Aquaculture Research, 2012, 1–6 doi:10.1111/j.1365-2109.2012.03248.

Ammonia tolerance of Litopenaeus vannamei (Boone)

larvae

Marı́a de Lourdes Cobo1, Stanislaus Sonnenholzner1, Mathieu Wille2 & Patrick Sorgeloos2
1
Escuela Superior Politécnica del Litoral (ESPOL), Centro Nacional de Acuicultura e Investigaciones Marinas (CENAIM),
Guayaquil, Ecuador
2
Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Ghent, Belgium

Correspondence: M de Lourdes Cobo, Centro Nacional de Acuicultura e Investigaciones Marinas (CENAIM), Escuela Superior Poli-
técnica del Litoral (ESPOL), Campus Gustavo Galindo Velasco, Km 30.5 vı́a Perimetral, P.O. Box 09-01-5863, Guayaquil, Ecuador.
E-mails: artemia@[Link]; mlcobo@[Link]

One of the most important limiting factors in

Abstract
intensive culture systems is the build-up of toxic
The tolerance of Litopenaeus vannamei larvae to nitrogenous waste. Ammonia is the main end-
increasing concentrations of total ammonia nitro- product of protein catabolism in crustaceans and
gen (TAN) using a short-term static renewal can account for 60–70% of nitrogen excretion
method at 26°C, 34 g L 1 salinity and pH 8.5 was with only small amounts of amino acids, urea and
assessed. The median lethal concentration (24 h uric acid (Chen & Kou 1996a,b). Of the nitroge-
LC50) for TAN in zoea (1-2-3), mysis (1-2-3) and nous compounds, ammonia is the most toxic with
postlarvae 1 were, respectively, 4.2-9.9-16.0; 19.0- nitrite and nitrate being less toxic to shrimp
17.3-17.5 and 13.2 mg L 1TAN (0.6-1.5-2.4; 2.8- (Chen, Liu & Lei 1990). In water, ammonia is
2.5-2.6 and 1.9 mg L 1 NH3-N). The LC50 values present in both ionized (NH4+) and un-ionized
obtained in this study suggest that zoeal and post- (NH3) state, with NH3 as the toxic form due to its
larval stages are more sensitive to 24 h ammonia ability to diffuse across cell membranes (Fromm &
exposure than the mysis stage of L. vannamei larvae. Gillete 1968; Emerson, Russo, Lund & Thurston
On the basis of the ammonia toxicity level (24 h 1975). The relative proportions of NH3 and NH4+
LC50) at zoea 1, we recommend that this level does depend on pH, temperature and to a lesser extent
not exceed 0.42 mg L 1 TAN – equivalent to on salinity (Bower & Bidwell 1978).
0.06 mg L 1 NH3-N – to reduce ammonia toxicity In penaeid shrimp, high concentrations of
during the rearing of L. vannamei larvae. ammonia may affect growth rates and survival,
and can in extreme cases cause mortality (Wickins
Keywords: Litopenaeus vannamei (Boone), ammo- 1976; Zin & Chu 1991; Chen & Lin 1992).
nia, toxicity, larval stages Ammonia damages the gills and reduces the abil-
ity of haemolymph to transport oxygen while
increasing oxygen consumption by tissues (Chien
Introduction
1992; Racotta & Hernández-Herrera 2000). Osmo-
Currently, the Pacific whiteleg shrimp Litopenaeus regulatory capacity decreases with increasing
vannamei (Boone) is the most important cultivated ammonia concentration and exposure time (Lin,
penaeid shrimp species in the world. Semi-inten- Thuet, Trilles, Mounet-Guillaume & Charmantier
sive and intensive shrimp culture systems are a 1993). Ammonia may also increase the moulting
common activity in many countries. However, frequency of shrimps (Chen & Kou 1992).
these culture practices usually result in degrada- Several studies have been carried out to deter-
tion of the culture water by uneaten food and mine toxicity levels of ammonia in different life
waste products of the shrimps. Thus, water quality stages of penaeid shrimp, such as Penaeus monodon
management and knowledge of water quality (Fabricius), Marsupenaeus japonicus (Bate), Farfan-
requirements are essential to any culture system. tepenaeus paulensis (Peréz-Farfante), Fenneropenaeus

© 2012 Blackwell Publishing Ltd 1

Ammonia tolerance of Litopenaeus vannamei larvae M de Lourdes Cobo et al. Aquaculture Research, 2012, 1–6

chinensis (Osbeck) and Litopenaeus setiferus (Bur- food EPIFEED-LHF® (Epicore, Eastampton, NJ,
kenroad) (Chin & Chen 1987; Chen & Lin 1991a, USA), FRIPPAK® (Inve Aquaculture, Dender-
b; Lin et al. 1993; Ostrensky & Wasielesky 1995; monde, SA, Belgium) and LARVA Z PLUS® (Zeigler,
Alcaraz, Chiappa-Carrara, Espinoza & Vanegas Gardners, PA, USA) were supplied.
1999). In L. vannamei, studies on the acute toxic-
ity of ammonia have been conducted on juveniles
Experimental design and toxicity tests
(Frı́as-Espericueta, Harfush-Melendez, Osuna-López
& Páez-Osuna 1999; Lin & Chen 2001) and on Short-term toxicity tests (24 h) were carried out to
post-larval stages (Magallón-Barajas, Servı́n, Porti- determine the acute total ammonia toxicity levels
lllo, Garcı́a & López 2006), but so far not on according to the method described by APHA,
larval stages. However, larviculture is a process of AWWA, WPCF (1992). Test solutions of ammonia
primary importance from which qualified larvae were prepared by dissolving 9.5518 g of Ammo-
must be produced to obtain maximum yields nium Chloride (NH4Cl) in 250 mL distilled water
during post-larval, nursery and grow-out culture. to make 10 000 mg L 1 stock solution and then
In traditional larviculture systems, water quality diluting it to the desired concentration. Ammonia
can rapidly degrade leading to increased mortality stock solution was added with an automatic pip-
and a higher incidence of diseases, particularly in ette (1–10 mL) directly to a series of 2-L glass
high density cultures. Therefore, the objective of beakers filled with 1 L of UV-treated seawater of
this study was to determine the acute toxicity of 34 g L 1 salinity. Flasks were placed into a con-
ammonia in the larval stages of L. vannamei, tainer filled halfway with water. The water bath
contributing to the optimization of water quality was heated to 26 ± 1°C. Before the start of the
management. actual toxicity tests, range-finding tests were
carried out to define concentrations for the final
toxicity test. The total ammonia nitrogen (TAN)
Materials and methods
nominal concentrations used for every larval sub-
stage are presented in Table 1. Water from two
Experimental animals and larval rearing
test flasks was sampled to determine actual
conditions
concentration of the testing solutions. Ammonia
Nauplii 5 (N5) of L. vannamei were obtained from analysis was performed according to the method
EGIDIOSA hatchery (San Pablo, Santa Elena Prov- described by Solórzano (1969). For every bioassay,
ince, Ecuador) and transported to the research facil- pH of the seawater was adjusted to 8.5 with a 2 N
ities of ESPOL-CENAIM Foundation. The nauplii sodium hydroxide (NaOH) solution. Groups of 20
were disinfected with 100 ppm of ARGENTYNE® larvae at each larval stage were taken randomly
(Argent, Redmond, WA, USA) for 1 min and accli- from the culture tank and transferred to the exper-
matized to the experimental conditions. N5 were imental units, once acclimatized for temperature.
stocked at 200 N5 L 1 in 4–500 L round-shaped All treatment concentrations had five replicates.
fibreglass tanks filled with sand-filtered and UV- Larvae were not fed and the water was not
treated seawater of 34 g L 1 and a pH of 8.1. Tem- renewed during the 24-h test, following the proce-
perature was maintained at 28 ± 1°C. Dissolved dure of Buikema, Niederlehner and Cairns (1982).
oxygen concentration was kept above 4 ppm. Lar- Mortality was checked after 24 h of exposure.
vae were fed with the microalgae Chaetoceros graci- Death was assumed when the larvae became
lis from N5 until PL1 (1-day postlarvae). From zoea immobile and showed no response. Based on the
2 (Z2) until PL1, also rotifers (Brachionus rotundifor- mortality, the 24-h Median Lethal Concentration
mis), frozen and live Artemia nauplii and artificial (LC50) and its 95% confidence intervals were cal-

Table 1 Nominal concentrations (mg L 1) of total ammonia nitrogen and ammonia NH3-N used for toxicity tests of
Litopenaeus vannamei (Boone) larval stages

Larval stages and sub-stages Nominal concentration of TAN (mg L 1) Nominal concentration of NH3-N (mg L 1)

Zoea 1, 2, 3 5.0, 7.5, 10.0, 12.5, 15.0, 20.0 0.7, 1.1, 1.5, 1.8, 2.2, 2.9
Mysis 1, 2, 3 and PL 1 5.0, 10.0, 12.5, 15.0, 20.0, 25.0, 30.0 0.7, 1.5, 1.8, 2.2, 2.9, 3.7,4.4

2 © 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–6

Aquaculture Research, 2012, 1–6 Ammonia tolerance of Litopenaeus vannamei larvae M de Lourdes Cobo et al.

culated using a Logistic regression using a JMP val development to 9.9 and 16.0 mg L 1 TAN
Statistical Package Program. The toxicity of un- (1.5 and 2.4 mg L 1 NH3-N) for Z2 and Z3
ionized ammonia (NH3-N) was calculated accord- respectively. Higher TAN LC50 were obtained for
ing to the equations proposed by Whitfield (1974), mysis sub-stages, being 19.0, 17.3 and
based on a salinity of 34 g L 1, a temperature of 17.5 mg L 1 TAN equivalent to 2.8, 2.5 and
26 ± 1°C and a pH of 8.5. The recommended 2.6 mg L 1 of un-ionized NH3-N for mysis 1,
levels to rear L. vannamei larvae were calculated mysis 2 and mysis 3, respectively. The 24-h LC50
using one-tenth of the 24-h LC50 values. for postlarvae 1 was 13.2 mg L 1 TAN and
1.9 mg L 1 NH3-N. The recommended levels for
rearing zoea stages were estimated to be between
Results
0.4 and 1.6 mg L 1 TAN (0.06–0.24 mg L 1
Survival of L. vannamei larvae exposed to increas- NH3-N). Mysis recommended levels were estimated
ing concentrations of TAN at the different larval to be from 1.73 to 1.90 mg L 1 TAN (0.25–
stages zoea 1 (Z1) to PL1 for 24 h are presented 0.28 mg L 1 NH3-N) and for PL1, values of 1.32
in Fig.1. High survival (80–100%) was observed and 0.19 mg L 1 for TAN and NH3-N were esti-
in the control group. Z1 showed lower survival mated respectively.
than the control from the lowest concentration
tested (5 mg L 1 TAN), whereas an increased tol-
Discussion
erance to ammonia was observed for Z2 and zoea
3 (Z3) sub-stages. Survivals above 50% were Several researchers have examined ammonia tox-
obtained for TAN concentrations of 10 and icity in various species of penaeid shrimp and at
15 mg L 1 for Z2 and Z3 respectively. Ammonia different developmental stages, especially for juve-
tolerance increased with larval developmental niles. Chen and Lei (1990) determined that for
stages, particularly at mysis, which displayed the P. monodon juvenile, toxicity of ammonia decreased
highest tolerance with survivals up to 70% for with exposure time. Chen and Lin (1992) observed
15 mg L 1 TAN. No variation was observed an increased susceptibility to ammonia as salinity
within mysis sub-stages. The increased ammonia decreased from 30 to 10 g L 1 in F. chinensis
resistance observed with larval development did juveniles. For Penaeus semisulcatus (De Haan) juve-
not extend to the PL1 stage, which showed a sur- niles exposed to different concentrations of ammo-
vival of 60% at 12.5 mg L 1 TAN. nia-N in a series of acute toxicity tests at four
LC50 values for TAN and their 95% confidence different water temperatures, Kir, Kumlu and
intervals for L. vannamei larval stages are shown Erodolgan (2004) found that lower temperatures
in Table 2. The 24-h LC50 for Z1 was 4.2 mg L 1 clearly increased tolerance of the shrimp to
TAN (0.6 mg L 1 NH3-N); this increased with lar- ammonia. Growth rates of M. japonicus juveniles

Zoea 1 Zoea 2 Zoea 3 Mysis 1 Mysis 2 Mysis 3 Postlarvae 1

100
90
80
70
Survival (%)

60
50
40
30
20
10
0
0 5 10 15 20 25 30 35
Total ammonia nitrogen (TAN) concentration (mg L–1)

Figure 1 Survival of Litopenaeus vannamei (Boone) larval stages from zoea 1 to postlarvae 1 exposed for 24 h to
increasing concentrations (mg L 1) of total ammonia nitrogen

© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–6 3

Ammonia tolerance of Litopenaeus vannamei larvae M de Lourdes Cobo et al. Aquaculture Research, 2012, 1–6

Table 2 The 24 h-LC50 values (mg L 1) of total ammo- zoea stages were the least tolerant and postlarvae
nia nitrogen (NH3-N + NH4+-N) and NH3-N for Litopena- the most tolerant to ammonia. In contrast, our
eus vannamei (Boone) larval stages and sub-stages. 95% results from short-term toxicity tests in L. vannamei
confidence intervals are shown between parentheses revealed that mysis stage was considerably more
resistant to ammonia than the other stages,
Larval stages
coinciding with the findings of Ostrensky and
and sub-stages 24-h LC50 TAN 24-h LC50 NH3-N
Wasielesky (1995) for F. paulensis larvae.
Zoea 1 4.2 (3.5–4.8) 0.6 (0.5–0.7) The LC50 values for un-ionized ammonia in
Zoea 2 9.9 (9.2–10.7) 1.5 (1.4–1.6)
[Link] zoea and mysis stages were higher
Zoea 3 16.0 (14.3–18.4) 2.4 (2.1–2.7)
Mysis 1 19.0 (18.1–20.0) 2.8 (2.7–2.9)
than those reported for P. monodon, M. japonicus
Mysis 2 17.3 (16.5–18.1) 2.5 (2.4–2.7) and F. chinensis (Chin & Chen 1987; Chen & Lin
Mysis 3 17.5 (16.7–18.4) 2.6 (2.5–2.7) 1991b; Lin et al. 1993; respectively), suggesting
Postlarvae 1 13.2 (12.1–14.2) 1.9 (1.8–2.1) that L. vannamei is more resistant to ammonia at
these stages. Similar 24-h LC50 values for ammo-
nia through the larval stages were obtained in this
exposed to different ammonia concentrations were study compared to those reported for F. paulensis
investigated by Chen and Kou (1992). The larvae (Ostrensky & Wasielesky 1995), and more-
authors concluded that ammonia had a stronger over the same pronounced decrease in ammonia
effect on weight rather than length. Studies in tolerance in the post-larval stage was also
L. vannamei juveniles have been conducted to observed. During the post-larval stage, the final
evaluate acute toxicity levels of ammonia (Frı́as- and most important metamorphosis occurs where
Espericueta et al. 1999) and at different salinity the larvae become juvenile shrimp. This could
levels (15–35 g L 1) (Lin & Chen 2001). Racotta explain the lower ammonia tolerance.
and Hernández-Herrera (2000) evaluated the met- Increase in pH levels favour the formation of the
abolic responses of ammonia exposure in L. vanna- more toxic un-ionized form of ammonia or enhance
mei juveniles, while Magallón-Barajas et al. (2006) the toxic effects (Colt & Armstrong 1981). Chen and
determined the daily variations in short-term Sheu (1990 a,b) reported that if the pH level gets
ammonia toxicity in L. vannamei postlarvae (1–30 higher than 8.2, increasing pH in a given ammonia
days old). solution could increase ammonia toxicity to P. mon-
Compared to juveniles, scarce information is odon M2 sub-stage and M. japonicus postlarvae. Sim-
available about acute toxicity levels of ammonia in ilar findings were observed by Chen and Chin
larval stages of penaeid shrimp. In Table 3, the (1989), when studying the effect of ammonia at dif-
24-h LC50 of un-ionized ammonia on several ferent pH levels in P. monodon postlarvae. The med-
penaeid species at larval stages are given. ian lethal time (LT50) values showed that ammonia
A progressive tolerance to ammonia with larval toxicity to P. monodon postlarvae increased as pH
development was observed for P. monodon (Chin & increased. Bower and Bidwell (1978) stated that the
Chen 1987), M. japonicus (Lin et al. 1993) and fraction of NH3 does not only depend on pH but also
F. chinensis (Chen & Lin 1991b), concluding that on temperature and to a lesser extent salinity. As

1
Table 3 The 24-h LC50 (mg L ) of un-ionized ammonia (NH3-N) in several penaeid species for early larval stages

Larval stages Test conditions

Postlarvae Temperature Salinity

Penaeid species Zoea Mysis (PL days old) pH (°C) (g L 1) Reference

Penaeus monodon 0.76 2.17 4.70 (PL6) 8.2 29.5 34 Chin and Chen (1987)
Marsupenaeus japonicus 0.97 1.08 1.98 (PL1) 7.9 25.0 36 Lin et al. (1993)
*
Fenneropenaeus chinensis 0.65 0.9 1.30 (PL4) 8.2 34 Chen and Lin (1991a,b)
†
Farfantepenaeus paulensis 1.79 2.91 1.40 (PL4) 25.0 28 Ostrensky and Wasielesky (1995)
Litopenaeus vannamei 1.47 2.63 1,94 (PL1) 8.5 26.0 34 This study

*Temperature for each larval stage: zoea (21°C); mysis (23°C); postlarvae (25°C).
†pH for each larval stage: zoea (8.24); mysis (7.90); postlarvae (8.1).

4 © 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–6

Aquaculture Research, 2012, 1–6 Ammonia tolerance of Litopenaeus vannamei larvae M de Lourdes Cobo et al.

pH and temperature rises, proportion of NH3 to for reviewing and giving comments on this
NH4+ increases and the toxicity of ammonia to manuscript.
animals increases. Magallón-Barajas et al. (2006)
investigated the daily variations in short-term (4 h)
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6 © 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–6