2017 年 42 巻 1 号 p. 7-15
Propanil stress response in the eel (Anguilla anguilla) was examined. Eels were exposed to 3.16 mg/L for 72 hr and allowed to recover for 96 hr. Plasma levels of cortisol, lactate dehydrogenase (LDH), alkaline phosphatase (AP), aspartate aminotransferase (AST), cholesterol, triglycerides, glucose, ammonium, lactate, albumin, and total proteins as well as electrolytes (chloride, sodium, potassium, calcium and phosphorus) were determined. As a consequence of exposure, cortisol, AP, AST, and LDH increased. A hyperglycemic condition, together with hyperlactemia, hypoalbuminemia, hypoproteinemia, hypercholesterolemia and hypertriglycemia was registered. Ammonium increased during exposure concomitantly to hyponatremia, hypochloremia, hypocalcemia, hypophosphatemia, and hypokatremia. During recovery, chloride, sodium, potassium, ammonium, albumin and LDH normalized. At the end of the experiment, fish still exhibited hyperglycemia and hyperlactemia. Hypercalcemia was observed. Cholesterol, triglycerides, AP, and AST did not recuperate. These findings are important for assessing potential risks for areas where fish are grown near intensive herbicide use (i.e., paddy fields).
The European eel (Anguilla anguilla) is a particular fish, specially monitored by EU protection directives.1) Member States are invited to practice freshwater restocking of yellow-stage (freshwater non-reproductive stage) eels for the purpose of improving the future escapement of the silver stage (sexual maturation stage) population mass to the seas. There is great interest in the toxic effects of pesticides on the yellow stage of this fish because there are numerous changes associated with profound alterations in the morphology and physiology of the animals to prepare them for the silver stage. Yellow eels pass most of their lives in localized freshwater areas, where they exhibit benthic habits, burying themselves in sediment. This, together with their carnivorous diet, greatly enhances the chance of exposure to pollutants.2)
Propanil (3,4-dichloropropioanilide) is a selective contact herbicide used to control barnyard grass (Echinochloa crus-galli) and broadleaf weeds. It is currently applied as a post-emergence herbicide in many paddy fields in wetlands regions of the world. Santos et al.3) monitored propanil in surface water samples from rice fields in the Ebro Delta area (Tarragona, Spain), following the agricultural application of 12 mg/L and half-life between 1.24 and 3.83 days after application. The same study reported that propanil half-lives under laboratory conditions of 35°C under light irradiation, with and without the addition of humic acid were 2.95 and 1.73 days, respectively (initial concentration of 10 µg/L). Concentrations of propanil of 50–100 µg/L in drainage waters have been reported in rice fields of the muda irrigation scheme in Kedah (Malaysia).4)
More than 95% of the sprayed herbicides reached the target species; however, the rest may affect non-target species, air, water, bottom sediments, and food. The environmental pollution caused by the application of herbicide in the rice field areas is heaviest in the water. Indiscriminate use of such pesticides could cause undesirable effects on the rice ecosystem and farmers’health.4) Changes in rice management practices have greatly reduced the concentration of pesticides in runoff waters. However, the alternative is not always as easy as expected. The addition of propanil mixed with the herbicide imazethapyr proved to be beneficial with economic returns increased by 29 to 70% in crop fields (Louisiana), even though the cost of treatment increased.5) The freshwater medium around agricultural areas is particularly sensitive to the influence of pesticides. In these areas sublethal effects are more subtle than acute. Sublethal effects do not cause death in the short term but do harm the individual, thus making it expend resources to survive in a state of altered equilibrium. The physiological changes associated with the fish response to environmental low-pesticide-levels not only provide a means of understanding environmental levels of pollutants in biological terms but also can be used as a model for the toxicity of vertebrates including man.
Pesticide toxicity is one of the main causes for the decline of fish populations in freshwater environments by increasing fish vulnerability, which may interfere with energy reserves and appropriate growing processes.6) The harmful effects (specially sublethal ones) retard the development of surviving individuals and/or exert a harmful influence on their normal metabolic processes.3) Propanil residue in the water during the first week following application may be sufficiently high to adversely affect fish populations if fish inhabit rice paddy floodwaters or if the residue passes over fish spawning and nursery grounds.7)
Stress is a mechanism of physiological compensation that an organism adopts in response to a stressing physical or chemical factor. Collectively, those compensations are called the General Adaptation Syndrome (GAS) because they form a group of stereotyped responses that do not differ from the original cause.6,8,9) The stress responses are adaptive and usually helps animals cope with changes in their environment. Under stress, fish are somewhat similar to other vertebrates in that they mobilize and use carbohydrates. If the stressful condition continues, the adrenal cortex is stimulated to release increased amounts of cortisol that sustain the changes caused by adrenalin and cause the mobilization of some body proteins into plasma amino acids and an assortment of other physiological changes. In addition, cortisol is related to ion balance, ion movement across membranes and plasma osmolality as well.9) Electrolytes are distributed in solution throughout all body fluids. The maintenance of constant internal ion concentrations is essential for the active regulation of water influx and ion efflux in fresh water. Any imbalances in the levels of these ions in animals lead to the impairment of various physiological activities. Ions such as calcium, sodium, and potassium play a significant role in keeping the hyperosmotic properties of freshwater fish to their medium.
The objective of the present study was to uncover the physiological sensitivity of young eels toward subacute short-term exposure to the herbicide propanil under controlled laboratory conditions. The stress response (the general adaptation syndrome) was used as a key indicator of herbicide poisoning. Furthermore, the intoxicated fish were allowed to recover in herbicide-free water. The selected parameters may be useful as a diagnostic test for propanil in intoxicated fish after spray operations in the field.
European eels, Anguilla anguilla, were obtained from a fish farm (Valencia, Spain). Yellow eels (47.75±18.20 g in weight; 29.81±4.07 cm in length) were selected to minimize the effects of sex variation because no sex differences are observed at this development stage. Fish were acclimatized to laboratory conditions for one week in 200-L glass aerated water tanks before starting the experiments.10) The tanks were supplied with a continuous flow of aerated and dechlorinated tap water (temperature: 20±1°C; total hardness: 240±10 mg/L as CaCO3 according to the Merck classification, Aquamerck 8039, Germany; pH 7.9±0.2 using a Crison pH-meter; alkalinity 4.0±0.5 mmol/L, Aquamerck 11109, Germany). The light/dark period was 12 hr/12 hr. Eels were feed once a day with fish food provided by the fish farmer. Feces were removed from the test aquaria every day. The University of Valencia (Spain) guarantees that all experimental fish used in the present study were maintained in the laboratory following national and institutional guidelines for the protection of animal welfare in accordance with our Institutional Animal Care and Use Committee. All animals were healthy during the acclimatization period. Mortality was observed neither during the acclimatization period nor during the experimental time.
2. Test chemicalTechnical grade propanil (3,4-dichloropropionanilide) used in the experiments was 80% pure (IVIA, Valencia, Spain). Stock solutions were prepared by dissolving propanil directly in the experimental water immediately before the experiments.
Gas chromatography analysis confirmed the presence of propanil in the water at the desired concentration over the entire exposure time.11)
3. Subacute short-term testAfter acclimatization, test animals were transferred to test aquaria (150-L volume) that were supplied with a continuous flow of dechlorinated tap water, maintaining the same abiotic conditions as well as the temperature and photoperiod as previously. Fish were fed once a day during the entire experiment with commercial fish food provided by the farmer at a level of 2% of their body weight per day. Uneated food and feces were removed every day.
The sublethal propanil concentration used 3.16 mg/L (1/10 of 96-hr LC50), was previously determined in our laboratory.12)
Experiments were carried out in a continuous flow-through system (Tª 22°C) based on OECD Guidelines10) and maintained by peristaltic pumps (D25 V Dinko, Barcelona, Spain) in 150-L aerated glass aquaria. Eels were exposed to propanil (3.16 mg/L) for 72 hr and then a recovery period of 96 hr in clean water was allowed. For the exposure period, the herbicide had been previously dissolved in water making a 4-L stock solution and this solution was supplied to a glass mixing chamber with tap water which was connected to a perfusor pump that generated a constant solution flow (2.44 mL/min). The outlet was connected to the 150-L aerated test aquarium. This diluted the pesticide to the desired concentration by a constant water flow. In this way, the aqueous test solution was renewed 3.7 times per day. This system was connected 24 hr before the start of the experiments to obtain balanced propanil contaminated water in the test aquaria.
After 2-, 24-, 48- and 72-hr exposure to propanil, five eels were randomly removed from the aquaria, rinsed with tap water and anaesthetized with MS-222 (ethyl 3-aminobenzoate, methanesulfonic acid salt 98%), Aldrich Chemical Corporation Inc. Milwaukee, WI, USA) at a concentration of 100 mg/L.12)
Blood was removed directly from the caudal vein with a heparinized syringe (1 mL) and centrifuged using an Eppendorf centrifuge (10 min, 4000×g, 4°C) within 30 min of collection. Finally the plasma samples were frozen (−80°C) until analysis.
An additional group of animals pre-exposed for 72-hr pre-exposed was transferred to a different aquaria with herbicide-free water for a recovery period of 96 hr in the same conditions as described above. At 48, 72 and 96 hr of this period (a total of 168 hr), five eels were removed, rinsed with tap water, anaesthetized, weighed, and measured, and their plasma was obtained and stored at −80°C.
Control groups were kept in 150-L test aquaria in the same experimental conditions but without propanil.
4. Analysis proceduresPlasma electrolytes (potassium, calcium and phosphorus), and metabolites (glucose, ammonium, lactate, cholesterol, triglycerides, total proteins and albumin), in conjunction with enzyme biomarkers lactate-dehydrogenase (LDH, EC 1.1.1.27), aspartate aminotransferase (AST, EC 2.6.1.1.), and alkaline phosphatase (AP, EC 3.1.3.1.) were determined using an automated system with adequate standards. All biochemical determinations were carried out using adequate diagnostic reagents. Methods provided by Kemia Científica S.A. (Spain), Spintrol Calibrator (Ref. 1002010) and Spintrol “H” Normal (Ref. 1002120) were used to calibrate the apparatus for plasma component control, respectively; using a Chemistry Profile Analyzer (Sinnowa B200, Full automatic biochemistry analyzer). The final temperature of measurements was 37±1°C, corresponding to the incubation CPA temperature. Plasma chloride and sodium were determined by a flame photometer (EOS flame). The final concentration of electrolytes was expressed as mEq/L. Cortisol hormone was measured by using the Cortisol Determination NovaTec Immunodiagnostic GmbH (DNOV001) provided by Kemia Científica S.A. (Spain). The color intensity was measured at λ 450 nm in a microtiter plate reader (Victor2V 1420-040, Perkin Elmer).
Metabolites were expressed as milligrams per 100 mL of volume. LDH, AP and AST activities were expressed as UI/L and cortisol was expressed as ng/mL of plasma.
5. Statistical analysisIndividual eels were grouped in intervals of time according to their exposure and recovery times. Mean values and standard deviations were calculated for each test group based on the values obtained for each individual from five fish. Variables (no transformed data) were tested for normality (Kolmogorov–Smirnov test with Lilliefors significance correction) and variance homogeneity (Levene’s test). These results were compared to determine the toxic effects of a treatment by one-way analysis of variance (ANOVA) and Dunnett’s Post Hoc test was used to find differences between experimental and control groups. Logarithm transformation was applied to the variables that failed Levene’s test. Tukey’s honestly significant difference (HSD) test was used to assess differences between the different control groups. The statistical analysis was performed using the Statistical Analysis System (SPSS 17.0) for Windows (SPSS, USA). The significance level was set at 0.05.
Table 1 shows the mean values of the selected parameters measured in control animals. Previous works12,13) showed that both laboratory conditions and fish handling had no effects on the physiological status of A. anguilla during the acclimatization period. Levels of the selected parameters were not affected in eels incubated without propanil for 168 hr.
| Parameters | Mean±SEM |
|---|---|
| Lenght (cm) | 29.33±1.44 |
| Weight (g) | 45.35±7.14 |
| Cortisol (ng/mL) | 18.45±1.54 |
| AP (nmol/min/mL) | 35.46±7.09 |
| AST (micromol/min/mL) | 0.21±0.01 |
| LDH (UI/L) | 563.6±51.85 |
| Albumin (mg/100 mL) | 1.56±0.03 |
| Lactate (mg/100 mL) | 0.6±0.089 |
| Ammonium (mg/100 mL) | 22±6.3 |
| Glucose (mg/100 mL) | 186.24±20.76 |
| Cholesterol (mg/100 mL) | 267.6±30.9 |
| Triglycerides (mg/100 mL) | 186.7±15.2 |
| Total proteins (g/100 mL) | 4.48±0.36 |
| Phosphorus (mg/100 mL) | 24.55±0.88 |
| Chloride (mEq/L) | 77±7.7 |
| Potassium (mEq/L) | 6±0.67 |
| Sodium (mEq/L) | 177±5.06 |
| Calcium (mg/100 mL) | 0.34±0.06 |
AP=alkaline phosphatase, LDH=lactate dehydrogenase, AST=Asp-Aminotransferase
Cortisol hormone (Fig. 1) increased in the plasma of exposed eels. Differences with control animals were statistically significant (p<0.05) after 24 hr of treatment. Eels exhibited increased levels of cortisol until 72 hr of permanency in the recovery period.
LDH levels increased in fish exposed to the herbicide (Fig. 1), this increase was statistically significant from 24–48 hr. The highest level of activity was found at 48 hr of exposure with a 240% increase over controls. From this time onward LDH activity tends to diminish. At the end of the recovery period LDH activity had recovered.
Alkaline phosphatase (AP) and AST significantly increased in the eels as a consequence of propanil exposure. Both activities remained high during the recovery period (Fig. 1).
Hyperglycemia (Fig. 2) was statistically significant after 24 hr herbicide exposure (+118%). Glucose levels continued to be increased during the overall experimental time including the recovery period. Great disturbances also were determined in lactate levels during the experiment (Fig. 2). Significant differences from control values were found after 2 hr (+100%). Pre-exposed eels also showed significant hyperlactemia (+133%) during the recovery period.
Significant hypercholesterolemia and hypertriglycemia were determined during exposure to propanil. Cholesterol levels increased in plasma by 34%–45% over control during exposure and triglycerides increased by 100% throughout the complete exposure phase. When eels were transferred to clean water the levels did not reach the same values as the corresponding controls, however a tendency of reduction was observed.
Hypoalbuminemia was registered in the exposed eels as compared with controls. The lowest level (−84%) of albumin was determined at 48 hr of exposure to the herbicide (Fig. 2). Albumin content recovered in pre-exposed eels when transferred to herbicide-free water. The total protein content also decreased by 40% during exposure to propanil. Normal values were re-established once fish were transferred into clean water.
In A. anguilla exposed to propanil for 72 hr disturbances on the plasma ionic profile of the individuals were also induced as shown in Fig. 3. These disturbances were already observable after 1 hr of exposure.
A gradual hyponatremia was exhibited in eels from the first hr of measurement and was statistically significant during propanil exposure but not during the recovery period.
Plasma chloride levels decreased rapidly, and this decrease was maintained during overall exposure. Once the eels were allowed to recover, plasma chloride levels tended to normalize by the end of this period.
The total calcium in the plasma of exposed eels decreased. Differences from controls animals were statistically significant after 2 hr of exposure. During the recovery period the total calcium increased in the plasma of eels pre-exposed to propanil.
Potassium levels also decreased during exposure; differences from control groups were statistically significant during the 72-hr exposure. During the recovery period fish potassium levels recuperated.
Plasma phosphorus levels decreased as consequence of propanil exposure. Differences from control animals were significant from 24 hr. The lowest level was determined after 72 hr of exposure (−48% of the control values). Once animals were allowed to recover, a tendency towards increased levels was observed, however, levels did not recuperate completely.
A significant increase in blood ammonium levels was registered in exposed eels since the first time of measurement (Fig. 3). The maximum increase was found after 24 hr of exposure. Levels were recovered in herbicide-free water.
Signs of poisoning were observed in eels exposed to the herbicide after the first hr of contact with the toxicant. During the first two hr, the behavior of exposed fish was similar to that of the control group. Behavioral changes started after that time and were typical of neural and respiratory poisoning as marked by agitation, muscular twitching, gyrating swimming movements and convulsions, followed by less spontaneous activity than controls, torpor and reduction in swimming performance, followed by diminished sensory activity and lethargy.12) In addition, animals exhibited the presence of petechiae and punctuate hemorrhages; however, they survived the stipulated exposure period. These clinical signs tended to disappear very slowly during the recovery period. The results agree with those found in fish exposed to propanil (0.4–3.8 mg/L)7) and other herbicides, such as molinate.3,14–16) Crossland17) reported lethargy and asthenia in fish as a common effect after amide herbicide exposure. Therefore, the presence of petechiae has an important biological significance since it could reveal the serious risk of eels to develop an anemic condition because of blood loss.16) Propanil is a very toxic pesticide to fish as compared to other herbicides. Low levels of propanil adversely affect the growth and survival of the juvenile Pimephales promelas fish; sublethal effects, such as swollen bodies with reddish zones of hemorrhaging along the visceral mass, were also reported.18)
Data of the present investigation indicated that the subacute propanil concentration tested, which is close to that used for agricultural purposes, might cause several disturbances in the stress response of the studied fish as well as in its general physiology. As we have already cited in the Introduction, Santos et al.3) monitored propanil in surface water samples from rice fields in the Ebro Delta area (Tarragona, Spain), following agricultural application of 12 mg/L with a half-life between 1.24 and 3.83 days after application. Propanil concentrations of 50–100 µg/L in drainage waters had been reported in the rice fields muda irrigation scheme of Kedah (Malaysia).4)
Our results and others are in agreement with a well-established pattern for exposure to chemical stressors. As a consequence of propanil intoxication, additional energy requirements became evident in fish, not only during exposure but also during the allowed recovery period.
Considering the observed cortisol level increased in exposed fish, the observed variation in plasma metabolites such as glucose, lactate and plasma proteins seems to be expected. The hyperglycemic status exhibited by treated eels should be correlated with a decrease in the glycogen content usually found in fish under stress conditions.8,19) This leads to the stimulation of processes, such as lipolysis or proteolysis, to use the degraded products as disposable for energy.
Elevated hepatic alanine aminotransferase (AlAT) activity is indicative of a higher utilization of alanine as a substrate for glyconeogenesis in fish liver cells that suggest a heavy drain on metabolites during stress to provide intermediates for the Krebs cycle.20) A mobilization of alanine and other amino acids as substrates to glyconeogenesis probably occurred in eels exposed to propanil. Previous studies have shown that AlAT activity increased in the livers of wild eels exposed to this herbicide.12) This seems to be a common fact in fish as a consequence of exposure to pesticides.20,21) Aminotransferases can be used as biomarkers of pesticide intoxication in experimental toxicology.8) Due to the fact that both are intracellular enzymes, an increase in blood levels could be indicative of tissue necrosis. Our results are in agreement with those of Bálint et al.22) and Oruç and Üner,23) who observed an increase in the plasma levels of ALT and AST in Cyprinus carpio exposed to pyrethroid deltamethrin and organophosphate azinphomethyl, respectively. An accelerated rate of protein catabolism would result in an increase of amino groups released from amino acids. Endogenous cortisol enhances hepatic glyconeogenesis from amino acids.24) These groups are converted to nitrogenous (urine) products as ammonium in fish in the detoxification process that takes place in the liver; therefore, an increase in ammonium levels will be detected.
Our results showed that albumin decreased in eels as a consequence of exposure to propanil. These results agree with those found in the Korean rockfish (Sebastes schlegeli) in response to cypermethrin.25) Albumin is the main protein in plasma’s colloidal osmotic role and one of the main carriers for plasma constituents. It is also a linked transport protein catabolized by metabolically active tissues. This protein is exclusively produced by the liver, which means that lowered levels of albumin may be used as an index of liver damage.26) Once animals were in herbicide-free water (recovery period), they were able to recover control levels of albumin. Furthermore, the decrease in total proteins and albumin levels in addition to the increase in AST activity suggested that the herbicide propanil had generalized effect in the whole body of animals, either in a direct or indirect way, related to protein metabolism in the eel. This would be interfering in the biosynthesis of lipoproteins from the membranes needed to repair injured biological systems. The modes of action of propanil, as a substituted amide, include the inhibition of RNA and protein synthesis, combined with enzyme inhibition of amylase, proteinase, dipeptidase, and other enzymes.27)
AP enzymes are involved in the generation of inorganic phosphorus needed to the DNA synthesis. Between others activities, these enzymes are implicated in the synthesis of proteins during the repair of injured tissues, a fact used in toxicology, as well.28) AP activity increased in the plasma of eels exposed to propanil in the present study. Propanil has been reported to be an inductor of lipid peroxidation in tissues such as liver and brain in conjunction with the hyperactivation of AP in mammals.29) Increased of susceptibility to lipid peroxidation could be related to the capacity of this herbicide to injure tissue. The authors observed a relationship between cytolysis in tissues, in conjunction with an increase in AP and AST activities. As AP is an intracellular enzymatic activity, an increase in AP levels measured in plasma is indicative of plasmatic membrane lysis, as observed in Cyprinus carpio and Oreochromis niloticus exposed to different herbicides such as glyphosate30,31) or trifluralin.32)
Lipids form the richest energy reserves, whose caloric content value is double that of an equivalent weight of carbohydrates or proteins. Therefore, the mobilization of lipid reserves indicates high-energy demands. Generally, the lipid content is mobilized to maintain homeostasis during toxicant exposure. The stimulation of lipid catabolism includes the formation of lipoproteins that are utilized to repair damaged cell and tissue organelles, to direct the utilization by cells for energy requirements, and to increase lipolysis. The increase of cholesterol and triglycerides in the plasma of eels exposed to propanil as reported in the present study, may cause diminished growth, survival probability, and reproduction as indicated in zebrafish exposed to the organophosphate parathion.33) The invertebrate Daphnia magna, exposed for more than 72 hr to various sublethal propanil concentrations showed significant reductions in lipid content.34) The authors cited reductions of 31% were determined in daphnids exposed to 0.55 mg/L for 120 hr. Other pesticides, such as endosulfan, have demonstrated their capacity to induce lipid catabolism in fish. Gill et al.35) observed that a depletion in the total lipid content in the muscle of Barbus conchonius exposed to endosulfan was correlated with hypercholesterolemia and fatty acids.
Pesticides are known for their ability to disrupt the structural integrity of fish gills. The uptake of gills plays an important role in the incorporation of pesticides via blood in other organs, such as the liver. Because of herbicide damage to gills, tissues receive less oxygen. The development of such internal hypoxic conditions may be ultimately responsible for a shift to the less efficient anaerobic metabolism. The increased LDH activity observed in the present study was indicative of the activation of processes of glycolysis and anaerobic metabolism to meet the required energy demands.8) This is correlated with high increases of lactate. LDH is a cytoplasm enzyme present in numerous tissues and forms the center of a delicate balance between catabolism/anabolism of carbohydrates. Lactate is the end product of glycolysis under hypoxic conditions. Its increase after an episode of propanil intoxication could suggest inadequate oxygen uptake and tissue damage. Recent studies have shown increases of more than 80% in LDH activities in the liver and skeletal muscle of wild eels exposed to this herbicide.12)
Freshwater fish regulate their blood and tissue osmolality by excreting excess water via the kidney making urine hipoosmotic and recovering lost salt by the active transport of ions from the water through the gills by chloride cells. Most likely, the malfunctioning of chloride cells due to herbicide exposure is the result of nonspecific damage to the gill epithelium as well as regulatory mechanisms of these epithelia.36) A temporary impairment of gills as a regulation system has already been observed in the European eel exposed to other herbicides, such as thiobencarb.13) As reported by those authors, the herbicide induced a rapid depletion of ATPase activities in both the gills and skeletal muscles of the European eel and an increased water content.
In the present study, plasma levels of sodium, chloride, potassium, calcium, and phosphorus decreased in eels exposed to propanil. These results suggest a lowered uptake of those ions through the gills: therefore, the levels will decrease in plasma. This fact is very important because eel osmoregulation will be seriously altered.
Hemolytic anemia, associated with severe hypophosphatemia, has been attributed clinically in humans and animals to the inability of erythrocytes to maintain the integrity of cell membranes in the face of ATP depletion, which leads to their destruction in the spleen.37) A transient hypophosphatemia would then reflect an impaired hematologic function in eels as a consequence of exposure to propanil. Phosphate deficiency also compromises oxygen delivery to tissues due to decreased erythrocyte 2,3-DPG and the resulting leftward shift in the oxygen-hemoglobin dissociation curve. The calcium level in plasma is greatly affected by the plasma level of inorganic phosphate. The symptoms have been associated with neurologic symptoms, such as lethargy in mammals and humans.37,38) The symptoms of toxicosis exhibited by propanil-exposed eels would be an indication of transient disorders in the plasma ion profiles of exposed eels.
A successful stress response (compensatory processes) in eels pre-exposed to propanil would be associated with the strict regulation of ion and water transport pathways within key osmoregulatory epithelia to enable animals to survive the stressing episode. Few data are available in the literature concerning the ionic profile in fish after exposure to pesticides. Literature indicates that cortisol interacts with hormones, such as prolactin, to restore the ion balance.6,9) Because of the experimental system used in the assays, our results must be considered to be the result of integrated compensatory responses in fish. The observed adverse effects induced by propanil are, in turn, the direct or indirect consequence not only of the propanil concentration but also of complex interactions among compensatory responses induced in exposed eels.
Cortisol is not only one of the primary stress hormones in fish but also an important stimulator of Na+/K+-ATPase activity in gills, intestines, and kidneys (osmoregulatory organs). The multiple functions of this hormone clearly demonstrate the intimate relationship between the stress response and osmoregulation in fish.9) Likely, when examining the effects of toxicants in vivo, two processes interfere with each other: the toxic actions of the chemicals themselves and the compensatory responses of the fish. This interference complicates the analysis of the effects of toxic substances. Successful compensation may obscure the negative effects of toxicants and lead to an underestimation of their impact on living organisms.3)
During the depuration period, fish still experienced stress consequences. Glucose, cortisol and lactate levels remained elevated, whereas others recuperated to their normal values. Calcium and phosphorus ions also were still disturbed. Studies of the plasma ionic profiles in freshwater fish exposed to pesticides are less extensive than those carried out with metals.36) Present results indicate that 96 hr was not long enough to observe recuperation to complete fitness, as major disturbances were still present in eels pre-exposed to propanil. The authors are unable to correlate these findings, due to the lack of information in the literature regarding both the ion profiles in the plasma of fish exposed to pesticides and regarding recovery aspects.
Low levels of pesticides can seriously damage immature fish. Successful compensation may lead to an underestimation of the true impact of pesticides on living organisms because the allocation of energy toward compensatory processes and cell-cycle acceleration limits the potential growth and reproduction of organisms and promotes aging.9)
In conclusion, our results clearly demonstrate that sublethal exposure to propanil induced a stress response in eels. Although cortisol’s functional response increased the fish’s physiological competence through a simultaneous wide range of important homeostatic mechanisms, the current results show that treated fish were forced to cope with a serious homeostatic crisis. Judging from the changes observed during the recovery period, fish still exhibited dysfunctions after exposure. In the long run, this altered fitness may have consequences for higher levels of biological organization, such as diminished growth or survival probability. These findings are a clear example of the potential effects of low pollutants released into fish environments. The selected parameters may be useful as a diagnostic test for propanil in intoxicated fish after spray operations in the field. Moreover, investigations into the effects of herbicides on fish have a diagnostic significance in evaluating the adverse effects of those pesticides, ultimately, to human health. Although propanil is currently forbidden in the European Union countries, it probably remains one of the most widely used herbicides in the World.
Authors want to thank Agrarian Laboratory of Generalitat Valenciana (Spain) for their help in the analysis of the propanil content in the water samples. Funding for this project was provided by General Directive of Research, Education and Science Ministry AGL2006-10840-C02-01 from NIDA Program, by Consellería de Empresa, Universidad y Ciencia GV06/359 from Generalitat Valenciana and by the grant UV-AE-10-24323 of University of Valencia (Spain).
