effect of ammonia and nitrite toxicity on ......ammonia and nitrite toxicity on litopenaeus vannamei...

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


nitrogen at 10 ppt salinity

30


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


salinity

35


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



42



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


concentration of TAN at 10 ppt

32


concentration of TAN at 15 ppt

33

4.4 Mortality of L. vannamei exposed to different

concentration of NO2 –N at 5ppt

37


concentration of NO2 –N at 10ppt

37


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



43



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

https://en.wikipedia.org/wiki/Carapace

https://en.wikipedia.org/wiki/Ocean

https://en.wikipedia.org/wiki/Juvenile_(organism)

https://en.wikipedia.org/wiki/Estuary

https://en.wikipedia.org/wiki/Rostrum_(anatomy)

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



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



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


at 10 ppt


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




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




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


NO2 –N at 10ppt


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


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.




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




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




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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