Lactate dehydrogenase activity as an effect criterion in toxicity tests with Daphnia magna straus
Introduction
In recent years, research has been undertaken into the development and validation of in vitro and in vivo tests based on biomarkers. These tests are able to detect toxic effects before alterations of important physiological functions, such as reproduction, growth or death occur (Janssen et al., 1993; Garle et al., 1994; Guilhermino et al., 1996; Blaise et al., 1997; Diamantino et al., 2000).
Lactate dehydrogenase (LDH) is a parameter widely used in toxicology and in clinical chemistry to diagnose cell, tissue and organ damage. However, the potential of this enzyme as an indicative criterion in invertebrate aquatic toxicity tests has been scarcely explored (Ribeiro et al., 1999). LDH is an important glycolytic enzyme being present in virtually all tissues (Kaplan and Pesce, 1996). Alterations of the normal LDH activity pattern were found after exposure to different concentrations of sodium bromide and 3,4-dichloroaniline (Guilhermino et al., 1994), copper gallium diselenide, copper indium diselenide and cadmium telluride (Morgan et al., 1995), cadmium (Hassoun and Stohs, 1996) or oxygen stress (Wu and Lam, 1997).
A number of procedures have been developed for measuring the total LDH activity in mammalian tissues. One of the most frequently used is the spectrophotometric method described by Vassault (1983) that quantifies the conversion of pyruvate to lactate. In this method, LDH activity is measured at 30°C as the amount of pyruvate consumed by continuously monitoring the decrease of absorbance due to the oxidation of NADH at 339 nm (Vassault, 1983). Two requirements of this technique severely limit its use in ecotoxicology: (i) a considerable volume of concentrate sample is needed and (ii) each sample takes about 4 min to be processed. In ecotoxicological studies, only a small amount of biological material is frequently available and a large number of samples should be analysed in a short period of time. In order to overcome these limitations, an adaptation to microplate technique of the Vassault method was developed and tested in our laboratory.
Daphnia magna is widely used as test organism in aquatic toxicology (Adema, 1978; Van der Meer et al., 1988; Soares et al., 1992). Toxicity tests with this species are required for the assessment of the potential impact of new chemicals on the aquatic environment (EEC, 1992). The objectives of this study were: (i) to optimize and standardize the conditions for the use of LDH activity as effect criterion in toxicity tests with the cladoceran D. magna, (ii) to develop both in vivo and in vitro LDH activity-based tests and (iii) to investigate the sensitivity of these tests. In order to achieve the first and second objectives, the influence of several parameters (e.g. number of animals per sample, nutritional status, age and presence/absence of eggs in the brood chamber) on LDH activity was investigated. In order to attain the third objective, the results from LDH tests were compared with those obtained in conventional acute toxicity tests, using zinc chloride as test substance.
Section snippets
Parent animals
Parent animals were cultured in hard water (ASTM, 1980) supplemented with an organic additive (Baird et al., 1989), in groups of 10 animals per 1000 ml of medium, and fed with the algae Chlorella vulgaris (0.322 mg carbon/daphnid/day). The photoperiod was 16 hL:8 hD and the temperature was 20°±1°C. All the experiments were carried out with 3rd to 5th brood neonates from the clone A (sensu Baird et al., 1989) except when the effects of the presence of eggs in the brood chamber and the age of
LDH determinations using a microplate technique
LDH activity was determined using an adaptation to microplate technique of the Vassault method (Vassault, 1983). Briefly, 250 μl of TRIS/NaCl/NADH solution, 40 μl of sample and 80 μl of TRIS/NaCl/pyruvate solution were successively introduced into each well of the microplate. After 30 s and during 5 min, the decrease of absorbance due to the oxidation of NADH was recorded each 20 s in a Labsystem Multiskan EX microplate reader at 340 nm. LDH activity was calculated from the slope of the absorbance
Number of animals per sample
No significant differences on LDH activity were found among samples prepared with 35, 45 and 55 juveniles and no differences in LDH determinations were found owing to the three groups of juveniles (NESTED ANOVA: F=1.5; d.f.=2, 6; P>0.05 and F=1.0; d.f.=6, 18; P>0.05, respectively).
Nutritional condition and age of juveniles
No significant differences in LDH activity were found between 24 h and 48 h old juveniles with food (t-Test: t=1.65, P=0.11) and no effects due to the presence of food were found (t-Test: t=2.00, P=0.06).
Presence of eggs in the brood chamber
Significant
Discussion
LDH is involved in the production of energy, being particularly important when a considerable amount of additional energy is rapidly required. A negative correlation between LDH activity and ambient oxygen levels for some aquatic organisms, suggesting a possible biochemical adjustment in response to the lowered oxygen levels (Wu and Lam, 1997). This probably occurs also in situations of chemical stress. Therefore, this enzyme may be a sensitive criterion both in laboratory and in biomonitoring
Acknowledgements
This work was supported by Fundação para a Ciência e a Tecnologia (Portugal), program PRAXIS XXI (BD/2872/94) and by the EU Project (TROCA-WET, contract: ERBIC 18/CT98/0264).
Teresa Cunha Diamantino. Research assistant in National Institute of Engineering and Industrial Technology (INETI), Lisbon, Portugal. Master degree in Ecotoxicology. Member in several research projects (national and international). Seven papers published in international scientific journals.
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Teresa Cunha Diamantino. Research assistant in National Institute of Engineering and Industrial Technology (INETI), Lisbon, Portugal. Master degree in Ecotoxicology. Member in several research projects (national and international). Seven papers published in international scientific journals.
Elisabete Almeida. Principal researcher of National Institute of Engineering and Industrial Technology (INETI) Lisbon, Portugal. Leader of LTR Laboratory of Surface Treatments and Coatings in INETI, Lisbon, Portugal. Co-ordinator, partner co-ordinator or member in several research projects (national and international). More than 30 published/in press papers in international scientific journals.
Amadeu M. V. M. Soares. Associate Professor and Chairman of the Scientific Council of the Department of Biology, University of Aveiro, Portugal. Chairman of the “Instituto do Ambiente e Vida”, University of Coimbra, Portugal, leading the Ecotoxicology Group. Coordinator and partner in several international and national research projects. Member of several Scientific European Advisory Committees in Toxicology and Ecotoxicology. 56 published papers in international scientific peer-reviewed journals.
Lúcia Guilhermino. Assistant Professor of the Institute of Biomedical Sciences of Abel Salazar, University of Porto, Portugal. Leader of the group of Ecotoxicology of the Centre of Marine and Environmental Research (CIMAR), Portugal. Co-ordinator, partner co-ordinator or member in several research projects (national and international). 20 published/in press papers in international scientific journals.