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EFFECT OF AMMONIA AND NITRITE TOXICITY ON
PACIFIC WHITE SHRIMP Litopenaeus vannamei
IN CULTURE SYSTEMS
SRIBIDYA WAIKHOM, B.F.Sc.
I.D.No. MFT 15055 (AEM)
DEPARTMENT OF AQUATIC ENVIRONMENT MANAGEMENT
SCHOOL OF FISHERIES RESOURCES AND ENVIRONMENT MANAGEMENT
FISHERIES COLLEGE AND RESEARCH INSTITUTE
TAMIL NADU FISHERIES UNIVERSITY
THOOTHUKUDI – 628 008
2017
EFFECT OF AMMONIA AND NITRITE TOXICITY ON
PACIFIC WHITE SHRIMP Litopenaeus vannamei
IN CULTURE SYSTEMS
Thesis submitted in part fulfilment of the requirements for the Degree of
Master of Fisheries Science in Aquatic Environment Management
to the Tamil Nadu Fisheries University, Nagapattinam
SRIBIDYA WAIKHOM, B.F.Sc.
I.D.No. MFT 15055 (AEM)
DEPARTMENT OF AQUATIC ENVIRONMENT MANAGEMENT
SCHOOL OF FISHERIES RESOURCES AND ENVIRONMENT MANAGEMENT
FISHERIES COLLEGE AND RESEARCH INSTITUTE
TAMIL NADU FISHERIES UNIVERSITY
THOOTHUKUDI – 628 008
2017
CERTIFICATE
This is to certify that the thesis entitled “Effect of ammonia and nitrite
toxicity on Pacific white shrimp Litopenaeus vannamei in culture systems”
submitted in part fulfillment of the requirements for the degree of Master of
Fisheries Science in Aquatic Environment Management to the Tamil Nadu
Fisheries University, Nagapattinam is a record of bonafide research work carried
out by Ms. Sribidya Waikhom, MFT 15055 (AEM) under my supervision and
guidance and that no part of this thesis has been submitted for the award of any
other degree, diploma, fellowship or similar titles or prizes and that part of the
thesis has been published in peer reviewed journal(s) and copy/copies
appended.
Place: Thoothukudi (Dr.S.AANAND)
Date : CHAIRMAN
RECOMMENDED
EXTERNAL EXAMINER
APPROVED BY
Chairman: Dr. S. Aanand
Members: 1. Dr. P. Padmavathy
2. Dr. M. Rosalind George
Place:
Date :
ACKNOWLEDGEMENTS
It is my proud privilege and pleasure to express my sincere appreciation to
my guide, Dr. S. Aanand, Assistant Professor and Head i/C, Erode Centre for
Sustainable Aquaculture, for his continuous guidance, interest, inspiration and
critical evaluation of the work during the entire duration of this dissertation.
I am greatly indebted to my advisory committee members
Dr. P. Padmavathy, Associate Professor and Head, Dept. of Aquatic
Environment Management, and Dr. M. Rosalind George, Professor and head,
Dept. of Fish Pathology and Health Management, for their assistance,
encouragement and valuable advice and also for critically correcting this
manuscript for improvements.
I am grateful to Dr. G. Sugumar, Dean, Fisheries College and Research
Institute, Thoothukudi for his everlasting encouragement throughout the research
period.
It is my privilege to extend my thanks to Dr. A. Srinivasan, Chairman,
School of Fisheries Resources and Environment Management,
Dr. V. Rani, Asst. prof., Dept. of Aquatic Environment Management and
Mrs. D. Manimekalai, Asst. prof, Department of Aquaculture, for their enormous
support and valuable suggestions throughout the research work.
I place on record my sincere thanks to Dr. Athithan Professor,
Professor, Dept. of Fish Nutrition and Feed Technology, for providing unlimited
access to the wetlab facility. Thanks also to Mr. Vijay Amirtharaj, Asst. Prof,
Dept. of Aquaculture for his unstinted support and help in providing animals and
other resources for successful completion of the lab experiments.
I wish to express my gratitude to Dr. Sujathkumar, Professor and PG
coordinator for his timely help.
I owe my deepest thanks to Mr. Magheswaran, Mr. Anto, Mr. Balaji,
Mr. Murugan and Mr. Suresh, who helped me in a multitude of ways to
complete various experiment related to this dissertation. I would also like to
express my sincere thanks to my fellow friends Miss. Deepika, Miss. Rajeswari,
Miss. Ruby and Mr. Ramesh Kumar for all their assistance and wishes. I am
also grateful to my senior Mr. Subburaj, juniors Mr. Deepak and Mr.
Jayapavithran, for all their assistance and wishes.
Finally, I wish to express my love, respect and feeling to my parents and
family for their endless love, support, encouragement, care and inspiration
throughout my life. Without their encouragement this research would not have
materialized.
Thanks to the almighty for his gracious blessings bestowed on me without
which it would not have been possible to complete this research work.
(SRIBIDYA WAIKHOM)
ABSTRACT
Title : Effect of ammonia and nitrite toxicity
on Pacific white shrimp Litopenaeus
vannamei in culture systems
Name of the student : Sribidya Waikhom
Degree : M.F.Sc.
Chairman : Dr. S. Aanand
Department : Department of Aquatic Environment
Management
School : School of Fisheries Resources and
Environment Management
College : Fisheries College and Research
Institute, Thoothukudi
Year and University : 2017, Tamil Nadu Fisheries
University, Nagapattinam
The present study was conducted to investigate the individual effects of
ammonia and nitrite toxicity on Litopenaeus vannamei juveniles at 5, 10 and 15
ppt salinity along with their haematological impacts and to asses stress levels on
L. vannamei, when exposed to sub lethal concentrations. The 48 h LC50 values
for TAN at 5, 10 and 15 ppt salinities were 16.61, 28.84 and 44.17 mg/L
respectively and 48 h LC50 values for nitrite-N (NO2-N) at 5, 10 and 15 ppt
salinities were 92.63, 136.79, 186.34 mg/L respectively. Based on the incipient
LC50 values and application factor of 0.1, safe value for rearing L. vannamei at
salinity levels of 5, 10 and 15 ppt for ammonia and nitrite were calculated to be
1.66, 2.88, 4.41 mg/L and 9.26, 13.67, 18.63 mg/L respectively.
Effect of ammonia and nitrite at sub lethal level on the shrimp haematology
was studied by observing blood glucose level and plasma protein. Both ammonia
and nitrite significantly increased blood glucose levels as well as total plasma
protein concentration in the treated groups. However the increase was not
significant (p>0.05).
Trials conducted to understand the effect of ammonia and nitrite under sub
lethal levels, showed increased activity of the enzymes Glutamate Oxaloacetate
Transaminase (GOT), Glutamate Pyruvate Transaminase (GPT) and Lactate
Dehydrogenase (LDH) in shrimp muscle. The enzymatic activities showed
significant difference (p<0.05) at different salinities except with GPT at 10 and 15
ppt salinities and GOT at 10 ppt; however, there was no significant difference
(p>0.05) between the control and treated groups. Enzymatic activity of LDH at
salinity of 10 ppt was significantly lower (p<0.05) than at salinities of 5 and 15
ppt.
CONTENTS
Chapter
No.
Title Page
No.
I. INTRODUCTION 1-6
II. REVIEW OF LITERATURE
2.1 Exposure to ammonia and nitrite
2.1.1 Ammonia toxicity
2.1.2 Effect of ammonia on survival and growth
2.1.3 Nitrite toxicity
2.1.4 Effect of nitrite on survival and growth
2.2 Haematology
2.3 Stress induced changes on enzyme
7-23
III. MATERIALS AND METHODS
3.1 Experimental animal
3.1.1 Test protocol
3.1.2 Ammonia and nitrite toxicity trials
3.1.3 Determination of LC50 of ammonia and nitrite
toxicity
3.2 Haematology
3.2.1 Measurement of total prptein concentration
in haemolymphs
3.2.2 Measurement of glucose in haemolymphs
3.3 Estimation of stress enzyme
3.3 Determination of Glutamate oxaloacetate
transaminase(GOT)
3.3.2 Determination of GPT
3.3.3 Determination of Lactate dehydrogenase
3.4 Statistical analysis
24-28
IV. RESULTS
4.1 Ammonia toxicity trials
4.2 Nitrite toxicity trials
4.3 Haematological parameter
4.4 Estimation of stress enzyme
V. DISCUSSION
5.1 Ammonia toxicity trials
5.2 Nitrite toxicity trials
5.3 Haematological parameter
5.4 Stress enzyme
VI. SUMMARY AND CONCLUSION
VII. REFERENCES
LIST OF TABLES
Table No. Title Page No.
4.1 Lethal concentration (LC) results for total ammonia
nitrogen (TAN) at 5 ppt
30
4.2 Lethal concentration (LC) results for total ammonia
nitrogen at 10 ppt salinity
30
4.3 Lethal concentration (LC) results for total ammonia
nitrogen at 15 ppt salinity
31
4.4 LC50 results for total ammonia nitrogen (TAN) at three
different salinities.
31
4.5 Lethal concentration (LC) results for NO2-N at 5 ppt
salinity
35
4.6 Lethal concentration (LC) results for NO2-N at 10 ppt
salinity
35
4.7 Lethal concentration (LC) results for NO2-N at 15 ppt
salinity
36
4.8 LC50 results for NO2-N at three different salinities. 36
4.9 Final mean haematological parameter ± SE of
Litopenaeus vannamei in sub-lethal concentration of
ammonia and nitrite. The column having same
alphabetical superscript are not significantly different at
(p>0.05).
40
4.10 Enzymatic activity of GPT, GOT and LDH in muscle of
Litopenaeus vannamei at 5 ppt
40
4.11 Enzymatic activity of GPT, GOT and LDH in muscle of
Litopenaeus vannamei at 10 ppt
42
4.12 Enzymatic activity of GPT, GOT and LDH in muscle of
Litopenaeus vannamei at 15 ppt
42
LIST OF FIGURES
Figure
No.
Title Page No
4.1 Mortality in L. Vannamei exposed to different
concentration of TAN at 5 ppt over 48 h
32
4.2 Mortality in L. Vannamei exposed to different
concentration of TAN at 10 ppt
32
4.3 Mortality in L. Vannamei exposed to different
concentration of TAN at 15 ppt
33
4.4 Mortality of L. vannamei exposed to different
concentration of NO2 –N at 5ppt
37
4.5 Mortality of L. vannamei exposed to different
concentration of NO2 –N at 10ppt
37
4.6 Mortality of L. vannamei exposed to different
concentration of NO2 –N at 15ppt.
38
4.7 Mean protein concentration of L. vannamei under sub-
lethal concentration of ammonia and nitrite.
41
4.8 Mean glucose level of L. vannamei under sub-lethal
concentration of ammonia and nitrite.
41
4.9 Mean enzymatic activity of GPT, GOT and LDH in
muscle of Litopenaeus vannamei at 5 ppt
43
4.10 Mean enzymatic activity of GPT, GOT and LDH in
muscle of Litopenaeus vannamei at 10 ppt
43
4.11 Mean enzymatic activity of GPT, GOT and LDH in
muscle of Litopenaeus vannamei at 15 ppt
44
LIST OF PLATES
Plate
No.
Title Between
pages
I Experimental animal
Experimental set up
24 & 25
II Stocking of animal to the experimental trough
Continous aeration during experimental trials
26 & 27
III Moribund and stress shrimps
Homogenization of muscle using pestle and mortar
28 & 29
I. INTRODUCTION
Aquaculture is a fast growing food sector which now accounts for almost
50% of world’s food fish production (FAO, 2006). With stagnating/declining
traditional fisheries, aquaculture promises the greatest potential to meet the
growing demand of aquatic food. Aquaculture not only provides a sustainable
source of aquatic food, but also provides meaningful livelihood to multitudes of
poor (FAO, 2006). Over the last two decades, aquaculture has gone through
major changes, from small scale home stead-level activities to large scale
commercial farming, exceeding landing from capture fisheries in many areas. The
decline in world fish catches has been the major driving force in the rapid growth
in fish and shellfish farming, or aquaculture. Further the ever bludgeoning human
population has also led to increased dependence on the farmed fish production
as an cheap source of animal protein to sustain the nutritional demand.
Aquaculture production has grown enormously in recent years and among
several species culture, Penaeid shrimps are one of the most important cultured
species worldwide especially in Asia due to their high economic value and export
(Sekar et al., 2014). Approximately more than 5 million metric tons of shrimps are
produced annually but the current global demand for both the wild and farmed
shrimp is approximately more than 6.5 million metric tons per annum
(Karthik et al., 2014). Despite high levels of shrimp production by culture, shrimp
farmers suffer significant economic losses in recent years due to disease
problems that have plagued the industry. Due to the continuous outbreak of this
WSSV disease in Penaeus monodon culture leading to loss of shrimp culture in
India. The farmers are seriously looking for alternative shrimp species for culture.
In 2008, the Coastal Aquaculture Authority (CAA) of India introduced a new
shrimp species Litopenaeus vannamei as an alternative Penaeid species in India
for culture and export. The penaeid shrimp, L. vannamei exhibits fast growth rate
and its culture period is significantly reduces compared to Penaeus monodon.
Thus, the Litopenaeus vannamei has quickly established itself as an alternative
to Penaeus monodon in shrimp farming sector in many countries such as,
East, Southeast and South Asia (Karuppasamy et al., 2013).
Litopenaeus vannamei commonly known as the Pacific white leg shrimp is
presently the most important cultivated shrimp species in the world
(Perez Farfante, 1997). They are widely distributed throughout tropical Pacific
waters, from Mexico to Peru. In many countries the common culture practices
being followed are semi-intensive and intensive culture system. Their ability to
thrive in low saline water makes them favourable species for inland aquaculture
system (Pan et al., 2007).
Temperature and salinity are two very important environmental factors in
the culture of this and other shrimp species. The optimal temperature for the
growth of L. vannamei has been reported to be size-specific, around 28–30ºC for
postlarvae (Ponce-Palafox et al., 1997), greater than 30ºC for small juveniles (5
g) and about 27ºC for subadults (Wyban et al., 1995). It is known that
L. vannamei can tolerate a wide salinity range from brackish water of 1–2 ppt to
hypersaline water of 50 ppt (Stern et al., 1990). Boyd (1989) considered salinity
of 15–25 ppt to be ideal for L. vannamei culture. But, in view of inconsistencies in
published information regarding salinity effects on shrimp survival and growth, the
optimum salinity for L. vannamei is still not conclusive. Significant effects of
temperature and salinity have been reported on survival (Ogle et al., 1992),
molting frequency, oxygen consumption (Villarreal et al., 1994;
Martinez-Pakacios et al., 1996), and growth of L. vannamei (Huang, 1983;
Wyban et al., 1995). L. vannamei do not require a diet as rich in protein as other
shrimp species (Briggs et al., 2004). L. vannamei is an open thelycum species
therefore spawning can be induced in captivity. L. vannamei can be cultured
successfully at densities as high as 400/m2 in high density recirculating systems
(Briggs et al., 2004; Browdy et al., 2005).
Currently, the Pacific whiteleg shrimp, Litopenaeus vannamei (Boone) are
mainly cultivated in semi-intensive and intensive shrimp culture systems.
However, these culture practices usually result in degradation of the culture water
by uneaten food and waste products of the shrimps. Thus, water quality
management and knowledge of water quality requirements are essential to any
culture system. In shrimp farming operations, one of the primary wastes of
concern is nitrogen, which appears as ammonia, nitrite and nitrate. Compared to
nitrate, both ammonia and nitrite are extremely toxic to shrimp. Ammonia and
nitrite levels should remain at negligible levels in mature ponds. Ammonia is the
main end product of protein catabolism in crustaceans and can account for
60–70% of nitrogen excretion with only small amounts of amino acids, urea and
uric acid (Chen and Kou 1996a, b). Because ammonotelism is so dominant in
aquatic gill-breathers, ammonia excretion rate typically has been used to
evaluate the effects of various factors on total nitrogen excretion by crustaceans.
But the importance of non-ammonia nitrogen excretion (amino acids, urea, etc.)
has rarely been scrutinized. Adequate knowledge about nitrogen excretion by
shrimp is required for successful design and operation of shrimp production
systems. The total ammonia concentration as nitrogen (TAN) is comprised of two
forms, unionized ammonia (NH3) and ionized ammonium (NH4+) (Armstrong et
al., 1978). These forms of the TAN are dependent on the pH, salinity, and
temperature (Bower and Bidwell, 1978). And the unionized ammonia is the more
toxic (Smart, 1978). The easiest way to determine the toxic effects of nitrate on
shrimp is to look at shrimp production numbers such as survival and growth.
Aquaculturists interested in preserving the health of their stocks can also
evaluate other physiological attributes, such as antennae, gills and the
hepatopancreas. Shrimp exposed to high concentrations of nitrate over a long
period of time exhibited shorter antennae length, gill abnormalities and lesions in
the hepatopancreas. Short antennae and gill abnormalities are often considered
early clinical signs of decreasing shrimp health.
In decapoda crustaceans, there are generally three types of circulating
haemocytes viz., hyaline cells, semi-granular cells and large granular cells
(Hose et al., 1990). They are involved in cellular immune responses, which
include phagocytosis and elimination of micro-organisms or foreign particles
(Bayne, 1990; Le Moullac et al,. 1997). Normal haemocyte counts have been
established for P. monodon (Tsing, 1989), Marsupenaeus japonicas
(Bachere et al., 1995) and L. stylirostris (Le Moullac et al., 1997). Haemocytes
are also associated with proteins like prophenoloxidase (proPO) and
phenoloxidase, which are involved in encapsulation, melanisation and functions
as non-self recognition systems (Johansson and Soderhall, 1989).
Ammonia, the end product of protein catabolism accounts for more than
half the nitrogenous waste released by decapod crustaceans (Regnault, 1987). It
has been reported that concentration of ammonia-N (un-ionised plus ionised
ammonia as nitrogen) increased directly with culture period, and might reach as
high as 46 mg/l in intensive grow-out ponds (Chen et al., 1988). Elevated
concentration of environmental ammonia has been reported to affect growth and
molting (Chen and Kou, 1992), oxygen consumption and ammonia excretion
(Chen and Lin, 1992). The 24 and 96 h LC50 of ammonia on L. vannamei
juveniles was 68.75 and 39.54 mg/l ammonia-N, respectively at 35 ppt (Lin and
Chen, 2001). Ammonia has also been reported to affect the immune response of
Litopenaeus stylirostris (Le Moullac and Haffner, 2000) and M. rosenbergii
(Cheng and Chen, 2002).
Nitrite is the most common pollutant in culture systems. Nitrite is formed
from ammonia and may be accumulated in aquatic systems as a result of
imbalances of nitrifying bacterial activity, Nitrosomonas sp. and Nitrobacter sp.
(Mevel and Chamroux, 1981). High levels of nitrite in water are potential factors
triggering stress in aquatic organisms (Lewis and Morris, 1986). The toxicity of
nitrite to crustaceans has been studied by several authors (Chen and Lee, 1997;
Cheng and Chen, 1999). Elevated environmental nitrite has been reported to
induce methaemocyanin formation, cause hypoxia in tissue and impair the
respiratory metabolism of penaeid shrimps (Chen and Nan, 1991;
Chen and Chen, 1992).
Nitrite is formed as an intermediate product either during bacterial
nitrification of ammonia or bacterial denitrification of nitrate. It has been reported
that the concentration of nitrite increases directly with culture period and might
reach as high as 20 mg/L in grow-out ponds of Pacific white leg shrimp,
L. vannamei (Tacon et al., 2002). Elevated concentration of environmental nitrite
has been reported to affect growth and moulting (Chen and Chen, 1992), oxygen
consumption and ammonia excretion (Cheng and Chen, 1989). The 24 and 96 h
LC50 (median lethal concentration) of nitrite in L. vannamei juveniles was 521 and
322 mg/L nitrite-N (nitrite as nitrogen), respectively at 35 ppt salinity
(Lin and Chen, 2003).
Therefore, the objective of the study was to determine the toxic levels of
ammonia and nitrite along with their haematological impacts and to asses stress
levels on L. vannamei.
Objectives:
1. To assess ammonia and nitrite toxicity in shrimp culture systems at different
salinities.
2. To study the LC50 and SAFE levels of ammonia and nitrite toxicity.
3. To study the haematological impact of ammonia and nitrite toxicity on Pacific
White Shrimp, L. vannamei.
4. To assess ammonia and nitrite stress induced changes on enzymes in Pacific
White Shrimp, L. vannamei.
II. REVIEW OF LITERATURE
Litopenaeus vannamei, commonly known as Pacific white leg shrimp/
White leg shrimp / Pacific white shrimp or King prawn, is the most important
cultivated shrimp species in the world (Perez Farfante, 1997). It grows to a
maximum length of 230 mm with a carapace length of 90 mm. Adults live in the
ocean, at depths of up to 72 m, while juveniles live in estuaries. The rostrum is
moderately long with 7-10 teeth on the dorsal side and 2-4 teeth on the ventral
side. Pacific white leg shrimp are widely distributed throughout tropical Pacific
waters, from Mexico to Peru. It is restricted to areas where the water temperature
remains above 20°C throughout the year. In many countries, the common culture
practices being followed are semi-intensive and intensive culture system. Their
ability to thrive in low saline water makes them favourable species for inland
aquaculture system (Pan et al., 2007). In intensive shrimp farming, build-up of
nitrogenous waste in the form of ammonia, nitrite and nitrate from uneaten food
and the waste products from the shrimp continuously degrade the culture
environment. Since ammonia and nitrite are extremely toxic to shrimp compared
to nitrate, control of ammonia and nitrite is second most important factor
impacting survival and growth of cultured organisms, following dissolved oxygen
(Ebeling et al., 2006). Ammonia which originates from excretion of cultured
animals and from ammonification of unconsumed food or organic detritus is the
most common toxicant. Nitrite, formed from ammonia by Nitrosomonas spp., is
rather more toxic than ammonia to crustaceans (Amstrong, 1979).
Ammonia and nitrite, the two main inorganic forms of nitrogen in a culture
system, may deteriorate water quality resulting in high mortality and low growth
rate in penaeids (Colt and Amstrong, 1998). In an intensive shrimp culture
system, ammonia and nitrite increase exponentially over time in grow-out ponds.
Ammonia and nitrite increase exponentially both in the hatchery and in grow out
farm, even with frequent water replacement (Chen et al., 1986, 1989). Therefore,
the accumulation of ammonia and nitrite may have detrimental effects on prawn
rearing.
2.1. Exposure to ammonia and nitrite
2.1.1. Ammonia toxicity
The cause of toxicity of ammonia is mainly based on the irritative
properties of the compound. Ammonia is the main end product of protein
catabolism in crustaceans and can account for 60–70% of nitrogen excretion with
only small amounts of amino acids, urea and uric acid (Chen and Kou, 1996a, b).
While mammals convert nitrogenous wastes into other forms of nitrogen such as
urea whereas fish and crustaceans excrete ammonia in an unaltered form. This
is possible since in natural conditions ammonia is instantly diluted to safe levels
by the surrounding water. Fish and crustaceans lack the ability to convert
ammonia to the less toxic, carbamoyl phosphate compound and therefore,
aquatic species are especially prone to toxic effects of ammonia at highly
concentrated levels.
In water, ammonia is present in both ionized (NH4+) and un-ionized (NH3)
state, with un-ionized NH3 as the toxic form due to its ability to diffuse across cell
membranes and ability to gain entry through the gills (Fromm and Gillete, 1968;
Emerson, Russo, Lund and Thurston, 1975). The lipid soluble, un-ionized form
can readily pass through cell membranes (Boardman et al., 2004), whereas the
ionized form does not readily cross hydrophobic microphores in the gill
membrane (Svobodova, 1993). The unionised ammonia can cause impairment of
cerebral energy metabolism, damage to gill, liver, kidney, spleen and thyroid
tissue in fish, crustaceans and mollusks (Smart, 1978).
Chronic un-ionized ammonia exposure may affect fish and other
organisms in several ways, viz., gill hyperplasia, muscle depolarization, hyper
excitability, convulsions and finally death (Ip et al., 2001). NH4+ is also toxic,
especially at low pH levels (Allan et al. 1990). Ammonia is oxidized to nitrite and
nitrate by Nitrosomonas and Nitrobacter bacteria (Sharma and Ahlert, 1977).
Ammonia and nitrite are the most common toxicants in culture systems and are
toxic to fish, molluscs and crustaceans (Colt and Armstrong, 1981).
The physiological changes in aquatic organisms due to ammonia
exposure vary. The effect of ammonia relates to site specific irritation.
Caglan et al. (2005) analyzed the gills of tilapia that had been exposed to chronic
ammonia tests and concluded that ammonia was responsible for gill hyperplasia
as well as lamella fusion. The hyperplasia and lamella fusion resulted in
restricted water flow over the gills, leading to respiratory stress on the organism.
Similarlly, epithelial pitting of the gills, were observed when rainbow trout were
tested and examined using scanning electron microscopy (Kirk and Lewis, 1993).
Exposure of Pacu fish to different concentrations of ammonia-N caused an
elevation in total hemoglobin and blood glucose (Barbieri and Bondioli, 2015).
The sub-lethal effects induced decrease in growth rate and resistance to
diseases and poor food conversion (Kuttchantran, 2013). In Nile tilapia
(Oreochromis niloticus), El-Sayed (2015) studied the effects of ammonium nitrate
on the hematological parameters and the serum attributes and found a parallel
disturbance in all parameters with increase of ammonia concentration.
In penaeid shrimp, high concentrations of ammonia may affect growth
rates, survival and in extreme cases cause mortality (Wickins, 1976;
Zin and Chu, 1991; Chen and Lin, 1992). Ammonia damages the gills and
reduces the ability of haemolymph to transport oxygen while increasing oxygen
consumption by tissues (Chien, 1992; Racotta and Hernandez-Herrera, 2000).
Osmoregulatory capacity decreases with increasing ammonia concentration and
exposure time (Lin, et al., 1993). Ammonia may also increase the moulting
frequency of shrimps (Chen and Kou, 1992). Ammonia is also thought to cause
damage to the central nervous system (Wright, 1995).
High ammonia content affects the immune system of
Marsupenaeus japonicus (Bate) (Jiang and Zhou, 2004) and L. Vannamei
(Liu and Chen, 2004). Reduced survival and growth because of sub lethal and
lethal effects of ammonia toxicity are relevant for aquaculture operations.
2.1.2. Effect of ammonia on survival and growth
A number of studies have been conducted on the lethal effects of
ammonia at various life stages of penaeid shrimps, such as Penaeus chinensis
(Chen and Lin, 1992), P. monodon (Chen and Lei, 1990), P. paulensis
(Ostrensky and Wasielesky, 1995), P. penicillatus (Chen and Lin, 1991),
Penaeus semisulcatus (Wajsbrot et al., 1990) and Metapenaeus ensis
(Nan and Chen, 1991). Lethal toxicity tests can be acute or chronic depending on
the time of exposure. In most cases, acute tests are performed over a period of
2 - 7 days while chronic tests are longer than 7 days. Concentrations leading to
50% mortality vary depending on the organism being tested.
Previous studies have shown that 48 h median lethal concentrations
(LC50) for ammonia-N to varying species of shrimp, ranged from 30 and 110 mg/L
TAN at full strength seawater depending on size and age (Chen et al., 1990a,
1990b; Ostrensky and Wasielesky, 1995; Frias Espericueta et al., 1999;
Kir and Kumlu, 2006). For Penaeus monodon and Metapenaeus macleayi
juveniles, LC50’s were determined using 96 h acute tests. The results showed the
respective LC50’s to be 1.69 and 1.39 mg/L NH3-N (Allan et al., 1990). Other
authors, through studies with various genera and species have concluded that
the toxicity of ammonia to specific species is dependent on time and
concentration. A study found that the tolerance level of Penaeus semisulcatus
post larvae (PLs) to ammonia-N decreased with decreasing salinity. Specifically,
the shrimp tested at 40 ppt salinity were tolerant to ammonia-N levels 2.9 times
higher than those at 15 ppt over 48 h (LC50’s of 32.5 and 11.2 mg/L TAN,
respectively) (Kir and Kumlu, 2006).
Elevated ammonia levels can also lead to reduced growth of species
raised in intensive aquaculture systems. Wickins (1976) showed that a
concentration of 0.45 mg/L NH3-N led to a 50% decrease in growth of five
species of penaid shrimp. The author also concluded that, a concentration of
above 0.10 mg/L NH3-N breached maximum acceptable levels for reduced
growth over a three week chronic test (Wickins, 1976). The median lethal
concentration of ammonia to Penaeus japonicus larvae has been reported by
Chen et al. (1989). Simillarly for juveniles has been reported by and by
Kou and Chen, 1991.
2.1.3. Nitrite toxicity
Nitrite is an intermediate product of ammonia either in the bacterial
nitrification of ammonia or in the bacterial denitrification of nitrate. It has been
reported that concentration of nitrite increased directly with culture period and
might reach as high as 4.6 mg/L nitrite-N (nitrite as nitrogen) in pond water
(Chen et al., 1989). Accumulation of nitrite in pond water may deteriorate water
quality, reduce growth, increase oxygen consumption, increase ammonia
excretion and even cause high mortality of shrimp (Chen and Chen, 1992;
Cheng and Chen, 1998). Elevated nitrite in water has also been reported to
increase the susceptibility of giant freshwater prawn, Macrobrachium rosenbergii
to pathogen Lactococcus garvieae (Cheng et al., 2002). Nitrite toxicity is not
related to site specific irritation. Instead, the toxicity of nitrite is a function of the
effects on the circulatory and immune systems of aquatic organism. Nitrite enters
the blood stream and inhibits the binding of oxygen to the iron molecule of
hemoglobin (Hargreaves, 1998).
The nitrite toxicity mechanism acts on the process of oxygen transport. In
other words, nitrite binds to hemocyanin, converting it into meta-hemocyanin,
which is unable to transfer oxygen to the tissues. Barbieri et al., (2014) observed
an increase in oxygen consumption and ammonia excretion in
Litopenaeus schmitti juveniles exposed to increasing concentrations of nitrite.
Previous studies have demonstrated that, the increase in nitrite in the
environment leads to nitrite accumulation in the hemolymph, which
immunosuppresses the L. Vannamei and increases their susceptibility to
Vibro alginolyticus infections (Tseng and Chen, 2004). Several researchers
evaluated the acute toxicity of the nitrogenous compounds in penaeid shrimps
(Lin and Chen, 2003; Gross et al., 2004; Campos et al., 2012 and
Barbieri et al., 2014).
Nitrite also competes with chloride for transfer across erythrocyte
membranes leading to the oxidation of haemoglobin to met-hemoglobin.
Consequently, excessive nitrite levels in culture systems can cause depressed
growth, increased susceptibility to disease and eventual mortality. However, this
competition with chloride decreases the detrimental effects of nitrite in marine
waters and makes nitrite more dangerous in freshwater aquaculture. In
crustaceans, ambient nitrite reduces their thermal tolerance and induces
methaemocyanin formation, causes hypoxia in tissues and diminishes the
respiration efficiency (Alcaraz and Carnegas, 1997).
2.1.4. Effect of nitrite on growth and survival
The acute lethal affects of nitrite on aquatic organisms is not as
pronounced as ammonia at low concentrations, yet its toxicity is still of concern.
The effects of nitrite stress on immune responses of Vibrio alginolyticus, was
examined by Tseng and Chen, 2004. They found that, shrimp exposed to nitrite
between 5 and 22 mg/L showed significantly reduced resistance to bacterial
infection. In another study that explored the acute effects of nitrite on
L. vannamei shrimp over 48 h revealed LC50’s of 142.2, 244.0, and 423.9 mg/L
nitrite-N for 15, 25, and 35 ppt salinity respectively (Lin and Chen, 2003).
Macrobrachium malcolmsonii juveniles were subjected to nitrite stresses in the
presence of the bacteria A. hydrophila. (Chand and Sahoo, 2006) concluded that
increased nitrite stress led to a reduction in immune response to A. hydrophila. In
aquacultural systems, an increase in ammonia concentration is followed by a
decrease in ammonia is indirectly proportional to nitrite, as NH3 is oxidized to
NO2. Gross et al. (2004) explored the acute effects of nitrite to L. vannamei in
low salinity waters. When reared in water with 2 ppt salinity, the 48 h LC value
was determined to be approximately 15 mg/L NO2-N (Gross et al., 2004),
significantly lower than seen in the Lin and Chen,( 2003) experiments. The
median lethal concentration (LC50) of ammonia and nitrite has been estimated for
penaeid shrimp postlarvae such as Penueus monodon, P. chinensis,
P. paulensis. and P. juponicus (Chin and Chen, 1987; Chen and Chin, 1988;
Chen and Lin, 1991; Lin et al., 1993; Ostrensky and Wasielesky, 1995).
The mean 48 h LC50 of un-ionized ammonia and nitrite for post larvae of
several penaeids has been estimated at 1.29 mg/L NH3-N (24 h mg/L ammonia-
N) and 170 mg/L nitrite-N (Wickins, 1976). The effect of nitrite has been widely
studied in freshwater animals (Lewis and Morris, 1986). In these organisms,
nitrite induces reversible methaemoglobin formation, which is unable to transport
oxygen to tissues (Russo, 1985). In crustaceans, incorporation of nitrite in
haemolymph may reduce haemocyanin levels. Nitrite has also been found to
oxidize the respiratory pigment (Needham, 1961). There are few studies
available on the toxic action of nitrite in marine organisms. There are direct
evidences that, P. setiferus post larvae are highly sensitive to ammonia and
nitrite on short-time and chronic exposures (Alcaraz and Carnegas, 1997). For
P. setiferus post larvae, nitrite was much less toxic than ammonia. The acute
toxicity of nitrite increased with time of exposure. The 24 h, 48 h and 72 h LC50
values for nitrite were 268.1, 248.8 and 167.3 mg/L nitrite-N. Thus, tolerance of
P. Setiferus post larvae to nitrite decreased 7 and 38% at 48 h and 72 h
exposure with respect to the 24 h LC50 values. The lethality of nitrite on the
juveniles of penaeid shrimp has been provided for fleshy shrimp
Fenneropenaeus chinensis (Chen et al., 1990c), P. monodon
(Chen and Lei, 1990), Fenneropenaeus penicillatus (Chen and Lin, 1991) and
Metapenaeus ensis (Chen et al., 1990b). The reported 96 h LC50 ranged from
37.71 to 54.76 mg/L for nitrite-N. However, little information is available on the
lethality of nitrite at different salinity levels for penaeid shrimp
(Chen and Lin, 1991). According to Lin and Chen (2003), there is an inverse
relationship between salinity and nitrite toxicity such that the toxicity increases
with the reduction in salinity, making juvenile of L. Vannamei more susceptible to
nitrite in hypo-osmotic conditions.
The environmental chloride can inhibit the uptake of nitrite and mortality
due to nitrite, suggesting a method of managing nitrite toxicity in aquaculture
production systems (Tomasso, 2012). The gills provide a selective interface
between the external and internal environment, constituting a multifunctional
organ responsible for gas exchange, ion transport, nitrogenous excretion, volume
adjustment, and acid–base regulation (Lucu and Towle, 2003). High levels of
nitrite in water are potential factors triggering stress in aquatic organisms
(Lewis and Morris, 1986).
The toxicity of nitrite to crustaceans has been studied by several authors
(Cheng and Chen, 1999; Chen and Lee, 1997). Elevated environmental nitrite
has been reported to induce methaemocyanin formation, cause hypoxia in tissue,
and impair the respiratory metabolism of penaeid shrimps (Chen and Nan, 1991;
Chen and Chen, 1992). However, very little is known about the effect of nitrite on
the crustacean immune system. Ambient nitrite-N of 1.59 mg/L has been
reported to decrease phagocytic activity of freshwater prawn,
Macrobrachium rosenbergii against Lactococcus garvieae, but increase the
respiratory burst of prawn. However, nothing is known regarding the effect of
nitrite stress on the immune response and pathogen resistance of penaeid
shrimps.
2.2. Haematology
Shrimp farming witnessed impressive growth in many developing countries
where this activity attained great economic and social importance. However, the
shrimp industry has always been affected by infectious diseases, mainly of
bacterial and viral etiology (Mohney et al., 1994; Hasson et al., 1995 and
Flegel, 1997) causing heavy loss of production. Therefore, sustainable shrimp
farming largely depends on health management and control of diseases in the
shrimp and immune system is a tool to assess the shrimp health
(Bachere et al., 1995a). Many authors had already studied the physiological
stress responses in crustaceans (Lorenzon et al., 2008;
Fotedar and Evans, 2011). Hemolymph chemistry has been the primary means
for assessing the effects of various stress inducing factors such as air exposure,
changes in temperature, salinity, low dissolved oxygen and other stressors
associated with fishing operations, live holding and transport.
Stress responses may either be primary, secondary or tertiary responses
(Iwama et al., 1999). Primary responses represent the initial neuroendocrine/
endocrine response to the body’s altered condition. In crustaceans, this involves
the rapid release of crustacean hyperglycemic hormone (CHH) from the sinus
gland, which acts to meet an increasing demand for energy (Fanjul-Moles, 2006).
This leads to secondary stress responses, such as elevated hemolymph glucose,
formed through the mobilization of intracellular glycogen (Patterson et al., 2007),
increased lactate, and a host of other physiological and hematological changes
that cascade from metabolic acidosis and the accumulation of metabolic end
products (Taylor and Whiteley, 1989; Whiteley and Taylor, 1992;
Paterson et al., 2005). Tertiary responses are whole-animal changes that occur
because of energetic repartitioning resulting from stress, such as reductions in
feeding, growth, predator avoidance, disease resistance and reproduction.
Elevated CHH and glucose are adaptive physiological responses that help
restore homeostasis in the body, while other physiological changes are
maladaptive, i.e. it may be a response to reduce stress, ultimately resulting in
reduced growth rate.
In crustacean immune defense system, haemocytes play a central role.
First, they remove any foreign particles in the hemocoel by phagocytosis,
encapsulation and nodular aggregation (Soderhall and Cerenius, 1992). Second,
haemocytes take part in wound healing by cellular clumping and initiation of
coagulation processes through the release of factors required for plasma gelation
(Johansson and Soderhall, 1989; Omori et al., 1989;
Vargas-Albores et al., 1998.), carriage and release of the prophenol oxidase
(proPO) system (Johansson and Soderhall, 1989; Hernandez-Lopez et al., 1996).
They are also involved in the synthesis and discharge in the haemolymph of
important molecules, such as α2-macroglobulin (α2M) (Rodriguez et al., 1995;
Armstrong et al., 1990), agglutinins (Rodriguez et al., 1995) and antibacterial
peptides (Destoumieux et al., 1997; Schnapp et al., 1996; Lester et al., 1997). A
hemogram consists of the total haemocyte count (THC) and the differential
haemocyte count (DHC). For the DHC, most researchers agree with the
identification of three cell types in penaeid shrimp namely large granule
haemocytes (LGH), small granule haemocytes (SGH) and agranular haemocytes
or hyaline cells (HC) (Rodriguez et al., 1995; Van de Braak et al., 1996).
Phagocytosis is generally recognised as a central and important way to
eliminate microorganisms or foreign particles (Bachere, 1995). Reactive oxygen
species are produced during phagocytosis. This phenomenon, known as
respiratory burst plays important role in microbicidal activity (Song and Hsieh,
1994). Phagocytosis is an important cellular defence mechanism and has been
reported in P. monodon to Vibrio harveyi and Bacillus
(Direkbunsarakom and Danayadol, 1998; Rengpipat, 2000). In addition,
clearance efficiency is considered as major humoral defence mechanism for
crustacean and has been observed in Pacific rock shrimp Sicyonia ingentis to
V. alginolyticus (Martin et al., 1993) and P. monodon to V. harveyi
(Destoumieux et al., 1999).
In mammalian phagocytic cells, the oxygen-dependent defence
mechanism results in the generation of reactive oxygen intermediates (ROIs) with
powerful microbicidal activity (Babior, 1984). In crustaceans, the demonstration of
respiratory burst is quite recent. Bell and Smith (1993) demonstrated the
generation of superoxide anions by haemocytes of the decapod Carcinus
maenas working with separated haemocyte fraction. Munoz et al. (2000)
demonstrated the production of superoxide anions (O2-) by haemocytes of the
white shrimp, Penaeus vannamei. Environmental contaminants can lead to non-
infectious diseases. Ahmad (1995) found evidence for oxidative stress-related
pathologies from pollutants in marine organisms. Aquatic organisms are
protected against ROIs by antioxidant enzymes and low molecular weight
scavengers (Winston and Giulio, 1991; Peters and Livingstone, 1996).
Crustaceans have an open circulatory system in which the haemolymph
carries out several physiological functions. One of these function is the transport
of molecules such as the respiratory protein (hemocyanin), which is the most
abundant molecule of the haemolymph (60% to 95 % of total protein)
(Djangmah, 1970), followed by the clotting protein and other humoral
components. Chisholm and Smith (1994) found a relation between the protein
concentration and water temperature, showing low plasma protein concentrations
when temperatures are at their lowest and highest in the year. The concentration
of total proteins is also related to the moult cycle of the shrimp. In P. japonicus,
Chen and Cheng, (1993) have reported lower levels of protein concentration
during post molt stage (41.37 mg/ml) as opposed to higher levels (74.90 mg/ml)
found in early pre-molt.
Hemolymph glucose is one of the traditional indicators of stress in lobsters
and crabs. Increase in glucose have been reported for a wide range of stressors,
including emersion, handling and disease in clawed lobsters
(Lorenzon et al., 2007; Basti et al., 2010), rock lobsters (Paterson et al., 2005)
and crabs (Barrento et al., 2009; Woll et al., 2010). Glycogen is the principal
reserve of carbohydrates for crustaceans and constitutes the primary source of
energy during intense or protracted exercise, therefore, high levels of glucose in
the hemolymph reveal increased energetic investment (Briffa and Elwood, 2001).
Giomi et al. (2008) showed that, the additive effect of high temperature on
emersion is strongly reflected in glucose concentration. However, recent studies
with both rock lobsters and clawed lobsters show that glucose concentrations can
increase or decrease rapidly depending upon duration of exposure to air and
elevated temperature (Ridgway et al., 2006; Basti et al., 2010). Many of the
physiological parameters discussed above are useful in understanding the
mechanisms involved in stress responses.
2.3. Stress induced changes on enzymes
Marine crustaceans are under the influence of numerous environmental
factors such as natural environmental changes, according to daily or seasonal
rhythms, environmental stress from contaminants or physico-chemical changes.
Sub-optimal temperature or unsuitable salinity level in water may interact in an
antagonistic, additive or synergistic manner with toxicants like ammonia, nitrite
and many others thereby causing changes in the tolerance capacity of aquatic
animals.
When an organism is subjected to stresses such as chemical, physical
and biological (i.e. pathogen infection) upon sudden shortage of oxygen,
abnormal oxidative reactions in the aerobic metabolic pathway result in the
formation of excess amounts of nassent oxygen and free radicals (sometimes
called ‘‘free radicals’’). These radicals can impair lipids, proteins, carbohydrates
and nucleotides (Yu, 1994), which are important parts of cellular constituents,
including membranes, enzymes and DNA. Radical damage can be significant
because it can proceed as a chain reaction. Consequently, mortality can occur
due to severe destruction by massive radicals generated from acute stresses or
long-term chronic stresses.
Fish respond to toxicants by altering their enzyme activities and the
inhibition or induction of these enzyme activities has been used to indicate tissue
damage (Nemcsok and Boross, 1982; Webb et al., 2005). Many enzymes like
carboxyl esterase (CE), lactate dehydrogenase (LDH), alkaline and acid
phosphates (ALP, ACP), glutamate oxaloacetate transaminase and glutamate
pyruvate transaminase (GOT and GPT) are measured as useful biomarkers to
determine cellular impairment and cell rupture. Transaminases such as GOT and
GPT play a vital role in protein and carbohydrate metabolism and act as an
indicator for tissue damage (Nemcsok et al., 1981; Nemcsok and Boross, 1982).
Aspartate aminotransferase (AST) or glutamate oxaloacetate
transaminase (GOT) and alanine aminotransferase (ALT) or glutamate pyruvate
transaminase (GPT) are enzymes involved in the transfer of amino groups from
one specific amino acid to another. Therefore, higher values indicate a greater
transfer of amino groups, or the greater metabolic waste of amino acids in the
tissue. AST and ALT activities are usually used as general indicators of the
functioning of vertebrate liver. High AST and ALT generally, but not definitively,
indicate a weakening or damage of normal liver function. AST and ALT may be
indirectly related to oxidant metabolites so they serve as indicators of oxidative
status. For finfish, AST and/or ALT have been used extensively in studies that
evaluate finfish response to toxins (heavy metal pollutants and pesticides), stress
caused by temperature changes, low oxygen, starvation, pH, ammonia, nitrite,
disease, health therapeutics monitoring and nutrition. The crustacean
hepatopancreas is assumed homologous to the mammalian liver and pancreas
(Gibson and Barker, 1979) and is responsible for major metabolic events,
including enzyme secretion, absorption and storage of nutrients, moulting and
vitellogenesis (Chanson and Spray, 1992). Several aminotransferases in different
tissues and organs including the hepatopancreas of crustaceans have been
studied, including AST and ALT in American spiny lobster, Homarus americanus
(Devereaux, 1986), kynurenine aminotransferase in tiger prawn,
Penaeus monodon (Meunpol et al., 1998), and D-alanine oxidase and
D-aspartate oxidase in several crustacean species (D’Aniello and Giuditta, 1980).
Lactate dehydrogenase (LDH) is also used as indicative criteria of
exposure due to chemical stress and anaerobic capacity of tissue (Diamantino et
al., 2001; Rendon-von Osten et al., 2005). LDH is present in all tissues and
normally associated with cellular metabolic action; it is used as potential marker
for assessing the toxicity of a chemical (Agrahari et al., 2007). Any changes in
protein and carbohydrate metabolism may cause change in LDH activity
(Abston and Yarbrough, 1976). Chemical stress alters the normal LDH activity
patterns (Diamantino et al., 2001). Elevated LDH activity in gills suggests that the
aerobic catabolism of glycogen and glucose has shifted towards the formation of
lactate, which may have adverse long-term effects on the organisms
(Szegletes et al., 1995). Increased release of LDH into the medium may indicate
damage in the integrity of cell membranes or heart muscle
(Nemcsok et al., 1984). Changes in food availability strongly affect LDH activity in
white muscle. However, LDH activity (and that of other metabolic enzymes) tends
to remain constant in brain, independent of changes in environmental food quality
or quantity (Yang and Somero, 1993; Kawall et al., 2002). LDH burst swimming
performance because its activity allows for the continuance of energy production
critical for muscle contraction during functional hypoxia. A decrease in LDH
activity because of low food availability directly impacts swimming performance,
causing a decline in the ability of an individual to escape from predators or
capture prey. Conversely, brain LDH activity, is usually low during starvation,
presumably to allow the individual to survive until conditions are more ideal for
active movement and growth. Thus, the measurement of alteration in the LDH
activity in gill, liver and kidney can be used as a biomarker indicating stress.
III. MATERIALS AND METHODS
3.1. EXPERIMENTAL ANIMAL
White shrimps, Litopenaeus vannamei were obtained from the King’s aqua
farm, Keezhavaipar, Tuticorin. The shrimps were acclimatized to laboratory
condition prior to experiment. The shrimps were divided randomly into three
groups and then adjusted gradually to three different salinity levels of 5, 10 and
15 ppt. The shrimps were reared for 1 week at these salinities with proper care.
The average total length of juvenile shrimps used was 51 mm. During this time,
they were fed with commercial food at the rate of 3% body weight. Acclimatized
and salinity adjusted shrimp were used for toxicity tests.
3.1.1. Test protocol
Active shrimp were selected and transferred by hand net into 35 L plastic
trough and maintained. Test agents NH4Cl or NaNO2 were added at
predetermined levels from a stock solution of 1000 mg/L stock solution. Test
solutions were then mixed well. Shrimps were not fed during the experiments and
no water changes were made. Continuous aeration was provided to each
experimental trough. Experimental trials was conducted with two replicates for
each treatment along with the controls. The shrimps were monitored for 48 h (end
point). Moribund shrimps were identified by the lack of response to the stimulus
by a glass rod. Dead shrimps were removed immediately.
3.1.2. Ammonia and nitrite toxicity trials
Toxicity of ammonia and nitrite were carried out at three different salinities
viz., 5, 10 and 15 ppt. Test solution dose for target concentration of TAN at 5, 10
and 15 ppt were 0, 10, 15, 20, 25, 30 ppm; 0, 10, 20,30, 40, 50 ppm and 0, 20,
30, 40, 50, 60 ppm respectively. Test solution dose for target concentration of
NO2-N at 5, 10 and 15 ppt were 0, 50, 60, 70, 80, 90,100 ppm; 0, 100, 120,140,
160 ppm and 0, 140, 160, 180, 200 ppm respectively.
3.1.3. Determination of LC50 of ammonia and nitrite toxicity
Lethal concentration at which 50% of the population mortality occurs within
48 h were determined by using probit analysis for both ammonia and nitrite
toxicity.
3.2. Haematology
For haematological study shrimps of 20 g size were exposed to sub-lethal
concentration of ammonia and nitrite for 15 days. Haemolymphs was collected
using 2 ml sterile syringe from the base of pleopod, walking legs and ventral
sinus. Collected blood was transferred into serological tubes with minute amount
of anticoagulant and was centrifuged for 10 min at 4oC. The supernatant was
used for the assay of protein and glucose.
3.2.1. Measurement of protein concentration in haemolymphs
Protein concentration in haemolymph was measured using protein diagnostic kit
(Coral Clinical systems, India) which was based on Biuret method of Gornall et al.
(1949). The kit had been designed based on the principle that protein in the alkaline
medium, bind with the cupric ions present in the Biuret reagent to form a blue-violet
coloured complex. This colour complex absorbs light at 550 nm. The intensity of the colour
formed is directly proportional to the amount of proteins present in the sample. Total
protein is calculated using the following
Total Protein (g dl-1) = Absorbance of Test/ Absorbance of Standard X 8
3.2.2. Measurement of glucose in haemolymph
For the determination of glucose in serum, glucose diagnostic kit (Coral
Clinical systems, India) was used which was designed based on Trinder (1969)
GOD/POD method. The kit works on the principle that glucose is oxidized to
gluconic acid and hydrogen peroxide in the presence of glucose oxidase.
Hydrogen peroxide further reacts with phenol and 4-aminoantipyrine by the
catalytic action of peroxidase to form a red colored quinoneimine dye complex.
Intensity of the colour formed is directly proportional to the amount of glucose
present in the sample.
Total Glucose (mg dl-1) = Absorbance of Test/ Absorbance of Standard X 100
3.3. Estimation of stress enzyme
Stress induced enzymes such as Glutamate Pyruvate Transaminase
(GPT), Glutamate Oxaloacetate Transaminase (GOT) and Lactate
Dehydrogenase (LDH) were examined in shrimps which were exposed on long
term stress condition for both ammonia and nitrite at three different salinities viz.,
5, 10 and 15 ppt respectively. For enzyme study, abdominal muscle was used.
Shrimps were exposed to sub-lethal concentration of ammonia and nitrite for 15
days. The tissues were homogenized in equal volumes of phosphate buffer
(0.1M, pH 7) in a glass homogenizer kept in cold condition and then centrifuged
at 15000 rpm for 15 minutes at 4oC. The supernatant was collected and used for
analysis of enzyme
3.3.1. Determination of Glutamate oxaloacetate transaminase (GOT)
GOT activity in muscle was determined using the GOT diagnostic kit
(Robonik India Pvt. Ltd.) based on IFCC method without pyridoxal phosphate,
kinetic UV. The method is based on the principle that GOT converts L-asparate
and α-ketoglutarate to oxaloacetate and glutamate Reitman and Frankel (1957).
GOT activity was measured by recording the change in absorbance per minute at
340 nm in Hach spectrophotometer DR 1900.
L- Aspartate + α – Ketoglutare GOT Oxaloacetate + L- Glutamate
Oxaloacetate + NADH + H+ MDH L- Malate + NAD+
3.3.2. Determination of GPT
GPT activity was carried out by using a diagnostic kit (Robonik India Pvt.
Ltd) based on IFCC method without pyridoxal phosphate, kinetic UV. The method
is based on the principle that GPT converts L-alanine and α-ketoglutarate to
pyruvate and glutamate described by Reitman and Frankel (1957). GPT activity
was measured by recording the change in absorbance per minute at 340 nm in
Hach spectrophotometer DR 1900.
L- Alanine + α- Ketoglutarate GPT Pyruvate + L- Glutamate
Pyruvate + NADH+ LDH L- Lactate + NAD+
3.3.3. Determination of Lactate dehydrogenase
LDH activity was carried out by using a diagnostic kit (Accurex biomedical,
India). This method was designed based on the principle that LDH catalyzes the
reduction of pyruvate by NADH to form lactate and NAD+ Thomas (1998). The
catalytic concentration is determined from the rate of decrease of NADH,
measured at 340 nm.
Pyruvate + NADH + H+ LDH Lactate + NAD+
3.4. Statistical analysis
The data obtained was further analyzed statistically and interpreted by
using suitable statistical method with Statistical Package for Social Sciences
(SPSS, version 16.0 for windows) and ANOVA, Analysis of Variance (one way
ANOVA) will be performed to determine the difference between the mean values
of different treatments. Differences in mean were compared by Duncan’s New
Multiple Range test (multiple range test) (Duncan, 1995) at P<0.05 level.
IV. RESULTS
Ammonia and nitrite toxicity towards shrimp were recorded from toxicity
studies at three different salinity viz., 5, 10 and 15 ppt. The trials were conducted
for 48 h at selected concentrations and the lethal concentrations were arrived
based on observations recorded during the 48 h study. The LC50 results for total
ammonia nitrogen (TAN) and NO2-N at three different salinities are presented in
Table 4.4 and 4.8.
4.1. Ammonia toxicity trials
The control group recorded 100% survival at all salinities after 48 h of
exposure. In the toxicity test at 5 ppt salinity, shrimps exposed at 10, 15, 20 and
25 mg/L of ammonia recorded a mortality rate of 15, 30, 55 and 95% respectively
after 48 h of exposure. However, at 30 mg/L of ammonia recorded 100%
mortality over 48 h of exposure. (Fig. 4.1)
In the trial conducted to study the ammonia toxicity at 10 ppt salinity
mortality rate of 5, 30 and 45% were recorded in shrimps exposed at 10, 20 and
30 mg/L of ammonia respectively over 48 h. Further trials conducted on shrimps
exposed to 40 and 50 mg/L of ammonia over same time interval, mortality of 65
and 90% were observed respectively (Fig. 4.2).
In the trial conducted to study the ammonia toxicity at 15 ppt salinity,
mortality of 10 and 25% were observed for 20, 30, mg/L of ammonia, respectively
over 48 h of exposure. Further experiments with higher concentrations of
Ammonia were
Table 4.1. Lethal concentration (LC) results for total ammonia nitrogen
(TAN) at 5 ppt
48 h LC results for TAN
end point TAN (mg/L) 95% confidence limit
Lower Upper
LC / EC 10 10.62 7.96 12.28
LC / EC 15 11.57 8.99 13.77
LC / EC 50 16.61 14.62 18.47
LC / EC 85 23.84 21.18 28.63
L C / EC 90 25.97 22.80 32.21
Table 4.2. Lethal concentration (LC) results for total ammonia nitrogen at 10
ppt salinity
48 h LC results for TAN
end point TAN (mg/L) 95% confidence limit
Lower Upper
LC / EC 10 13.33 7.87 17.35
LC / EC 15 15.45 9.89 19.48
LC / EC 50 28.84 23.91 34.50
LC / EC 85 53.83 43.15 81.84
L C / EC 90 62.39 48.51 92.69
Table 4.3. Lethal concentration (LC) results for total ammonia nitrogen at 15
ppt salinity
48 h LC results for TAN
end point TAN (mg/L) 95% confidence limit
Lower Upper
LC / EC 10 21.06 12.03 26.72
LC / EC 15 24.27 15.41 29.75
LC / EC 50 44.17 37.76 54.30
LC / EC 85 80.41 62.47 116.83
L C / EC 90 92.64 69.29 148.69
Table 4.4. LC50 results for total ammonia nitrogen (TAN) at three different
salinities.
Test chemical Total ammonia as nitrogen
at different salinities
48 h LC50 (with 95% confidence
limits) (mg/L)
TAN 5 ppt 16.61 (7.96 - 12.28)
TAN 10 ppt 28.84 (23.91 - 34.50)
TAN 10 ppt 44.17 (37.76 - 54.30)
Fig. 4.1. Mortality in L. Vannamei exposed to different concentration of TAN
at 5 ppt over 48 h
Fig. 4.2. Mortality in L. Vannamei exposed to different concentration of TAN
at 10 ppt
Fig. 4.3. Mortality in L. Vannamei exposed to different concentration of TAN
at 15 ppt
conducted and mortality rates of 40, 55 and 75% were recorded for shrimps
exposed to 40, 50 and 60 mg/L of ammonia respectively, over same time interval
(Fig. 4.3). Table 4.1 - 4.3 provides the lethal concentration at different percent
mortalities (LC10- 90) of ammonia exposure.
4.2. Nitrite toxicity trials
In the toxicity test with nitrite at 5, 10 and 15 ppt salinity, no mortalities
were recorded in the control groups. In test at 5 ppt salinity, no mortality were
recorded for shrimps exposed to 50, 60 and 70 mg/L of NO2-N over 48 h.
However, mortality rate of 30, 40 and 65% were observed for shrimps exposed at
80, 90 and 100 mg/L of NO2-N respectively for the same time intervals (Fig. 4.4).
At 10 ppt salinity, shrimps exposed at 100, 120, 140 and 160 mg/L of NO2-
N recorded mortality rates after 48 h were 10, 25, 65 and 75% respectively. In
toxicity test at 15 ppt salinity, no mortality were observed in the control. In the
experimental trials shrimp exposed to 140, 160, 180 and 200 mg/L of NO2-N
mortality rates after 48 h were observed to be 10, 25, 40 and 65% respectively
(Fig. 4.5 - 4.6). Table 4.5 - 4.7. provides the lethal concentration at different
percent mortalities (LC10- 90) of nitrite exposure.
Table 4.5. Lethal concentration (LC) results for NO2-N at 5 ppt salinity
48h LC results for NO2-N
end point NO2-N (mg/L) 95% confidence limit
Lower Upper
LC / EC 10 67.56 59.93 78.30
LC / EC 15 71.76 64.34 81.11
LC / EC 50 92.63 83.40 129.93
LC / EC 85 119.56 103.22 133.12
L C / EC 90 126.99 106.79 158.98
Table 4.6. Lethal concentration (LC) results for NO2-N at 10 ppt salinity
48h LC results for NO2-N
end point NO2-N (mg/L) 95% confidence limit
Lower Upper
LC / EC 10 105.87 88.42 115.39
LC / EC 15 111.19 95.72 119.92
LC / EC 50 136.79 128.21 147.67
LC / EC 85 168.29 154.14 202.47
L C / EC 90 176.75 160.05 219.49
Table 4.7. Lethal concentration (LC) results for NO2-N at 15 ppt salinity
48h LC results for NO2-N
end point NO2-N (mg/L) 95% confidence limit
Lower Upper
LC / EC 10 140.86 109.09 154.20
LC / EC 15 148.61 121.72 160.55
LC / EC 50 186.34 174.43 211.07
LC / EC 85 233.63 207.72 333.93
L C / EC 90 246.47 215.51 373.92
Table 4.8. LC50 results for NO2-N at three different salinities.
Test chemical NaNO2 at different salinities
48 h LC50 (with 95% confidence limits) (mg/L)
NO2-N 5 ppt 92.63 (83.40 - 129.93)
NO2-N 10 ppt 136.79 (128.21 - 147.67)
NO2-N 15 ppt 186.34 (174.43 - 211.07)
Fig. 4.4. Mortality of L. vannamei exposed to different concentration of
NO2 –N at 5ppt
Fig. 4.5. Mortality of L. vannamei exposed to different concentration of
NO2 –N at 10ppt
Fig. 4.6. Mortality of L. vannamei exposed to different concentration of
NO2 –N at 15ppt.
4.3. Haematological parameter
The mean final hematological parameters (blood glucose and total protein
concentration) of Litopenaeus vannamei in sub-lethal concentration of ammonia
and nitrite are presented in the Table 4.9 and Figs. 4.7- 4.8. The blood glucose
was showing significant difference (p<0.05) when compared to the controls and
the treated groups. While, total protein concentration shows no significant
difference (p>0.05) between the control and treated groups. The blood glucose
increases significantly in ammonia when compared to the controls groups. The
blood glucose and total protein concentration of haemolymph was found highest
in test with ammonia (86.98 mg dl-1 and 6.67 g dl-1). While the control groups
shows lowest (77.38 mg dl-1 and 5.932 g dl-1).
4.4. Estimation of stress enzyme
The mean enzymatic activity of stress enzyme GOT, GPT and LDH of
Litopenaeus vannamei at three different salinity of ammonia and nitrite are
presented in the Table 4.10 – 4.12 and Figs 4.9 – 4.11. The enzymatic activity
showed significant difference (p<0.05) in the different salinities except for GPT at
10 and 15 ppt salinity and GOT at 10 ppt there was no significant difference
(p>0.05) between the controls groups and treatments.
Enzymatic activity of LDH at 10 ppt was significantly lower (p<0.05) in
comparison with salinities of 5 and 15 ppt. While GPT and GOT level was found
to be highest in 5 ppt (20.95 UL-1), (20.08 UL-1) and lowest in 15 ppt (13.09 UL-1),
(12.21 UL-1) respectively.
Table 4.9. Final mean haematological parameter ± SE of Litopenaeus
vannamei in sub-lethal concentration of ammonia and nitrite. The column
having same alphabetical superscript are not significantly different at
(p>0.05).
Parameter
Treatments
Control
Nitrite
Ammonia
Total protein concentration
(g dl-1)
5.932±0.09
6.134±0.15
6.676±0.23
Glucose (mg dl-1)
77.38±1.75a
83.296±1.46ab
86.98±1.20b
Table 4.10. Enzymatic activity of GPT, GOT and LDH in muscle of
Litopenaeus vannamei at 5 ppt
Parameter Control Ammonia Nitrite
GPT 11.34±0.8a 20.95±1.7b 20.08±0.8b
GOT 9.6±0.8a 20.08±0.8b 18.34±0.8b
LDH 28.375±4.0a 101.36±4.0b 85.14±4.0b
Fig 4.7. Mean protein concentration of L. vannamei under sub-lethal
concentration of ammonia and nitrite.
Fig. 4.8. Mean glucose level of L. vannamei under sub-lethal concentration
of ammonia and nitrite.
Table 4.11. Enzymatic activity of GPT, GOT and LDH in muscle of
Litopenaeus vannamei at 10 ppt
Parameter Control Ammonia Nitrite
GPT 9.6±0.8a 18.33±0.8b 18.33±2.6b
GOT 6.98±1.7a 18.33±2.6b 17.46±1.7b
LDH 16.22±0a 85.14±4.0c 68.92±4.0b
Table 4.12. Enzymatic activity of GPT, GOT and LDH in muscle of
Litopenaeus vannamei at 15 ppt
Parameter Control Ammonia Nitrite
GPT 6.11±0.8 13.96±1.7 13.09±2.6
GOT 2.62±0.8a 15.71±1.7b 12.21±1.7b
LDH 16.22±0a 52.71±4.0c 36.49±4.0b
Fig. 4.9. Mean enzymatic activity of GPT, GOT and LDH in muscle of
Litopenaeus vannamei at 5 ppt
Fig. 4.10. Mean enzymatic activity of GPT, GOT and LDH in muscle of
Litopenaeus vannamei at 10 ppt
Fig. 4.11. Mean enzymatic activity of GPT, GOT and LDH in muscle of
Litopenaeus vannamei at 15 ppt
V. DISCUSSION
5.1. Ammonia toxicity trials
Several researchers have examined ammonia toxicity in various species of
penaeid shrimp and at different developmental stages, especially for juveniles.
Chen and Lei (1990) determined that toxicity of ammonia for P. monodon juvenile
decreased with exposure time. Chen and Lin (1992) observed an increased
susceptibility to ammonia as salinity decreased from 30 to 10 g L -1 in F.
Chinensis juveniles. Growth rates of Metapenaeus japonicus juveniles exposed
to different ammonia concentrations were investigated and concluded that
ammonia had a stronger effect on weight rather than length (Chen and Kou,
1992).
The experimental trials conducted under the present study indicated that
salinity affects the tolerance levels of Litopenaeus vannamei to ammonia. The
tolerance to ammonia concentration increased with increase in salinity. At higher
salinities, shrimp exhibited higher survival rate with respect to ammonia toxicity.
Mortality rate increased with increasing ammonia concentrations and exposure
time at lower salinities. The ammonia present as NH3-N increases with pH and
temperature but decreases with salinity (Losordo et al., 1992;
Sampaio et al., 2002). Similar results were also reported for other shrimp species
by Chin and Chen (1987), Frias-Espericueta et al. (1999) and
Lin and Chen (2001). The fact that toxicity of ammonia increased as salinity
decreased may primarily be due to higher uptake of ammonia at low salinity.
The toxicity experiments carried out on L. vannamei in this study revealed
that 48 h LC50 values at 5, 10 and 15 ppt for TAN were 16.61, 28.84 and
44.17 mg/L, respectively. The study conducted for 48 h LC50 values for other
shrimp species ranged between 32.5 mg/L for Penaeus semisalcatus
(Kir and Kumlu, 2006) and 43.1 mg/L for P. paulensis
(Ostrensky and Wasielesky, 1995). In present study, the 48 h LC50 for
L. vannamei was observed to be higher (44.17 mg/L) than those reported for
other species.
The incipient LC50 values for ammonia to L. Vannamei at three different
salinities were 16.61, 28.84 and 44.17 mg/L respectively. Sprague (1969, 1971)
pointed out the effects of a given toxicant could be described in terms of “safe
level”, that can be obtained using an application factor of 0.1. The calculated safe
level in the present study would be below 1.6, 2.8 and 4.4 mg/L TAN at 5, 10 and
15 ppt respectively.
5.2. Nitrite toxicity trials
Acute toxicity of nitrite on various aquatic organisms has been widely
studied and reviewed by Lewis and Morris (1986) and Tomasso (1994). A study
conducted by (Wickins, 1976) reported that the 48 h LC50 for larvae of seven
species of penaeid shrimp was 170 mg/L nitrite.
Most of the previous study reported LC50 values of nitrite for penaeid
shrimp at different salinity levels. Penaeid shrimp are generally cultured
intensively in a semi-static environment with varying salinity, from 17 ppt to 34 ppt
(Chen and Wang, 1990). Chen and Lin (1991) reported that the toxicity of nitrite
on F. Penicillatus juveniles increased by 103 to 150% as salinity decreased from
34 ppt to 25 ppt.
Researchers who have conducted nitrite studies at low salinities reported
that the 48 h LC50 of nitrite to L. vannamei juveniles to be around 143 mg/L
NO2-N with a 95% confidence interval of 137.6 to 148.4 (Lin and Chen, 2003)
and 154 mg/L NO2-N with a 95% confidence interval of 146 to 163 (Schuler et
al., 2010). This is comparable with the effect of nitrite observed in the present
study due to overlapping confidence intervals. The calculated LC50 with 95%
confidence Limits for this study were 92.63 (83.40 - 129.93) mg/L NO2-N at 5 ppt,
136.79 (128.21 - 147.67) mg/L NO2-N at 10 ppt and 186.34 (174.43 - 211.07)
mg/L NO2-N at 15 ppt, respectively. This difference could be accounted for due to
the stock of shrimp purchased or size of the shrimps or due to variance in water
parameter.
5.3. Haematological parameter
Blood is a readily available body fluid and one of the most important fluids
of the body whose combinations are fluctuating and changing under the influence
of different physiological states (Ballarin et al., 2004). Blood parameters are used
as physiological indicators of stress during internal and external environment
changes of the fish (Cataldi and Mandich, 1998). Crustaceans, like other
invertebrates mainly rely on the innate immune defence mechanism, mediated by
the circulating haemocytes (Powell and Rowley, 2007). The blood glucose was
showing significant difference (p<0.05) when compared to the controls and the
treated groups while total protein concentration shows no significant difference
(p>0.05) between the control and treated groups. The blood glucose and total
plasma protein concentration of haemolymph was found maximum (86.98 mg dl-1
and 6.67 g dl-1) in test with ammonia concentration 4.41 mg/L TAN, while the
control groups shows lowest (77.38 mg dl-1 and 5.932 gl-1).
In the present study, the haemolymph glucose content increased
prominently at 4.41 mg/L of ammonia and 18.63 mg/L nitrite respectively. Similar
observations were reported in other crustaceans as a result of toxicity due to
heavy metals, environmental changes and pesticides (Hall and van Ham, 1998;
Racotta, 1998). During stress, shrimps use carbohydrate as a source of energy
and this reflects in elevation of glucose level in the haemolymph as a means to
combat the stress exerted by the pesticides (Paterson, 1993). A possible reason
for this phenomenon could be due to the transport of glucose component from
hepatopancreas and muscle towards hemolymph, resulting in the reduction of
glycogen reserves in these organs. Breakdown of glycogen as a result of
glycogenolysis for energy production through glycolytic pathway to meet high
energy demands due to pesticide and heavy metal toxicity was reported in
invertebrates.
Elevated blood glucose levels and decreased glycogen content in
hepatopancreas and muscle were reported in Macrobrachium malcolmsonii
(Saravana and Geraldine,1997) Scylla serrata (Kulkarni and Kulkarni,1989)
Oziotelphusa senex senex (Sreenivasalu and Bhagyalakshmi, 1994)
Uca marionis (Yeragi, et al., 2000) and in Daphnia magna (De Coen et al., 2001)
exposed to heavy metals and pesticides, indicated that shrimps could detect
hypoxia by moving glucose before aerobic pathways are used and this response
could be a strategy to prepare for anoxia (Taylor and Spicer, 1987).
In the present study, increased plasma proteins was observed in
L. vannamei when external medium contain elevated levels of ammonia and
nitrite. In decapods, elevation of plasma proteins was reported in
Barytelphusa guerini (Reddy, 1991) and Macrobrachium malcolmsonii exposed to
endosulfan (Saravana and Geraldine,1997, 2001). The observed increase in
haemolymph plasma protein of L. vannamei could be realized as a method to
maintain the homeostasis of the animal due to the loss of certain enzymes in
tissues which have precise physiological functions in the normal animal
(Saravana and Geraldine, 1997, 2001).
5.4. Stress enzymes
The enzymatic activity showed significant difference (p<0.05) in different
salinities of ammonia and nitrite except for GPT at 10 and 15 ppt salinity and
GOT at 10 ppt there was no significant difference (p>0.05) between the control
and treatments. Enzymatic activity of LDH at 10 ppt was significantly lower
(p<0.05) in comparison with the activity at salinity of 5 and 15 ppt. While GPT and
GOT level were found to exhibit highest activity in 5 ppt (20.95 UL-1), (20.08 UL-1)
and lowest activity in 15 ppt (13.09 UL-1), (12.21 UL-1) respectively. It is generally
accepted that an increase of these enzyme activities in the extracellular fluid or
plasma is a sensitive indicator of even minor cellular damage (Van der et al.,
2003; Palanivelu et al., 2005) Thus, the measurement of transaminase activities
in blood plasma of fish can be used as indicator for toxicity. Therefore it is
possible to conclude that the test agent ammonia and nitrite exerted a negative
influence on GOT, GPT and LDH activities in shrimp muscle. Activities of these
enzymes may not be as great in crustacean haemolymph as in fish and other
marine animals where the blood is the adequate tissue for enzymatic
determinations of GOT and GPT (Renquing, 1990).
LDH is a tetrameric enzyme recognized as a potential marker for
assessing the toxicity of a chemical. The elevated levels of LDH observed in the
hemolymph in the present study with L. vannemei might be due to the release of
isozymes from the destroyed tissues. LDH is an important glycolytic enzyme in
biochemical systems and is inducible by oxygen stress.
VI. SUMMARY AND CONCLUSION
Toxicity trials on Litopenaeus vannemei, were conducted for 48 h with
different concentrations of ammonia and nitrite at three different salinities viz., 5,
10 and 15 ppt. The half lethal concentrations (LC50) were calculated for both
ammonia and nitrite at respective salinities using Probit analysis. The nominal
concentrations of ammonia for the test solutions were 0, 10, 15, 20, 25 and 30
ppm at 5 ppt; 0, 10, 20,30, 40, 50 ppm at 10 ppt and 0, 20, 30, 40, 50, and 60
ppm at 15 ppt. The recorded 48 h LC50 values of ammonia at three different
salinities were 16.61, 28.85 and 44.18 mg/L respectively.
The nominal concentrations of nitrite for test solutions were 0, 50, 60, 70,
80, 90 and 100 ppm at 5 ppt; 0, 100, 120,140 and 160 ppm at 10ppt and 0, 140,
160, 180 and 200 ppm at 15 ppt. The recorded 48 h LC50 values of nitrite in three
different salinities were 92.63, 136.80 and 186.34 mg/L respectively.
Haematological studies were carried out at 15 ppt salinity at the sub
lethal concentration of ammonia and nitrite for 15 days. The study revealed the
glucose level to be 86.98 and 83.296 mg dl-1 and total protein concentration to be
6.676 and 6.134 g dl-1 respectively. The blood glucose showed significant
difference (p<0.05) between control and the treated groups while total protein
concentration showed no significant difference (p>0.05) between the control and
treated groups. The blood glucose in test animals increased significantly when
exposed to ammonia in the control groups. The blood glucose and total protein
concentration of haemolymph were found highest in test animals exposed to
ammonia (86.98 mg dl-1 and 6.67 g dl-1 respectively) while the control groups
showed lowest (77.38 mg dl-1 and 5.932 g dl-1). The elevation of plasma proteins
could be realized as a means to maintain the homeostasis of the animal due to
the loss of certain enzymes in tissues which have precise physiological functions
in normal animals.
To assess the stress levels on L. vannamei, as exhibited by stress
induced enzymes such as GPT, GOT and LDH, shrimps were exposed long term
(15d) to sub lethal concentrations of ammonia and nitrite. The activity of the
selected enzymes were assessed in abdominal muscle. The shrimps were
exposed to sub-lethal concentrations of ammonia and nitrite at three different
salinities viz., 5, 10 and 15 ppt and the enzyme activities were recorded. The
activities of GPT, GOT and LDH for ammonia at 5, 10 and 15 ppt were 20.95,
20.08, 101.36 U L-1; 18.33, 18.33, 85.14 U L-1 and 13.96, 15.71, 52.71 U L-1
respectively. The activities of GPT, and LDH for nitrite at 5, 10 and 15 ppt were
20.08, 18.34, 85.14 U L-1; 18.33, 17.46, 68.92 U L-1 and 13.09, 12.21, 36.49 U L-1
respectively.
The enzymatic activities showed significant difference (p<0.05) at
different salinities except for GPT at 10 and 15 ppt salinity and GOT at 10 ppt,
wherein no significant difference (p>0.05) between the control groups and
treatments was recorded. LDH activity at 10 ppt was significantly lower (p<0.05)
in animals exposed to different toxicities at salinities of 5 and 15 ppt. On the other
hand GPT and GOT levels were found to be highest in 5 ppt (20.95 UL-1), (20.08
UL-1) and lowest in 15 ppt (13.09 UL-1), (12.21 UL-1) respectively.
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