Aquaculture 165 Ž1998.

233–242

A simple stress test for Penaeus Õannamei

postlarvae
Tzachi M. Samocha a,) , Horacio Guajardo b,
Addison L. Lawrence c , Frank L. Castille c , Michael Speed d ,
David A. McKee b, Kim I. Page e
a
Texas Agricultural Experimental Station, Shrimp Mariculture Project, Texas A&M UniÕersity System, 4301
Waldron Rd., Corpus Christi, TX 78418, USA
b
Texas A&M UniÕersity-Corpus Christi, 6300 Ocean Dr., Corpus Christi, TX 78412, USA
c
Texas Agricultural Experimental Station, Shrimp Mariculture Project, Texas A&M UniÕersity System, 1300
Port St., Port Aransas, TX 78373, USA
d
Texas A&M UniÕersity-College Station, College Station, TX 77843, USA
e
Harlingen Shrimp Farms, Route 2 Box 300K, Centerline Rd., Los Fresnos, TX 78566, USA
Accepted 13 April 1998

Abstract

The effects of age on resistance to low salinity and formalin stress were determined in early
Penaeus Õannamei postlarvae ŽPL. with acute static bioassays. Two-hour median lethal concentra-
tions Ž2 h-LC 50 . of formalin were 274, 288, 298 and 293 ppm for PL ages of 1, 2, 3 and 4 days
ŽPL 1 –PL 4 ., respectively. After PL 4 , resistance to formalin increased with age to 374, 497 and
598 ppm for PL 5 , PL 6 and PL 7 , respectively. For exposure to low salinities, 2 h-LC 50 decreased
from 16.8 ppt for both PL 1 and PL 2 to 14.3, 10.3. 8.3, 4.5. and 3.0 ppt for PL 3 , PL 4 , PL 5, PL 6
and PL 7 , respectively. Based on salinity decreases or differentials, 2 h-LC 50 increased from 12.9
and 11.8 ppt for PL 1 and PL 2 , respectively, to 14.2, 18.8, 19.5, 23.3 and 24.9 ppt for PL 3 , PL 4 ,
PL 5 , PL 6 and PL 7 , respectively. A practical stress test to evaluate the hardiness of a hatchery
produced P. Õannamei PL was proposed, using exposure to a single concentration of formalin or
single reduced salinity as a stressor. Because resistance to formalin and reduced salinity increased
with age, different stressor concentrations are recommended for PL ages with different sensitivi-
ties. The proposed stress tests, which are rapid, inexpensive and simple, can be used by shrimp
hatcheries as a quality control procedure. q 1998 Elsevier Science B.V. All rights reserved.

Keywords: Stress tests; Penaeus Õannamei; Salinity; Formalin; Postlarvae quality

)
Corresponding author.

0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

PII S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 2 6 4 - 6
234 T.M. Samocha et al.r Aquaculture 165 (1998) 233–242

1. Introduction

Although there is no universally-accepted method of determining PL quality, many

different criteria have been suggested for the evaluation of PL quality. Yunker Ž1989.,
Bauman and Jamandre Ž1990., Villalon Ž1991. and Samocha and Lawrence Ž1992.
report that PL activity in the larval rearing tanks, PL survival, body pigmentation and
muscle development can be used to determine PL quality. Other morphological condi-
tions such as integumental fouling, debris accumulation on appendages, body deformi-
ties and muscle opaqueness, have also been suggested as tools for evaluating PL quality
ŽHirono, 1989; Bauman and Jamandre, 1990; Villalon, 1991; Samocha and Lawrence,
1992.. Time needed to complete larval metamorphosis, survival during larval develop-
ment and PL size have been recommended as useful criteria for PL quality evaluation
ŽWilkenfeld et al., 1984; Kuban et al., 1985; Samocha et al., 1989; Smith et al., 1992..
Bray and Lawrence Ž1992a,b. reviewed different tests and standards proposed for
seedstock quality evaluation.
Acute stress tests have been described to distinguish between healthy and weak PL
ŽMaugle, 1988; Baybay, 1989; Tackaert et al., 1989; Bauman and Jamandre, 1990;
Bauman and Scura, 1990; Nietes, 1990; Gomez et al., 1991. and used by commercial
hatcheries to evaluate PL’s hardiness. Samocha et al. Ž1993. summarized procedures for
PL stress tests. In most procedures, PL are subjected to sudden changes in salinity or to
selected chemical solutions. However, because the response to osmotic or chemical
stress may be dependent upon the age and nutritional status of the PL ŽTackaert et al.,
1989; Arellano, 1990; Gomez et al., 1991; Rees et al., 1994., there is not a standard
stress test to identify good quality PL. A standardized stress test needs to be developed
to enable comparison of PL quality between different sources of PL.
Hirono Ž1989. described two separate stress tests used in Ecuador for Panaeus
Õannamei. Water salinity and temperature are lowered instantaneously to 10 ppt and
208C from the shipping conditions and survival is monitored after 24 h. However, the
author did not specify survival that suggests good PL quality. Villalon Ž1991. recom-
mended exposure to a combined temperaturersalinity stress test for hatchery produced
P. Õannamei. In his test, three groups of 100 PL are transferred from the larval rearing
tank Ž33 ppt and 298C. into buckets with 15 l of water at 5 ppt and 208C. After 1 h, all
live PL are counted and the average survival for the three replicates is calculated.
Survival above 60% is considered indicative for a good PL quality.
Bauman and Jamandre Ž1990. suggested two stress tests for 14-day-old PL ŽPL 14 1 .
and older: a 15-ppt salinity drop and exposure to a formalin solution of 100 ppm Ž37%
aqueous solution, wrw.. They suggest that 100% survival in stress tests can serve as a
practical method to establish good PL quality and readiness for sale.
Clifford Ž1992. recommends a standardized methodology for evaluating hatchery-pro-
duced PL. The procedure calls for simultaneous reductions in salinity and temperature to

1
This term is used to describe the postlarval age rather than the developmental stage Že.g., culture
conditions such as water temperature, salinity and feed availability will determine whether stage 5 postlarva is
five-day-old or older..
 T.M. Samocha et al.r Aquaculture 165 (1998) 233–242 235

20 ppt and 108C, respectively, for 4 h. PL exposure to formalin challenge of 100–150

ppm is another variation suggested by the author. Survival of 80–100% suggests high
quality PL, 60–79% survival is considered acceptable and survival below 60% is
considered not adequate for pond stocking. In addition, Clifford suggested that weak PL
exhibiting low survival in the stress test should be kept longer in the hatchery to
improve their strength.
In contrast to juvenile and adult penaeid shrimp that are capable of both hypoosmotic
and hyperosmotic regulation over a wide range of salinities ŽCastille and Lawrence,
1981., the osmoregulatory capabilities of larval shrimp are limited. The ability to
osmoregulate and thus survive at low salinities develops after metamorphosis to PL.
Concurrent with the ability to osmoregulate is the increased tolerance of PL to salinity
changes. Increased salinity tolerance with PL age has been reported in both P. japonicus
ŽCharmantier et al., 1988. and P. Õannamei ŽAQUACOP et al., 1991.. If salinity
tolerance increases with PL age and salinity tolerance is to be used as an indicator of PL
hardiness, age must be considered as a variable in salinity stress tests for PL.
For acute static bioassays, median lethal concentrations ŽLC 50 . can be calculated by a
variety of methods. Ward and Parrish Ž1982. report that the most widely-used methods
are the probit ŽFinney, 1971. and moving average ŽLichtfiel and Wilcoxon, 1949.. Reish
and Oshida Ž1987. suggest the arithmetic graphic, logarithmic and probit methods. The
advantage of the probit method is that it provides the most reliable statistical treatment
of the data and helps comparisons between experiments with statistical validity ŽReish
and Oshida, 1987..
For many applications, stress tests can be simplified to reduce requirements for time,
equipment, supplies, labor and analytical resources. Using treatments, for which LC 50
values are reliably known, simplified stress tests can be developed from expected
survival to single concentrations of test solutions or conditions. Simplified stress tests
using a single concentration for all PL ages have been described by Bauman and
Jamandre Ž1990., Villalon Ž1991. and Clifford Ž1992.. However, if resistance to stress
changes with age, different levels or concentration of stress may be appropriate for each
age of PL.
The objectives of this study were to Ž1. determine the effect of age of early PL on
resistance to salinity and formalin stress and Ž2. develop a simple and rapid stress test
for evaluating the hardiness of P. Õannamei PL, based on expected survival to a single
stressor concentration for each age of PL.

2. Materials and methods

Acute static bioassays were conducted to determine LC 50 for low salinities and
formalin at each age of PL from PL 1 to PL 7 . Tests were conducted at the Harlingen
Shrimp Farm Hatchery ŽLos Fresnos, TX, USA. with PL reared from Hypodermal and
Hematopoietic Necrosis Virus-free P. Õannamei broodstock. Each stress test was
conducted with a new population of PL collected from a single larval rearing tank ŽLRT.
stocked with larvae from several spawning females. Water temperature in LRT varied
between 27 and 318C.
236 T.M. Samocha et al.r Aquaculture 165 (1998) 233–242

In the salinity bioassays, exposure salinities were decreased with age as resistance to
low salinities increased. PL 1 were exposed to salinities of 10, 12, 13, 14, 15, 16, 17 and
18 ppt, PL 2 to 14, 15 and 16 ppt, PL 3 to 12, 14, 15 and 16 ppt, PL 4 to 5, 10, 14 and 15
ppt, PL 5 to 0.5, 1, 2, 3, 4, 5, 6 and 7 ppt, PL 6 to 0.5, 1, 2, 3, 4, 5, 6, 8 and 10 ppt and
PL 7 to 0.5, 1, 2, 3, 4, 5 and 6 ppt. Bottled distilled water was used to adjust the salinity
of the hatchery water. Salinity was measured by a temperature compensated refractome-
ter Žmodel A-165, Aquafauna Biomarine, Hawthorne, CA, USA.. For the salinity
bioassays, LC 50 were reported both as exposure salinities and salinity differentials
Ždifference in LRT salinity and exposure salinity. because of differences in LRT
salinities Ž27 to 30 ppt..
For the formalin bioassays, exposure concentrations were increased with age as
resistance to formalin increased. PL 1 were exposed to formalin concentrations of 150,
200, 300, 400 and 500 ppm, PL 2 to 100, 300, 400 and 500 ppm, PL 3 to 100, 200, 300,
400 and 500 ppm, PL 4 to 100, 300, 400, 500 and 600 ppm, PL 5 to 200, 250, 300, 400,
500, 600, 750 and 1000 ppm, PL 6 to 100, 250, 300, 400, 500, 750 and 1000 ppm and
PL 7 to 250, 400, 500, 750 and 1000 ppm. Test solutions were prepared by mixing
hatchery seawater with 40% Žwrv. formaldehyde solution stabilized in 10–15% methanol
ŽCatalogue a: F77-20, Fisher Scientific, Fair Lawn, NJ, USA..
Bioassays were initiated by pouring 10 PL onto a small strainer and rinsing the PL
off the strainer into a 250-ml beaker with 100 ml of test solution. Five replicate beakers
were used for each treatment. All bioassays were conducted at temperatures between 25
and 278C. Initially, shrimp mortality was recorded after 0.5, 1, 2 and 3 h. When an
optimal response-time was established, based on PL mortality, the number of observa-
tions was reduced to one observation 2 h from test initiation. The criteria for death were
lack of movement and absence of any response to gentle pressure with a probe. Mean
survival rates, for the same PL age, at different exposure times were analyzed by
One-way ANOVA and Student–Newman–Keuls multiple comparison procedures to
detect significant statistical differences among treatments using SPSS software ŽNorusis,
1993.. The results are reported as means with 95% confidence intervals ŽCI. and
reported in the original units. To analyze survival trends over time for the two stressors,
the average percent survival of the grouped concentrations and PL ages was compared
against each exposure period Ž0.5, 1, 2 and 3 h.. Differences among means were
analyzed for significance at P s 0.05. PL survivals after exposure to different salinities
or salinity differentials were transformed to a natural logarithm ŽLog N. for a better fit.
For each age of PL, probit-analysis was used to establish the median lethal concentration
after 2-h exposure Ž2 h-LC 50 . and its 95% confidence limits using SPSS software
ŽNorusis, 1993..
Assuming a stress test could be conducted without replication with a large number of
PL, the Estimated Sample Size for Population Proportion formula ŽTriola, 1992. was
used to calculate the number of PL needed to conduct the test with a 95% CI for
survival with an error of "0.05, "0.10 and "0.14. From the calculated number of PL
needed and the expected survivals based on the LC 50 determinations in this study, the
expected number of surviving PL can be estimated for a simplified stress test. Note that
the error of "0.14 was chosen because the Estimated Sample Size for that error is 50.
In a simplified stress test, comparison of numbers of surviving PL to the expected
 T.M. Samocha et al.r Aquaculture 165 (1998) 233–242 237

numbers will compare hardiness of PL in the stress test to the hardiness of PL in this
study. Recommended exposure concentrations were adjusted from the LC 50 values
determined in this study to simplify testing. Ranges of survivors Ž95% CI. were
calculated from expected survivals for each sample size, stressor and age of PL.

3. Results

A summary of PL survival, in four exposures periods, at different salinities and

formalin solutions is presented in Fig. 1. The Student–Newman–Keuls procedure
showed significant differences Ž P - 0.05. between PL survival after 0.5, 1 and 2 h for
formalin, but failed to show significant differences between 2- and 3-h exposures.
Significant difference in PL survival for salinity stressors was found only among the
0.5-h and the other three exposure periods. For both stressors, PL survival showed a
decrease trend with increasing exposure time.
After PL age of four days, tolerance to formalin increased with the age of PL. For
exposure to formalin, 2 h-LC 50 increased from 293 ppm for PL 4 to 598 ppm for PL 7
ŽTable 1.. For PL 1 to PL 4 , 2 h-LC 50 increased only from 274 ppm to 293 ppm. After PL
age of 2 days, tolerance to low salinities increased with the age of PL. For exposure to

Fig. 1. The effect of different salinities and formalin concentrations under four exposure periods, on the
survival of Penaeus Õannamei postlarvae in stress test study under controlled conditions. Note: Each point in
this figure represents the average over the age and the concentration of all observations at a given exposure
period.
238 T.M. Samocha et al.r Aquaculture 165 (1998) 233–242

Table 1
Effect of age of Penaeus Õannamei PL on tolerance to formalin and low salinity
Age of PL Ždays. 2 h-LC 50
Formalin Žppm. Salinity Žppt. Salinity decrease Žppt.
1 274 16.8 12.9
2 288 16.8 11.8
3 298 14.3 14.3
4 293 10.0 18.8
5 374 8.3 19.5
6 497 4.5 23.3
7 598 3.0 24.9

Table 2
Recommended exposure concentration and expected survival for formalin stress test of one- to seven-day-old
Penaeus Õannamei postlarvae
PL age Recommended Expected 95% CI a for expected number of surviving PL
Ždays. exposure Žppm. survival Ž%.
nb s 50 nb s100 nb s 400
1 300 40 13–27 30–50 140–180
2 300 40 13–27 30–50 140–180
3 300 50 18–32 40–60 180–220
4 300 50 18–32 40–60 180–220
5 400 40 13–27 30–50 140–180
6 500 50 18–32 40–60 180–220
7 600 50 18–32 40–60 180–220
a
Confidence Interval.
b
Sample size.

Table 3
Recommended exposure salinities and expected survival for low salinity stress test of one- to seven-day-old
Penaeus Õannamei postlarvae
PL age Recommended Expected 95% CI a for expected number of surviving PL
Ždays. exposure Žppt. survival Ž%.
nb s 50 nb s100 nb s 400
1 17 50 18–32 40–60 180–220
2 17 50 18–32 40–60 180–220
3 14 50 18–32 40–60 180–220
4 10 50 18–32 40–60 180–220
5 8 50 18–32 40–60 180–220
6 5 55 21–35 45–65 200–240
7 3 50 18–32 40–60 180–220
a
Confidence Interval.b Sample size.
 T.M. Samocha et al.r Aquaculture 165 (1998) 233–242 239

Table 4
Recommended salinity decrease and expected survival for low salinity stress test of one- to seven-day-old
Penaeus Õannamei postlarvae
PL age Recommended Expected 95% CI a for expected number of surviving PL
Ždays. exposure Žppt. survival Ž%.
nb s 50 nb s100 nb s 400
1 13 50 18–32 40–60 180–220
2 13 50 18–32 40–60 180–220
3 14 50 18–32 40–60 180–220
4 19 50 18–32 40–60 180–220
5 19 55 21–35 45–65 200–240
6 23 55 21–35 45–65 200–240
7 25 50 18–32 40–60 180–220
a
Confidence Interval.
b
Sample size.

different salinities, 2 h-LC 50 increased from 16.8 ppt for PL 2 to 3.0 ppt for PL 7 ŽTable
1.. Based on salinity differential, 2 h-LC 50 increased from 11.8 ppt for PL 2 to 24.9 ppt
for PL 7 ŽTable 1.. For PL 1 to PL 2 , 2 h-LC 50 did not increase for either low salinity or
salinity decrease.
Recommended formalin concentrations, salinities and salinity decreases in the pro-
posed simplified stress test are given in Tables 2–4 for each age of PL. Also indicated in
these tables are the expected percentage of survival for each age and concentration.
Confidence limits Ž95%. for expected survival are given as ranges of the number of PL
surviving for different samples sizes.

4. Discussion

The 96-h exposure is the most common time used in acute static bioassays ŽReish and
Oshida, 1987.. Organisms are usually not fed and solutions are not changed during the
exposure. For the organisms used in this study, where resistance to the stressor solutions
changed with age, a shorter exposure time was necessary. In addition, shorter exposure
times decrease the time needed to conduct the stress tests. Preliminary observations
showed that within an hour after death, the appearance of PL changed from transparent
to opaque. Consequently, mortality after exposure period greater than an hour was easier
to evaluate than mortality after exposure times less than an hour. Under the conditions in
this study, the exposure time of 2 h was short enough to allow rapid determination of
stress resistance and long enough to ease evaluation of PL mortality. This exposure time
resulted in adequate and reproducible mortality at the concentrations and PL ages tested.
In this study, formalin tolerance increased from PL 4 to PL 7 and salinity tolerance
increased from PL 2 to PL 7 . Similar increases in salinity tolerance with age have been
reported by other researchers. Charmantier et al. Ž1988. found that salinity tolerance in
P. japonicus increases from PL 1 to PL 6 . The lethal salinity that kills 50% of PL at 258C
Ž24 h-LS 50 as defined by these authors. is 25 ppt for PL 1 and 7–10 ppt for PL 6 or older.
240 T.M. Samocha et al.r Aquaculture 165 (1998) 233–242

AQUACOP et al. Ž1991., in their research with P. Õannamei PL 2 through PL 20 ,

concluded that tolerance to salinity changes increases with PL age. In contrast to PL
stress tests that use single stressor concentrations for all PL ages ŽBauman and
Jamandre, 1990; Villalon, 1991; Clifford, 1992., this study suggests that different
stressor concentrations should be used for each PL age.
In addition, this study provides direct evidence that PL 7 are more resistant to stress
than younger PL. This suggests that PL 7 would be a more optimal age for harvest,
shipping and stocking than would be younger PL.
Based upon the results from this study, the following stress test is proposed for use
with either formalin or decreased salinity: Ž1. select a desired error to find the number of
PL needed; Ž2. using the concentrations recommended in Tables 2 and 3, place the
determined number of PL in a single container containing the stressor solution at a
density of 100 PL ly1 ; and Ž3. after 2 h, count the number of live PL. If the number of
live PL is below the expected number found in Tables 2–4 for the corresponding age
and sample size, then the PL are less hardy than the PL used in this study. If the number
of live PL is above the expected number, then the PL are more hardy than the PL used
in this study. For example, to evaluate the quality of PL 7 with a minimum error of
"0.10, a sample size of 100 PL would be needed. The PL should be placed in 1 l of test
solution Ž600-ppm formalin, 3-ppt salinity, or a 25-ppt drop in salinity.. If the number of
live shrimp, after a 2-h exposure, is between 40 and 60, the hardiness of the PL would
be comparable to those used for determinations of 2 h-LC 50 in this study.
The stress test proposed in this study is simple, rapid, inexpensive and does not
require specialized equipment or extensive scientific skill. It could be used either at a
hatchery or in the field to evaluate PL hardiness. In the hatchery, formalin and salinity
stress tests can be important tools to establish quality standards and to improve hatchery
practices.
If PL hardiness is indicative of future growth and survival performance, stress tests
may become important indicators of PL quality. Poor survival and lack of predictability
in the growout phase are major obstacles in developing a stable shrimp farming industry
ŽFegan, 1992; Samocha et al., 1993.. If PL quality affects variability in growth and
survival during the growout phase, PL quality can be a key factor for successful shrimp
farming ŽSamocha and Lawrence, 1992.. Shrimp farmers and PL producers need a rapid
and simple procedure to evaluate PL quality with respect to future growth and survival
ŽCastille et al., 1993.. Characterizing good PL quality can benefit shrimp farmers by
increasing crop profits ŽClifford, 1992. and help hatchery managers to identify and
assess the effects of factors affecting PL quality ŽBriggs, 1992; Fegan, 1992; Samocha
and Lawrence, 1992..
Evidence that stress tests have a predictive value in determining PL viability in the
growout phase is limited. Although Bauman and Jamandre Ž1990. reported better results
in growout ponds with P. monodon PL that had passed stress tests, their observations
were not supported by any statistical tests.
In this study, only young PL ŽPL 1 to PL 7 . were evaluated. Further research with
older PL is warranted to determine if stress resistance continues to increase with age.
Field testing is needed to establish the relationship between PL hardiness and seedstock
quality. Records of PL stress test performance should be retained with hatchery data
 T.M. Samocha et al.r Aquaculture 165 (1998) 233–242 241

Že.g., nauplii sources, spawn size, percent hatch, survival to PL 1 , time needed to
complete the metamorphosis, etc.. to establish correlation between these parameters and
PL performance in the growout.

Acknowledgements

This research was funded in part under Grant No. H-8158, Oceanic InstituterGulf
Coast Research Laboratory Consortium through the Texas Agricultural Experiment
Station, Texas A & M University System; Grant No. 95-38808-1424 from the U.S.
Department of Agriculture, Cooperative State Research Service, awarded to Oceanic
Institute and the Gulf Coast Research Laboratory Consortium. The authors thank
Harlingen Shrimp Farms, Los Fresnos, TX, USA for providing the animals and the
space to conduct the research.

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