BIOASSAY: 24hr TOLERANC TEST

F117 Lab Exercise 3

Cordoves, Epalan, Murata, Nacis, Tabasin

March 19, 2014

INTRODUCTION
Tilapia is considered as one of the most widely cultured fish in the Philippines. Last
year, tilapia ranked as the 2nd highest volume of production when it reached 318 thousand
metric tons output in 2013, or a growth of 3.18 percent next to milkfish (BAS, 2013). The
booming growth of tilapia industry is mainly contributed to its diverse adaptation and
distinct biological characteristics which make it as an ideal species for aquaculture farming.
They exhibit many qualities including resistance to handling and disease, efficient
conversion of low protein diets, ease of breeding, and high palatability which suit them for
culture.
In terms of its environmental requirements, tilapia can tolerate a wider range of
environmental conditionsincluding factors such as salinity, dissolved oxygen,
temperature, pH, and ammonia levels than most cultured freshwater fishes can. Most
tilapia are relatively euryhaline (can tolerate a wide range of salinities) but all species are
tolerant to brackish water. They are thermophilic fish and known to tolerate a wide range
of water temperature. The temperature range for normal development, reproduction and
growth of tilapia is about 20 to 35C, depending on fish species (Balarin and Haller, 1982).
Tilapia can tolerate lower lethal temperature for most species from 10 to 110C for a few
days (Reyes, 1984) while all species can tolerate high water temperature.
In general, all tilapia are highly tolerant of low dissolved oxygen concentration for
they can grow well at dissolved oxygen level of 1 - 3 ppm. In terms of feeding, tilapias feed
on variety of phytoplankton as their primary food items. They are cannibalistic and will
feed on their prey if food is not abundant (Reyes, 1984).
Although tilapia exhibits high resistance to diseases, it is inevitable that some
species are still susceptible to certain infestations of parasites. For this reason, aquaculture
farmers tend to use chemical treatments, involving the use of substances as control
measures. Copper sulfate for example is widely used as an algacide and for control of some

of the protozoan ectoparasites and monogenetic trematodes. However, the use of such
treatment poses high potential risk and toxicity to the fish. In addition, copper sulfate is not
approved by the FDA for treatment of fish wherein once consumed by humans may pose
health risk (www.massmind.org. 2014).
The toxicity of the copper ion varies with the species and age of fish; however,
tilapia are, in general, relatively tolerant of copper. Treatment dosages range between 0.1
to 0.2 ppm copper (www.massmind.org, 2014). Once treated, copper will concentrate in
many different organs in fish and mollusks. Heavy metal exposure on fish caused increased
mucus like secretion over gills, excessive excretion, anorexia and increased fin movement
(Ezeonyejiaku et. al, 2011). Furthermore, copper sulfate is more toxic to invertebrates than
fish.
In aquaculture practices, the conduct of certain test to measure potential risks of
chemicals is important. A biological assay is a procedure involving use of the responses of
aquatic organisms to detect or measure the presence or effect of one or more subtances,
wastes, or environmental factors, alone or in combination (Lio Po et. al, 2001). In this
study, the use of tolerance test or the traditional LC50 test as an example of short-term
bioassay is considered before treating fish with substances such as copper sulfate or
formalin. It is essential for it determines the different concentration levels of a chemical
used and its effects to the physiological mechanisms of the fish.
The purpose of this activity is to be able to familiarize, understand and acquire
knowledge in conducting bioassay or tolerance test for fish. Specifically, it aims to:

determine approximate toxic range of concentration of copper sulphate using rangefinding test of bioassay.

assess the tolerance of tilapia fry on the varying concentration of copper sulphate
(CuSO4.5H2O) and its behavioral response and changes as the most sensitive
indication of potential toxic effects.

gather and monitor data of the parameters to be taken during the course of the
experiment such as temperature and survival rate of the tilapia.

determine median lethal concentration (LC50) of copper sulphate using Reed

Muench method or Finneys Probit Analysis

METHODOLOGY
Materials:
Tilapia fry

Copper sulphate

Analytical balance

Ruler

Glass containers

Thermometer

Graduated cylinder

Beaker

Freshwater

Scoop net

Aerators

Methods:
1. Obtain tilapia fish samples from the Freshwater Aquaculture Station (FAS) and place in
a maintenance aquarium with aeration to expose in a conditioning period at room
temperature.
2. Prepare eight (8) clean and uniform sized glass containers and fill-up with two (2)
liters of freshwater. The water should not be polluted or contaminated with wastes
from any sources. Then, set-up uniform aeration system for each container in order to
keep the amount of oxygen not less than 6 mg/l.
3. Place ten (10) healthy Tilapia fry per container randomly using scoop net. Allow one
(1) hour for acclimatization.
4. Prepare stock solution of copper sulphate and dilute to the desired concentrations
using distilled water as diluents. Test solutions must be freshly prepared. Also, avoid
unnecessary exposure to air and light. Make sure that the test chemical is dissolved
completely before adding into the test container.
5. Three (3) different concentrations of copper sulphate solution will be used plus a
control group (no chemical added) to determine the range-finding test. Total volume of
the experimental water must be two (2) liters. Then, add the chemical gradually to the
respective container near aeration (0 hour).
6. Record the temperature (initial) in each test container. Rinse well the thermometer
after every use to avoid contamination.
7. Monitor and observe fish behavior and survival rate at the following intervals: every 15
minutes (for the 1st hour), every hour (2nd 6th hour), at 12th hour and at the end of the
24th hour. Record swimming behavior, body coloration, mucus production, ventilation
and mortality within the 24-hour duration. Remove the dead samples using scoop net
and discard properly.
8. Get ten (10) representative samples and measure each total length and body weight.
9. At the end of 24th hour, record the temperature (final) in each test container and
terminate the experiment.
10. Discard all the treated samples and dispose properly in the designated areas. Clean all
the used materials and return to the respective places.
11. Solve for the mortality rates in each concentration as well as its median lethal
concentration (LC50) values, standard error and 95% confidence interval.

Collection of fish samples

(tilapia fry)

Preparation of test containers

and aeration set-up

Control (0ppm)
1

Swimming behavior
Body coloration

A (1 ppm)

B (5 ppm)

C (10 ppm)

Selection & transfer of fish

samples (tilapia fry)
Addition of copper sulphate
at designated concentration

Ventilation
Measure initial temperature

Mortality/ survival rate

Collection of 10
representative samples

Measure body weight

Measure total length

Monitoring

Every 15 mins (1st hour)

Every hour (2nd-6th hour)

On the 12th hr

Measure final temperature

Computations
LC50
Standard error
95% Confidence Interval
Figure 1. Schematic diagram of tolerance test procedure

At the end of 24th hr

RESULTS AND DISCUSSIONS

Tilapia (Oreochromis sp.) fry were subjected for 24 hr tolerance test against varying
level of copper sulphate concentrations. Table 1 shows the recorded body weight and total
length of ten representative samples of tilapia fry. The average weight of fish samples was
0.0210g having approximately 1 cm total length.
Table 1. Total length and body weight of representative fish samples

Sample No.

Weight (g)

Total Length (cm)

0.0191

0.0217

1.1

0.0248

1.1

0.0184

0.0253

1.1

0.0157

0.0143

0.9

0.0206

1.1

0.0246

1.1

10

0.0252

1.2

Total

0.2097

10.6

Mean

0.0210

1.06

Copper sulfate pentahydrate (CuSO4.5H2O) was used in the preparation of stock

solution. Copper sulfate pentahydrate crystals are considered 100% active and granular
copper sulfate is widely used as an algacide and for control of common ectoparasites.
According to www.massmind.org (2014), the solubility and thus the toxicity of the copper
ion is strongly influenced by the presence of calcium and carbonate (measured as
alkalinity) and the pH of the water. In freshwater copper treatment should not be used if
the total alkalinity (as CaCO3) is unknown. If total alkalinity is < 50 ppm or waters pH is <
7.2 copper treatment should only be used if it has been preceded by a bioassay. While in
marine waters or freshwater of total hardness above 200 ppm copper sulfate is rapidly
precipitated and should not be used unless a citrated or chelated form of copper is applied.

The following computations were considered in preparing the stock solution.

Copper (II) Sulfate Pentahydrate
Cu
S
O
O
H

=
=
=
=
=

(CuSO4.5H2O)

1 x 63.546
=
1 x 32. 067 =
4 x 15.9994 =
5 x 15.9994 =
10 x 1.0079 =
CuSO4.5H2O =
249.69 / 159.61 =

63.546
32.067
63.9976
79.997
10.079
249.69
1.56

CuSO4 = 159.61

Given the above calculations, it can be assumed that inn 1.56 g CuSO4.H2O there is
1.00 g CuSO4 and 1.00 g CuSO4 in 1 Liter water equals 1000 ppm CuSO4. Therefore, 1.56 g
CuSO4.5H2O in 1 Liter water will give 1000 ppm CuSO4.
To prepare 100 ppm CuSO4 in 1000 ml water, weigh 0.156 g CuSO4.H2O and add the
chemical to 1000 ml distilled water. Using the formula below, the sample calculation shows
the volume of stock solution needed for each desired copper sulphate concentrations (shown
in Table 1):
C1V1 = C2V2

Where:
C1 = 100 ppm (stock solution)
V1 = volume needed (from the stock solution to be transferred to the glass
jars in order to achieve the desired concentration)
C2 = desired concentration
V2 = 2000 ml (total volume in the glass jar)

V1 = 5 ppm x 2000 ml = 100 ml

100 ppm
Table 2. Calculated volume of stock solution for each desired concentration
Desired concentration
Volume from stock Volume of water in
Total Volume (ml)
(ppm)
solution (ml)
the set up (ml)

0
1.0
5.0
10.0

0
20
100
200

2000
1980
1900
1800

2000
2000
2000
2000

In bioassay, the recommended measures or indices or relative toxicity are 48 and 96

h LC. However, this study only followed the short-term: range-finding test which is a smallscale exploratory test to determine approximate toxic range of concentration, usually
within 24 h. It also determines the median lethal concentration (LC50). The widely used
methods for calculating an LC50 and confidence limit are probit, logit, moving average and
Lithcfield-Wilcoxon. Other methods are straight line interpolation and the Reed-Muench
method (Lio Po et. al, 2001)
The Reed-Muench method of estimating LC50 was used in this experiment. This
method artificially increases the number of organisms subjected to a given level of a
stimulus. It is assumed that any organism responding to a given concentration of a drug
would respond to lower concentrations. Table 3 shows the set of data computed
particularly the cumulative percent mortality using Reed-Muench method while Figure 2
shows the mortality versus concentration plot.
Table 3. Calculated percent mortality using Reed-Muench method
Conc.
(ppm)

Log dose
(ln)

Number
Dead

Number
Alive

Accum.
Dead

Accum.
Alive

Total

Cumulative
% Mortality

20

18

45

47

4.26%

20

12

14

27

41

34.15%

1.61

20

16

30

19

49

61.22%

10

2.30

20

15

35

15

50

70.00%

Figure 2. Graph showing cumulative % mortality versus concentration plot

Mortality vs Concentration

Cumulative % Mortality

80.00%
70.00%
60.00%
50.00%
40.00%
30.00%

Cumulative %
Mortality

20.00%
10.00%
0.00%
0

10
Concentration

15

Computations:

To solve for LC50:

To solve for LC75:

*LC75 cannot be performed using Reed Muench method since the given data
is only limited to 70% mortality (24 hrs).

To solve for Standard Error (S.E.):

*Standard error cant be computed due to undetermined LC75 value for R

To solve for 95% Confidence Interval (C.I.):

*Confidence interval cant be determined since standard error is unknown

The obtained median lethal concentration (LC50) is 2.57ppm. Based on our

gathered data, Reed Muench method is not suitable to determine the standard error and
confidence interval for this case. This can be accounted for a limited range of copper
sulphate concentration. Further test on concentrations higher than 10 ppm should be
considered to be able to contain 75% mortality rate. Other factor might be the greater
tolerance of tilapia fry on copper sulphate solution in which intermediate type of bioassay
would be more appropriate to study its sensitivity. However, our group considered other
methods of determining LC50. We used Finneys Probit Analysis LC50 Determination
Method using Statistical Package for Social Sciences (SPSS) Program 20.
Probit Analysis (Finney, 1971) is commonly used in toxicology to determine the
relative toxicity of chemicals to living organisms. This is done by testing the response of an
organism under various concentrations of each of the chemicals in question and then
comparing the concentrations at which one encounters a response. The following are the
outputs obtained using probit:

Table 4. 24 h lethal concentrations of copper sulphate to tilapia

Test Animal
Tilapia (Oreochromis sp.)

LC50

LC5

LC75

LC95

2.651 ppm
.048 ppm
13.781 ppm
147.606 ppm
(1.083-4.499) (.000-0.246) (7.346-1.586) (37.566-16203.861)

S.E.
.195

Table 4 shows the 24 hours toxicity of copper sulphate to tilapia fry with Figure 3
displaying the Probit line graphs of the toxicity data for the test fish species. The coefficient
of correlation was also computed and was sought to be 0.998. This value is close to the
maximum value (1.00) which means that the linearity is near excellent. Using probit, an
LC50 value of 2.651 ppm was obtained which is relatively higher compared to ReedMuench estimated LC50. It is due to the higher observed responses than the expected
responses, thereby increasing the probability of each concentration (see Table 5).
Moreover, the probit analyses were able to measure the LC75 value as well as the standard
error and 95% confidence intervals of a given probability.
Toxicity of copper sulphate varies with species and ages of tilapia and their
susceptibility rates to the test chemical. Ezeonyejiaku et. al (2011) studied the toxicity of
copper sulphate on healthy adult tilapia (Oreochromis niloticus) within 96 hrs tolerance
test and they reported a 58.837 ppm LC50 value. In addition, Mance (1987) indicated that
the juvenile fish are generally more susceptible to metal than adult fish.

Figure 3. Probit line graph of toxicity of copper sulphate to tilapia

Table 5. Cell Counts and Residuals of tilapia

concentration

Number of
Subjects

Observed
Responses

.000

41

14

14.143

-.143

.345

1.609

49

30

29.519

.481

.602

2.303

50

35

35.326

-.326

.707

Number

PROBIT

Expected
Residual
Responses

Probability

The parameter estimates of the Probit analysis, and Chi-Square test for the toxicity
of the copper sulphate to tilapia fry (Oreochromis sp.) is shown in Table 6 and 7
respectively. The table shows that copper sulphate is significantly toxic to tilapia fry
(P0.05). Several studies (Oyewo, 1998; Otitloju, 2001) also supported such account that
Tilapia sp. is said to be highly sensitive to copper sulphate in relation to other aquatic
animals that has been subject to the same treatment.
Table 6. Parameter estimates of the Probit analysis for tilapia
95% Confidence Interval
Parameter
Estimate Std. Error
Z
Sig.
Lower Bound
Upper Bound
PROBITa

concentration

.409

.117

3.492

.000

.180

.639

Intercept

-.399

.195

-2.051

.040

-.594

-.204

a. PROBIT model: PROBIT(p) = Intercept + BX (Covariates X are transformed using the base 2.718 logarithm.)
Table 7. Chi-Square tests for 24 hrs LC50 values of tilapia

PROBIT

Pearson Goodness-of-Fit
Test

Chi-Square

dfb

Sig.

.032

.858a

a. Since the significance level is greater than .050, no heterogeneity factor is used in
the calculation of confidence limits.
b. Statistics based on individual cases differ from statistics based on aggregated cases.

In terms of mortality rate, an erroneous data might be considered on the number of

dead fish observed in 10 ppm concentration. As the concentration increases, the number of
expected dead samples must also be increasing upon increase in exposure and time.
However, our data shows higher death values on 1ppm and 5 ppm than 10 ppm. Perhaps,
other factors (i.e. stress factors, low aeration) contributed to the increase mortality rate
between 1 and 5ppm containers. Such irregularities might affect the reliability of the
results of this study. The rapid action of copper in killing fish may be due to its toxic effect

on the respiratory function of fish by acting as uncoupler of oxidative phosphorylation at

the mitochondrial level (Andrews et al., 1983)
The temperature was also measured right after copper sulphate was added and at
the end of the 24 hr period (see Figure 4). The decrease in temperature was only observed
on the control set-up while no change in temperature was perceived on the treated
samples. Temperature as a parameter does not affect the behavioral response as well as the
mortality rate of the tilapia fry. Stress factor brought about by change in temperature is not
considerable enough to contribute significant effects since Tilapia species are known to
tolerate a wide range of water temperature.

Temperature
29.5
29
Celsius

28.5
28
27.5
27
26.5
26
0 ppm 0 ppm 1 ppm 1 ppm 5 ppm 5 ppm 10 ppm 10 ppm
1
2
1
2
1
2
1
2
Initial Temperature (0 hr)

Final Temperature (24th hr)

Figure 4. Record temperature at 0 hr and 24th hour for each concentration

Behavioural responses are important to survival because they are necessary to

perform essential life functions (Little et al., 1985). The following are the behavioral
changes observed among the fish species exposed to various concentrations of copper
sulphate as follow:
Control Group
No observable behavioral changes and mortality among fish fry was observed
during bioassay. From the start, the fish were normally and actively swimming. However,

at the end of 24 hrs, the fry were observed as weak but still of normal swimming behavior.
Overall, two (2) out of 20 fish was found dead at the end of the experiment.
Experimental Groups
For 1 ppm set-up, sluggish swimming behavior was observed two (2) hours after
the copper sulphate solution was added. As the exposure increased, the fish was observed
weak, with minimal movement or swimming activity and mostly stayed at the bottom until
the experiment was terminated. After 12 hrs, high number of dead fish was observed in
replicate 2.
For 5 ppm set-up, unsteady swimming pattern with jerky movements and
sluggishness were observed on the fish fry after an hour of exposure to copper sulphate.
After six (6) hours, the group observed a very small amount of blue cottony sediments in
the container. Like in the first set-up, fry usually became less active and sank at the bottom
motionless. At the end of 24th hour, 5 ppm set-up obtained the highest mortality rate
among all concentrations used in this experiment.
For 10 ppm set-up, there was loss of balance and exhaustion. Faster behavioral
anomalies were recorded at the start of the experiment. After long period of exposure, fish
remained inactive/motionless at the bottom, some were near the aerators. Also, this set-up
gained 2nd lowest mortality which was supposed to have higher behavioral response and
mortality.
CONCLUSION
The experiment shows the behavioral anomalies and subsequent death of fish
exposed to the heavy metal toxicant (copper sulphate). For this case, Finneys Probit
analysis is more effective than Reed-Muench method of estimating LC50. Statistical analysis
reveals that copper sulphate concentration at an estimate of 2.651 ppm ranging from
1.083-4.499 ppm (LC50) is significantly toxic to tilapia fry (Tilapia sp.) Moreover, the use of
intermediate bioassay would be more effective to consider its sensitivity since most are
relatively tolerant to copper sulphate. Lastly, temperature as a parameter in this study does
not affect the behavioral response as well as the mortality rate of the tilapia fry.

REFERENCES
Andrews, P., J. Thyssen and D. Lorke 1983. The biology and toxicology of molluscicides,
bayluscide. Pharmacol. Ther., 19: 245-295.
Balarin, J.D. and Haller, R. D. 1982. The intensive culture of tilapia in tanks, raceways and
cages. In: Muir, J. F. and Roberts, R. J. (eds) Recent Advances in Aquaculture. Croom
helm, London and Canberra, and Westview Press, Boulder, Colorado, pp. 267-355
Bureau of Agricultural Research. 2013. Fisheries Situationer January-December 2013.
Department of Agriculture, Philippines.
Ezeonyejiaku, C.D., Obiakor, M.O. and Ezenwelu, C.O. 2011. Toxicity of copper sulphate and
behavioural locomotor response of tilapia (Oreochromis niloticus) and catfish
(Clarias gariepinus) species. Online J. Anim. Feed Res., 1(4): 130-134.
Finney, D. J. 1971. Probit Analysis. Cambridge University Press, London.
Lio-Po, G.D., C.R. Lavilla, and E.R. Cruz-Lacierda (eds.). 2001. Health Management in
Aquaculture. SEAFDEC Iloilo, Philippines
Little, E. E., B. A. Flerov and N. N. Ruzluinskaya 1985. Behavioural approaches in aquatic
toxicity: A review 72-98. In: Toxic Substances in the Aquatic Environment : An
International Aspect, p. 32, P. M. Mehrle Jr, Gray R. H. and Kendall R. L. (Eds).
American Fisheries Society Water Quality Section, Bethesda, Maryland.
Otitoloju, A.A. 2001. Joint action toxicity of heavy metals and their bioaccumulation by
benthic animals in the Lagos lagoon. Ph.D. Thesis, submitted to the Department of
Zoology University of Lagos, 231p.
Oyewo, E.O. 1998. Industrial Sources and Distribution of Heavy Metals in Lagos Lagoon and
their Biological Effects on Estuarine animals. Ph.D. Thesis Department of Marine
Science. University of Lagos, 274pp.

Reed, L.J. and H. Muench. 1938. A simple method of estimating fifty percent end points.
Amer. J. Hygiene. 27:493-497.
Reyes, R. A. 1984. Basic Biology of Tilapia. Philippine (BFAR) Freshwater Aquaculture
Externsion Training Manual. Vol. III. Technology.
www.massmind.org. 2014. Tilapia Topic: Disease Treatment: Copper Sulfate. Retrieved at:
http:// /techref/other/pond/tilapia/coppersulfate.htm"> Tilapia Topic: Disease
Treatment: Copper Sulfate</A, March 16, 2014.