerance limits were conducted in 10-l polyethylene tanks containing 5-l test solutions.
Each tank contained 10 test shrimps, and water was aerated continuously by air-stone
with a blower. A group of 10 shrimps was used as control for each salinity levels. Each
test solution was renewed daily, in accordance with the static renewal method for

Table 1
ANOVA table of survival of L. Õannamei juveniles exposed to different concentrations of ammonia-N Ž C . at
control, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150 mg ly1 , different salinity levels Ž S . at 15‰, 25‰ and
35‰ and after various exposure times Ž t . at 12, 24, 36, 48, 60, 72, 84 and 96 h
Source df Sum of square Mean square F Pa
))
Model 263 132.994 0.506 4.08
))
C 10 70.039 7.004 56.46
))
S 2 0.447 0.222 1.79
))
T 7 16.622 2.375 19.14
))
C=S 20 4.494 0.225 1.81
))
C=t 70 20.560 0.294 2.37
))
S=t 14 1.674 0.120 0.96
))
C=S=t 140 19.160 0.137 1.10
Error 264 32.750 0.124
Total 527 165.745
a
P - 0.01.
112 Y.-C. Lin, J.-C. Chen r J. Exp. Mar. Biol. Ecol. 259 (2001) 109–119

Fig. 1. The LT50 Žmedian lethal time, hour required to half the population. of ammonia-N ŽA. and NH 3 -N ŽB.
to L. Õannamei juveniles exposed to different concentrations of ammonia-N at 15‰, 25‰ and 35‰.
 Y.-C. Lin, J.-C. Chen r J. Exp. Mar. Biol. Ecol. 259 (2001) 109–119 113

toxicity tests ŽBuikema et al., 1982.. Each bioassay was conducted in duplicate. During
the experiment, shrimps were fed a diet Žprotein 39%. designed for P. monodon by
Tairoun Feedstuff twice a day Ž10:00 and 18:00. at 15% of body weight per day. Water
temperature was maintained at 23 " 18C, dissolved oxygen at 6.2 " 0.4 mg ly1 , pH
ranged from 7.95 to 8.12 with an average of 8.05.
Observations were usually made at 12-h interval up to 96 h. The LT50 Žmedian lethal
time, hour required to kill half the production. was determined. Death was assumed
when shrimps were immobile and showed no response to touch with a glass rod. Dead
shrimps were removed daily. The concentration response of test organisms was deter-
mined by plotting probit transformed percent mortality against log concentration ŽFin-
ney, 1971.. The LC50 value Žmedian lethal concentration. of ammonia-N and NH 3-N
and their 95% confidence limits were calculated with a computer program ŽTrevors and
Lusty, 1985.. The estimated probit line and results of a X 2 ŽChi-square. test for

Table 2
Relationship between probit of mortality Ž Y . and log ammonia-N as mg ly1 Ž X . at various exposure times at
15‰, 25‰ and 35‰ for L. Õannamei juveniles
Time Žh. Y s aq bX na X 2b X 2 calculated X 2 df 0.95
15‰
12 Y sy7.261q6.217X 4 0.925 0.879 2 5.937
24 Y sy10.229q8.574 X 4 0.995 0.102 2 5.937
36 Y sy6.559q7.099 X 4 0.929 2.135 2 5.937
48 Y sy6.468q7.093 X 3 0.987 0.226 1 3.747
60 Y sy7.099q7.753 X 3 0.996 3.747 1 3.747
72 Y sy6.704q7.766 X 3 0.994 0.204 1 3.747
84 Y sy6.069q7.725 X 3 0.999 0.030 1 3.747
96 Y sy7.751q9.109 X 3 0.958 1.226 1 3.747

25‰
12 Y sy10.699q7.745 X 4 0.750 6.467 2 5.937
24 Y sy12.357q9.485 X 4 0.986 0.523 2 5.937
36 Y sy11.641q9.617X 4 0.996 0.126 2 5.937
48 Y sy11.175q9.585 X 3 0.998 0.019 1 3.747
60 Y sy10.976q9.620 X 3 0.952 1.124 1 3.747
72 Y sy7.220q7.422 X 3 0.777 3.723 1 3.747
84 Y sy10.807q10.018 X 3 0.737 9.413 1 3.747
96 Y sy8.665q8.817X 3 0.788 5.903 1 3.747

35‰
12 Y sy11.627q8.115 X 4 0.887 2.935 2 5.937
24 Y sy6.926q6.524 X 4 0.955 0.422 2 5.937
36 Y sy10.343q8.724 X 5 0.907 4.039 3 7.775
48 Y sy8.779q7.960 X 4 0.956 1.186 2 5.937
60 Y sy20.336q15.109 X 4 0.982 0.763 2 3.747
72 Y sy7.219q7.268 X 3 0.803 2.919 1 3.747
84 Y sy3.471q5.146 X 3 0.685 3.341 1 3.747
96 Y sy5.650q6.670 X 3 0.899 1.440 1 3.747
a
Number of concentration for calculation.
b
Coefficient of determination.
114 Y.-C. Lin, J.-C. Chen r J. Exp. Mar. Biol. Ecol. 259 (2001) 109–119

Fig. 2. LC50 Žmedian concentration. of ammonia-N ŽA. and NH 3 -N ŽB. to L. Õannamei juveniles exposed to
different concentrations of ammonia-N in 15‰, 25‰ and 35‰.
 Y.-C. Lin, J.-C. Chen r J. Exp. Mar. Biol. Ecol. 259 (2001) 109–119 115

goodness of fit were computed. Relationship among survival, salinity, concentration of

ammonia-N and exposure time was tested with the statistical system ŽSAS, 1988. using
the General Linear Model procedure.

3. Results

Actual concentrations of ammonia in test solution were within 5% of nominal

concentrations. No shrimp died in the control solutions of all salinity levels. No shrimp
died in 20-mg ly1 ammonia-N solution at 25‰ and 35‰ when exposed for 96 h and in
20-mg ly1 ammonia-N solution at 15‰ when exposed for 60 h either. Survival of the L.
Õannamei juveniles among concentration of ammonia-N and salinity interaction, among
concentration of ammonia and exposure time interaction, and among salinity and
exposure time interaction were significantly different ŽTable 1..
The LT50 data of ammonia-N and NH 3-N for L. Õannamei juveniles are depicted in
Fig. 1. Statistical analysis indicated that LT50 had a negative exponential relationship
with concentrations of ammonia-N and NH 3-N. The LT50 of L. Õannamei juveniles
was 34, 44 and 54 h when exposed to 50 mg ly1 ammonia-N at 15‰, 25‰ and 35‰,
respectively.
The probit of mortality for a L. Õannamei juvenile exposed to ammonia-N was
linearly related to log ammonia-N at various exposure times ŽTable 2.. An X 2 test
indicated that values of Chi were less than the values in those tables, suggesting that the
assumed lines were satisfactory ŽTrevor and Lusty, 1985..
The LC50 values of ammonia-N and NH 3-N at different period exposure time for L.
Õannamei juveniles are shown in Fig. 2. At 24, 48, 72 and 96 h, LC50 values of
ammonia-N were 59.72, 40.58, 32.15 and 24.39 mg ly1 at 15‰; 66.38, 48.83, 43.17 and
35.47 mg ly1 at 25‰; and 68.75, 53.84, 44.93 and 39.54 mg ly1 at 35‰. Those of
NH 3-N were 2.95, 2.00, 1.59, 1.20 mg ly1 at 15‰; 2.93, 2.16, 1.91 and 1.57 mg ly1 at
25‰ and 2.78, 2.18, 1.82 and 1.60 mg ly1 at 35‰. The LC50 values of ammonia-N and
NH 3-N declined sharply during the first 60 h. Resistance of L. Õannamei juveniles to
ammonia-N was 89–95% less at 25‰ than that at 35‰, and 69–88% less at 15‰ than
that at 25‰.

4. Discussion

Toxic effects of ammonia on various organisms have been reported: fresh teleosts
ŽAlabaster and Lloyd, 1982., different stages of penaeid larvae, and the Asafety levelB of
ammonia for P. indicus ŽJayasankar and Muthu, 1983., P. japonicus ŽChen et al., 1989.,
P. monodon ŽChin and Chen, 1987., P. paulensis ŽOstrensky and Wasielesky, 1995. and
P. setiferus ŽAlcaraz et al., 1999.. Toxic effects of ammonia on the juveniles of different
species of penaeid shrimps are given in Table 3. Reported 96 h LC50 of ammonia-N
ranged from 23.70 mg ly1 for P. semisulcatus ŽWajsbrot et al., 1990. to 45.58 mg ly1
for P. monodon ŽChen and Lei, 1990.. Reported 96 h LC50 of NH 3-N ranged from 0.87
mg ly1 for M. ensis ŽNan and Chen, 1991. to 2.47 mg ly1 for P. chinensis ŽChen and
116 Y.-C. Lin, J.-C. Chen r J. Exp. Mar. Biol. Ecol. 259 (2001) 109–119

Table 3
The 96 h LC50 of ammonia-N and NH 3 -N on the several species of penaeid shrimps
Species Salinity Ammonia-N NH 3 -N Reference
Ž‰. Žmg ly1 . Žmg ly1 .
P. chinensis
Ž36.1 mm. 10 28.18 1.94 Chen and Lin Ž1992.
20 38.87 2.46
30 42.44 2.47
Ž39.6 mm. 33 37.00 1.53 Chen et al. Ž1990.

P. monodon
Ž35.4 mm. 20 45.58 1.29 Chen and Lei Ž1990.

P. pauleneis
Ž5.45 g. 28 38.72 1.10 Ostrensky and Wasielesky Ž1995.

P. penicillatus
Ž35.8 mm. 25 24.88 0.99 Chen and Lin Ž1991.
34 29.77 1.11

P. semisulcatus
Ž0.35–2.4 g. 23.7 Wajsbrot et al. Ž1990.

L. Õannamei
Ž22 mm. 15 24.39 1.20 Present study
25 35.40 1.57
35 39.54 1.60

M. ensis
Ž10.7 mm. 25 35.59 0.87 Nan and Chen Ž1991.

Lin, 1992.. At 25‰, L. Õannamei juveniles are considered to be more tolerant to

ammonia as compared to P. penicillatus.
Chin and Chen Ž1987. reported that 24-h LC50 on P. monodon was 6.0, 8.48, 24.04
and 52.11 mg ly1 ammonia-N and 0.54, 0.76, 2.17 and 4.70 mg ly1 NH 3-N for
nauplius, zoea, mysis and postlarvae, respectively. Chen and Lei Ž1990. reported that
24- and 96-h LC50 on P. monodon juveniles were 94.46 and 45.58 mg ly1 for
ammonia-N, and 2.68 and 1.29 mg ly1 for NH 3-N, respectively. These data suggest that
tolerance to ammonia of penaeid shrimp increase with age.
Most of the previous papers documented LC50 values of NH 3-N and ammonia-N on
penaeids at one salinity level. Penaeids are generally cultured intensively in a semi-static
environment with salinity varying from 17‰ to 34‰ ŽChen et al., 1988; Chen and
Wang, 1990.. Chen and Lin Ž1991. reported that the 96-h LC50 of ammonia on P.
penicillatus juveniles was 24.88 and 29.77 mg ly1 for ammonia-N and 0.99 and 1.11
mg ly1 for NH 3-N at 25‰ and 34‰, respectively. Chen and Lin Ž1992. reported that
the 96-h LC50 of ammonia on P. chinensis juveniles was 28.18, 38.87 and 42.44 mg
ly1 for ammonia-N, and 1.94, 2.46 and 2.47 mg ly1 for NH 3-N at 10‰, 20‰ and 30‰,
respectively. The present study indicated that toxicity of ammonia-N to L. Õannamei
juveniles increased by 119–162% as salinity decreased from 35‰ to 15‰. The fact that
 Y.-C. Lin, J.-C. Chen r J. Exp. Mar. Biol. Ecol. 259 (2001) 109–119 117

toxicity of ammonia increased as salinity decreased is considered due to higher uptake

of ammonia at low salinity. Therefore, shrimp farmers should note the toxicity differ-
ence of ammonia at different salinity levels when marking pond management decisions.
Chen and Lin Ž1992. reported that the LC50 value for NH 3-N and ammonia-N on P.
chinensis juveniles at 10‰ were 70–90% less than those at 20‰ and 30‰, and the
LC50 for NH 3-N at 20‰ was 85–90% less than that at 30‰ after 12–60 h of exposure.
The present study indicated that the LC50 for ammonia-N at 15‰ were 79–89% less
than those at 35‰, and the LC50 for ammonia-N at 25‰ were 91–97% less than those
at 35‰ after 12–96 h. In other words, toxicity of ammonia to P. chinensis, P.
penicillatus and L. Õannamei juveniles is significantly higher for those reared at 10‰ or
15‰ than those reared at 30‰ or 35‰. Ponce-Palafox et al. Ž1997. reported that the
survival and growth were the best at around 33‰ to 40‰ for juvenile L. Õannamei
among the salinities tested. The present study indicated that there is a relationship
between the better growth and increment to the tolerance to ammonia in higher salinity.
The toxicity of ammonia-N to L. Õannamei juveniles increased with exposure time.
The tolerance of L. Õannamei juveniles to ammonia-N decreased sharply by 56.7% and
74.0% after 48 and 96 h as compared to 24 h LC50 at 15‰. The tolerance of L.
Õannamei juveniles to ammonia-N decreased sharply by 52.0% and 64.7% after 48 and
96 h as compared to 24 h LC50 at 35‰. Similar phenomenon was obtained previously
in other penaeid shrimps. Chen and Lin Ž1991. observed that the tolerance of P.
penicillatus juveniles decreased by 37.8% and 50.5% after 48 and 96 h as compared to
24 h LC50 at 25‰. Chen and Lin Ž1992. reported that the tolerance of P. chinensis
juveniles to ammonia-N decreased by 19.2% and 36.4% after 48 and 96 h as compared
to 24 h LC50 at 30‰. The increase of ammonia-N toxicity over time was abrupt in P.
Õannamei as compared to that in P. chinensis and P. penicillatus.
Based on the 96-h value and an empirical application factor 0.1 ŽSprague, 1971., the
Asafety levelB for rearing L. Õannamei juveniles was calculated to be 0.12 mg ly1
NH 3-N Ž2.44 mg ly1 ammonia-N. in 15‰, 0.16 mg ly1 NH 3-N Ž3.55 mg ly1
ammonia-N. in 25‰, and 0.16 mg ly1 NH 3-N Ž3.95 mg ly1 ammonia-N. in 35‰. The
safety level of NH 3-N and ammonia-N at 15‰ was 75% and 61% less than that at 35‰,
respectively, and the safety level of ammonia-N at 25‰ was 90% less than that at 35‰.

Acknowledgements

This research was given to Y.C. Lin ŽGrant number: NSC 89-2815-C019-014-B. as a
support by the National Science Council. We appreciate Dr. S.Y. Cheng, Mr. C.J. Pei
and Mr. Y. Lee for their assistance in the experiment. [SS]

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