UNIVERSITY OF COPENHAGEN

DEPARTMENT OF PLANT AND ENVIRONMENTAL SCIENCES

TOXICOLOGY AND ECOTOXICOLOGY 2022

Poster title

Names of authors

Introduction (max 100 words)

• Why is this experiment important?
• …

Objectives (max 50 words)

• Think about keywords (e.g., in vitro, test animal, chemical names,
chemical classes, mode of action, among others)
• …

Materials and methods

• Ideally a summarized scheme with main ”ingredients”, main steps,
how you did data treatment (including R)

Results and discussion

• First the results
• Usually text with bullet points, straight to the point, saying what you
can see in the figures. E.g.: ”As can be seen from Figure 3, ”there
was a more effective inhibition of the AchE activity by methiocarb
than malathion.”
• Then, discussion, two to three bullet points on why you have the
results you have and what you would have done different
• E.g., ”We cannot explain our results as we expected that (…)
• OR ”As expected, the organophosphate did this, and the
carbamate did that, which makes sense because…”
• …

Conclusion
• One to two bullet points on the main result of your experiment

References
• References for literature IC50s
• References for hypothesis
• References for SOP
UNIVERSITY OF COPENHAGEN
DEPARTMENT OF PLANT AND ENVIRONMENTAL SCIENCES
TOXICOLOGY AND ECOTOXICOLOGY 2022

In vitro toxicity test for malathion and methiocarb

on Gammarus pulex
By Annika Grubbe Eising, Carlos Mariño Goday & Thea Simmelhack Eggen

Introduction Mode of action

 Methiocarb (carbamate) and malathion (organophosphate) are
insecticides that inhibit acetylcholinesterase (AChE) in the target
organism[1][2]. When applied the run-off can pollute water bodies, causing
adverse effects in aquatic organisms. Both insecticides are toxic to non-
target species, and it is suggested the same mechanism of AChE
inhibition applies[3]
 Malathion needs to be biotranformed[4] and binds to AChE irreversibly[3] ​
 Methiocarb binds to AChE reversibly[3]
Objectives ​
 Establish two IC50 values for methiocarb and malathion respectively
 Test hypothesis  Organophosphate will have far lower effect than in
vivo

Method
r a t
bst ATCh Thiocholine Subtract background absorbance
Su e
x ATC DTN Linear regression on ΔAbs/t
mi
h B Lam
AChE ber
DTNB Abs t- Bee
r
AChE

Enz
3 Replicates act
2-nitro-5-benzoic
+ Controls acid

Methiocarb Measure absorbance Calculate enzyme activity (Excel)

Malathio for 15 min at 412 nm & fit to log-logistic model (R)

Results and discussion

Figure 1. Normalized AChE activity (mol/min*mg protein) as a function of Figure 2. Normalized AChE activity (mol/min*mg protein) as a function of
malathion concentration (µg/L). Data shown as mean (n=3) and described with a methiocarb conc. (µg/L). Data shown as unique datapoints and described with a
log-logistic dose-response curve (Eqn: ) log-logistic dose-response curve (Eqn: )

 Malathion IC50 = 0.0027 (-0.00686-0.0234) µg/L  IC50 << literature  Methiocarb IC50 = 87.16 (23.03-151.23) µg/L  IC50 >> expected
value (4,16µg/L)  Methiocarb data (pilot) not as reliable  no replicates, wide range of
 Malathion  Toxicity after biotransformation  Expected IC50 conc.
 Malathion curve different why?  Inhibition malathion >> inhibition methiocarb  Opposite of expected

Conclusions
 Unexpected results most likely due to errors in preparing the plates
(solution concentration) and solutions (enzyme extract and subtrate mix
not preserved in cold and too much incubation time)
 Deficiencies in experiment execution prevent from extracting a sound
conclussion whether in vitro tests are reliable alternatives to in vivo tests
References​
1.​ Boran, M. et al (2007). Acute Toxicity of Carbaryl, Methiocarb, and Carbosulfan to the
Rainbow Trout (O mykiss) and Guppy (P reticulata). Turk J Vet Anim Sci, 31(1), 39-45.
2. DrugBank (n.d.): Malathion [online]. [cited 19th of October]. Updated January 02, 2022.
Available online: https://go.drugbank.com/drugs/DB00772
3.Mdeni, N. L. et al. (2022). Analytical Evaluation of Carbamate and Organophosphate
Pesticides in Human and Environmental Matrices: A Review. Molecules, 27(3). MDPI.
https://doi.org/10.3390/molecules27030618
4. Malathion Technical Fact Sheet. (n.d.). Retrieved September 29, 2022, from
http://npic.orst.edu/factsheets/archive/malatech.html#mode