1. Ecotoxicological assays as a means of
monitoring vulnerable aquatic ecosystems
A. Kungolos1, V. Tsiridis2, M. Petala2, C. Emmanouil1, S.
Kipouros1, D. Antoniadis3, G. Drakopoulos3, P. Dimakos1 and
K. Perakis1
1Departmentof Planning and Regional Development, University of
Thessaly, 38334 Volos, Greece
2Department of Civil Engineering, Aristotle University of
Thessaloniki, 54006, Thessaloniki, Greece
3NGO Epidro, Massalias 16, 10680 Athens, Greece

2. How to determine the hazard resulting from
pollution?
Chemical approach
Chemical analyses of the pollutants
Biomonitoring
Bioassays: effects of chemicals, natural waters,
pollutants, wastes,…on aquatic and terrestrial living
organisms

3. Why do we need Monitoring?
Water quality may be affected by:
 Spills of oil and industrial products from tanks, pipelines
 Pesticides from agricultural area, leaching pathogens
 Endocrine disrupting chemicals
 Neurotoxins, hepatotoxins from algae blooms
 Contamination from terrorist attack (toxins, microbes,
viruses, radioactive compounds)
 Accidents, sabotage etc.

4. Why do we need Biomonitoring?
 It is impossible to analyze all chemical substances, which are
brought into nature by human
 Scientists suggest that there are more than 100 000 harmful
chemicals
 Even with the most advanced instruments it is not yet
possible to detect them all
 With the help of biomonitoring, a much broader view of the
possible dangerous effects can be detected

5. Toxicity testing used in our Lab in the
University of Thessaly, Greece
 15 min inhibition test of the photobacterium Vibrio
fischeri
 72 h growth inhibition test of the microalae
Pseudokirchneriella subcapitata
 24 or 48 h immobilization test of the crustacean
Daphnia magna

6. Photobacterium Vibrio fischeri (Microtox test)
Bioluminescence inhibition
Normal bioluminescence
Photobacterium Vibrio fischeri
Contaminated sample is
added!!
Bioluminescence
measurement at exposure
Microtox M500 analyzer time 5, 15 and 30 min

7. Microalgae Pseudokirchneriella subcapitata
(Algaltoxkit F)
mobilization
Immobilized algae
Measurement of the optical
density (at 670 nm) after 72 h

8. Crustacean Daphnia magna
(Daphtoxkit F magna)
Hatching of the
ephippia
Exposure for
24 h at 20 oC
Addition of the samples and live Measurement of the
organisms in multiwell test plate immobilized/dead
organisms

9. Applications of ecotoxicity testing
Toxicity tests have been used for:
 Marine and fresh waters biomonitoring
 Toxicity testing of wastewaters and soils
 Toxicity testing of fly ash leachates
 Toxicity assessments of pure compounds, heavy metals and
pesticides
 Wastewater treatment plant applications

10. Correlation between the toxicity of lignite fly ash leachates towards
D. magna and the concentration of Cr(VI) detected in the leachates
Correlation coefficient, r= 0.961
Level of significance, p<0.01

11. Investigation of the interactive toxic effects
of chemicals on live test organisms
MODE OF INTERACTION
•Additive if there is no significant difference between
expected and observed effect
• Synergistic if the observed effect is significantly
higher
• Antagonistic if the observed effect is significantly
lower

12. Expected and observed effects
Expected
by a model based on the theory of probabilities
P(E) = P1 + P2 – P1P2/100
where
P(E): the theoretically expected additive effect of a
binary chemical mixture
Pi : the effect caused by a certain concentration of a
chemical when it acts alone
Observed
The measured effect of the binary chemical mixture

13. Combined effect of Cu2+ and Zn2+ on V. fischeri
(Synergistic effect)
100 Cu 2+ Zn 2 +
Ex pe cte d (m g /L ) (m g /L )
O bs e rve d A: 0.117 0.432
B : 0.117 0.864
80 C: 0.117 1.727
D: 0.168 1.727
E : 0.252 0.432
F: 0.252 0.864
60
Eff e c t, %
G : 0.252 1.727
40
20
0
A B C D E F G
C onc e ntr ation c ombinations

14. Comparison between theoretically expected and observed immobilization
for the combined effect of metalaxyl-M and copper on D. magna
(antagonistic action)
100
M eta la x y l-M Cu
E xp ected
(m g /L ) (m g /L )
O b served A : 10 0 .0 5
B: 10 0 .1 0
80
C : 30 0 .0 5
D : 30 0 .1 0
Im m ob ilization , %
E: 60 0 .0 5
60 F: 60 0 .1 0
40
20
0
A B C D E F
C on cen tration com b in ation s

15. Current project on ecotoxicity testing
Ecotoxicity testing of selected lake waters and treated wastewater
effluents
- Lakes Koronia and Karla
- Treated wastewater efluents from Tyrnavos region
- Ecotoxicity testing using
the photobacterium Vibrio fischeri,
the crustacean Daphnia magna and
the algae Pseudokirchneriella subcapitata
The research is funded by Ministry of Environment, Energy and
Climatic Change/Green Fund programme: Environmental Research-
Innovation- National Cooperations 2012”: “Improvement of
monitoring water quality with the use of ecotoxicological assays”

16. Environmental
Chemical analyses + Bioassays = Hazard
Assessment
 The only way to get an “integrated” answer to the hazard caused by
pollutants to the environment, is to expose organisms of the
receiving environment to these pollutants
 Chemical analysis in many cases deals with a restricted number of
compounds due to financial and technical limitations
 For a “real hazard” monitoring, phenomena such as bioavailability,
synergistic or antagonistic effects on the biota have to be examined
 A single toxicity test does not tell whether other important groups
of biota are affected by exposure to the same samples. It is thus
necessary to use a battery of tests with species representative of the
different links in the trophic chains

17. SECOTOX 2013 & CEMEPE Conference
(Conference on Environmental Management, Engineering,
Planning and Economics)
Myconos, June 24 – 28, 2013
THANK YOU
FOR YOUR ATTENTION!