Toxicity of Sodium Molybdate and Sodium Dichromate to Daphnia Magna Straus Evaluated in Acute, Chronic, and Acetylcholinesterase Inhibition Tests Teresa C
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Ecotoxicology and Environmental Safety 45, 253}259 (2000) Environmental Research, Section B doi:10.1006/eesa.1999.1889, available online at http://www.idealibrary.com on Toxicity of Sodium Molybdate and Sodium Dichromate to Daphnia magna Straus Evaluated in Acute, Chronic, and Acetylcholinesterase Inhibition Tests Teresa C. Diamantino,*,-,1 Lucia Guilhermino,? Elisabete Almeida,- and Amadeu M. V. M. Soares* *Instituto do Ambiente e Vida, Departamento de Zoologia, Largo Marque' s de Pombal, Universidade de Coimbra, 3000 Coimbra, Portugal; -Instituto Nacional de Engenharia e Tecnologia Industrial, Estrada do Pac,o do Lumiar, 1699 Lisboa Codex, Portugal; and ?Instituto de Cie' ncias BimeH dicas de Abel Salazar, Departamento de Estudos de Populac,oJ es, LaboratoH rio de Ecotoxicologia/Centro de Investigac,a8 o Marinha e Ambiental, Universidade do Porto, Lg Prof. Abel Salazar, 2, 4050 Porto, Portugal Received March 18, 1999 spread application in numerous industrial processes, chro- As a result of a widespread application in numerous industrial mium is a contaminant of many environmental systems processes, chromium is a contaminant of many environmental systems. Chromium and their compounds are toxic to both (Cohen et al., 1993). Industrial uses of chromium include invertebrates and vertebrates and, for this reason, there has been metal "nishing industry, production of paints and pigments, a search for suitable and less toxic alternatives. Molybdenum tanning, wood preservation, chromium chemicals produc- compounds have been studied as alternative to chromium tion, pulp and paper production, and metal smelting (Paw- compounds for some industrial applications. The toxicity of lisz et al., 1997). One of the major uses of environmentally chromium is well known but the e4ects of molybdenum and hazardous chemicals is in the metal "nishing industry. So- molybdenum mining on natural populations and communities of dium dichromate is the most important of the industrial freshwater invertebrates have not often been studied. However, chromium chemicals; it is used as the starting compound for chromium, and molybdenum (and their compounds) are included almost all chromium compounds (Anger et al., 1986) and is in the same list (List II) of European Union dangerous substan- "rmly established in the metal "nishing "eld (Biestek and ces. In this study, the acute and chronic e4ects of sodium molyb- Weber, 1976). One of the environmental problems of these date and sodium dichromate to Daphnia magna Straus were evaluated. Furthermore, in vitro and in vivo e4ects of these two industries is the elimination of hexavalent chromium com- metals on acetylcholinesterase (AChE) activity of D. magna pounds, which are used extensively for the passivation of Straus were investigated. LC50 values determined at 48 h were zinc, and for zinc-based coatings and surfaces. The elimina- 0.29 and 2847.5 mg L21 for chromium (as sodium dichromate) tion of these toxic chemicals from metal processing, parti- and molybdenum (as sodium molybdate), respectively. No signif- cularly in the automobile industry where chromates are icant in vitro e4ects of both metals on AChE were found. How- widely used for the protection of zinc-plated parts, is con- ever, both toxicants inhibited AChE in vivo at concentrations sidered a priority within the European Union (SI, 1997). under the respective 48-h LC50 values. Both sodium dichromate Molybdenum compounds are used in catalysis, luber"z- and sodium molybdate inhibited the reproduction and growth of ation, #ame retardancy and smoke suppression, pigments, D. magna, but the concentrations inducing signi5cant e4ects agriculture, and corrosion inhibition (Sebenik et al., 1990). were di4erent for the two chemicals. Sodium molybdate had At high concentrations, molybdenum is toxic to many or- signi5cant lower toxicity to D. magna Straus than sodium dich- ganisms, including mammals and freshwater invertebrates romate. ( 2000 Academic Press Key Words: sodium molybdate; sodium dichromate; acute and (Khangarot, 1991). The e!ects of molybdenum or molyb- chronic e4ects; AChE, Daphnia magna. denum mining on natural populations or communities of freshwater invertebrates have not often been studied (Whiting et al., 1994). Based on toxicological studies from 1. INTRODUCTION the literature, some authors have been studying alternatives to chromium hexavalent compounds for passivation of zinc Heavy metals are widely recognized as highly toxic and coatings based on molybdenum compounds (Tang et al, dangerous to organisms (Mance, 1987). As a result of wide- 1994; Wharton et al., 1996; Almeida et al., 1998). Despite the di!erences of toxicity between chromium and molybdenum 1 To whom correspondence should be addressed at IMP/LTR, Instituto reported in the literature, both metals (and their com- Nacional de Engenharia e Tecnologia Industrial, Estrada do Pac,odo Lumiar, 1699 Lisboa Codex, Portugal. Fax: #351-21-7160901. E-mail: pounds) are included in the List II of European dangerous [email protected]. substances directive (CEC, 1976). 253 0147-6513/00 $35.00 Copyright ( 2000 by Academic Press All rights of reproduction in any form reserved. 254 DIAMANTINO ET AL. Daphnia magna is widely used as a test organism in test was carried out for 48 h. The criterion of toxic e!ect was aquatic toxicology (Adema, 1978; Meer et al., 1988; Soares death, recognized by immobilization after stimulation by et al., 1992). Bioassays involving this species are required for a bright light. In chronic tests, the organisms were cultured the assessment of the potential impact of new chemicals on in ASTM medium with an organic additive (Baird et al., the aquatic environment (Soares et al., 1991). Acute and 1989), one animal per 1000 ml of medium, and fed with the chronic bioassays with this species are the most widely used algae C. vulgaris (0.322 mg carbon/daphnia/day). Each test test methods for the toxicity assessment of chemical com- was carried out for 21 days. Animals were transferred to pounds and e%uents. In acute tests the measured parameter newly prepared test solutions three times a week (i.e., every is death, while in chronic tests the inhibition of normal other day). One control and six toxicant concentrations reproduction and growth are the most used endpoints for with 10 replicates for each treatment were used. Endpoints toxicity evaluation. However, chronic tests are particularly were reproduction, growth, and mortality. In both acute time-consuming and expensive. For this reason, in recent and chronic tests, temperature and photoperiod were as decades there have been e!orts to develop and validate described above. alternative methods to conventional toxicity tests (Blaise et al., 1997; Tylor et al., 1998). 2.2. In Vitro AChE Inhibition Test Enzymes are known to play a crucial role in vital func- tions of all organisms. The inhibition of cholinesterases has Tests were performed as described in Guilhermino et al. been widely used as a speci"c biomarker for organophos- (1996) with the following modi"cations: animals were cul- phate and carbamate pesticides, being considered a &&gold tured for 48 h in the presence of food; homogenates were standard biomarker'' (Peakall, 1994). However, studies pub- stored at !203C for a maximum of 1 week. The incubation lished in recent years indicate that some metals (including procedure was performed according to Herbert et al. chromium) might also inhibit these enzymes, under both in (1995/1996). In each experiment, homogenate samples vivo and/or in vitro conditions (Labrot et al., 1996; Payne et (495 ll) were incubated at 203C with 5 ll of each test solu- al., 1996; Guilhermino et al., 1998). tion, for 30 min. For each compound, a water control and In this study, the toxicity of sodium dichromate and "ve concentrations of the metallic element were used (12.5, sodium molybdate to D. magna was evaluated using conven- 25, 50, 75, and 100 mg L~1). Three replicates per treatment tional 48-h acute tests, in vivo and in vitro acetylcholines- were used. The activity of AChE was determined, in tripli- terase (AChE) inhibition assays, and 21-day chronic tests. cate, by the Ellman method (Ellman et al., 1961) adapted to Furthermore, the e!ect of the color of sodium dichromate in microplate (Herbert et al., 1995/1996). The protein content the results of an in vitro AChE inhibition test was also of the samples was determined according to the Bradford investigated. technique (Bradford, 1976) with the modi"cations described by Herbert et al. (1995/1996). The activity of AChE in each sample was presented as the mean of the three determina- 2. MATERIALS AND METHODS tions performed and expressed in units (U) per milligram of protein (1 U"1 nmol of substrate hydrolyzed per In the following sections total chromium and molyb- minute). A Labsystems Multiskan MS microplate reader denum concentrations refer to the concentrations of the was used. metallic element in the medium. Sodium dichromate solutions presented an orange color Parent animals were cultured in ASTM hard water interfering with both AChE and protein determinations. (ASTM, 1980) with an organic additive (Baird et al., 1989), For this reason, a correction test for color interference as in groups of 10 animals per 1000 ml of medium, and fed with described above but without Daphnia homogenate was per- the algae Chlorella vulgaris (0.322 mg carbon/daphnia/day). formed in parallel. The objective of this procedure is to The photoperiod was 16 h light : 8 h dark and the temper- account for eventual e!ects due to the color of the chro- ature was 20$13C. mium solutions. 2.1. Acute and Chronic Tests 2.3. In Vivo AChE Inhibition Tests Tests were carried out in ASTM hard water (ASTM, Tests were carried out according to Guilhermino et al. 1980), with animals from a single clone (clone A, sensu; (1996) with the following modi"cations: 60 animals were Baird et al., 1989) and initiated with third- to "fth-brood used per treatment, in groups of 20 per 1000 ml of test neonates ((24 h).