Acute toxicity test with Daphnia magna to

evaluate landfill leachate toxicity

Quelhas A. T. 1,2, Simoes-Cardoso J. 1,2, Azevedo R. M.2, Fraga M. S.2, Catalo M. B.2,
Amaral A.V.G.2, Pais V.N.2, Barros R.B.2, Martins A. S. P.2, Peralta C. C.2, Sousa A.C.A.1, Pastorinho M.R.1,3
1CICS-UBI

Health Sciences Research Centre, University of Beira Interior, Covilh, Portugal; 2Department of Chemistry, University of Beira Interior,
Covilh, Portugal; 3Faculty of Health Sciences, Department of Medicine, University of Beira Interior, Covilh, Portugal

INTRODUCTION

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MATERIALS AND METHODS

Waste volume is growing faster than the worlds population, and management of wastes is a
matter of considerable human concern[1]. Landfilling is still widely used and it is a popular
method for the ultimate disposal of solid waste materials.
Daphnia magna (female, 2 -5 week)

According to European Union legislation about waste management, landfills should be the last
resource for urban wastes [2] because the leachate products coming from landfills contain
extremely toxic chemicals to the environment. For this reason, landfill leachate toxicity
evaluation is a matter of the utmost importance, being also mandatory under the EU
legislation.

Potassium dichromate (K2Cr2O7) for

test validation

Landfill leachate

Generation of Daphnia magna

neonates (N3-N5)
0.1

0.2

0.39

0.78

3.2

1.6

0.8

0.2

0.4

mg/mL

Preparation of dilutions with the

reference toxic (K2Cr2O7)
1.56

3.13

6.25

12.5

25

Preparation of leachate dilutions

Transference of 5 neonates (<24h)
for each test vessel

50 mL of test solutions per test

vessel

Fig.1 Example of a landfill. Photo source: www.environment.gov.au 04-07-2015

There are several tests to evaluate toxicity but regulatory agencies, such as OECD, recommend
the use of acute tests with invertebrates as first screening method for the toxicity assessment
of chemicals to wildlife and humans [3].

The objective of this work is to evaluate the toxicity of a leachate from an intermunicipal
sanitary landfill using the aquatic invertebrate Daphnia magna as test organism.

Daphnia magna neonates are exposed to different concentrations for 24 hours.

Immobility was registered and the EC50 (immobilization) calculated using sigmaplot software (through sigmoidal regression using 3 parameters).

RESULTS AND DISCUSSION

1 hour analysis
0%

0%

0%

0%

0%

10%

0%

50%

100%

100%

0%

0,1%

0,2%

0,39%

0,78%

1,56%

3,13%

6,25%

12,5%

25%

Immobilization rate
Leachate concentration

24 hours analysis
0%

0%

0%

10%

0%

10%

40%

100%

100%

100%

0%

0,1%

0,2%

0,39%

0,78%

1,56%

3,13%

6,25%

12,5%

25%

Immobilization rate
Leachate concentration

Results obtained after 1 hour of exposure and 24 hours. Only the 24 hours data was used to calculate the EC50.
No. Daphnids
immobilized
after 24 hours

29.06 Toxic Units

Very toxic
Leachate EC50: 3.44%

With EC50 value, its possible to calculate the TU factor (Toxic Units),
which represents the toxicity index of any compound and allows the
comparison with the previous results or other substances.
The toxicity index was calculated with the following formula:
=1/(50 )100 =1/3.44100 = 29.06

Test with a reference substance

According to OECD recomendations: potassium dichromate (K2Cr2O7)
[K2Cr2O7]
(mg/L)
3.2
1.6
0.8
0.4
02
0

No. Initial
Daphnids
25
25
25
25
25
25

1 hour later 24 hours later

(immobile) (immobile)
8
25
2
25
1
25
1
17
0
14
0
0

No. Daphnids
immobilized
after 24 hours

[K2Cr2O7] (mg/L)

Leachate concentration (%)

DISCUSSION AND CONCLUSIONS

Considering the TU obtained (29.06) and according to the classification proposed by Pablos et al., this leachate is
considered very toxic, because the TU value is between 10 and 100 TU [5].
*According to OECD Guideline4 a reference substance may be tested for EC50 as a means of assuring that the test
conditions are reliable. At 24 hours, the concentration of the reference substance (K2Cr2O7) necessary to immobilize
50% of the individuals, i.e. EC50, should be between 0.6 and 2.1 mg/L. In this case the EC50 for the K2Cr2O7 was 0.2345
mg/L which means that the organisms were too sensitive to be used in this test.
Not reliable

EC50 K2Cr2O7

0.2345 mg/L

0.6 2.1 mg/L

There are some factors that can explain why the organisms were too sensitive, such as:
The adults daphnids that were used to provide the neonates were transported from Aveiro University and the
transport was not performed in the optimal conditions since the temperature was too high. T > 21C.
Daphnia should be fed every day and they were not fed the two days before the trials.

K2Cr2O7 EC50: 0.2345 mg/mL

Daphnids too sensitive*

The tested leachate has a toxic effect on this organism and it seems to depend on the exposure time. The calculated
EC50 for the leachate is 3.44% although the real value would be higher since the Daphnia were too sensitive. This also
shows the importance of using a reference substance.

REFERENCES
1 - Kalka J. Landfill Leachate Toxicity Removal in Combined Treatment with Municipal Wastewater; The
Scientific World Journal; doi: 10.1100/2012/202897
2 - Diverting waste from landfill, European Environment Agency report no 7/2009. ISSN 1725-9177
3 - Guilhermino, L.; Diamantino, T.; Silva, M.; Soares, A.. Acute Toxicity Test with Daphnia magna: An
Alternative to Mammals in the Prescreening of Chemical Toxicity?; Ecotoxicology and Enviromental Safety;
doi:10.1006/eesa.2000.1916
4 - OECD guideline for testing of chemicals. Daphnia sp., Acute immobilization Test. 202 adopted: 13 April
2004
5 - Pablos MV, Martini F, Fernndez C, Babn MM, Herraez I, Miranda J, Martnez J, Carbonell G, SanSegundo L,
Garca-Hortigela P, Tarazona JV (2011) Correlation between physicochemical and ecotoxicological approaches
to estimate landfill leachates toxicity. Waste Management 31:1841- 1847.

ACKNOWLEDGMENTS
The authors would like to thank the Faculty of Sciences and Faculty of Health Sciences from University of Beira
Interior, and the Health Sciences Research Centre of University of Beira Interior. Thanks are also due to Dr.
Isabel Lopes from Aveiro University for providing adult daphnids to set up the culture and to Dr. Annabel
Fernandes from the Chemistry Department (UBI) for providing the landfill leachate.