Download Effect of organophosphates malathion and temephos on

NATO SfP 982590 project workshop,
Dubrovnik, Croatia – Sep 23-26, 2010
Characterisation of
hazardous chemical
contamination – from
environmental
chemistry and
toxicology to risk
assessment
Effect of organophosphates malathion
and temephos on cholinesterase
activity in the earthworm Eisenia fetida
(Oligochaeta, Lumbricidae)
Mirna Velki, Sandra Stepić, Davorka Jarić,
Branimir K. Hackenberger
Department of Biology, Josip Juraj Strossmayer University in Osijek,
Ljudevita Gaja 6, HR-31000 Osijek, Croatia
Earthworms
 common test organisms and good indicator species for
ecotoxicological research
 they have highly differentiated organs and tissues and an immune
system that is comparable with that of vertebrates
 they can be exposed to
pesticides either through
dermal contact or by
ingestion
Biomarkers
 many biomarkers have been identified in environmental pollution
dealing with pesticides and organochlorine contaminants
 acetylcholinesterase (AChE) activity is routinely used as a
biomarker of the exposure to organophosphates and carbamates
compounds
 many unsolved questions in biomarker applicability in real time
environmental pollution monitoring
 effects of small so called sublethal doses
Experiment
 adult earthworms (Eisenia fetida) were exposed to the sub-lethal
concentrations of organophosphates malathion and temephos
 contact filter paper test procedure
 ChE activity was determined according to the method of Ellman et
al. (1961)
Experiment
Malathion
 one of the most often used
organophospahte insecticide in the
world
 insecticide and acaricide with contact,
gut and respiratory action
 in insects and mammals is
metabolically converted to its
structurally-similar metabolite,
malaoxon
 malaoxon inhibits AChE resulting in
respiratory, myocardial and
neuromuscular transmission
impairment
structure of malathion
Experiment
Temephos
 nonsystemic organophosphorus insecticide used to
control mosquito, midge, and black fly larvae
 it is used in lakes, ponds, and wetlands
 only organophosphate with larvicidal use
 temephos affects the central nervous system
through inhibition of cholinesterase
 in larvae, this results in death before reaching the
adult stage
structure of temephos
Experiment
PESTICIDE
CLASS OF PESTICIDE
TYPE OF PESTICIDE
MODE OF ACTION
MALATHION
organophosphate
acaricide,
insecticide
indirect inhibitor of
cholinesterase
TEMEPHOS
organophosphate
insecticide
(larvicidae)
cholinesterase inhibitor
Experiment
 PRELIMINARY EXPERIMENT
 Determination of EC50 and LC50 values
 Choosing the concentrations for final experiments
 FINAL EXPERIMENT
 Determination of ChE activity
MALATHION
TEMEPHOS
0.05
ng/cm2
0.1
ng/cm2
0.2
ng/cm2
0.4
ng/cm2
0.6
ng/cm2
1
ng/cm2
4
ng/cm2
8
ng/cm2
0.025
ng/cm2
0.01
ng/cm2
0.05
ng/cm2
0.12
ng/cm2
0.25
ng/cm2
0.05
ng/cm2
1.24
ng/cm2
2.5
ng/cm2
Results
Concentration-response curves for 24, 48, and 72 h of exposure to different
temephos concentrations with mortality as an endpoint, calculated from the
first-preliminary experiment. Curves representing: (white) 24h, (grey ) 48 h,
and (black) 72 h of exposure.
Results
Activity of ChE measured in E. fetida earthworms after exposure to
different concentrations of temephos for 2 h.
*Significantly different vs. control activity (p<0.05).
Results
Activity of ChE (nmol min-1 mg-1)
50
*
45
*
40
35
30
*
25
20
15
10
5
0
0
0.05
0.1
0.2
0.4
0.6
1
4
8
Concentration of malathion (ng cm -2)
Activity of ChE measured in E. fetida earthworms after exposure to
different concentrations of malathion for 2 h.
*Significantly different vs. control activity (p<0.05).
low dose stimulation,
high dose inhibition
opposite effect in small doses
(sub-lethal, sub-effect)
compared to large doses
 Role of hormetic effect?
 Importance of the hormetic effect?
INTERPRETATION OF RESULTS, ESPECIALLY
IN INTERPRETATION OF ENVIRONMENTAL
MONITORING
↓
ENVIRONMENTAL RISK ASSESSMENT
 Response to:
 Low-level concentrations
 Time!
Objective of biomonitoring
 early detection of the biologically relevant concentrations
of pollutants
 early detection of the effect of pollutants on biological
systems
 Important to detect any variation from BENCHMARK
VALUES for test organisms
 Mathematical models which describe response on
presence of pollutant often show high sensitivity on so
called starting conditions
↓
Mixtures!
Is hormesis doubtful?
Why is hormetic effect difficult to prove?


Small concentrations!
(researchers usually work with concentrations in range of
expected response)
Biomarkers often show relatively large variance
(especially measured in situ organisms; in vivo)
↓
using cell cultures could result in more accurate results and more
precise determination of hormetic effect
Hormetic effect is NOT doubtful!
1.
2.
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...
...and many others in which hormesis has been described,
but not defined!
1.
2.
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4.
Bilgihan, A., Turkozkan, N., Isman, F., Kilinc, M., Demirsoy, S., 1998. The effect of
different doses of epidermal growth factor on liver ornithine decarboxylase and Na-K
ATPase activities in newborn rats. Gen. Pharmacol. 31(2), 261-263.
Ono, T., Inoue, M., Rashid, M.H., Sumikawa, K., Ueda, H., 2002. Stimulation of peripheral
nociceptor endings by low dose morphine and its signaling mechanism. Neurochem. Int.
41(6), 399-407.
Nyitrai, P., Bóka, K., Gáspár, L., Sárvári, E., Lenti, K., Keresztes, A., 2003.
Characterization of the stimulating effect of low-dose stressors in maize and bean seedlings.
J. Plant Physiol. 160(10),1175-1183.
Lambrou, G.I., Vlahopoulos, S., Papathanasiou, C., Papanikolaou, M., Karpusas, M.,
Zoumakis, E., Tzortzatou-Stathopoulou, F., 2009. Prednisolone exerts late mitogenic and
biphasic effects on resistant acute lymphoblastic leukemia cells: Relation to early gene
expression. Leuk. Res. 33(12), 1684-1695.
Conclusions
 Some dose-response curves can show an inverted Ushape characteristic for hormesis as it was seen by
temephos and malathion in earthworms.
 This hormetic-like effect could be important for health
status of an earthworm as well for correct
interpretation of biomonitoring results.
Conclusions
 The significance of hormetic effect detection:
 1) in environmental biomonitoring, where very low
concentrations are usually expected, it is obvious that
hormetic effect can theoretically be anticipated as a
marker of exposure to such concentrations
 2) when earthworms are used for toxicological
parameters research, models that include hormetic
effect should be taken into account
THANK YOU FOR YOUR ATTENTION!
Questions... ?