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Effect of dichlorvos on cholinesterase activity of the European sea bass (Dicentrarchus labrax)
Pesticide Biochemistry and Physiology 75 (2003) 61–72 www.elsevier.com/locate/ypest
Effect of dichlorvos on cholinesterase activity of the European sea bass (Dicentrarchus labrax) I. Var o,a,* J.C. Navarro,a F. Amat,a and L. Guilherminob,c a Instituto de Acuicultura de Torre de la Sal (CSIC), 12595 Ribera de Cabanes, Castell on, Spain Instituto de Ci^ encias Biom edicas de Abel Salazar, Laborat orio de Ecotoxicologia, Dept. de Estudos de Populacßo~es, Universidade do Porto, Lg do Prof. Abel Salazar, 2, 4099-003 Porto, Portugal Centro Interdisciplinar de Investigacßa~o Marinha e Ambiental, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
b
c
Received 8 March 2002; accepted 3 August 2002
Abstract In this study, the acute toxicity of the organophosphorous pesticide (OP) dichlorvos and both in vitro and in vivo effects of dichlorvos on cholinesterase (ChE) activity of the European sea bass (Dicentrarchus labrax) were investigated. The characterisation of ChE and the ‘‘normal’’ range of activity in brain and muscle of non-exposed fish were determined in a first phase of the study. Acetylthiocholine was the substrate preferred of both brain and muscle ChE. Eserine sulphate and BW284C51 significantly inhibited the brain and muscle enzyme activity at low concentrations (lM range). Iso-OMPA had a significant effect in muscle, but not in brain tissue. These results suggest that acetylcholinesterase (AChE) is the predominant ChE form in brain tissue. In contrast, both acetylcholinesterase and butyrylcholinesterase seem to exist in muscle. Using acetylthiocholine as substrate, the ‘‘normal’’ range of fingerling head and muscle ChE were 58:05 2:11 and 118:03 8:67 U/mg protein, respectively. Corresponding values for juveniles were 43:32 4:42 and 19:44 2:44 U/mg protein for brain and muscle, respectively. Dichlorvos significantly inhibited the activity of ChE in the selected tissues, both in vitro and in vivo conditions. Differences in ChE sensitivity were found in relation to the age of the fish and the tissue analysed. The present study also showed that fingerlings of the European sea bass are relatively resistant to in vivo acute (96 h) dichlorvos exposure to concentrations between 0.125 and 1 mg/L, being able to tolerate high percentages of head ChE inhibition (37% and 76%) without lethal effects. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Marine fish; European sea bass; Cholinesterases (ChE); Biomarkers; Dichlorvos; Toxicity
1. Introduction Dichlorvos is an organophosphate pesticide (OP) commonly used in fish farming to eradicate crustacean ectoparasites. It is specially used in the
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Corresponding author. Fax: +34-96431950. E-mail address: [email protected] (I. Var o).
treatment of sea lice (Lepophtheirus salmonis and Caligus elogatus) on commercial salmon farms. When a chemical product is used in bath treatments to control ectoparasites of cultivated fish, it is absorbed mainly through the gills and body surface, and may produce both lethal and sub-lethal effects on the fish. Dichlorvos is a direct-acting inhibitor of acetylcholinesterase (AChE) [1], the enzyme that degrades the neurotransmitter
0048-3575/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0048-3575(03)00019-1
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acetylcholine in cholinergic synapses. The inhibition provokes an accumulation of acetylcholine in synapses with disruption of the nerve function that can end in the death of the organism. The activity of cholinesterases (ChE) has been widely used as a biomarker of exposure to OP and carbamate (CB) pesticides in both vertebrates and invertebrates. The relationship between OP and/or CB exposure and ChE inhibition has been studied in several species of aquatic organisms, including molluscs [2,3] crustaceans [4–7] and fish [8–16]. All these studies have shown the sensitivity of ChE as a specific biomarker to diagnose exposure and effects of OP and CB pesticides. Although ChE activity is widely used as specific biomarker for anticholinesterase pesticides, its use in a particular species requires the characterisation of the enzyme(s) present in the tissues to be analysed and the determination of the activity range that may be considered as ‘‘normal’’ for non-exposed individuals [17,18]. Vertebrate ChEs are divided into two main classes, AChE and butyrylcholinesterase or pseudocholinesterase (BChE). The properties of ChE may differ from species to species and may also display variations in different tissues of the same species. Historically, fish brain and muscle tissues were though to contain mostly AChEs, while BChEs were though to be the predominant forms in liver and plasma. However, in some species of marine fish, the presence of both AChE and BChE in muscle tissue has been described [14,19]. Since more than one ChE may be present in the tissues of marine fishes and these ChEs may show different sensitivities to anticholinesterase agents, it is important to perform the biochemical characterisation of the ChE present in the species and in the tissues to be studied before their use as biomarker [18]. The acute toxicity of dichlorvos to fish has been previously determined by a number of researchers. Its toxicity for freshwater and estuarine fish is moderate to high, although it does not bioaccumulate in fish [20]. For freshwater and estuarine fish, 96h-LC50 values range from 0.2 to 12 mg/L [1]. For marine fish, the toxicity was estimated to be more than 4 mg/L for adults and pre-adults of Atlantic salmon (Salmo salar) [21]. However, Sievers et al. [22], did not find mortality in 100 g salmon at dichlorvos concentrations between 1 and 5 mg/L after 24 h of exposure. Several authors have been reporting significant inhibition of ChE activity in fish at sub-lethal concentrations of dichlorvos [14,15,23,24].
To the best of our knowledge, no previous studies on the effect of dichlorvos on ChE activity of the European sea bass (Dicentrarchus labrax), a common species of marine fish cultured in the Mediterranean, have been carried out. Since dichlorvos is being used in bath treatments against crustacean ectoparasites in marine-farmed fish, the main objective of this study was to investigate the effects of this pesticide on the European sea bass (Dicentrarchus labrax). Therefore, the 50% lethal concentration (LC50) value of dichlorvos to this species was determined, and both in vivo and in vitro effects on ChE activity of fingerlings and juveniles induced by exposure to sub-lethal concentrations of this chemical was investigated. To reach this objective, in a first phase of the study, the characterisation of the ChE present in brain and muscle was performed and the ‘‘normal’’ range of activity in both tissues of non-exposed fish was determined.
2. Materials and methods 2.1. Chemicals Acetylthiocholine iodide, s-butyrylthiocholine iodide, propionylthiocholine iodide, iso-OMPA (tetraisopropyl pyrophosphoramide), eserine sulphate, BW284C51 (1,5-bis(4-allyldimethyammoniumphenyl) pentan-3-one dibromide), 5, 50 -dithiobis(2-nitrobenzoic acid) (DTNB) and bovine c-globulinÕs were obtained from Sigma Chemicals. Dichlorvos was obtained from Dr. Ehrenstorfer Reference Materials (Germany). 2.2. Experimental fish Fingerlings (weight ¼ 5:62 1:63 g, length ¼ 7:88 0:72 cm) and juveniles (weight ¼ 103:9 27:96 g, length ¼ 19:93 1:26 cm) of the European sea bass (Dicentrarchus labrax) were reared at the Instituto de Acuicultura de Torre de la Sal (CSIC), Castell on (Spain). They were kept in 500 L fibreglass tanks filled with seawater and supplied with continuous aeration under room temperature (22–28 °C) and natural photoperiod. Fish were fed ad libitum with commercial food pellets (Pro-Aqua, Spain). 2.3. Cholinesterase characterisation ChE characterisation of European sea bass was determined in juveniles. Fish were killed on
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ice before the brain and a piece of dorsal muscle (100 mg) being quick removed and kept on ice until the homogenates were prepared. Tissues were homogenised in 1 ml of ice-cold phosphate buffer (0.1 M, pH 7.2), using a tissue disrupter ultraturrax T-8 (IKA, Germany), centrifuged (at 8500 rpm for 5 min at 4 °C) and the supernatant was stored at )20 °C for no more than three weeks, until enzymatic analysis could be performed. Cholinesterase characterisation was initiated by measuring the enzyme activity at concentrations ranging from 0.01 to 20.5 mM of the substrates acetylthiocholine iodide (ATC), s-butyrylthiocholine iodide (BUT) and propionylthiocholine iodide (PROP). At least three replicates of each substrate were performed. Eserine sulphate, iso-OMPA, and BW284C51 were used as specific inhibitors of ChE, BChE, and AChE, respectively. Eserine sulphate and iso-OMPA were dissolved in ethanol, while BW284C51 was dissolved in bidistilled water. For cholinesterase assays, the supernatants were diluted in 0.1 M phosphate buffer at 1:10 (v:v) for head samples and at 4 mg/ml for muscle samples. Before ChE activity determinations, 0.490 ml of diluted supernatant were incubated for 30 min with 0.010 ml of each inhibitor, at room temperature (20–22 °C). For each inhibitor, the concentrations prepared ranged from 1.56 to 800 lM for eserine sulphate and BW284C51, and from 0.125 to 16 mM for isoOMPA. Controls were incubated with 0.010 ml of bidistilled water. Controls with 0.010 ml of ethanol were included for assays with eserine sulphate and iso-OMPA.
method [27] adapted to microplate, using c-globulin as standard and a wavelength of 600 nm.
2.4. Enzyme activity
2.6. Acute toxicity tests based on mortality
ChE activity was determined at room temperature (20–22 °C), in triplicate, by the Ellman method [25] adapted to microplate as described in Guilhermino et al. [26], using acetylthiocholine as substrate. In each assay, 0.05 ml of diluted supernatant and 0.250 ml of the reaction solution (phosphate buffer 0.1 M + 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) 10 mM + substrate) were used. The absorbance increase at 405 nm was followed for 5 min using a Bio-Rad microplate reader. The activity of ChE was expressed as the mean of the determinations performed (at least three independent samples) and was expressed as Units (U) per mg of protein (1 U ¼ nmol of substrate hydrolysed per min). The protein content of the samples was measured by the Bradford
Acute toxicity tests for determination of LC50 values were carried out following the recommendations of OECD [28]. Before the test, fingerlings were transferred to 10 L glass aquaria filled with filtered (0.2 lm) seawater, and exposed to a range of dichlorvos concentrations from 0.125 to 12.5 mg/L for 96 h, in static conditions. The medium was changed daily to assure the desired concentration of pesticide in the seawater. A stock solution of dichlorvos in acetone was prepared and the necessary amount of the stock solution added to the glass aquaria filled with filtered seawater. Duplicate groups of 4 fish were kept in separate 10 L glass aquarium filled with filtered seawater containing the desired pesticide concentration. Control fish were kept in 10 L glass aquaria filled
2.5. ChE in vitro tests In vitro ChE inhibition tests were carried out with both fingerlings and juveniles of the European sea bass, in independent experiments. Fish were fed and maintained under standard conditions of culture as described before. Feeding was stopped 24 h before fish were sampled for ChE assays. After killing the fish on ice, the brain and muscle tissues were rapidly removed and kept on ice until the preparation of the homogenates, as described before. The brain tissue of fingerlings was impossible to remove because the small size of the fish, so, the whole head without eyes was isolated from each animal and prepared and stored as described before. Also, the small amount of muscle associate to the head was carefully removed to avoid any influence on head ChE measurements. Prior to enzyme determinations, 0.490 ml of diluted supernatant was incubated for 30 min with 0.010 ml of different dichlorvos concentrations, at room temperature (20–22 °C). The nominal concentrations of dichlorvos tested were 1, 2, 4, 8, 16, and 32 mg/L. Controls were incubated with 0.010 ml of bidistilled water, and additional controls incubated with 0.010 ml of acetone solution, were also included. Between three juveniles and six fingerlings of sea bass per treatment were used for each tissue sampled. Three ChE determinations were performed per sample as previously described for the enzyme activity, using acetylthiocholine as substrate.
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with filtered seawater. A control with acetone was also included in which fish were exposed to the same amount of acetone as in the highest concentration tested (12.5 mg/L). Fish were maintained in a temperature and photoperiod controlled room (20 1 °C; 12 hL:12 hD). They were not fed during the test period and the medium was changed daily, to maintain water quality and pesticide concentrations. The effect criteria was mortality. Dead specimens were removed twice a day.
regression probit module of SPSS Systems (SPSS, 1989–1992) at 95% of confidence, was used. Differences among treatments were tested by one-way Analysis of Variance (ANOVA). For in vivo tests, no-observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) values were determined by TukeyÕs test for multiple comparisons [29], with a significance level of 0.05.
2.7. ChE in vivo tests
3.1. Characterisation of ChE
In vivo ChE tests were conducted only for head and muscle tissues of fingerlings, the most sensitive stage, because it is known that, in general, the increase of size and age reduce the fish sensitivity to toxicants. Duplicate groups of 4 fish were exposed to five nominal pesticide concentrations (0.125, 0.5, 1, 2, and 4 mg/L) in 10 L glass aquaria for 96 h, under the conditions of temperature and photoperiod described for acute tests. Fish were not fed during the exposure time (96 h). The concentrations of the pesticide used in ChE in vivo tests were based on 96h-LC50 values previously determined. A stock solution of the pesticide was prepared in acetone and the necessary amount of the stock solution added to the aquarium filled with filtered seawater to prepare the different treatments. Controls with acetone were also included as described above. At the end of the test, live fish were killed on ice before removal of head and muscle and the preparation of homogenates for ChE assays, as described before. The whole head (without the eyes and the musculature associate to the head), and a piece of muscle (50 mg) tissue were isolated from each animal, prepared, and stored as described above. The ChE activity was performed as previously described for enzyme activity determinations. The ‘‘normal’’ range of ChE activity of head or brain and muscle tissues was determined in nonexposed fish (both fingerlings and juveniles). Homogenates were prepared as previous described, and ChE activity was determined using acetylthiocholine as substrate.
ChE activity of brain and muscle as a function of different substrates is shown in Fig. 1. The results obtained for both tissues indicate that ChE shows preference for acetylthiocholine as substrate. A high inhibition of ChE activity in the descending part of the ATC curve, thus under excess of substrate conditions, was observed only for brain tissue. Values of ChE activity measured in both tissues using butyrylthiocholine and propionylthiocholine as substrates were considerably lower than the correspondent values obtained with ATC. The effects of eserine sulphate, isoOMPA and BW284C51 on ChE activity of brain and muscle of the European sea bass are shown in Figs. 2–4, respectively. Eserine sulphate had a significant inhibitory effect on both brain and muscle ChE activity at low concentrations (106 M range) (see Fig. 2; brain: F ¼ 278.9, df ¼ 11, 107, p 6 0:05; muscle, F ¼ 616.5, df ¼ 11, 239, p 6 0:05). Brain ChE activity was not significantly affected by iso-OMPA (see Fig. 3, F ¼ 1.2, df ¼ 9, 119, p P 0:05), while muscle ChE activity was significantly affected by this chemical (see Fig. 3, F ¼ 20.1, df ¼ 9, 119 p 6 0:05), with an inhibition ranging between 15 and 22%. In contrast, the inhibitor BW284C51 produced a significant effect on ChE activity (see Fig. 4; brain: F ¼ 466, df ¼ 10, 98, p 6 0:05; muscle, F ¼ 37.8, df ¼ 10, 175, p 6 0:05) for both tissues. However, the inhibition of ChE was higher in the brain (<8% of control activity in all the concentrations tested) than in the muscle with an enzyme activity lower than 8% only in the highest concentrations tested (400 and 800 lM).
2.8. Statistical analysis
3.2. ChE in vitro tests
To calculate LC50 values, 50% in vitro inhibition concentration values (IC50) and 50% in vivo inhibition concentration values (EC50), the
In vitro exposure to dichlorvos affected the ChE activity in both head or brain and muscle tissues of fingerlings and juveniles (Figs. 5 and 6,
3. Results
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Fig. 1. ChE activity of the European sea bass brain and muscle tissues as a function of the different substrates, acetylthiocholine iodide (ATC), s-butyrylthiocholine iodide (BUT), and propionylthiocholine iodide (PROP). Values are means SD. (N ¼ 3 and three enzymatic determinations per replicate).
Fig. 2. Effect of Eserine sulphate on ChE activity of the European sea bass brain (j) and muscle tissues () (N ¼ 3 and three enzymatic determinations per replicate). 00 ¼ ethanol control. H I Indicates significant differences from control (p < 0:05).
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Fig. 3. Effect of Iso-OMPA on ChE activity of the European sea bass brain (j) and muscle tissues () (N ¼ 3 and three enzymatic determinations per replicate). 00 ¼ ethanol control. I Indicates significant differences from control (p < 0:05).
Fig. 4. Effect of BW284C51 on ChE activity of the European sea bass brain (j) and () (N ¼ 3 and three enzymatic determinations per replicate). H I Indicates significant differences from control (p < 0:05).
respectively). For fingerlings, IC50 values estimated for head and muscle ChE activity were 0.89 mg/L (95% confidence limits: 0.39–2.05) and
4.25 mg/L (95% confidence limits: 1.6–10.8), respectively. In contrast, for juveniles, no differences on ChE sensitivity to dichlorvos were found
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Fig. 5. In vitro effect of dichlorvos on ChE activity of brain (j) and muscle () tissues of fingerlings of European sea bass (N ¼ 3–6 and three enzymatic determinations per replicate). 00 ¼ acetone control. H I Indicates significant differences from control (p < 0:05).
Fig. 6. In vitro effect of dichlorvos on ChE activity of brain (j) and muscle () tissues of juveniles of European sea bass (N ¼ 3 and three enzymatic determinations per replicate). 00 ¼ acetone control. H I Indicates significant differences from control (p < 0:05).
between brain and muscle ChE activity, with IC50 values of 7.4 mg/L (95% confidence limits: 7.3–7.5) and 9.9 mg/L (95% confidence limits: 9.9–10.1) for brain and muscle, respectively. No significant effects of the solvent at the maximal concentration tested (32 mg/L) was observed for fingerlings or juveniles ChE (see Figs. 5 and 6).
3.3. Acute toxicity test based on mortality The 96h-LC50 value obtained for fingerlings was 3.5 mg/L (95% confidence limits: 0.06–5.31). The survival of the control fish in the acute toxicity test based on mortality was 100%. However, fish exposed to the highest pesticide
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concentrations tested, 12.5 and 8 mg/L, resulted in 100% of mortality after 5 h of the start of the test. 3.4. ChE in vivo tests The effects of in vivo exposure to dichlorvos on head and muscle ChE activity of fingerlings are shown in Fig. 7. Dichlorvos-induced significant alterations on ChE activity in both tissues (head, F ¼ 70.1, df ¼ 6, 28, p 6 0:05; muscle, F ¼ 43.7, df ¼ 6, 29, p 6 0:05) after 96 h of exposure. Also, a significant effect on ChE activity of the solvent at the maximal concentration tested (12.5 mg/L) was observed. The NOEC and LOEC values were quite similar for both tissues analysed. NOEC values for head and muscle were <0.125 and 0.125 mg/L, respectively. LOEC values were 0.125 mg/L for head and 0.5 mg/L for muscle. The EC50 values were 0.31 mg/L (95% confidence limits: 0.55–16.28) for head and 1.33 mg/L (large 95% confidence limits) for muscle. The values of ‘‘normal’’ range of fingerlings ChE activity using acetylthiocholine as substrate were 58:05 2:11 U/mg protein for head and 118:03 8:67 U/mg protein for muscle. Corresponding values for juveniles were 43:32 4:42 U/mg protein for brain and 19:44 2:44 U/mg protein for
muscle. Significant differences in the ‘‘normal’’ range of ChE activity were found between the two age groups of fish (fingerlings, t ¼ )7.4; juveniles, t ¼ 4.7, p 6 0:05) and the tissues tested (head/brain, t test t ¼ 3.2 p < 0:05; muscle t test t ¼ 11.9, p 6 0:05).
4. Discussion The contribution of non-specific esterases to the measured activity has been estimated using the compound eserine sulphate, which is considered a specific inhibitor of ChE. ChE activity of selected tissues was almost completely inhibited (96–99% inhibition) by eserine sulphate at concentrations in the lM range, thus, within the range considered typical of ChE activity (concentration 106 – 105 M) [30–32]. This result indicates that the enzymatic activity measured in our experimental conditions was mainly due to ChE and not to other types of esterases. The ChE of the two tissues studied showed a preference for acetylthiocholine as substrate over butyrylthiocholine and propionylthiocholine. ATC and PROP are not selective substrates. A typical AChE will show high activities with ATC
Fig. 7. In vivo effect of dichlorvos on ChE activity of head (j) and muscle () tissues of fingerlings of European sea bass (N ¼ 3–6 and three enzymatic determinations per replicate). 00 ¼ acetone control. H I Indicates significant differences from control (p < 0:05).
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and low activities with PROP and BUT, while the reverse is true for normal BChE. Inhibition of brain ChE activity occurred at high concentrations of ATC. Also, ChE activity was sensitive to BW284C51, and showed no sensitivity to isoOMPA. These results indicate that the predominant form present in the brain tissue is AChE. Muscle ChE behaviour towards iso-OMPA and BW284C51, specific inhibitors of BChE and AChE, respectively, was different. Muscle ChE showed low substrate inhibition with increasing concentrations, moderate but significant sensitivity (between 15 and 22% inhibition) to iso-OMPA, and a high inhibitory effect of BW284C51 was observed (72% inhibition at 50 lM to 95% inhibition at 800 lM). These results suggest that both AChE and atypical BChE may be present in the muscle of sea bass, as Sturm et al. [14] reported for three marine teleost fish. These results are also in agreement with other studies carried out on several freshwater, estuarine, and marine fish, in which BChE has been found in muscle [13,14,33]. In contrast, in some teleost fish AChE seems to be the predominant form in head and muscle tissues, while BChE predominates in liver and plasma [16], as found in other studies. The present study shows that the enzyme activity from the selected tissues of fingerlings and juveniles was inhibited by in vitro exposure to dichlorvos. A greater sensitivity of the ChE activity of head and muscle tissues of fingerlings to dichlorvos relatively to juveniles was found. Thus, in fingerlings, a ChE inhibition between 58% and 40% was obtained for head and muscle tissues, respectively, at the lowest concentration tested (1 mg/L), whereas, in juveniles, the inhibition of ChE activity, at 1 mg/L, varied only between 9% and 7% for brain tissue and muscle tissues, respectively. This age-related sensitivity is in agreement with previous studies carried out with different fish species [10,34]. In general, an increase in size, age, and biomass are known to reduce the fish sensitivity to toxicant exposure [35]. Differences in ChE sensitivity to dichlorvos between both head and muscle tissues of fingerlings were found. The 96h-LC50 mortality value obtained (3.5 mg/L; 95% confidence limits. 0.06– 5.31) was about 4 times higher than the 96h-IC50 (0.89 mg/L) value estimated for head ChE inhibition. In muscle, the 96h-LC50 and 96h-IC50 (4.25 mg/L) values were quite similar. Thus, in fingerlings head tissue ChE was much more sensitive than muscle ChE. Differences in the relationship between ChE inhibition in brain and
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muscle tissues and mortality have been previously found in fish, for example, a higher sensitivity of brain ChE in comparison to muscle tissue has been previously reported by Fulton et al. [33] in the estuarine fish mummichog (Fundulus heteroclitus). A comparison of the 96h-LC50 values published for several teleost fish species [1] indicates that fingerlings of the European sea bass are more resistant to dichlorvos exposure than the most part of the other species of estuarine and freshwater fish studied. However, the comparison with fathed minnow (Pimephales promelas) or with mosquito fish (Gambusia affinis) of similar size indicates that sea bass fingerlings are more sensitive to dichlorvos, since 96h-LC50-values of 12 and 5.3 mg/L have been reported [1] for both species, respectively. In agreement with Sievers et al. (1995), who found that 100% of 100 g salmon (Salmon salar) survived after 24 h of exposure to 1, 3, and 5 mg/L of dichlorvos, no mortality of fingerlings (5 g) between 1 and 4 mg/L of dichlorvos after 24 h of exposure, was observed in this study. In vivo exposure of fingerlings to dichlorvos significant affected ChE activity in both head and muscle. Head ChE activity was more sensitive to this pesticide than muscle ChE. More than 35% inhibition of head ChE was detected at the lowest concentration tested (0.125 mg/L). It is well accepted that a 20% or greater inhibition of ChE in birds, fish, and invertebrates indicates exposure to OP insecticides [35]. Some animals are able to survive with more than 50% of ChE inhibition but this is an indication of a life-threatening situation [36]. In fish, inhibition of brain AChE between 60–70% can result in death [37]. Also, lethal effects have been described at >70% of brain ChE inhibition in most species of fish [16]. However, other authors found values P70% of reduction in brain ChE activity not to be lethal [38,39]. The current experiments show that fingerlings surviving to 96 h of exposure to 4 mg/L of dichlorvos presented a reduction of about 62% and 51% ChE activity of head and muscle, respectively. Moreover, strong inhibition of head (76%) and muscle (61%) ChE activity was found after 96 h of exposure to 1 mg/ L of dichlorvos, without mortality. Significant head inhibition (37%) was also observed at 0.125 mg/L, a concentration well below that causing mortality (LOEC for mortality was 2 mg/ L in this study). Similar results were obtained by Coppage and Matthews [38] for pinfish (Lagodon rhomboides) and Sancho et al. [40] for European
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eel (Anguilla anguilla). These findings suggest that sea bass fingerlings are able to tolerate high levels of head and muscle ChE inhibition before death occurs. The relationship between mortality and ChE inhibition induced by dichlorvos indicates differences for the two tissues analysed. Although, the NOEC and LOEC values for ChE activity were quite similar for both tissues, 96h-EC50 value for head was lower than that for obtained for muscle. Also, the 96h-LC50 value (3.5 mg/L; 95% confidence limits. 0.06–5.31) was 11 times greater than the 96h-EC50 value (0.31 mg/L) for head, while the 96h-EC50 value for muscle was only about 3 times greater than the 96h-EC50 value calculated (1.33 mg/L). In the current study, the brain of the juveniles had higher ChE activity than muscle. This result is consistent with other studies carried out in different fish species, in which brain tissue showed higher ChE activity than muscle, whole blood or plasma tissues [39,41,42]. On the contrary, in fingerlings, the highest ChE activity was obtained in muscle. This is in agreement with the results obtained by other researchers, including Bretaud et al. [43] who found that skeletal muscle presented higher ChE activity than brain tissue in juveniles of goldfish (Carassius auratus) of size 5 g exposed to three different pesticides. Likewise, Bonne and Chambers [8] and Carr et al. [9] demonstrated that muscle ChE of mosquito fish was more sensitive than brain to chlorpyrifos exposure. This may be partially due to the fact that in the present study brain ChE activity in fingerlings was measured using whole-head homogenates rather than isolate brain homogenates as in juveniles, that is the specific neurological tissue. The results obtained with different substrates and specific inhibitors indicate that in the European sea bass,the predominant ChE form in brain seems to be AChE, whereas in muscle, both AChE and BChE seem to be present. ChE activity of fingerlings exposed in vitro to dichlorvos was significantly reduced in both head and muscle tissues, in all the concentrations tested. However, for juveniles, ChE activity of both tissues was only significantly reduced at the highest concentrations tested (from 8 to 32 mg/L). Also, differences in ChE sensitivity were found in relation to the age of fish and the tissue analysed. The present study shows that fingerlings of the European sea bass are relatively resistant to acute dichlorvos exposure. In vivo exposure to dichlorvos of fingerlings, significantly reduced the enzyme activity of both head and muscle, in all concentrations tested,
except in muscle tissue at the lowest concentrations (0.125 mg/L). However, fingerlings are quite resistant to sublethal pesticide concentrations. A concentration of 1 mg/L dichlorvos has been found to produce a high degree of ChE inhibition in head (76%) and muscle (61%) of fingerlings, without mortality after 96 h of exposure. Therefore, it can be stated that ChE activity of head and muscle tissues of the European sea bass seems to be a sensitive biomarker to dichlorvos exposure. For a safe use of this insecticide in the control of ectoparasites in farmed fish as the European sea bass, more experimental work should be performed to determine the concentration and time of exposure to dichlorvos that do not induce significant sub-lethal effects on ChE activity of fish, and the time necessary to ChE recovery.
Acknowledgments This work was supported by ‘‘Plan Nacional I þ D project MAR98-0871C02-01’’ and by ao para a Ci^encia e Tecnologia–Portu‘‘Fundacß~ gal’’ (project CONTROL, contract PDCTM/PP/ MAR/15266/1999). We thank to M.E. PerezOlivares for technical support in the IATS, CSIC, Castell on, Spain.
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Effect of dichlorvos on cholinesterase activity of the European sea bass (Dicentrarchus labrax) I. Var o,a,* J.C. Navarro,a F. Amat,a and L. Guilherminob,c a Instituto de Acuicultura de Torre de la Sal (CSIC), 12595 Ribera de Cabanes, Castell on, Spain Instituto de Ci^ encias Biom edicas de Abel Salazar, Laborat orio de Ecotoxicologia, Dept. de Estudos de Populacßo~es, Universidade do Porto, Lg do Prof. Abel Salazar, 2, 4099-003 Porto, Portugal Centro Interdisciplinar de Investigacßa~o Marinha e Ambiental, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
b
c
Received 8 March 2002; accepted 3 August 2002
Abstract In this study, the acute toxicity of the organophosphorous pesticide (OP) dichlorvos and both in vitro and in vivo effects of dichlorvos on cholinesterase (ChE) activity of the European sea bass (Dicentrarchus labrax) were investigated. The characterisation of ChE and the ‘‘normal’’ range of activity in brain and muscle of non-exposed fish were determined in a first phase of the study. Acetylthiocholine was the substrate preferred of both brain and muscle ChE. Eserine sulphate and BW284C51 significantly inhibited the brain and muscle enzyme activity at low concentrations (lM range). Iso-OMPA had a significant effect in muscle, but not in brain tissue. These results suggest that acetylcholinesterase (AChE) is the predominant ChE form in brain tissue. In contrast, both acetylcholinesterase and butyrylcholinesterase seem to exist in muscle. Using acetylthiocholine as substrate, the ‘‘normal’’ range of fingerling head and muscle ChE were 58:05 2:11 and 118:03 8:67 U/mg protein, respectively. Corresponding values for juveniles were 43:32 4:42 and 19:44 2:44 U/mg protein for brain and muscle, respectively. Dichlorvos significantly inhibited the activity of ChE in the selected tissues, both in vitro and in vivo conditions. Differences in ChE sensitivity were found in relation to the age of the fish and the tissue analysed. The present study also showed that fingerlings of the European sea bass are relatively resistant to in vivo acute (96 h) dichlorvos exposure to concentrations between 0.125 and 1 mg/L, being able to tolerate high percentages of head ChE inhibition (37% and 76%) without lethal effects. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Marine fish; European sea bass; Cholinesterases (ChE); Biomarkers; Dichlorvos; Toxicity
1. Introduction Dichlorvos is an organophosphate pesticide (OP) commonly used in fish farming to eradicate crustacean ectoparasites. It is specially used in the
*
Corresponding author. Fax: +34-96431950. E-mail address: [email protected] (I. Var o).
treatment of sea lice (Lepophtheirus salmonis and Caligus elogatus) on commercial salmon farms. When a chemical product is used in bath treatments to control ectoparasites of cultivated fish, it is absorbed mainly through the gills and body surface, and may produce both lethal and sub-lethal effects on the fish. Dichlorvos is a direct-acting inhibitor of acetylcholinesterase (AChE) [1], the enzyme that degrades the neurotransmitter
0048-3575/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0048-3575(03)00019-1
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acetylcholine in cholinergic synapses. The inhibition provokes an accumulation of acetylcholine in synapses with disruption of the nerve function that can end in the death of the organism. The activity of cholinesterases (ChE) has been widely used as a biomarker of exposure to OP and carbamate (CB) pesticides in both vertebrates and invertebrates. The relationship between OP and/or CB exposure and ChE inhibition has been studied in several species of aquatic organisms, including molluscs [2,3] crustaceans [4–7] and fish [8–16]. All these studies have shown the sensitivity of ChE as a specific biomarker to diagnose exposure and effects of OP and CB pesticides. Although ChE activity is widely used as specific biomarker for anticholinesterase pesticides, its use in a particular species requires the characterisation of the enzyme(s) present in the tissues to be analysed and the determination of the activity range that may be considered as ‘‘normal’’ for non-exposed individuals [17,18]. Vertebrate ChEs are divided into two main classes, AChE and butyrylcholinesterase or pseudocholinesterase (BChE). The properties of ChE may differ from species to species and may also display variations in different tissues of the same species. Historically, fish brain and muscle tissues were though to contain mostly AChEs, while BChEs were though to be the predominant forms in liver and plasma. However, in some species of marine fish, the presence of both AChE and BChE in muscle tissue has been described [14,19]. Since more than one ChE may be present in the tissues of marine fishes and these ChEs may show different sensitivities to anticholinesterase agents, it is important to perform the biochemical characterisation of the ChE present in the species and in the tissues to be studied before their use as biomarker [18]. The acute toxicity of dichlorvos to fish has been previously determined by a number of researchers. Its toxicity for freshwater and estuarine fish is moderate to high, although it does not bioaccumulate in fish [20]. For freshwater and estuarine fish, 96h-LC50 values range from 0.2 to 12 mg/L [1]. For marine fish, the toxicity was estimated to be more than 4 mg/L for adults and pre-adults of Atlantic salmon (Salmo salar) [21]. However, Sievers et al. [22], did not find mortality in 100 g salmon at dichlorvos concentrations between 1 and 5 mg/L after 24 h of exposure. Several authors have been reporting significant inhibition of ChE activity in fish at sub-lethal concentrations of dichlorvos [14,15,23,24].
To the best of our knowledge, no previous studies on the effect of dichlorvos on ChE activity of the European sea bass (Dicentrarchus labrax), a common species of marine fish cultured in the Mediterranean, have been carried out. Since dichlorvos is being used in bath treatments against crustacean ectoparasites in marine-farmed fish, the main objective of this study was to investigate the effects of this pesticide on the European sea bass (Dicentrarchus labrax). Therefore, the 50% lethal concentration (LC50) value of dichlorvos to this species was determined, and both in vivo and in vitro effects on ChE activity of fingerlings and juveniles induced by exposure to sub-lethal concentrations of this chemical was investigated. To reach this objective, in a first phase of the study, the characterisation of the ChE present in brain and muscle was performed and the ‘‘normal’’ range of activity in both tissues of non-exposed fish was determined.
2. Materials and methods 2.1. Chemicals Acetylthiocholine iodide, s-butyrylthiocholine iodide, propionylthiocholine iodide, iso-OMPA (tetraisopropyl pyrophosphoramide), eserine sulphate, BW284C51 (1,5-bis(4-allyldimethyammoniumphenyl) pentan-3-one dibromide), 5, 50 -dithiobis(2-nitrobenzoic acid) (DTNB) and bovine c-globulinÕs were obtained from Sigma Chemicals. Dichlorvos was obtained from Dr. Ehrenstorfer Reference Materials (Germany). 2.2. Experimental fish Fingerlings (weight ¼ 5:62 1:63 g, length ¼ 7:88 0:72 cm) and juveniles (weight ¼ 103:9 27:96 g, length ¼ 19:93 1:26 cm) of the European sea bass (Dicentrarchus labrax) were reared at the Instituto de Acuicultura de Torre de la Sal (CSIC), Castell on (Spain). They were kept in 500 L fibreglass tanks filled with seawater and supplied with continuous aeration under room temperature (22–28 °C) and natural photoperiod. Fish were fed ad libitum with commercial food pellets (Pro-Aqua, Spain). 2.3. Cholinesterase characterisation ChE characterisation of European sea bass was determined in juveniles. Fish were killed on
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ice before the brain and a piece of dorsal muscle (100 mg) being quick removed and kept on ice until the homogenates were prepared. Tissues were homogenised in 1 ml of ice-cold phosphate buffer (0.1 M, pH 7.2), using a tissue disrupter ultraturrax T-8 (IKA, Germany), centrifuged (at 8500 rpm for 5 min at 4 °C) and the supernatant was stored at )20 °C for no more than three weeks, until enzymatic analysis could be performed. Cholinesterase characterisation was initiated by measuring the enzyme activity at concentrations ranging from 0.01 to 20.5 mM of the substrates acetylthiocholine iodide (ATC), s-butyrylthiocholine iodide (BUT) and propionylthiocholine iodide (PROP). At least three replicates of each substrate were performed. Eserine sulphate, iso-OMPA, and BW284C51 were used as specific inhibitors of ChE, BChE, and AChE, respectively. Eserine sulphate and iso-OMPA were dissolved in ethanol, while BW284C51 was dissolved in bidistilled water. For cholinesterase assays, the supernatants were diluted in 0.1 M phosphate buffer at 1:10 (v:v) for head samples and at 4 mg/ml for muscle samples. Before ChE activity determinations, 0.490 ml of diluted supernatant were incubated for 30 min with 0.010 ml of each inhibitor, at room temperature (20–22 °C). For each inhibitor, the concentrations prepared ranged from 1.56 to 800 lM for eserine sulphate and BW284C51, and from 0.125 to 16 mM for isoOMPA. Controls were incubated with 0.010 ml of bidistilled water. Controls with 0.010 ml of ethanol were included for assays with eserine sulphate and iso-OMPA.
method [27] adapted to microplate, using c-globulin as standard and a wavelength of 600 nm.
2.4. Enzyme activity
2.6. Acute toxicity tests based on mortality
ChE activity was determined at room temperature (20–22 °C), in triplicate, by the Ellman method [25] adapted to microplate as described in Guilhermino et al. [26], using acetylthiocholine as substrate. In each assay, 0.05 ml of diluted supernatant and 0.250 ml of the reaction solution (phosphate buffer 0.1 M + 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) 10 mM + substrate) were used. The absorbance increase at 405 nm was followed for 5 min using a Bio-Rad microplate reader. The activity of ChE was expressed as the mean of the determinations performed (at least three independent samples) and was expressed as Units (U) per mg of protein (1 U ¼ nmol of substrate hydrolysed per min). The protein content of the samples was measured by the Bradford
Acute toxicity tests for determination of LC50 values were carried out following the recommendations of OECD [28]. Before the test, fingerlings were transferred to 10 L glass aquaria filled with filtered (0.2 lm) seawater, and exposed to a range of dichlorvos concentrations from 0.125 to 12.5 mg/L for 96 h, in static conditions. The medium was changed daily to assure the desired concentration of pesticide in the seawater. A stock solution of dichlorvos in acetone was prepared and the necessary amount of the stock solution added to the glass aquaria filled with filtered seawater. Duplicate groups of 4 fish were kept in separate 10 L glass aquarium filled with filtered seawater containing the desired pesticide concentration. Control fish were kept in 10 L glass aquaria filled
2.5. ChE in vitro tests In vitro ChE inhibition tests were carried out with both fingerlings and juveniles of the European sea bass, in independent experiments. Fish were fed and maintained under standard conditions of culture as described before. Feeding was stopped 24 h before fish were sampled for ChE assays. After killing the fish on ice, the brain and muscle tissues were rapidly removed and kept on ice until the preparation of the homogenates, as described before. The brain tissue of fingerlings was impossible to remove because the small size of the fish, so, the whole head without eyes was isolated from each animal and prepared and stored as described before. Also, the small amount of muscle associate to the head was carefully removed to avoid any influence on head ChE measurements. Prior to enzyme determinations, 0.490 ml of diluted supernatant was incubated for 30 min with 0.010 ml of different dichlorvos concentrations, at room temperature (20–22 °C). The nominal concentrations of dichlorvos tested were 1, 2, 4, 8, 16, and 32 mg/L. Controls were incubated with 0.010 ml of bidistilled water, and additional controls incubated with 0.010 ml of acetone solution, were also included. Between three juveniles and six fingerlings of sea bass per treatment were used for each tissue sampled. Three ChE determinations were performed per sample as previously described for the enzyme activity, using acetylthiocholine as substrate.
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with filtered seawater. A control with acetone was also included in which fish were exposed to the same amount of acetone as in the highest concentration tested (12.5 mg/L). Fish were maintained in a temperature and photoperiod controlled room (20 1 °C; 12 hL:12 hD). They were not fed during the test period and the medium was changed daily, to maintain water quality and pesticide concentrations. The effect criteria was mortality. Dead specimens were removed twice a day.
regression probit module of SPSS Systems (SPSS, 1989–1992) at 95% of confidence, was used. Differences among treatments were tested by one-way Analysis of Variance (ANOVA). For in vivo tests, no-observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) values were determined by TukeyÕs test for multiple comparisons [29], with a significance level of 0.05.
2.7. ChE in vivo tests
3.1. Characterisation of ChE
In vivo ChE tests were conducted only for head and muscle tissues of fingerlings, the most sensitive stage, because it is known that, in general, the increase of size and age reduce the fish sensitivity to toxicants. Duplicate groups of 4 fish were exposed to five nominal pesticide concentrations (0.125, 0.5, 1, 2, and 4 mg/L) in 10 L glass aquaria for 96 h, under the conditions of temperature and photoperiod described for acute tests. Fish were not fed during the exposure time (96 h). The concentrations of the pesticide used in ChE in vivo tests were based on 96h-LC50 values previously determined. A stock solution of the pesticide was prepared in acetone and the necessary amount of the stock solution added to the aquarium filled with filtered seawater to prepare the different treatments. Controls with acetone were also included as described above. At the end of the test, live fish were killed on ice before removal of head and muscle and the preparation of homogenates for ChE assays, as described before. The whole head (without the eyes and the musculature associate to the head), and a piece of muscle (50 mg) tissue were isolated from each animal, prepared, and stored as described above. The ChE activity was performed as previously described for enzyme activity determinations. The ‘‘normal’’ range of ChE activity of head or brain and muscle tissues was determined in nonexposed fish (both fingerlings and juveniles). Homogenates were prepared as previous described, and ChE activity was determined using acetylthiocholine as substrate.
ChE activity of brain and muscle as a function of different substrates is shown in Fig. 1. The results obtained for both tissues indicate that ChE shows preference for acetylthiocholine as substrate. A high inhibition of ChE activity in the descending part of the ATC curve, thus under excess of substrate conditions, was observed only for brain tissue. Values of ChE activity measured in both tissues using butyrylthiocholine and propionylthiocholine as substrates were considerably lower than the correspondent values obtained with ATC. The effects of eserine sulphate, isoOMPA and BW284C51 on ChE activity of brain and muscle of the European sea bass are shown in Figs. 2–4, respectively. Eserine sulphate had a significant inhibitory effect on both brain and muscle ChE activity at low concentrations (106 M range) (see Fig. 2; brain: F ¼ 278.9, df ¼ 11, 107, p 6 0:05; muscle, F ¼ 616.5, df ¼ 11, 239, p 6 0:05). Brain ChE activity was not significantly affected by iso-OMPA (see Fig. 3, F ¼ 1.2, df ¼ 9, 119, p P 0:05), while muscle ChE activity was significantly affected by this chemical (see Fig. 3, F ¼ 20.1, df ¼ 9, 119 p 6 0:05), with an inhibition ranging between 15 and 22%. In contrast, the inhibitor BW284C51 produced a significant effect on ChE activity (see Fig. 4; brain: F ¼ 466, df ¼ 10, 98, p 6 0:05; muscle, F ¼ 37.8, df ¼ 10, 175, p 6 0:05) for both tissues. However, the inhibition of ChE was higher in the brain (<8% of control activity in all the concentrations tested) than in the muscle with an enzyme activity lower than 8% only in the highest concentrations tested (400 and 800 lM).
2.8. Statistical analysis
3.2. ChE in vitro tests
To calculate LC50 values, 50% in vitro inhibition concentration values (IC50) and 50% in vivo inhibition concentration values (EC50), the
In vitro exposure to dichlorvos affected the ChE activity in both head or brain and muscle tissues of fingerlings and juveniles (Figs. 5 and 6,
3. Results
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Fig. 1. ChE activity of the European sea bass brain and muscle tissues as a function of the different substrates, acetylthiocholine iodide (ATC), s-butyrylthiocholine iodide (BUT), and propionylthiocholine iodide (PROP). Values are means SD. (N ¼ 3 and three enzymatic determinations per replicate).
Fig. 2. Effect of Eserine sulphate on ChE activity of the European sea bass brain (j) and muscle tissues () (N ¼ 3 and three enzymatic determinations per replicate). 00 ¼ ethanol control. H I Indicates significant differences from control (p < 0:05).
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Fig. 3. Effect of Iso-OMPA on ChE activity of the European sea bass brain (j) and muscle tissues () (N ¼ 3 and three enzymatic determinations per replicate). 00 ¼ ethanol control. I Indicates significant differences from control (p < 0:05).
Fig. 4. Effect of BW284C51 on ChE activity of the European sea bass brain (j) and () (N ¼ 3 and three enzymatic determinations per replicate). H I Indicates significant differences from control (p < 0:05).
respectively). For fingerlings, IC50 values estimated for head and muscle ChE activity were 0.89 mg/L (95% confidence limits: 0.39–2.05) and
4.25 mg/L (95% confidence limits: 1.6–10.8), respectively. In contrast, for juveniles, no differences on ChE sensitivity to dichlorvos were found
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Fig. 5. In vitro effect of dichlorvos on ChE activity of brain (j) and muscle () tissues of fingerlings of European sea bass (N ¼ 3–6 and three enzymatic determinations per replicate). 00 ¼ acetone control. H I Indicates significant differences from control (p < 0:05).
Fig. 6. In vitro effect of dichlorvos on ChE activity of brain (j) and muscle () tissues of juveniles of European sea bass (N ¼ 3 and three enzymatic determinations per replicate). 00 ¼ acetone control. H I Indicates significant differences from control (p < 0:05).
between brain and muscle ChE activity, with IC50 values of 7.4 mg/L (95% confidence limits: 7.3–7.5) and 9.9 mg/L (95% confidence limits: 9.9–10.1) for brain and muscle, respectively. No significant effects of the solvent at the maximal concentration tested (32 mg/L) was observed for fingerlings or juveniles ChE (see Figs. 5 and 6).
3.3. Acute toxicity test based on mortality The 96h-LC50 value obtained for fingerlings was 3.5 mg/L (95% confidence limits: 0.06–5.31). The survival of the control fish in the acute toxicity test based on mortality was 100%. However, fish exposed to the highest pesticide
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concentrations tested, 12.5 and 8 mg/L, resulted in 100% of mortality after 5 h of the start of the test. 3.4. ChE in vivo tests The effects of in vivo exposure to dichlorvos on head and muscle ChE activity of fingerlings are shown in Fig. 7. Dichlorvos-induced significant alterations on ChE activity in both tissues (head, F ¼ 70.1, df ¼ 6, 28, p 6 0:05; muscle, F ¼ 43.7, df ¼ 6, 29, p 6 0:05) after 96 h of exposure. Also, a significant effect on ChE activity of the solvent at the maximal concentration tested (12.5 mg/L) was observed. The NOEC and LOEC values were quite similar for both tissues analysed. NOEC values for head and muscle were <0.125 and 0.125 mg/L, respectively. LOEC values were 0.125 mg/L for head and 0.5 mg/L for muscle. The EC50 values were 0.31 mg/L (95% confidence limits: 0.55–16.28) for head and 1.33 mg/L (large 95% confidence limits) for muscle. The values of ‘‘normal’’ range of fingerlings ChE activity using acetylthiocholine as substrate were 58:05 2:11 U/mg protein for head and 118:03 8:67 U/mg protein for muscle. Corresponding values for juveniles were 43:32 4:42 U/mg protein for brain and 19:44 2:44 U/mg protein for
muscle. Significant differences in the ‘‘normal’’ range of ChE activity were found between the two age groups of fish (fingerlings, t ¼ )7.4; juveniles, t ¼ 4.7, p 6 0:05) and the tissues tested (head/brain, t test t ¼ 3.2 p < 0:05; muscle t test t ¼ 11.9, p 6 0:05).
4. Discussion The contribution of non-specific esterases to the measured activity has been estimated using the compound eserine sulphate, which is considered a specific inhibitor of ChE. ChE activity of selected tissues was almost completely inhibited (96–99% inhibition) by eserine sulphate at concentrations in the lM range, thus, within the range considered typical of ChE activity (concentration 106 – 105 M) [30–32]. This result indicates that the enzymatic activity measured in our experimental conditions was mainly due to ChE and not to other types of esterases. The ChE of the two tissues studied showed a preference for acetylthiocholine as substrate over butyrylthiocholine and propionylthiocholine. ATC and PROP are not selective substrates. A typical AChE will show high activities with ATC
Fig. 7. In vivo effect of dichlorvos on ChE activity of head (j) and muscle () tissues of fingerlings of European sea bass (N ¼ 3–6 and three enzymatic determinations per replicate). 00 ¼ acetone control. H I Indicates significant differences from control (p < 0:05).
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and low activities with PROP and BUT, while the reverse is true for normal BChE. Inhibition of brain ChE activity occurred at high concentrations of ATC. Also, ChE activity was sensitive to BW284C51, and showed no sensitivity to isoOMPA. These results indicate that the predominant form present in the brain tissue is AChE. Muscle ChE behaviour towards iso-OMPA and BW284C51, specific inhibitors of BChE and AChE, respectively, was different. Muscle ChE showed low substrate inhibition with increasing concentrations, moderate but significant sensitivity (between 15 and 22% inhibition) to iso-OMPA, and a high inhibitory effect of BW284C51 was observed (72% inhibition at 50 lM to 95% inhibition at 800 lM). These results suggest that both AChE and atypical BChE may be present in the muscle of sea bass, as Sturm et al. [14] reported for three marine teleost fish. These results are also in agreement with other studies carried out on several freshwater, estuarine, and marine fish, in which BChE has been found in muscle [13,14,33]. In contrast, in some teleost fish AChE seems to be the predominant form in head and muscle tissues, while BChE predominates in liver and plasma [16], as found in other studies. The present study shows that the enzyme activity from the selected tissues of fingerlings and juveniles was inhibited by in vitro exposure to dichlorvos. A greater sensitivity of the ChE activity of head and muscle tissues of fingerlings to dichlorvos relatively to juveniles was found. Thus, in fingerlings, a ChE inhibition between 58% and 40% was obtained for head and muscle tissues, respectively, at the lowest concentration tested (1 mg/L), whereas, in juveniles, the inhibition of ChE activity, at 1 mg/L, varied only between 9% and 7% for brain tissue and muscle tissues, respectively. This age-related sensitivity is in agreement with previous studies carried out with different fish species [10,34]. In general, an increase in size, age, and biomass are known to reduce the fish sensitivity to toxicant exposure [35]. Differences in ChE sensitivity to dichlorvos between both head and muscle tissues of fingerlings were found. The 96h-LC50 mortality value obtained (3.5 mg/L; 95% confidence limits. 0.06– 5.31) was about 4 times higher than the 96h-IC50 (0.89 mg/L) value estimated for head ChE inhibition. In muscle, the 96h-LC50 and 96h-IC50 (4.25 mg/L) values were quite similar. Thus, in fingerlings head tissue ChE was much more sensitive than muscle ChE. Differences in the relationship between ChE inhibition in brain and
69
muscle tissues and mortality have been previously found in fish, for example, a higher sensitivity of brain ChE in comparison to muscle tissue has been previously reported by Fulton et al. [33] in the estuarine fish mummichog (Fundulus heteroclitus). A comparison of the 96h-LC50 values published for several teleost fish species [1] indicates that fingerlings of the European sea bass are more resistant to dichlorvos exposure than the most part of the other species of estuarine and freshwater fish studied. However, the comparison with fathed minnow (Pimephales promelas) or with mosquito fish (Gambusia affinis) of similar size indicates that sea bass fingerlings are more sensitive to dichlorvos, since 96h-LC50-values of 12 and 5.3 mg/L have been reported [1] for both species, respectively. In agreement with Sievers et al. (1995), who found that 100% of 100 g salmon (Salmon salar) survived after 24 h of exposure to 1, 3, and 5 mg/L of dichlorvos, no mortality of fingerlings (5 g) between 1 and 4 mg/L of dichlorvos after 24 h of exposure, was observed in this study. In vivo exposure of fingerlings to dichlorvos significant affected ChE activity in both head and muscle. Head ChE activity was more sensitive to this pesticide than muscle ChE. More than 35% inhibition of head ChE was detected at the lowest concentration tested (0.125 mg/L). It is well accepted that a 20% or greater inhibition of ChE in birds, fish, and invertebrates indicates exposure to OP insecticides [35]. Some animals are able to survive with more than 50% of ChE inhibition but this is an indication of a life-threatening situation [36]. In fish, inhibition of brain AChE between 60–70% can result in death [37]. Also, lethal effects have been described at >70% of brain ChE inhibition in most species of fish [16]. However, other authors found values P70% of reduction in brain ChE activity not to be lethal [38,39]. The current experiments show that fingerlings surviving to 96 h of exposure to 4 mg/L of dichlorvos presented a reduction of about 62% and 51% ChE activity of head and muscle, respectively. Moreover, strong inhibition of head (76%) and muscle (61%) ChE activity was found after 96 h of exposure to 1 mg/ L of dichlorvos, without mortality. Significant head inhibition (37%) was also observed at 0.125 mg/L, a concentration well below that causing mortality (LOEC for mortality was 2 mg/ L in this study). Similar results were obtained by Coppage and Matthews [38] for pinfish (Lagodon rhomboides) and Sancho et al. [40] for European
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eel (Anguilla anguilla). These findings suggest that sea bass fingerlings are able to tolerate high levels of head and muscle ChE inhibition before death occurs. The relationship between mortality and ChE inhibition induced by dichlorvos indicates differences for the two tissues analysed. Although, the NOEC and LOEC values for ChE activity were quite similar for both tissues, 96h-EC50 value for head was lower than that for obtained for muscle. Also, the 96h-LC50 value (3.5 mg/L; 95% confidence limits. 0.06–5.31) was 11 times greater than the 96h-EC50 value (0.31 mg/L) for head, while the 96h-EC50 value for muscle was only about 3 times greater than the 96h-EC50 value calculated (1.33 mg/L). In the current study, the brain of the juveniles had higher ChE activity than muscle. This result is consistent with other studies carried out in different fish species, in which brain tissue showed higher ChE activity than muscle, whole blood or plasma tissues [39,41,42]. On the contrary, in fingerlings, the highest ChE activity was obtained in muscle. This is in agreement with the results obtained by other researchers, including Bretaud et al. [43] who found that skeletal muscle presented higher ChE activity than brain tissue in juveniles of goldfish (Carassius auratus) of size 5 g exposed to three different pesticides. Likewise, Bonne and Chambers [8] and Carr et al. [9] demonstrated that muscle ChE of mosquito fish was more sensitive than brain to chlorpyrifos exposure. This may be partially due to the fact that in the present study brain ChE activity in fingerlings was measured using whole-head homogenates rather than isolate brain homogenates as in juveniles, that is the specific neurological tissue. The results obtained with different substrates and specific inhibitors indicate that in the European sea bass,the predominant ChE form in brain seems to be AChE, whereas in muscle, both AChE and BChE seem to be present. ChE activity of fingerlings exposed in vitro to dichlorvos was significantly reduced in both head and muscle tissues, in all the concentrations tested. However, for juveniles, ChE activity of both tissues was only significantly reduced at the highest concentrations tested (from 8 to 32 mg/L). Also, differences in ChE sensitivity were found in relation to the age of fish and the tissue analysed. The present study shows that fingerlings of the European sea bass are relatively resistant to acute dichlorvos exposure. In vivo exposure to dichlorvos of fingerlings, significantly reduced the enzyme activity of both head and muscle, in all concentrations tested,
except in muscle tissue at the lowest concentrations (0.125 mg/L). However, fingerlings are quite resistant to sublethal pesticide concentrations. A concentration of 1 mg/L dichlorvos has been found to produce a high degree of ChE inhibition in head (76%) and muscle (61%) of fingerlings, without mortality after 96 h of exposure. Therefore, it can be stated that ChE activity of head and muscle tissues of the European sea bass seems to be a sensitive biomarker to dichlorvos exposure. For a safe use of this insecticide in the control of ectoparasites in farmed fish as the European sea bass, more experimental work should be performed to determine the concentration and time of exposure to dichlorvos that do not induce significant sub-lethal effects on ChE activity of fish, and the time necessary to ChE recovery.
Acknowledgments This work was supported by ‘‘Plan Nacional I þ D project MAR98-0871C02-01’’ and by ao para a Ci^encia e Tecnologia–Portu‘‘Fundacß~ gal’’ (project CONTROL, contract PDCTM/PP/ MAR/15266/1999). We thank to M.E. PerezOlivares for technical support in the IATS, CSIC, Castell on, Spain.
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