Laboratory work no.

Laboratory of Ecotoxicology and LCA

Department of Environmental Chemistry, ICT Prague

Acute toxicity test on brine shrimp

(Artemia salina)

1.

Testing organism
1.1. Characteristic of organism

Brine shrimp eggs are almost exclusively produced in USA, e.g. by Ocean Star
International, Inc. company, and imported in tins. The eggs of brine shrimps are picked up in
Great Salt Lake in Utah. Eggs are washed by fresh water, dried and vacuum stored in tins.
Commonly they are used as feed for aquarium fish.
Though brine shrimp is an organism living in saline water, it is found only in lakes but
not in seas. The advantage of the test is quite homogeneity in eggs and in freshly born
individuals, respectively. Through the freshly natal individuals are immediately applied in
testing, difficult cultivation is not demanded.

a)
Fig. 1 a) Hatched Artemia nauplius

b)
Fig. 1 b) Cysts of Artemia

1.2. Storage
Unopened tin of eggs shall be stored in cold and dry place. Hatching performance of
embryos in cysts1 is guaranteed at about 1 year. Cysts up to 4 years old can be however
applied well. Opened tin has to be stored in undamaged plastic bag closed properly by elastic
band and put in fridge. Cysts must be protected against water and air moisture.

Cysts are dormant embryos in capsules. They are very resistant they survive both short-time freezing
and boiling point of water.

Laboratory work no. 6

Laboratory of Ecotoxicology and LCA

Department of Environmental Chemistry, ICT Prague

1.3. Hatching of eggs / cysts

Put little amount of cysts into saline laboratory water (1.2-3.0 % NaCl). It is
recommended to keep the water in mild motion, best by aeration with mild air flow, which
gives enough air to the organisms at the same time. It is necessary to regulate the aeration in
such way to allow the cysts circulate in water column but not to lay at the bottom or water
surface. Hatching performance of quality cysts usually approximate to 100 %. Optimum
temperature for hatching is 27-29 C, where hatching occurs approximately after within 18
hours. By lowering the temperature to 25 C sensitivity of the nauplii is not influenced
whereas the hatching time is prolonged. Cysts hatch after 24 hours at this temperature, which
is often more comfortable for laboratory practice. Non-hatched eggs usually lay at the bottom
and empty capsules float at the water surface. Vital brine shrimps are attracted by light which
allows to aggregate them to one place using dot-lit lamp and harvest them afterwards.

2.

Material
Dilution marine water
Tab. 1: Composition of laboratory marine water (g/l)

Crystallic salts
Stock solution ZR1 (20 ml/l)

Stock solution ZR2 (10 ml/l)

3.

NaCl
MgSO4.7H2O
MgCl2.6H2O
NaBr
KCl
CaCl2
NaHCO3
SrCl2.6H2O
H3BO3
NaF

23,960
10,346
325,0
51,45
29,80
29,98
20,10
2,70
0,60
0,42

Test procedure

Freshly hatched individuals, so called nauplii, are used for testing. Hatched individuals
are to be replaced from a beaker into dishes with given concentrations of tested sample using
pipette. Brine shrimps are not fed during the test. Tested sample is not aerated. Mortality of
nauplii is noticed as endpoint. Test duration usually makes 24 hours. Nevertheless, it is
recommended to determine the mortality also after longer period.

Laboratory work no. 6

Laboratory of Ecotoxicology and LCA

Department of Environmental Chemistry, ICT Prague

3.1. Basic test

Within this test percentage of died naplii of Artemia salina grown in different
concentrations is measured to determine the EC50 value. The aim of basic test should be also
determination of other values (NOEC, LOEC, LC).
Tested concentrations of examined sample diluted by laboratory marine water has to
be prepared. The concentration rate is made up by usual way, according to OC0 and OC100
values. 5 ml of tested solution is put into Petri dish with diameter of 60 mm. Samples are
tested in 2-3 replicates. 10 nauplii of brine shrimps are put into each Petri dish. According to
large water surface and enough air at the same time, it is possible to cover the dishes. No
aeration is done. Temperature of solutions should be in range of 22-25 C. Mortality of
nauplii has to be checked after 24 h and after 48, 72, eventually after 96 and 120 h. The test is
therefore focused on mortality of brine shrimps versus toxicant concentration in time. After
120 hours usually all brine shrimps die due to leaking feed.
Together with the tested sample 1 control as minimum is run. After given time died
brine shrimps are counted.
3.2. Test procedure by laboratory work

Hatching of eggs (run by assistant a day before)

o a high beaker is filled up to approximately 2/3 with marine water
o addition of about a tea spoon of frozen dry brine shrimp eggs
o the beaker with eggs is put into cultivator for 24 h with established aeration

Test start
o dilution of water leachate with marine dilution water according to Tab. 3.
o getting of empty cysts using micropipette and replacement of 10 individuals
into each testing dish
o addition of solutions of 2 concentration rates, including 2 controls into dishes
o location of the dishes into cultivator
o test is carried out by light (lamp in cultivator)

Counting and evaluation after 24 and 48 hours

visual counting of dead brine shrimps (laying at bottom)

Laboratory work no. 6

Laboratory of Ecotoxicology and LCA

Department of Environmental Chemistry, ICT Prague

Tab. 2: Artemia salina test conditions

Testing organism:
Artemia salina
Colour:
light
Size:
up to 0.5 mm
Hatching performance:
minimum 90%
Number of individuals in 1 dish:
10
Endpoint:
mortality
Test conditions:
Stable temperature and light (incubator)
Replicates:
2
Volume of tested concentration:
5 ml in 1 Petri dish
Temperature:
25 28 C
Exposition length:
48 h (for laboratory use different length possible)
Illumination:
continuous light
Chemicals:
initial solution of tested sample, laboratory dilution marine water
Instruments and equipment:
Petri dishes, beakers, micropipettes, incubator

For the laboratory purposes only basic test will be done, to spare time and material.
Before testing pH, conductivity and oxygen content in the leachate have to be determined.
Water leachate of a soil is to be diluted using dilution medium in following ratios:
1:10, 1:20, 1:40, 1:80 (see Tab. 3).
Test is carried out using two replicates.
Tab. 3: Preparation of sample concentration rate

Dilution
1:10
1:20
1:40
1:80

Leachate volume [ml]

0,45
0,24
0,12
0,06

Medium volume [ml]

4,55
4,76
4,88
4,94

Laboratory work no. 6

4.

Laboratory of Ecotoxicology and LCA

Department of Environmental Chemistry, ICT Prague

Evaluation of the test results

Make a graph on organism mortality (in %) related to logarithm of concentration of tested

compound.
Using the values on died individuals in given concentrations determine the percent of
mortality according following formula:

Mmct =
Where:
Mmct
NMm
N0

N Mm
100
N0

is mortality of individuals in time t [%]

is average number of died individuals
is initial number of living individuals put into every concentration at the test start

Assess the EC10, EC50, EC90 values using non-linear regression, where mortality is related
to decimal logarithm of concentration. Individual EC values should be determined for each
replicate separated and average value should be determined afterwards. EC50 values cannot
differ more than 30 %.

5.

References

DVOK, P., UCMAN, E., ERVENKOV, D., KONEK, K., BLECHOV, R. (1999): Utilization of bioassay with
Artemia salina in pharmacotoxicology. In: Collection of abstracts from 9th conference on Toxicity and biodegradability of wastes and chemicals significant in aquatic environment. Sol, Czech Republic,13. -15. 9.1999.
(In Czech)
DVOK, P. (1995): Modified test with A. salina for observation of effect of interactions of heterogeneous compounds. In: Collection of abstracts from 7th conference on Toxicity and biodegradability of wastes and chemicals significant in aquatic environment. Milenovice, Czech Republic. (In Czech)