A neurophysiological detector (NPD) is a hybridization of olfactory system neurons of the fish crucian carp, Carassius carassius L., with a computerized electronic device connected to a high-performance liquid chromatograph (HPLC). This system makes it possible to measure neurophysiological activities in the olfactory system of C. carassius L. after exposure of this fish to alarm pheromones. The construction of the system was presented for the first time at the 3rd International Symposium on Separation in Bio Sciences SBS 2003 in I. Brondz, et al., The Fish Olfactory System Used as an In-Line HPLC Neurophysiologic Detector NPD, 3rd Int. Symposium on Separation in Bio Sciences SBS 2003: A 100 Years of Chromatography, 13-18 May, Moscow, Russia, 2003, Abstract O- 27, p. 95. A complete paper was published in I. Brondz, et al., Neurophysiologic Detector (NPD)—A Selective and Sensitive Tool in High-Performance Liquid Chromatography, Chromatography B: Biomedical Sciences and Applications, Vol. 800, No. 1-2, 2004, pp. 41-47, and the hybridization of living cells with an electronic device has been discussed (I. Brondz, et al., International Scientific-Technical Conference Sensors Electronics and Microsystems Technology (SEMST-1), 1-5 June, (Odessa), Ukraine, 2004, Plenum Lecture, Abstract p. 17; I. Brondz, et al., The European Chemoreception Research Organization ECRO 2004 Congress, 12-15 September, (Dijon), France, 2004, Abstract P-3; and I. Brondz, et al., Biosensors as Electronic Compounds for Detector in the High-Performance Liquid Chromatography (HPLC), Electronic Components and Systems, Vol. 3, No. 103, 2006, pp. 25-27). In the present study, an HPLC equipped with an NPD was used to assess the influence of organophosphate (OP) pesticides on olfactory sensory nerves and the modification of nerve signals from the olfactory organ. The results show that exposure of the olfactory system to OP pesticides can lead to disruption of normal reflexes and to significant suppression of individual sexual activity and, as a result, to the suppression of a population.
This paper is primarily concerned with the use of neurons from the olfactory epithelium (OE) and olfactory bulb (OB) of the fish Carassius carassius L. as a physiologically specific and sensitive detection tool [
The simultaneous recording of both the Rt of physiologically active substances and their specific influence on neurons from the OE and OB makes it possible to study the nature of OPs as disruptors of sexual activity and species reproduction. Correlations between the signals from a DAD and NPD give undisputable evidence that behavioral changes of the fish C. carassius L. reflect structural changes in the signals from the olfactory organ after exposure to OPs. In addition to the direct toxicity of OPs as endocrine disruptors on the endocrine system of mammals [
Joy V. Browne wrote, “The chemosensory system is one of the earliest emerging systems in fetal development…” [
In fishes, the sensory organs responsible for smell detection and information processing have been studied for nearly a century [
The information generated as electrochemical signals in cells of the OE are conducted via the olfactory nerves (ONs) to the OB, and via the olfactory tract to the olfactory lobes of the forebrain [
The recording of electrical nervous activity of a single neuron or a few neurons with identical electrical activity patterns was described by Døving in [
The behavioral effects of alarm pheromones from fish skin extract have been thoroughly studied and are described in [
The work-up reprocessing of fish skin extract shows that at least two individual substances were present under every peak with Rt of 5.454 and 19.077. By reprocessing, it was possible to clean up the active alarm substances with Rt of 4.889 and 20.900 from the ballast. Homogeneity analyses of UV spectra by scanning of these two peaks with Rt 4.889 and 20.900 were performed and confirmed 95% homogeneity. Additional analyses of the fluorescence spectra were performed. The substances had distinct fluorescence. This finding supports the
hypothesis that the substances under these two peaks with Rt 4.889 and 20.900 were derivatives of pterins or folates; however, additional studies should be performed using mass spectrometry. The nature of alarm substances as pterins was proposed earlier by (Hüttel, 1941) [
The HPLC fractions with Rt from 19.9 min to 21.9 min were collected and combined. This liquid was introduced through thin polyethylene tubing into an aquarium with C. carassius L. The fish demonstrated behavior described previously in [
The study by Scholz et al. [
Other external observations of fish behavior after exposure to diazinon and other OP pesticides strongly support these conclusions and describe an array of other disorders in fish behavior after exposure to OP toxins. In water, under the influence of sun and oxygen from atmosphere, diazinon oxidizes to diazoxon, and the latter is more toxic in all ways than the former.
Generally, OPs are phosphate esters of many important biologically active molecules such as DNA, RNA, and cofactors, and are constituents of insecticides, herbicides, and warfare nerve agents. The general molecular formula is given in
In agriculture and the military, the term OPs has a specific meaning as insecticides and warfare nerve agents, respectively, with anti-acetylcholinesterase enzyme activity. Pesticides and OPs such as sarin and VX (nerve agent) irreversibly inactivate acetylcholinesterase.
Philippe de Clermont and Wladimir P. Moshnin (1854) in Adolphe Wurtz’s laboratory in Paris synthesized tetraethyl pyrophosphate (TEPP), the first cholinesterase inhibitor [
Sarin, or GB, is an organophosphorus compound used as a chemical weapon and has been classified as a weapon of mass destruction in UN Resolution 687. A nonlethal dose of sarin causes neurological damage in both the peripheral and central nervous systems in insects and vertebrata (including fish and mammals).
OPs are anti-acetylcholinesterase agents that attack the peripheral and central nervous systems. Their action has been described as “The primary target for OP inhibition is acetylcholinesterase (AChE)” [
inhibitors of acetylcholinesterase, which degrades the neurotransmitter acetylcholine. Preventing acetylcholi- nesterase activity through exposure to OP leads to an accumulation of a high concentration of acetylcholine in the synaptic cleft or the synaptic junction. Nerve impulses are continually transmitted to muscles, excretory cells, and the brain. OPs can affect the brain through axoplasmic transport to ONs [
Crucian carp, C. carassius L. (20 - 35 g) were caught in a small lake in the vicinity of the rural city Ski, Norway (the lake is a nature reserve in summer). The fish were transported to the aquaria facilities at the Norwegian Drug Control and Drug Discovery Institute (NDCDDI) AS, Ski, Norway, for stabilization over one month and where they were fed three times a week. Fish were initially anesthetized with benzocaine (45 mg/L) and immobilized by intraperitoneal injection of saffan (Schering-Plough Animal Health, Welvyn Garden City, UK) at 24 mg/kg body weight. To prevent drying and to avoid any unforeseen movement during the experiment, the fishes were wrapped in a wet paper cloth and fixed by two steel rods, which were fastened to the upper parts of the orbital bones [
The surgical procedure has been described in [
The active substances were introduced directly into the flow by microvalve 12 or by reconstructed Rheodyne injector 4, which allowed smooth switching of the flow between column 1 and column 2 and the injection of the biologically active substances. The change in flow of APW to APW with 10 ppm of diazinon and reversion of flow from APW with 10 ppm of diazinon to APW was performed by using Rheodyne injector 4. The exposuretime of the OE to APW with 10 ppm of diazinon in the nasal cavity of fish was 1.0 min. Relaxation between experiments was performed using APW for 6.0 min.
The control measurements were performed using only APW before every experiment. Every experiment used only one fresh fish that had not been exposed to chemicals for the one-month stabilization in the aquaria facilities. A typical recording of single-unit activity (SUA) at a recording site in the OB using only mobile phase APW is shown in
Two HPLC Brownlee™ conventional columns 250 mm in length, 4.6 mm i.d. (PerkinElmer Instruments (Shelton, USA)) were used in the experiments. The support phase of the columns was removed, and all parts of the columns were cleaned with ethanol and distilled water. The columns were packed with river sand as the support phase using vibration, and sintered filters were installed on both sides of the columns.
Preparation of the Support Phase (River Sand)
River sand was obtained from a small lake in the vicinity of the rural city Ski, Norway (the lake is a nature reservation in summer). The sand was washed with distilled water, dried, defatted with n-hexane p.a. quality (Merck), washed with 1 M NaOH p.a. quality (Merck) for 1 h, washed with 1 M HCl p.a. quality (Merck) for 1 h, and finally washed with deionized water. The columns were installed in the HPLC and were flushed with APW for 1 h before the experiments.
Each fish was prepared as described in paragraphs 2.1.1 and 2.1.2 and in publications [
Two of the 19 experiments failed because of mistakes during surgery. Figures 6(a)-(d) show characteristic results. At the start of every experiment, the basal nervous activity at the measured site was measured by passing APW from bottle 1 through the nasal cavity for 10 min. The experiment started when the nervous activity was stable. Experiments 1, 3, and 5 were as follows: 10 min at a flow rate of 0.5 mL/min of APW from bottle 1 (characteristic results are shown in
microvalve 12, as shown in
Several studies have reported observations of behavioral changes in fishes exposed to OPs [
The belief that living creatures except hominids lack intelligence or even cognitive abilities and live only on the basis of Pavlovian reflexes is not correct. If we accept that fish lacks a well-developed memory and what are at present unknown cognitive abilities, it is impossible to explain certain phenomena; for example, how salmon and other fishes can find their hatching places in rivers and lakes and return there from their feeding places by traveling many thousands of kilometers in the open oceans.
Only disruption of cognitive abilities [
To understand the toxic effects of OPs and other pesticides on the populations of salmon and other migrating fishes, several other factors should also be considered as the transport of OPs to the brain [
The mechanism of behavioral changes in fish after exposure to diazinon was visualized as a change from nervous attraction signals to sex pheromones in the brain as attractants to alarm nervous signals of repulsion and danger.
The changes toward “not return to the hatching places in the rivers and lakes” maybe a result of abnormal nerve signals from the OE to the brain and/or brain damage [
The suppression of the production of a broad spectrum of bioactive substances by pesticides [
The same toxic mechanisms as observed in fishes are applicable to all living creatures including humans [