Developing of a neurotoxicity biomarker for use in surface waters based on acetylcholinesterase of Cyprinus carpio L. | Developpement d'un biomarqueur de neurotoxicite en eau douce base sur l'acetylcholinesterase de Cyprinus carpio L.

Using specific inhibitors (BW284C51, Eserine, I50 OMPA) it was proved that brain cholinesterase of common carp is acetylcholinesterase (AChE) which has five molecular forms. Acetylcholinesterases (AChE : EC. 3.1.1.7) are used as specific biomarkers of organophosphorous (OP) and carbamate (C) insecticides. Incubation of the common carp (Cyprinus carpio L.) brain AChEs with the active ingredients of chlorfenvinphos, chlorpyrifos, malathion, malaoxon (OP) and carbofuran (C) showed a broad range of enzyme affinity and different in vitro inhibition rates (p). Carbofuran performed the highest inhibitory activity on AChE (Ki = 878467; p=49 ), followed by chlorfenvinphos (Ki = 54037; p=40), malaoxon (Ki = 588400; p=3.9) chlorpyriphos (Ki =48040; p=0.76), and malathion (non oxidized form) (Ki=4675). In vitro experimentation with the same pesticides allowed to determine the I50 values (concentrations inhibiting 50/ of the enzyme activity) ranging from 0.00000041 to 0.000812 M, both in single and joint inhibitory action. When studying the combined activity of pesticide mixtures, synergistic inhibitory efficiency was detected especially in case of the "chlorfenvinphos-carbofuran" and "atrazine-chlorfenvinphos" combinations. Tributylzin chlorid (TBT) had only slight inhibitory impact on the AChE activity with I50 = 700 ppm. However, the mixture chlorfenvinphos-TBT was antagonistic, as well as the combination carbofuran-TBT has not showed any interaction on AChE. The heavy metal pollutants (iron and copper) did not inhibit the AChE activity at 0.001 M. Nevertheless, when using them in combination with insecticides at 0.000001 M, they enhanced the efficiency of carbofuran, chlorfenvinphos and atrazine. After the in vitro inhibition experience, living one-year-old carp individuals were exposed for 96 hours to different doses of insecticides and subsequently their AChE activity was measured in the brain. The highest concentration of carbofuran (2.44 mg per liter) chlorfenvinphos (2.9 mg per liter) and diazinon (2.5 mg per liter) killed all the test animals after already 4 hours though there was no statistical difference between the control and the treatments. It is to mention, however, that the lowest doses after 96 hour exposure inhibited significantly the brain AChE. Chlorfenvinphos was found to be the most potent and chlorpyrifos the least active in vivo inhibitor. After 96 hour in vivo exposure of carps to a mixture of Birlan (chlorfenvinphos: 0.24 ppb) and Curater (carbofuran: 3 ppb), and to their mixture of half of these concentrations, the AChE activity decreased. The highest decrease was observed with low concentration mixtures (34/ compared to control group). The recovery of AChE activity of carps exposed to the aforementioned mixtures occurred more rapidly in the higher concentrations (38 days) than in lower concentrations, althought the recovery time of initial activity was 15 days when treated by chlorfenvinphos only. The high sensitivity of common carp brain AChE to insecticides and the long recovery time of its activity, make it a potentially good biomarker for monitoring our surface waters.