E. Sancho

Glutathione-dependent resistance of the European eel Anguilla anguilla to the herbicide molinate

Eels of species Anguilla anguilla were exposed to 5/4 LC50 (41.8 mg/l) of the herbicide molinate for 96 h in a time to death (TTD) test. Glutathione content (GSx, GSH, GSSG), glutathione reductase (GR) and gamma-glutamyl transpeptidase (gamma-GT) activities were determined in the liver and muscle tissues of dead and surviving (intoxicated) animals and compared to control values (non-exposed eels). TTD was positively correlated to hepatic GSH, GSH:GSSG ratio, hepatic and muscular GR, but negatively correlated to muscular GSH, which was severely depleted. Furthermore, glutathione and enzyme activities were intercorrelated, especially GSH and GR. These results indicate that eels which were able to induce GR activity, increase GSH and maintain the GSH:GSSG ratio in the liver showed an extended survival under the oxidative stress generated by molinate than those that lost glutathione homeostasis.

Acute, chronic and sublethal effects of the herbicide propanil on Daphnia magna

Acute and chronic toxicity tests with propanil were conducted on Daphnia magna. The 24 and 48 h LC50 were 43.74 and 5.01 mg/l respectively. Chronic toxicity tests were carried out using sublethal propanil concentrations (0.07, 0.10, 0.21 and 0.55 mg/l) during 21 days. The effect of propanil on survival, reproduction and growth of D. magna organisms was monitored. The parameters used to evaluate herbicide effect on reproduction were: mean total young ones per female, mean brood size, time to first reproduction, mean number broods per female and intrinsic rate of natural increase (r). Survival and growth (body length) were also determined after 21 days of exposure to the herbicide. Reproduction was significantly reduced when propanil concentration increased in the medium. The intrinsic rate of natural increase (r) decreased with increasing concentrations of propanil especially in those animals exposed to 0.55 mg/l. However, growth as well as survival of the exposed organisms only decreased in daphnids exposed to the highest propanil concentration tested. The maximum acceptable toxicant concentration (MATC) was calculated for D. magna exposed to the herbicide using as parameter of evaluation the intrinsic rate of natural increase (r). The interpolation of these results gave MATC values of 0.08 mg/l herbicide. We have derived the EC50 values for some selected parameters on D. magna exposed to propanil. EC50 values indicated that reproductive parameters were very sensitive of the effect of propanil on daphnids. Finally, the daphnids were exposed to the same sublethal herbicide concentrations as in the chronic study and the effect of the toxicant on filtration and ingestion rates was determined. Feeding rates of D. magna declined with increasing propanil concentrations. The effective propanil concentrations at which feeding rates were reduced to 50% of that in controls (EC50) were also calculated.

Disturbances in energy metabolism of Daphnia magna after exposure to tebuconazole

This study was conducted to investigate the change of some biochemical parameters in the aquatic invertebrate Daphnia magna following exposure to the fungicide tebuconazole and to determine the most sensitive biomarker among the ones tested in this species. Four biochemical biomarkers (protein, glycogen, lipids and caloric content) were correlated with feeding behaviour studies of D. magna after fungicide exposure. Juveniles of D. magna were exposed to four sublethal concentrations of tebuconazole (0.41, 0.52, 0.71 and 1.14 mgL(-1)) for 5d. Daphnid samples were taken from each test and control group at 24, 48, 72, 96 and 120 h after the start of the experiment. Tebuconazole EC(50) values were calculated on D. magna in our laboratory as 56.83 and 40.10 mgL(-1) at 24 and 48 h, respectively. Results showed that daphnid energy content decreased as tebuconazole concentration increased, especially after 96-120 h of exposure to 0.52 mgL(-1) and higher fungicide concentrations. The data suggest that tebuconazole is moderately toxic to D. magna but also that it seriously impairs the metabolic functions, resulting in alterations in biochemical constituents. In the D. magna feeding study, algae feeding rates were inhibited after fungicide exposure. Such findings indicate the importance of feeding studies in laboratory toxicity test as well as their relationship with others studies. The results emphasize the importance of considering different kind of biomarkers to identify and evaluate the biological effect of a fungicide in the aquatic environment. Although the biochemical biomarkers used resulted good indicators of tebuconazole toxicity, feeding rates in D. magna decreased after only 5h exposure to the fungicide resulting in the most sensitive parameter of daphnid fungicide exposure.

Short-term exposure to sublethal tebuconazole induces physiological impairment in male zebrafish (Danio rerio)

The aim of the present study was to assess the physiological response of male zebrafish Danio rerio to the fungicide tebuconazole and recovery in fungicide-free water. Acute toxicity tests were carried out and the median lethal concentration (LC(50)) from 24 to 96 h was calculated. The fish were exposed to a sublethal fungicide concentration of 230 microg/L for 7 or 14 days and allowed to recover for 7 or 14 more days, respectively. Whole-body levels of vitellogenins, triglycerides, cholesterol, glucose, lactate and proteins as well as the activities gamma-glutamil transpeptidase (gamma-GT), alanin aminotransferase (AlAT), alkaline phosphatase (AP) and lactate dehydrogenase (LDH) were assayed; corpulence factor (k) was also calculated. Fish exhibited significant increase of vitellogenins (Vtg), which continued to increase after 14 days of recovery. Levels of glucose, lactate, cholesterol and triglycerides increased after 7 and 14 days of exposure. Finally, cholesterol and glucose recovered after 14 days of recovery whereas triglycerides and lactate continued to be elevated. Proteins and k remained unaltered the entire experiments. AAT, AlAT and AP enhanced during exposure and did not recover at the end (except AlAT). A longer recovery period should be necessary to re-establish fish physiology. These results alert about the multiple disruptive physiological actions that tebuconazole may have on fish.

In vivo inhibition of AChE activity in the European eel Anguilla anguilla exposed to technical grade fenitrothion.

European eel (Anguilla anguilla) were exposed to sublethal fenitrothion concentrations in a continuous flow-through system for 4 days. Plasma acetylcholinesterase (AChE) activity was evaluated after 2, 8, 12, 24, 32, 48, 56, 72 and 96 h pesticide exposure. AChE activity in the plasma of the eel decreased as concentration of fenitrothion increased. Pesticide induced significant inhibitory effects on the AChE activity of A. anguilla ranging from 51% inhibition at sublethal concentration of 0.02 ppm to 57% inhibition at sublethal concentration of 0.04 ppm. Eel were exposed to both fenitrothion concentrations for 96 h and then allowed a period of recovery in pesticide-free water. Following 1 week of recovery, the AChE activity for those animals previously exposed to fenitrothion was still different from the controls. Animals transferred to clean water showed plasma AChE activities reduced in a 34 and 51% when previously exposed to 0.02 and 0.04 ppm pesticide, respectively. This is probably due because regeneration of the enzyme is mainly by the novo synthesis after exposure to organophosphates and levels need a large extent to recover.

Thiobencarb toxicity and plasma AChE inhibition in the European eel

The acute toxicity of the herbicide thiobencarb (S-4-chlorobenzyl diethylthiocarbamate) was determined for the European eel (Anguilla anguilla). The 24, 48, 72 and 96 hours median lethal concentrations (LC50) were 25.7, 21.7, 17.0 and 13.2 mg/L, respectively. Fish were also exposed to a sublethal thiobencarb concentration (1/60 LC50-96 hr = 0.22 mg/L) during 96 hours in a flow-through system and then an elimination period of 192 hours in clean water was allowed. Eels were removed and blood samples taken out at each exposure time and recovery period in order to evaluate AChE activity. Thiobencarb induced significant inhibitory effects on plasma AChE activity of A. anguilla from the first contact time with the toxicant. This inhibition (under 50% activity) was maintained during the entire exposure period (96 hours) and even those animals transferred to clean water showed plasma AChE activity different from the controls. Differences between total and specific AChE activity were detected during the exposure period. Total AChE activity in the plasma from animals transferred to a medium free of toxicant recovered its normal value while specific AChE activity remained depressed (< 50%) until five days later.

Biochemical stress response in tetradifon exposed Daphnia magna and its relationship to individual growth and reproduction.

Environmental risk assessment of chemicals toxicity requires the use of costly and labor-intensive chronic data and short-term tests provide additional information. Energy budget is used by the animals for their growth, reproduction, and metabolism and it is reduced in case of toxic stress. Tetradifon acaricide is frequently used in the European Mediterranean region and it is implicated in aquatic environmental pollution. Previous studies showed that the EC(50)-24 h of tetradifon on Daphnia magna was 8.92 mg/L. Based on that, D. magna were exposed to sublethal tetradifon concentrations of 0.10, 0.18, 0.22 and 0.44 mg/L for five days in order to investigate their effect on intermediate metabolism. Caloric content was determined as biomarker of tetradifon toxicity. Results were analyzed using one-way analysis of variance (ANOVA) and Duncans significant difference test was used to find differences between groups (alpha was set at p=0.05). Daphnids energy content decreased as tetradifon concentration increased. At 120-h caloric content was depleted >51% at pesticide concentrations of 0.18 mg/L and higher. In order to determine a possible link between the 5-d test and the 21-d chronic test, animals under short-term test were exposed to the same pesticide concentrations known to cause adverse effects on reproduction, growth and survival. Results from the present study indicated a good correlation between the proposed 5-day test and daphnid energy budget. Comparison between both, caloric content results and the chronic effect values obtained using life-table studies, suggested a good fit between them. These studies can be used as earlier, predictive and useful tests with comparable results to the classic chronic ones. Our results indicate that caution must be done about the use of tetradifon in the aquatic environment.

Effects of tetradifon on Daphnia magna during chronic exposure and alterations in the toxicity to generations pre-exposed to the pesticide.

Daphnia magna was exposed to some nominal sublethal tetradifon concentrations (0.10, 0.18, 0.22 and 0.44 mg/l) during 21 days. Chronic toxicity tests were carried out using neonates of F(1) first brood (F(1)-1st) and F(1) third brood (F(1)-3rd) offspring generations from parentals (F(0)) pre-exposed to the pesticide. The effect of tetradifon on survival, reproduction and growth was monitored for the selected daphnid generations. The parameters used to evaluate pesticide effect on reproduction were: mean total young per female, mean brood size, time to first reproduction, mean number broods per female and intrinsic rate of natural increase (r). Survival and growth (body length) were also determined after 21 days of exposure to the pesticide. Reproduction was significantly reduced when tetradifon concentration increased in the medium. This effect was higher in F(1)-1st and F(1)-3rd offsprings compared to the parental generation (F(0)) daphnids. The intrinsic rate of natural increase (r) decreased with increasing concentrations of tetradifon especially in those animals from F(1)-3rd. However, the growth of the exposed organisms decreased in the same order of magnitud in all the generations tested. Survival was not affected after exposure to the selected tetradifon concentrations. The maximum acceptable toxicant concentration (MATC) was calculated for F(0), F(1)-1st and F(1)-3rd generations exposed to the pesticide using as parameter of evaluation the intrinsic rate of natural increase (r). The interpolation of these results gave MATC values of 0.13 mg/l pesticide for F(0) and F(1). The EC50 values have been derived for some selected parameters on D. magna exposed to tetradifon. EC50 values decreased in F(1)-1st and F(1)-3rd generations compared to the parental generation F(0), these results mean that less amount of toxicant would be necessary to reduce the selected parameters to 50% in the first and third broods of D. magna. Expanding the reproduction tests to several broods of a parental generation revealed important information on chronic toxicity that could be used in the protection of aquatic environment.

Physiological effects of tricyclazole on zebrafish (Danio rerio) and post-exposure recovery.

Short-term effects of tricyclazole on male zebrafish (Danio rerio) physiology were examined joint to the degree of recovery after exposure. Fish were exposed to 142 microg/L (1/100 LC(50)-96 h) of tricyclazole for 7 (Exp.1) and 14 days (Exp.2) and then allowed to recover for 7 or 14 more days, respectively. Whole-body triglycerides, cholesterol, glucose, lactate and total proteins were measured as well as the aspartate aminotransferase (AAT), alanine aminotransferase (AlAT), alkaline phosphatase (AP) and lactate dehydrogenase (LDH) activities as biomarkers of intermediary metabolism; gamma-glutamyl transpeptidase (gammaGT) as biomarker of oxidative detoxification processes and vitellogenin (Vtg) concentration as endpoint for endocrine disruptor effect were also determined. Corpulence factor (k) was calculated. Fungicide exposure in zebrafish resulted in an increased of triglycerides, cholesterol, glucose and lactate levels, however the total protein content did not change. LDH, AlAT and AAT enhanced while AP activity decreased. Corpulence factor (k) also decreased. At the end of the recovery periods cholesterol and glucose levels recovered whereas triglycerides and lactate continued to elevate. Induced disorders on the selected enzymes remained and did not recover at the end of experiments. Fish exhibited significant increases of Vtg during the overall experimental times as a consequence of the fungicide exposure. These findings are of importance in the assessment of the potential risk of new fungicides as tricyclazole on aquatic ecosystems.

Changes in carbohydrate metabolism in the eel anguilla anguilla, during short‐term exposure to diazinon

The present communication deals with the effect of diazinon (0.042 mg/L) on the energy metabolism of the european eel Anguilla anguilla. Analysis of various parameters such as glycogen, lactate and glucose was made on different eel tissues after 6, 24, 48, 72 and 96 hr of diazinon exposure. Liver and muscle glycogen content decreased significantly (p < 0.05) from 6 to 96 hr pesticide exposure. Mean blood glucose values were elevated significantly after exposure to this sublethal diazinon concentration. Liver, blood and muscle lactate levels in fish increased during all exposure time being the highest at 96 hr exposure. Diazinon intoxicated fish showed signs of pesticide poisoning as muscular twitching, gyrating swimming movements and diminished sensory activity but survived the stipulated exposure period. The results are discussed in relation to the stress effect produced by the pesticide and the related responses of the fish.