Multi-Marker Approach for Evaluating Environmental Impacts of Neonicotinoids on Aquatic Ecosystems

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Multi-marker approach for the
evaluation of environmental
impacts of 
insecticides
 on aquatic
ecosystems
 
David Somogyvári (david.somogyvari@gmail.com)
PhD student
University of Pannonia, Department of Limnology, Veszprém
Eötvös Loránd Research Network Balaton Limnological Research Institute,
 Ecophysiologycal and
Environmental Toxicologycal Research Group
, Tihany
 
Supervisors:
 
Prof. Dr. Judit Padisák 
(University of Pannonia)
Dr. János Győri (
Eötvös Loránd Research Network)
 
Introduction: Neonicotinoids
 
Neonicotinoids are considered among the most important
classes of pesticide active ingredients currently used in agricultural crop protection.
Neonicotinoids mimic the action of
 
acetylcholine (ACh), one of the main excitatory
neurotransmitters of the central nervous system and the primary mechanism of action is their
strong binding to the postsynaptic
 
nicotinic acetylcholine receptors (nAChRs) showing a more
selective pharmacological/
 
toxicological profile to arthropod than vertebrate receptors.
 
Recent detection of clothianidin in surface waters has
 
raised interest in characterizing the
potential impacts of
 
this insecticide to aquatic organisms as well.
 
Evaluation of
 
locomotor activity as a potential biomarker of exposure of
environmental stressors was suggested to monitor freshwater
 
ecosystems
.
 
N
e
o
n
i
c
o
t
i
n
o
i
d
s
 
 
Toxi
c
 metabolit
es
  - imidacloprid / desnitro/imidacloprid
                                                    - thiamethoxam - clotianidin
 
„Third generation”  pesticides
Nitro
- and 
cyano
-substitution
 
Wos   2014  n =  206               
Wos
  
2015      =   196
 
Kohinor  -  
imidacloprid
Mospilan -  
acetamiprid
Calypso  -    
thiacloprid
Actara
 -   thiamethoxam
Apacs - 
      clothianidin
 
1mg
 
1g
 
1
μ
g
 
Insects
 
Artemia !
 
Fish
 
Molluscs
 
Daphnia !
 
Toxicity of neonicotinoids – aquatic  
(
μ
g/l)
 
Imidacloprid in soil -  
13.05 to 71.8 ug/l
   Jaume 2015Egy
 
Neonicotinoids in surface water: Aquatic animals
 
Land water content:
Chronic toxicity
Chronic toxicity
Acute toxicity
Acute toxicity
Measured Evironmental Concentration
Measured Evironmental Concentration
fish
 
Aim
 
S
tudy the 
behaviour patterns of
 Gammarids 
by
measuring distance of swimming, immobilization
time and pleopod beating 
using 
neonicotinoid
insecticides.
Determination of LC50 of Gammarids exposed to
neonics.
 
Aquatic model animal and
breedeing conditions
D
ikerogammarus villosus
 
 
 
 
Locomotor activity assay
 
This assay was carried out with three groups, one
control and two treated groups, in each group ten
animals were selected. The concentration of APACS
50WG was 3.9 ng l−1 dissolved in Balaton water.
Individual animals were placed in 6-well plates,
and measurements of swimming behaviour were
performed by video tracking system, taking short
(90 s) records using a camera. The videos were
analysed with Fiji ImageJ 1.52p software. The
programme recorded the coordinates and
automatically reconstructs the trajectories of the
test animals swimming within the ring of the whole
of the plate
.
 
Locomotor activity test
 
Behaviour test 1
 
Behaviour test 2
 
Behaviour test 3
 
Determination of LC50 concentration
 
Conclusion
 
We conclude that at a low concentration of neonicotinoid the oxidative damage evoked
by
 
the neonicotinoid impairs the intracellular compensatory mechanisms, yielding to
increased activity against the
 
neonicotinoid-induced toxicity. Our results underline
 
the
importance of non-standard aquatic toxicity studies
 
using 
Gammarides
.
 
Future plans
 
 
Investigate 
other behavioural patterns (etc. beating of pleopods of
gammarids, distance of swimming) using clothianidin and other neonics.
 
Quantification of LOEC of Gammarides using neonics.
 
 
Our results are expected confirm or exclude that neonicotinoids, at
concentrations commonly found in surface and groundwater, alter a
variety of physiological and pathological processes in animal model
systems.
 
The chronic effects of neonicotinpoids and the metabolites on
biochemical, reproductive, and survival parameters in a non-target
animals are described to predict long-term effects on an ecosystem.
 
Based on biomarker and behaviour studies, we provide more realistic
assessment of impacts and exposure of aquatic organisms to neonicotinoids
present in water bodies.
 
Results obtained to this class of systemic and persistent ingredients serve
as a tool for modelling of the environmental fate of bioactive
contaminants in the ecosystem.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Acknowledgement
 
Hungarian Scientific Research Fund No OTKA K112712.
Identification of neurotoxic biologically active compounds
and their metabolites and chemical / ecotoxicological
characterization
 
of their effects
 
 
References
 
 
Benzidane Y et al (2010) Effect of thiamethoxam on cockroach locomotor activity is
associated with its metabolite clothianidin. Pest
Manag Sci 66(12):1351–1359. https://doi.org/10.1002/ps.2022
Felten V et al (2008) Physiological and behavioural responses of Gammarus pulex
exposed to acid stress. Comp Biochem Physiol C
Toxicol Pharmacol 147(2):189–197. https://doi.org/10.1016/j.
cbpc.2007.09.006
Tomizawa M, Casida JE (2005) Neonicotinoid insecticide toxicology: mechanisms of
selective action. Annu Rev Pharmacol
Toxicol 45(1):247–268. https://doi.org/10.1146/annurev.pharm
tox.45.120403.095930
Wallace WG, Estephan A (2004) Differential susceptibility of horizontal and vertical
swimming activity to cadmium exposure in a
gammaridean amphipod (Gammarus lawrencianus). Aquat Toxicol 69(3):289–297.
https://doi.org/10.1016/j.aquatox.2004.05.010
 
Thank You for your kind attention!
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Dear Audience! I would like to present some results of last year of our research in Balaton Limnological Intitute.

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Neonicotinoids, a class of insecticides, are widely used in agriculture but pose environmental risks to aquatic organisms. This study by David Somogyvári explores the effects of neonicotinoids on aquatic ecosystems using a multi-marker approach, focusing on the toxicity levels and potential impacts on various aquatic species. The research highlights the importance of monitoring neonicotinoid exposure and its effects on freshwater environments for better environmental management strategies.


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  1. Multi-marker approach for the evaluation of environmental impacts of insecticides on aquatic ecosystems David Somogyv ri (david.somogyvari@gmail.com) PhD student University of Pannonia, Department of Limnology, Veszpr m E tv s Lor nd Research Network Balaton Limnological Research Institute, Ecophysiologycal and Environmental Toxicologycal Research Group, Tihany Supervisors: Prof. Dr. Judit Padis k (University of Pannonia) Dr. J nos Gy ri (E tv s Lor nd Research Network)

  2. Introduction: Neonicotinoids Neonicotinoids are considered among the most important classes of pesticide active ingredients currently used in agricultural crop protection. Neonicotinoids mimic the action of acetylcholine (ACh), one of the main excitatory neurotransmitters of the central nervous system and the primary mechanism of action is their strong binding to the postsynaptic nicotinic acetylcholine receptors (nAChRs) showing a more selective pharmacological/ toxicological profile to arthropod than vertebrate receptors. Recent detection of clothianidin in surface waters has raised interest in characterizing the potential impacts of this insecticide to aquatic organisms as well. Evaluation of locomotor activity as a potential biomarker of exposure of environmental stressors was suggested to monitor freshwater ecosystems.

  3. Neonicotinoids Third generation pesticides Nitro- and cyano-substitution Nicotine Kohinor - imidacloprid Mospilan - acetamiprid Calypso - thiacloprid Actara - thiamethoxam Apacs - clothianidin Toxic metabolites - imidacloprid / desnitro/imidacloprid - thiamethoxam - clotianidin Wos 2014 n = 206 Wos2015 = 196

  4. 2 Toxicity of neonicotinoids aquatic ( g/l) 1.5 1 Insects Molluscs Fish 0.5 Daphnia ! Artemia ! 0 1 10 100 1000 1mg 10000 100000 1000000 1g 1 g Taxon LD50 ug/l 2.1 5.75 5.9 13 30.4 59 65.4 100 350 540 6000 10000 43900 85000 211000 280000 241000 361000 reference Chen et al., 2010 Stoughton et al. (2008 Sanches-Bajo and Goka 2012 Song and Brown 1999 Sanches-Bajo and Goka 2012 Barbee and Stout 2009 Stoughton et al. (2007 Ashauer 2011 Mineau and Palmer Nyman et al Nyman et al 2014 Dondero et al 2010 Morrisey et al 2015 Ashauer 2011 Tomlin1997 Anderson (USEPA 2003) Tisler et al 2009 Song and Brown 1998 Ceriodaphnia Chironomus insect "geometric mean" Aedes cladoceras general Crayfish Procambarus Hyalella azteca (amphipoda) Gammarus Gammarus snail: Lymnaea snail: Planorbella mussel Mytilus Daphnia "geometric mean" Daphnia fish: rainbow trout fish: Cypronus carpio fish: Danio rerio Artemia salina Imidacloprid in soil - 13.05 to 71.8 ug/l Jaume 2015Egy

  5. Neonicotinoids in surface water: Aquatic animals Measured Evironmental Concentration EC50 Daphnia [mg L-1] k rnyezeti konc. LOEL hal LOEL Daphnia LC50 hal fish Land water content: = kr nikus toxicit s Chronic toxicity = akut toxicit s Acute toxicity acetamiprid Country Sample containing neonicotinoids [%] acetamiprid thiacloprid thiamethoxam mg L-1 Sweden 9 0 3 0,002 0,2 1 25 50 75 100 125 150 500 Switzerland - 36 60 Canada 0 5 - 70 thiamethoxam USA 1 17 25 35 47 Japan 50 - 100 mg L-1 0,002 0,2 1 25 50 75 100 125 150 500 Australia 70 80 27 thiacloprid mg L-1 0,002 0,2 1 25 50 75 100 125 150 500

  6. Aim Study the behaviour patterns of Gammarids by measuring distance of swimming, immobilization time and pleopod beating using neonicotinoid insecticides. Determination of LC50 of Gammarids exposed to neonics.

  7. Aquatic model animal and breedeing conditions Dikerogammarus villosus

  8. Locomotor activity assay This assay was carried out with three groups, one control and two treated groups, in each group ten animals were selected. The concentration of APACS 50WG was 3.9 ng l 1 dissolved in Balaton water. Individual animals were placed in 6-well plates, and measurements of swimming behaviour were performed by video tracking system, taking short (90 s) records using a camera. The videos were analysed with Fiji ImageJ 1.52p software. The programme recorded the coordinates and automatically reconstructs the trajectories of the test animals swimming within the ring of the whole of the plate.

  9. Locomotor activity test

  10. Behaviour test 1

  11. Behaviour test 2

  12. Behaviour test 3

  13. Determination of LC50 concentration Total number of organisms Number of dead animals Corrigated death % PROBIT corrigated death % Concentration Log 10 concentration Death % 50 100 150 250 1.70 2 2.18 2.40 10 10 10 10 3 7 9 10 30.00 70.00 90.00 10.00 30 70 90 100 4.48 5.52 6.28 7.33 LC50=decimal-based exponentiation of intersection intersection LC50 (ng/l) 1.84 69.18

  14. Conclusion We conclude that at a low concentration of neonicotinoid the oxidative damage evoked by the neonicotinoid impairs the intracellular compensatory mechanisms, yielding to increased activity against the neonicotinoid-induced toxicity. Our results underline the importance of non-standard aquatic toxicity studies using Gammarides.

  15. Future plans Investigate other behavioural patterns (etc. beating of pleopods of gammarids, distance of swimming) using clothianidin and other neonics. Quantification of LOEC of Gammarides using neonics. Our results are expected confirm or exclude that neonicotinoids, at concentrations commonly found in surface and groundwater, alter a variety of physiological and pathological processes in animal model systems. The chronic effects of neonicotinpoids and the metabolites on biochemical, reproductive, and survival parameters in a non-target animals are described to predict long-term effects on an ecosystem. Based on biomarker and behaviour studies, we provide more realistic assessment of impacts and exposure of aquatic organisms to neonicotinoids present in water bodies. Results obtained to this class of systemic and persistent ingredients serve as a tool for modelling of the environmental fate of bioactive contaminants in the ecosystem.

  16. Acknowledgement Hungarian Scientific Research Fund No OTKA K112712. Identification of neurotoxic biologically active compounds and their metabolites and chemical / ecotoxicological characterization of their effects

  17. References Benzidane Y et al (2010) Effect of thiamethoxam on cockroach locomotor activity is associated with its metabolite clothianidin. Pest Manag Sci 66(12):1351 1359. https://doi.org/10.1002/ps.2022 Felten V et al (2008) Physiological and behavioural responses of Gammarus pulex exposed to acid stress. Comp Biochem Physiol C Toxicol Pharmacol 147(2):189 197. https://doi.org/10.1016/j. cbpc.2007.09.006 Tomizawa M, Casida JE (2005) Neonicotinoid insecticide toxicology: mechanisms of selective action. Annu Rev Pharmacol Toxicol 45(1):247 268. https://doi.org/10.1146/annurev.pharm tox.45.120403.095930 Wallace WG, Estephan A (2004) Differential susceptibility of horizontal and vertical swimming activity to cadmium exposure in a gammaridean amphipod (Gammarus lawrencianus). Aquat Toxicol 69(3):289 297. https://doi.org/10.1016/j.aquatox.2004.05.010

  18. Thank You for your kind attention!

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