Mechanism of Ammonia toxicity in Prawns by venturetechsg

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									Mechanism of Ammonia toxicity in Prawns

Introduction

The aim of this study is to determine the effects of ammonia on the physiological condition of the
prawn. In natural aquatic communities, animals are at risk of ammonia toxicity arising from urban
and agricultural runoffs and biological waste released from farm animals (Randall and Tsui 2002). In
the intensive cultivation of prawns in the aquaculture industry, ammonia toxicity is a major concern
as about 40-90% of the nitrogenous waste products that are continuously excreted from crustaceans
consist of ammonia (Chen and Nan, 1992; Pan et al. 2003). In addition, the decomposition of organic
waste matter or excess feed (Ajaz et al. 2007) also contributes to elevated ammonia levels in culture
medium. Hence the understanding of ammonia toxicity will help in formulating strategies to protect
against ammonia toxicity, which will ensure optimal prawn survival and production efficiency
during aquaculture.

        Ammonia toxicity arises due to the presence of abnormally high levels of total ammonia,
consisting of the non-ionised ammonia (NH3) and ionized ammonia (NH4+) in an organisms body
which results in the breakdown of normal physiological functions. In aquatic organisms, the
lipophillic ammonia-N is known to be more toxic due to its ability to diffuse across the gill
epithelium and penetrate the haemocyanin more readily than the lipophobic NH4+ ( Randall and
Wright 1987; Chen and Kou 1993). However, there have been reports that high levels of NH4+can
have equally detrimental effects on prawns at low pH (Armstrong et al. 1978). This study focuses on
the toxic effects of ammonia on osmoregulation of prawn and the alteration of prawn immunity
coupled to the production of reactive oxidation species (ROS).

Effects of ammonia on osmoregulation

The ammonia in the prawn’s body only becomes toxic when the ammonia is forced to accumulate in
the body of and beyond the tolerable concentration specific to various prawn species. Under normal
conditions, the prawns’ excretory mechanisms should function to remove ammonia. The three main
mechanisms known for prawns include the outward diffusion of NH3 via the gill epithelium, the
exchange of NH4+ for Na+ from the medium (Armstrong et al. 1973) and the detoxification of
ammonia to urea via ureotelic excretion (Chen and Cheng, 1993a). At high ambient ammonia levels
accumulation of haemolymph ammonia level within the prawn becomes inevitable. However, this
excess of ammonia is unable to be removed due to the unfavourable diffusion gradient set up.

a) Diffusion of NH3
Diffusion of ammonia out of the haemolymph into the surrounding aquatic environment via the gill
epithelium is one of the principal routes of nitrogenous waste excretion by crustaceans (Freire et al.
2008). This form of excretion is known as ammoniotelic excretion and is most probably crucial in
protecting the prawn against ammonia toxicity. This excretion mechanism only functions because
haemolymph NH3 concentration is usually much higher than ambient water concentrations (Kinne,
1976), and hence can be easily removed by simple diffusion.

       The extent of ammonia excretion and uptake by prawns exposed to different ambient NH3 or
NH4+ concentrations varies among species, size classes and nutrition status (Chen and Cheng,
1993a). A study on Paneaus chinensis found that ammonia-N excretion ultimately decreased when
exposed to increased ambient ammonia-N in the range of 0.965-5.087 mg/l (Chen and Lin, 1992).

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Chen and Kou’s (1991) and Chen and Cheng’s (1993a) studies of Paneaus japonicus found that
increasing ammonia-N concentrations to 10 mg/l or 5.121 mg/l for 24 hours respectively, led to the
reversal of the direction of NH3 diffusion, which began to enter the haemolymph from the
surrounding water.

       From the various studies on the different species of prawns under varying ammonia
conditions, we conclude that normal outward diffusion of ammonia from haemolymph to water
increases with increasing ambient ammonia concentration until the optimal rate of diffusion is
achieved. However, beyond a threshold level of ammonia, ammonia excretion decreases with further
increases in ammonia concentration.

b) Na+/ NH4+ exchange
An important mechanism involved in driving ammonia excretion is the Na+/ NH4+ exchange system
which involves the influx of Na+ and efflux of NH4+ .This exchange of sodium ions for ammonium
ions is coupled to the action of Na+/K+-ATPase, which is normally involved in maintaining
membrane potentials. In the study by Chen and Nan (1992) of P. chinensis, initial increases in
ammonia-N levels would increase in Na+/K+-ATPase. This is to allow increase in Na+ in the external
media to maintain the diffusion gradient for the exchange of NH4+ for Na+. However, with further
increases of ambient ammonia to 20 mg/L, ammonia-N excretion became inhibited and Na+/K+-
ATPase activity decreased (Chen and Nan, 1992). Since Na+/K+-ATPase activity is known to
increase during the moulting process as seen from M. nipponense, decreases in its activity will most
likely restrict prawn growth( Wang et al. 2003), which might in turn increase prawn mortality.
Another study by Armstrong et al. (1978) reported similar results where high ambient ammonia
levels inhibited Na+ /NH4+ exchange in the freshwater prawn, Macrobrachium rosenbergii at
different pH values. Hence, we can see that as ammonia level builds up beyond a certain level,
excretion via diffusion and exchange via the Na+/NH4+ would be drastically affected.

        While at high ambient ammonia, Chen and Nan (1992) documented that ammonia-N
excretion was inhibited and Na+/K+-ATPase activity decreased as shrimps were exposed to ambient
ammonia-N at 10 and 20 mg/1. This indicated the malfunctioning of Na+ influx and NH4+ efflux
which led to significant NH4+ uptake (Chen and Nan, 1992) and loss of Na+ in the haemolymph.
Another study by Armstrong et al. (1978) investigated the same effect of ammonia on the freshwater
prawn, M. rosenbergii at different pH values and suggested similar results where high ambient
ammonia levels inhibited the Na+ influx rate and affected NH4+ efflux. To add, this decrease in ions
would lead to the decrease in haemolymoh osmolarity. Study of penaeids showed that Na+ and Cl-
accounted for majority of the haemolymph osmolarity (Castille and Lawrence, 1981 and Chen and
Chen, 1996). Hence, we can see that as ammonia level builds up beyond a certain level, excretion
via diffusion and exchange via the Na+/NH4+ would be affected.

c) Ureotelic excretion
Studies have shown that certain species of prawns have evolved an alternative strategy for
preventing ammonia toxicity other than via the Na+/ NH4+ exchange system or ammoniotelic
excretion. The paeneid prawns for example (Chen and Cheng, 1993a) have evolved the ability to
convert ammonia to the less toxic urea. This mechanism is known as ureotelic excretion. Studies by
Chen and Lin (1995) have shown that at high ambient ammonia-N concentrations, there was a
decreasing ammonia excretion. We postulate that similar to how fish and other crustaceans function
physiologically, there is a possibly that ammonia is converted to urea to be excreted. In the study by
Chen and Cheng (1993a), they reported that haemolymph pCO2 (partial pressure of carbon dioxide)

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of P. juponicus decreased with increased ambient ammonia-N in the range of 0.055 -20.335 mg/l.
This suggested that CO2 had reacted with ammonia to form urea, where it is then excreted from
prawns. Hence, under increasing ammonia stress from the environment, when the levels of NH3 rise
to levels that Na+/NH4+ exchange system, detoxification via urea excretion in prawns becomes
paramount.

Effects of ammonia on prawn immunity

a) Reduction of phenoloxidase activity
Crustaceans exposed to environmental stress, such as microbial diseases, tend to respond by
stimulating phagocytosis by their haemocytes (Moullac and Haffner, 2000). This is usually
accompanied by a respiratory burst which leads to the formation of the highly microbicidal reactive
oxygen intermediates (ROIs) such as superoxide anion(O2-), to counter the stress (Moullac and
Haffner, 2000). Apart from biological stress, several studies have found that respiratory bursts occur
in prawns in response to chemical stresses such as elevated ammonia levels (Chun and Chen, 2004;
Cheng and Chen, 2002,; Liu and Chen, 2004). Hence, this suggests a relation between ambient
ammonia levels, and prawn immunity. Studies on Litopenaeus vannamei (Chun and Chen, 2004) and
Macrobrachium rosenbergii (Cheng and Chen, 2002) have found these prawns to have reduced
immunity to microbial infections when exposed to elevated ammonia levels. Both studies found that
prawns had decreased phenoloxidase(PO) activity and increased mortality to the bacteria when
exposed to relatively higher ammonia levels . L. vannamei were more susceptible to Vibrio
alginolyticus when exposed to ammonia-N levels above 5.24mg/L for 7 days, while, M. rosenbegii
had increased mortality with ammonia increasing ammonia-N concentrations after 72 hours (Cheng
and Chen, 2002)

b) Production of Reactive oxidative species (ROIs)
In the crustacean immunity system, PO is a haemolymph enzyme which forms part of the
prophenoloxidase(proPO) pathway that is involved in activating cellular defense responses such as
phagocytosis, encapsulation and haemocyte coagulation (Johansson and K. Sö             ll,
                                                                                derhä 1989; Moullac
and Heffner, 2000). PO is found in the inactive form as proPO in crustacean haemocytes,
specifically, in the granular and semi-granular cells (Johansson and K. Sö       ll,
                                                                           derhä 1989). The semi-
granular cells are known to have phagocytotic abilities (Johansson and K. Sö         ll,
                                                                               derhä 1989) and PO
found in these cells serves to remove foreign microbial polysaccharides such as fungal -1,3- glucan
or bacterial lipopolysaccharides via melanization through the proPO activation system (Johansson
and K. Sö        ll,
           derhä 1989; Cheng and Chen, 2002). The study on L. vannamei found that the
phagocytosis of V.alginolyticus decreased significantly in this species after exposure to higher
concentrations of 11.21 and 21.22 mg/L ammonia-N. This could be due to decreased haemocyte
counts due to haemocyte immobilization in the gills or reduced haemocyte turnover rates upon
exposure to increasing ammonia stress (Moullac and Heffner, 2000). Consequentially, PO activity is
reduced and hence prawn immunity lowered.

        In response to environmental stress, the respiratory burst may only provide a temporary
defense against toxicity by stress elements. If the stress is extreme or prolonged, the numerous ROIs
generated will cause a build up of oxidative stress which may lead to mortality of the prawns.
(Cheng and Wang, 2001, Chun and Chen, 2004). Normally, the prawn’s own anti-oxidant
mechanisms such as ROI scavenger enzymes will be able to remove the ROIs resulting from its
immune response. However, studies have shown that elevated ambient ammonia levels reduce the
resistance of prawns to oxidative stress by reducing the activity of these ROI scavenging enzymes.

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Chun and Chen (2004) found that increasing ambient ammonia concentrations led to the decreased
activity of the ROI scavenging enzyme, superoxide dismutase (SOD) and the accumulation of the
ROI, superoxide anion (O2-). Meanwhile, Wang et al (2004) found that elevated concentrations of
ammonia led directly to the production of another ROI, hydrogen peroxide. Hence, ROI production
could be beneficial in the short run as small increases in the production of O2- may increase the
cytoxic immunity of the prawn (Chun and Chen, 2004), possibly increasing the prawn’s survival rate
more than the removal of toxic ROIs would. However, when ammonia levels become too high, the
accumulation of high ROI levels prove too toxic to the organism and may undermine the immune
response and overall survival.

Dietary supplements to increase ammonia resistance

Interestingly, studies have found that introducing Vitamin C into the diet of the prawn, either
directly or indirectly, can increase its resistance to ammonia-induced production of harmful
oxidizing agents in the tissues. Vitamin C was introduced indirectly into Penaeus vannamei diet by
feeding them with live Artemia franciscana nauplii enriched with L-ascorbyl-2-polyphosphate
(Wang et al 2006) and directly by the addition of Vitamin C into Macrobrachiurn nippones feed
(Wang et al. 2005). In both studies, compared to those fed the Vitamin C-deficient diet, prawns fed
with the enriched diets produced significantly less ROIs and had less drastic decreases in anti-
oxidant enzyme activities upon exposure to elevated ambient ammonia levels. Anti-oxidant enzymes
such as SOD, catalase and glutathione reductase are important in removing the numerous ROIs
produced during high ammonia stress. The increased resistance to ammonia may also help improve
immunity to microbial agents. Moreover, Vitamin C is particularly beneficial to aquaculture systems
as supplementation of cultured prawn diet with Vitamin C may reduce ammonia excretion of prawn
by about 47% and hence improve the carrying capacity of the water (Wang et al. 2003).

        Another substance known to increase resistance to ammonia stress is astaxanthin.
Astaxanthin is known to have the ability to scavenge oxygen radicals in cells and hence reduce
cellular damage and enhance resistance to ammonia (Pan et al. 2003) Tiger prawn Penaeus monodon
Fabricius juveniles fed diets supplemented with astaxanthin were found to exhibit improved
antioxidant defense abilities and 15–20% higher survival rates than control shrimp (Pan et al. 2003),
when subjected to ammonia stress. Thus, this indicates that the shrimp’s resistance to ammonia
stress can be improved by dietary astaxanthin.

Conclusion

Elevated ammonia levels may cause chemical toxicity to cells by altering their osmoregulatory
functions or increasing the risk of biological toxicity from harmful microbial agents. This can be
seen from the fact that high concentrations of ammonia disrupt the diffusion of NH3 out of the
haemolymph, Na+/ NH4+ exchange and ureotelic excretion. In addition, elevated ammonia levels
have also led to reduced immunity to microbial infections due to increased oxidative stress and
decreasing the levels of superoxide dismutse and phenoloxidase activites. Supplementation of diet
with antioxidants like Vitamin C and astaxanthin has been found to improve the resistance of prawns
to ammonia stress and enhance immunity. Strategies to remedy the breakdown of osmoregulatory
functions would most likely involve the reduction of ammonia levels in the aquatic medium. This
could be accomplished by natural biodegradation using nitrifying bacteria and introducing plant
species capable of detoxifying ammonia.


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