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Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
ACUTE TOXICITY OF AMMONIA ON
Macrobrachium tenellum
(SMITH) LARVAE
Gerardo FIGUEROA-LUCERO
1
, María Cecilia HERNÁNDEZ-RUBIO
2
* y
Miguel de Jesús GUTIÉRREZ-LADRÓN DE GUEVARA
2
1
Planta Experimental de Producción Acuícola. Departamento de Hidrobiología. DCBS. Universidad Autónoma
Metropolitana-Iztapalapa. Av. San Rafael Atlixco 186, Colonia Vicentina. C. P. 09340. México, D. F.
2
Laboratorio de Hidrobiología Experimental, Depto. de Zoología, Escuela Nacional de Ciencias Biológicas,
IPN. Prol. M. Carpio esq. Plan de Ayala s/n. Col. Sto. Tomás. 11340. México, D.F. Apdo. Postal 4-132. 06400.
México
*Corresponding author; cecheru@yahoo.com.mx
(Recibido abril 2011, aceptado enero 2012)
Keywords: river shrimp, prawn, zoea, tolerance
ABSTRACT
The prawn shrimp
Macrobrachium tenellum
is a potential species for culture in México.
The effect of ammonia on larvae was evaluated to provide basic information on safe
levels for larviculture. A 72 h static assay was performed on 5 days old
M. tenellum
larvae. The nominal concentrations tested ranged from 2.89 to 185.48 mg NH
4
-N/L
which represent 0.103 to 6.585 mg NH
3
-N/L at 20 g/L salinity, 28 ºC and pH 7.79. LC
50
for 12, 24, 48 and 72 h were 2.939 ± 0.505, 0.749 ± 0.301, 0.477 ± 0.163 and 0.409
± 0.068 mg NH
3
-N/L, respectively. These results suggest that
M. tenellum
exhibits a
slightly higher tolerance to ammonia in the zoea stage when compared to most of the
prawn and shrimp species.
Palabras clave: camarón de río, langostino, zoea, tolerancia
RESUMEN
El langostino
Macrobrachium tenellum
es una especie potencial para cultivo, en
México. El efecto del amonio en larvas se evaluó para proveer información básica de
los niveles seguros para el cultivo en esta etapa de desarrollo. Un ensayo estático de
72 h se realizó con larvas de
M. tenellum
de cinco días de edad. Las concentraciones
nominales probadas fueron desde 2.89 a 185 mg NH
4
-N/L, que equivalen a 0.103 hasta
6.585 mg NH
3
-N/L a 20 g/L de salinidad, 28 ºC y pH 7.79. Las LC
50
a 12, 24, 48 y 72
h fueron 2.939 ± 0.505, 0.749 ± 0.301, 0.477 ± 0.163 y 0.409 ± 0.068 mg NH
3
-N/L,
respectivamente. Estos resultados sugieren que
M.
tenellum
presenta una tolerancia
ligeramente más alta al amonio en el estado de zoea que otras especies de langostinos
y camarones.
Rev. Int. Contam. Ambie. 28 (2) 145-150, 2012
G. Figueroa-Lucero
et al.
146
INTRODUCTION
Macrobrachium tenellum
(Smith) is a freshwater
prawn, from the Paci±c coast rivers of America. In
Mexico, it is a commercially important resource,
particularly in the state of Guerrero, and it is consi-
dered suitable for mass culture (Guzmán 1987) even
through the capture and growth of wild postlarvae
(Martínez
et al
. 1980).
Ammonia is the main excretory product in
crustaceans (Hartenstein 1980, Cavalli
et al
. 2000).
This is an end product of amino acid catabolism
originated from excretion and organic matter de-
composition. Crustaceans excrete 60-70 % of nitro-
gen as ammonia through their gills through passive
diffusion and the rest is made up of small amounts
of ammonic acid, urea and uric acid (Chen and Kou
1996). High ammonia concentrations in tanks stoc-
ked in high densities of larvae is a potential danger
to aquatic organisms due to high toxicity (Chin
and Chen 1987, Ostrensky and Wasielesky 1995),
which may cause death or slow down prawn growth
rate at sublethal levels (Wickins 1976, Armstrong
et al
. 1978, Daniels
et al
. 1992, Miranda-Filho
et
al.
2009). In aqueous solution, ammonia can be
present in ionized (NH
4
+
) and/or unionized (NH
3
)
form, condition that is pH, temperature and salinity
dependent.
In crustaceans, elevated ammonium concentra-
tion might produce hemolymph alcalinization as a
consequence of increased internal concentration of
ammonium (Campbell 1973, Chen and Kou 1993,
Chen and Lin 1995, Mugnier and Justou 2004). Other
reported effects are respiratory inhibition (Alcaraz
et
al
. 1999, Malassen and Valenti 2005), reduction of
osmoregulatory capacity (Young-Lai
et al
. 1991, Lin
et al
. 1993, Mugnier and Justou 2004) and reduction
of survival (Mallasen and Valenti 2005, Naqvi
et al
.
2007, Schuler
et al
. 2010, Barbieri 2010, Liao
et al
.
2011).
In animals with gills, sensitivity to ammonia is
greater during the early developmental stages, becau-
se gill surface ratio to body weight is bigger and also
because the physiological detoxifying mechanisms
are still immature (Rand and Petrocelli 1985).
By understanding tolerances of
Macrobrachium
tenellum
(Smith) to ammonia its culture system can
be improved to optimized survival. In this study LC
50
values were obtained for larvae at various exposure
times, to increase our knowledge about the water
quality requirements of this species for aquaculture
systems.
MATERIALS AND METHODS
A static bioassay was performed to assess the acute
toxicity of ammonia (LC
50
values) on
M. tenellum
larvae over a period of 72 h, with toxic renewal.
The experiment was designed to assess the effect of
different concentrations of ammonia on survival
of larvae. The different concentrations of ammonia
were obtained by ±rst making a stock solution of
reagent grade ammonium chloride (NH
4
Cl, Baker
TM
).
Test concentrations of ammonia were then made up as
total ammonia nitrogen (TAN) by measuring a speci-
±ed quantity of NH
4
Cl, dissolving it in culture water
in a volumetric ²ask and then making up the solution
with more culture water to 5 L in a plastic container.
Stock solutions with measured ammonia concentra-
tions were then further diluted with brackish water
(20 g/L, Reefsalt
TM
of Seachem
TM
) according to the
individual concentrations required for each treatment.
Concentrations tested ranging from 2.89 to 185.48 mg
NH
4
-N/L (0.103, 0.206, 0.412, 0.823, 1.540, 3.292,
6.585 mg NH
3
-N/L). Ammonia was measured using
a Hach Model DR-2000 spectrophotometer (Hach
Company, Ames, Iowa, USA). The concentrations of
unionized ammonia (NH
3
-N) were calculated accor-
ding to the equations of Thurston, Khoo and Whit±eld
modi±ed by Boueres (2001) based on salinity 20 g/L,
water temperature 28 ºC and pH 7.8. Each treatment
was stocked with ±ve days old larvae, at zoea III stage,
obtained from a single ovigerous
M. tenellum
female
reared in laboratory ponds. Three replicates of ten
larvae each were used for treatment. For the assay,
larvae were placed in 250 mL beakers containing 150
mL of test solution without aeration. Salinity was 20
g/L, pH 7.79 and temperature 28 ºC. Larvae were fed
on
Artemia
nauplii before and during the experiment.
Food debris was removed from the beakers daily to
prevent decomposition. Test solutions in the beakers
was completely replaced every 24 h with new solution
prepared fresh each day
Mortality of larvae was recorded after 1, 2, 3, 6, 12,
24, 48 and 72 h of exposure, following the parameters
established by Armstrong
et al
. (1976), considering
ceasing of the heartbeat as death sign for the ±rst 24
hours, and opacity and lack of movement after 24 h.
Statistical analysis
One-way ANOVA was used to investigate the
effect of ammonia concentration on survival and
comparisons amongst means were made using T post
hoc test (Sokal and Rholf 1981).
ACUTE TOXICITY OF AMMONIA ON
Macrobrachium tenellum
(SMITH) LARVAE
147
The reported LC
50
values and 95% confdence
limits were obtained on the statistical so±tware EPA
Probit Analysis Program ver. 1.5.
RESULTS
There was no mortality in control treatments
during the experiment. Neither was observed any
deaths, during the frst six hours o± exposure ±or all
ammonia concentrations. All larvae exposed to 5.796
mg NH
4
-N/L (0.206 mg NH
3
-N/L) and 11.593 mg
NH
4
-N/L (0.412 mg NH
3
-N/L) survived ±or 24 and
12 h, respectively.
However, exposure to total ammonia had a signi-
fcant e±±ect (ANOVA, P< 0.01) on larvae survival,
causing mortality in concentrations as low as 2.898
mg NH
4
-N/L (0.103 mg NH
3
-N/L) at 48 h (
Fig. 1
).
A mortality o± 100 % was observed at concentra-
tions o± 43.37 mg NH
4
-N/L (1.540 mg NH
3
-N/L) and
92.74 mg NH
4
-N/L (3.292 mg NH
3
-N/L) a±ter 48 h.
and in 185.48 mg NH
4
-N/L (6.585 mg NH
3
-N/L) at
24 h (
Table I
).
The LC
50
values obtained decreased with increa-
sing exposure time, ±rom 75.95 mg NH
4
-N/L (2939
mg NH
3
-N/L) ±or 12 h, 23.98 mg NH
4
-N/L (0.749 mg
NH
3
-N/L) ±or 24 h, 14.25 mg NH4-N/L (0.477 mg
NH
3
-N/L) ±or 48 h, and 12.66 mg NH
4
-N/L (0.409
mg NH
3
-N/L) ±or 72 h exposure (
Table II
).
DISCUSSION
Previous studies have shown that ionized and
unionized ammonia toxicity varies with water pH
(Armstrong
et
. 1978), with development stage (Neil
et al
. 2005), and among decapods species (Allan
et al
.
1990), but also with temperature, salinity, atmosphe-
ric pressure and dissolved oxygen (Allan
et al
. 1990).
In ammonia toxicity assays with fshes, toxic
concentrations are expressed as unionized ammo-
nia only. Nevertheless, it has been shown that both
±orms o± ammonia are toxic. At a higher pH, am-
monia is predominantly in the unionized ±orm and
TABLE I.
PERCENT MORTALITY OF
M. tenellum
LARVAE
EXPOSED TO DIFFERENT AMMONIA-N AND
NH
3
-N CONCENTRATIONS, AT DIFFERENT
EXPOSURE TIME AND 20 g/L OF SALINITY
NH
4
+
-N
(mg/L)
NH
3
-N
(mg/L)
Exposure time (h)
12
24
48
72
2.898
0.103
0.0
0.0
6.6
6.6
5.796
0.206
0.0
0.0
6.6
6.6
11.593
0.412
0.0
16.6
30
43
23.185
0.823
3.3
56.6
80
90
43.37
1.540
10
86.6
100
100
92.74
3.292
70
93.3
185.48
6.585
83.3
100
TABLE II.
ESTIMATED LC
50
(mg/L) VALUES AND THEIR
95 % CONFIDENCE LIMITS FOR NH
3
-N
h
Conc. NH
3
-N
(mg/L)
95% Confdence limits
Lower
Upper
12
LC
50
2.939
2.434
3.603
24
LC
50
0.749
0.448
1.252
48
LC
50
0.477
0.314
0.715
72
LC
50
0.409
0.341
0.490
100
80
60
40
20
0
1
72
48
24
12
0
2
3
4
5
Concentration
6
7
72
24
0
Time (h)
Survival /%)
Fig. 1.
Average survival ±or larvae in stage III, during the 72 h exposition
period to di±±erent concentrations o± total amonia, at pH 7.79. 1: 2.898
(0.103), 2: 5.796 (0.206), 3: 11.593 (0.412), 4: 23.185 (0.823), 5: 43.37
(1.540), 6: 92.74 (3.292), 7: 185.48 (6.585) mg/L NH
4
-N (NH
3
-N)
G. Figueroa-Lucero
et al.
148
it is responsible for the toxicity; the opposite occurs
at a low pH, when NH
4
is the main form present
(Armstrong
et al
. 1978). An increase in ammonia
toxicity with increased pH has been reported in
Macrobrachium rosenbergii
and other crustaceans,
during larval and juvenile stages (Noor-Hamid
et al
.
1994, Mallasen and Valenti 2005, Neil
et al
. 2005).
In brackish water species, salinity exerts an im-
portant effect on ammonia internal concentration.
Research experiments have shown that sodium has
a lower af±nity than NH
4
for the enzyme responsible
for the active transport into the intracellular milieu.
Apparently the Km values for Na
+
transport are ten-
fold higher in marine species compared to freshwater
species (Shaw 1960). Barbieri (2010) observed that
Litopenaeus schmitti
juveniles experienced an increa-
se in susceptibility to TAN up 69 % as the salinity
decreased from 35 g/L to 5 g/L for 96 h exposure.
The present study was performed at 20 g/L salinity
(5 166 mg Na
+
/L), ammonia concentrations considered
toxic ranged from 9.39 to 85.16 mg/L. The ratio of
NH
4
+
to Na
+
was 0.0018-0.016: 1. For
Macrobrachium
rosenbergii
larvae (Armstrong
et
al
. 1978), it was de-
termined a 0.01-0.02: 1 ratio, at 12 g/L salinity. Those
results agree with our results, since an increase from
12 to 20 g/L, approximately, produces a hundredfold
increase in ammonia toxicity. In freshwater decapods
larvae it has been determined an inverse relationship,
Shaw (1960) obtained a ratio of 10:1.
Several toxic effects of ammonia on crustacean
decapod larvae and adults have been reported. For
M. rosenbergii
larvae, development slowed down
and mortality rate increased in alkaline water (pH
9) with increasing ammonia concentration and larval
tolerance to high ammonia and pH levels decreased
for the last zoeal stages (Mallasen and Valenti 2005).
Ammonia stress has been associated with decreased
haemolymph osmotic concentrations in
Penaeus
japonicus
(Chen and Chen 1996), changes in nitroge-
nous excretion in
M. rosenbergii
adult prawns (Chen
and Kou 1996), decreased survival and slowed down
larval development in
P. monodon
(Noor-Hamid
et
al
. 1994), changes in oxygen consumption (Alcaraz
et
al
. 1999, Barbieri 2010) and decreased growth
(Armstrong
et al
. 1978, Chen and Kou 1992).
Recent studies with other decapods species
report that tolerance to ammonia decreases for the
last zoeal stages or even in later development stages
(Mallasen and Valenti 2005), but the opposite has
also been found (Chin and Chen 1987, Ostrensky and
Wasielesky 1995). In this experiment the tolerance to
ammonia concentrations was tested on zoea III larvae
in order to control for other environmental factors, i.
e., larval nutritional status or damage associated to
larvae handling during the rearing period.
These results suggest that
M. tenellum
exhibits a
slightly higher tolerance to ammonia in the zoea stage
when compared to most of the prawn and shrimp spe-
cies complied in Ostrensky and Wasielesky (1995).
Sprague (1969, 1971) pointed out the effects of a
given toxicant could be described in terms of “safe le-
vel”, that can be obtained using an application factor
of 0.1. According to our results, safe level would be
below 0.6 mg/L for TAN and 0.075 NH
3
-N/L on the
basis of the 24 h LC
50
value at pH 7.79 and 20 g/L
salinity for
Macrobrachium tenellum
larval rearing
under controlled conditions. The results suggest TAN
and unionized ammonia must be daily measured since
a little increase, combined with an increase pH, could
result in a high mortality.
ACKNOWLEDGMENTS
This research was partly supported by the project
from Banco de Germoplasma de Recursos Genéticos
Acuáticos y Fauna Silvestre: Etapa I, UAMI, partly
by the project CONACyT-130200 from UAM-IPN
(México) and partly by the project SIP-20111206
(Secretaría de Investigación y Postgrado, IPN,
México).
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