Sub-lethal effects of acetone on Daphnia magna

Transcription

1 Ecotoxicology (2008) 17: DOI /s Sub-lethal effects of acetone on Daphnia magna Barbara Leoni Æ Roberta Bettinetti Æ Silvana Galassi Accepted: 29 November 2007 / Published online: 23 December 2007 Ó Springer Science+Business Media, LLC 2007 Abstract There is increasing concern about the sub-lethal effect of hydrophobic chemicals in the water medium. Even though acetone is a commonly used solvent in toxicity testing, few studies have focussed on its chronic toxicity to Daphnia magna and the available results are often contradictory. In this study, acetone was tested on D. magna in a 21-day exposure experiment and the effects on mortality, fertility and morphology of exposed organisms (F 0 ) and offspring (F 1 F 2, reared without acetone) were evaluated. No significant reduction of survival was observed with increasing concentrations, and no significant reduction in fecundity in any treatment group in terms of average number of daphnids per mother was observed. Abnormal development of second antennae was observed on F 1 from F 0 exposed to 79 mg l -1 solvent. The ET50 of acetone on the number of mothers that produced deformed offspring over time was 12.5 days. Our results suggest that the acetone concentration should not exceed 7.9 mg l -1, which is 10 times less than the allowed concentration as determined by OECD chronic assays on D. magna. More attention should be paid to small, water-soluble molecules usually considered of low concern for chronic toxicity because they might affect other metabolic pathways. B. Leoni (&) Dipartimento di Scienze dell Ambiente e del Territorio, Università degli Studi di Milano Bicocca, Piazza della Scienza 1, Milan 20126, Italy barbara.leoni@unimib.it R. Bettinetti Dipartimento di Scienze Chimiche e Ambientali, Università degli Studi dell Insubria, Via Valeggio 11, Como 22100, Italy S. Galassi Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria 26, Milan 20133, Italy Keywords Acetone Ecotoxicity test Long-term exposure Abnormalities Daphnia magna Introduction Many aquatic bioassays rely on organic solvents to solubilize the so-called difficult substances, toxicants that are relatively hydrophobic and lipophilic (ECETOC 1996; Rufli et al. 1998; Kahl et al. 1999). Several standard international protocols (e.g., OECD, US EPA, ASTM) recommend a set of solvents, providing the criteria for their selection and specifying their maximum allowed concentrations in exposure media. Lethal effects should not be observed within the recommended concentrations of organic solvents and dispersants; however, sub-lethal effects on organisms cannot be excluded and their use may make the results of ecotoxicological tests unreliable (Rayburn et al. 1991a; Calleja and Persoone 1993; El Jay 1996). It is therefore necessary to determine the maximum allowable concentration (MAC) of each solvent to avoid any interference with observed results, for each specific case and species. Among the solubilizing agents, acetone is one of the most commonly used and recommended delivery systems for water-insoluble chemicals in aquatic tests due to its great affinity for water and low toxicity (LeBlanc and Suprenant 1983; OECD 1999; Morris and Wolf 2006). A recent paper reviewing the available data on the toxic effects of acetone on aquatic invertebrate species (Hutchinson et al. 2006) demonstrated that this solvent has a very low acute toxicity: the 48h IC50 (half inhibitory concentration) for Daphnia magna ranges from 9,000 to 31,000 mg l -1 (Canton and Adema 1978; LeBlanc and Suprenant 1983; Cowgill and Milazzo 1991). However,

2 200 B. Leoni et al. few studies have assessed the chronic toxicity of acetone on this model organism and the available results are contradictory. LeBlanc and Suprenant (1983) documented survival in 28-day tests and reported a MAC between 1,100 and 2,200 mg l -1. Cowgill and Milazzo (1991) used an 11- day brood test and found that the NOEC value for survival in D. magna was less than 403 mg l -1 and that NOEC values for reproduction ranged between 3,110 mg l -1 (for total progeny and mean brood size) and 5,180 mg l -1 (for total brood number). Zhang and Baer (2000) showed that an acetone concentration of 79 mg l -1 influenced reproductive parameters in D. magna, with an increase in fecundity and male production under conditions of limited food availability. Hutchinson et al. (2006) hypothesized that the solvent is an additional nutrient source for the microbial community, which may increase food availability. In contrast, Comber et al. (1993) did not observe any increase in D. magna fecundity exposed to the same acetone concentration under similar experimental conditions. There is increasing concern about the sub-lethal effect of hydrophobic chemicals (OECD 2000) in the water medium, and it is essential that the solvent threshold concentration that does not interfere with ecotoxicological endpoints be unequivocally assessed. The aim of the present study was to test acetone effects on D. magna using a 21-day exposure experiment performed in accordance with OECD Guideline 211 (OECD 1998). Concentrations up to the maximum allowed value of 100 ll l -1 corresponding to 79 mg l -1 (OECD 2000) were tested. In addition to the effects on mortality and fertility of exposed organisms (F 0 generation), the effects on offspring (F 1 and F 2 generations) reared in the water medium without the addition of acetone were evaluated by microscopic observations of daphnids (F 1 generation) during different developmental stages. Materials and methods The organisms used in this study were derived from a single clone of D. magna Straus obtained from the Istituto Superiore di Sanità, Rome (courtesy of Dr. Silvia Marchini). Daphnids were cultured (40 ind l -1 ) in aerated commercial mineral water (conductivity 435 ls cm -1 at 20 C, ph 7.2 at 20 C, 306 mg l -1 HCO 3 -, 48.2 mg l -1 Ca 2+, 29.4 mg l -1 Mg 2+ ). The culture medium was renewed and offspring discarded three times a week. Brood daphnids were renewed every 4 weeks and replaced with neonatal organisms. Several generations were reared before the commencement of the experiment. Cultured daphnids were fed with a suspension of the unicellular green alga Pseudokirchneriella subcapitata ( cells ml -1 ) and the yeast Saccharomyces cerevisiae ( cells ml -1 ) three times a week. Culture and experimental solutions were maintained at 20 ± 2 C under a 16-h light:8-h dark photoperiod. These conditions assure continuous amictic parthenogenetic reproduction in cultures (Frey 1982). The algae were cultured in ISO 8692/89 medium within a 2-l flask at 20 ± 2 C under continuous light and were shaken by bubbling air. Algae were harvested during their exponential growth and added directly to the breeding and exposure containers after centrifugation. Cell density was determined using a Burker counting chamber under a bright-field light microscope. To evaluate the toxicity of acetone a chronic test was carried following the guideline n. 211 (OECD 1998). Daphnids (\24 h old) were exposed to 20 ml of 7.9, 47 or 79 mg l -1 acetone (Carlo Erba Reagenti) solutions in mineral water in 50 ml bottles for 21 days. Each treatment and the control consisted of replicates each containing a single daphnid. Test solutions were renewed and daphnids fed three times a week with Saccharomyces cerevisiae ( cells ml -1 ) and Pseudokirchneriella subcapitata (0- to 8-day-old daphnids: cells ml -1, 9- to 15-day-old daphnids: cells ml -1, 16- to 21- day-old daphnids: cells ml -1 ). Daphnids (F 0 generation) usually began to release broods of parthenogenetically reproduced offspring (F 1 generation) on approximately day 7. Survival and offspring production were assessed daily after the first brood. Offspring were counted, removed and reared in new bottles in the same manner as F 0 individuals for another 21 days without the presence of acetone in order to produce the F 2 generation. Endpoints included survival, offspring production, total number of live neonates (fecundity), total number of broods, mean brood size and frequency of broods, including morphological observations of organisms at the end of the experiment. All of the generations (F 0,F 1 and F 2 ) were examined microscopically (49, 109, 409 and 609) to identify developmental abnormalities. Pictures of normal and abnormal developmental features were taken using a digital camera (Olympus Camedia C-7070) installed on the microscope (Wild Leitz GMBH). The number of individuals analysed varied among treatments and depended on the number of embryos. Statistical significance for all analyses was assessed using a one-way ANOVA (p B 0.05). Results and discussion The effects of acetone on daphnid survival, reproduction and morphological development were investigated in a 21- day chronic test (OECD 1998). A slight but not significant reduction of survival was observed in F 0 individuals exposed to 47 and 79 mg l -1 acetone concentrations,

3 Sub-lethal effects of acetone on Daphnia magna 201 representing 85% and 90% of survival in control trials, respectively. No mortality was observed in the control or at an acetone concentration of 7.9 mg l -1 (Table 1). Since mortality of parent animals for any treatment did not exceed 20% and the mean number of live offspring produced per parent animal surviving by the end of the test in control beakers was C60 daphnids (Table 1), the chronic exposure can be considered valid (OECD 1998). However, an effect on fecundity was observed in terms of percentage of mothers producing C60 total daphnids in 21 days: more than 20% of F 0 generation individuals exposed to the two highest acetone concentrations (47 and 79 mg l -1 ) produced \60 daphnids by the end of the test. Therefore, although no significant reduction of fecundity (ANOVA: F = 0.025, p = 0.99), total number of broods (ANOVA: F = 0.212, p = 0.89) and brood size (ANOVA: F = 0.475, p = 0.70) was found in any treatment group when comparing the average number of daphnids per mother produced by acetone-exposed animals to control organisms (Fig. 1), a test should not be considered valid if the acetone concentrations of 47 and 79 mg l -1 allowed by the OECD guideline 211 (OECD 1998) were used as a carrier for a chemical to be tested by a chronic assay. Adverse effects on reproduction are so small that they were not highlighted by previous studies (Cowgill and Milazzo 1991; Comber et al. 1993; also see Table 1 The effects of acetone on D. magna reproduction during chronic exposure (21 days) Control 7.9 mg 47 mg 79 mg F 0 F 1 F 1 F 1 F Dead Dead Dead F 74.0 (±8.3) 72.4 (±14.3) 73.5 (±18.3) 73.2 (±13.9) MTBN 4.8 (±0.4) 4.8 (±0.9) 4.5 (±0.8) 4.7 (±1.0) MBS 15.1 (±1.7) 15.3 (±2.2) 16.0 (±1.7) 15.8 (±2.1) F 0 : maternal organisms exposed to 0 (Control), 7.9, 47 and 79 mg l -1 acetone. F 1 : offspring produced over time. F: fecundity (mean ± standard deviation). MTBN: mean of total brood number (±standard deviation). MBS: mean brood size (±standard deviation) (ind) offspring Live Control 79 mg 47 mg 7.9 mg exp. exp. exp. exp days Fig. 1 Cumulative number of offspring over time for the control and groups exposed to different solvent concentrations (mg l -1 ). All data represent mean numbers of offspring per female (broken lines). Exponential curves are the best fit for the growth data Introduction ), which probably used Daphnia magna with a higher fecundity than that exhibited by our clone. Further effects were observed on F 1 generation daphnids born from F 0 mothers exposed to 79 mg l -1 of acetone and reared in a water medium without the addition of acetone. Most of these individuals showed an abnormal development of the antennae. The large biramous second antennae are the most conspicuous feature of the head of cladocerans and are positioned near the posterior part of the head (Fig. 2). The second antenna consists of a single basal article, the basipodite. Two branches or rami arise from the distal end of the basipodite. They consist of four (exopodite) and three (endopodite) cylindrical segments and bear large plumose natatory setae. The presence and size of setae on antennal segments varies in different taxa. Segments are usually counted beginning from the base of the branches. In Daphnia sp., there are three setae on distal segments, one seta on proximal and medial segments of the endopodite and on the third segment of the exopodite. Of the other segments, the first (basal) and second of the four-segmented rami (exopodite) do not have setae (Fig. 2a, b). The morphological antennal abnormalities associated with continuous exposure of the maternal organism to acetone involved both branches and ranged from minor malformations, such as a reduced number of the rami segments and terminal setae, to severely underdeveloped antennae with a stump of branches and weak setae (Fig. 2c h). The abnormalities observed were consistent with aberrations during late stages of embryo maturation (i.e., stages 4 6; LeBlanc et al. 2000; Kast-Hutchenson et al. 2001;

4 202 B. Leoni et al. Fig. 2 Developmental abnormalities elicited by acetone among the F 1 generation. (a, b) Organisms with normally developed second antennae of the control group at 21 days; arrows identify structures that exhibit abnormalities in individuals of acetone-exposed groups. (c h) Daphnids with deformed second antennae at 21 days resulting from continuous exposure of maternal organisms to 79 mg l - 1 of acetone; (d, f) reduced number of rami segments and terminal setae; (c, e, f, g) severely underdeveloped antennae with a stump of branches and weak setae a c setae Normal Antennae II 500 µm setae endopodite endopodite exopodite exopodite basipodite basipodite Antennae II with abnormalities 500 µm b d 500 µm e f g h Zhang et al. 2003). During normal embryonic development, stage 4 embryos h after deposition into the brood chamber show well-developed second antennae still confined within the second embryonic membrane. At stage 5 the second antennae extend from the embryo s body, but associated setae are not fully developed. After 48 h (stage 6) the second antennae and their setae are well developed and animals are freely swimming (Kast-Hutchenson et al. 2001).

5 Sub-lethal effects of acetone on Daphnia magna 203 The percentage of offspring with abnormal antennae increased with increasing acetone exposure time (Table 2). No underdeveloped antennae were observed in daphnids of the first broods, though they were observed in the second broods approximately after 9 days. By the 21st day, 88.9% of maternal organisms exposed to 79 mg acetone l -1 produced deformed offspring (Table 2) and 54.3% of all neonates presented underdeveloped second antennae (Table 2). These abnormalities seem to have no effect on survival or reproductive capability of F 1 organisms since they produced morphologically normal daphnids (F 2 generation) that were numerically comparable to those of the controls (data not shown). It seems that acetone is not directly embryotoxic to daphnid embryos, but that embryotoxicity appears after maternal individuals are exposed for a minimum of 9 days. The median effect time (ET50) of acetone on the number of mothers that produced deformed offspring with time was 12.5 days with a range of (ET50 calculated using the EPA programme). The percentage of offspring with deformed antennae referred to the total number of daphnids produced by their own mother does not significantly change with time (Fig. 3) being in a range 42 63% (ANOVA p = 0.395). These results support the hypothesis that acetone elicits embryotoxicity by interfering in a constant way with a metabolic pathway linked to the maternal provisioning of certain constituents necessary for the normal development of embryos. The toxicokinetics of acetone are dose-related in humans and rodents. Once acetone has been absorbed it is extensively metabolized. Metabolism follows a hepatic pathway at low concentrations, while at higher concentrations the metabolism proceeds through an extrahepatic pathway followed by excretion (Casazza et al. 1984; Thornalley 1996). Table 2 Incidence of abnormalities associated with maternal organism exposure to acetone over time Days % F 0 %F F 0 : percentage of maternal organisms exposed to 79 mg l -1 acetone that produced abnormal offspring in 21 days. F 1 : cumulative percentage of abnormal offspring in 21 days % (AO /TOMA) ANOVA: F (8,46) =1.08; p= days Fig. 3 Percentage mean (±standard deviation) of the cumulative number of daphnids (F 1 ) with deformed antennae (AO abnormal offspring) relative to the total number of offspring produced by their own mother (TOMA) during 21 days As a general pattern, the gluconeogenic pathways lead to the production of glucose with subsequent liberation of carbon dioxide (ATSDR 1994). The best-known effects of acetone ingestion are the diabetes-like symptoms of hyperglycaemia and glycosuria (Opresko 1995). In humans, an oral dose of 2.2 g kg -1 of pure acetone significantly increases blood glucose levels (Gitelson et al. 1966). In addition to being used as a caloric source, and also causing hyperglycaemia in crustaceans, acetone is associated with changes in levels of the hyperglycaemic hormone family (CHH) (LeBlanc 2007). These hormones regulate several metabolic activities (Keller and Sedlmeier 1988), oocyte maturation (Tensen et al. 1989) and molting (Chang et al. 1990). In particular, MIH (Molt-inhibiting hormone) negatively regulates ecdysteroid synthesis, signaling molecules involved in the control of processes such as molting and embryo development (Chang 1985; Mu and LeBlanc 2002, 2004; Gunamalai et al. 2004). MIH maintains ecdysteroid levels at low circulating concentrations during the intermolt phase, while MIH levels prior to molting decrease and result in an increase in circulating ecdysteroid levels. During the early stage of development, crustacean embryos use ecdysteroids of maternal origin packaged into eggs (Subramonian 2000). Perturbations in ecdysteroid signaling have resulted in embryo abnormalities ranging from early partial development arrest to incomplete development of antennae and shell spines (LeBlanc 2007). Many well-known endocrine disruptors such as 4-nonylphenol (Shurin and Dodson 1997; LeBlanc et al. 2000), propiconazole (Kast-Hutchenson et al. 2001), bisphenol A (Mu et al. 2005) and the fungicide fenarimol (Mu and LeBlanc 2002) exhibit anti-ecdysteroidal activity in D. magna by lowering endogenous ecdysteroid levels and interfering with the normal development of antennae, causing incomplete development of second antennae and antennal setae. Acetone most likely does not act as a hormone agonist/antagonist, but might interfere with ecdysteroid synthesis.

6 204 B. Leoni et al. We are not aware of any studies on the embryotoxic effects of acetone on the metabolic pathways of aquatic invertebrates subjected to chronic exposure. As far as vertebrates are concerned, Korhonen et al. (1983) reported that 5 ml of acetone injected into chicken eggs did not increase the percentage of malformations in embryos that failed to survive. No evidence of teratogenicity was seen in chick embryos at lower acetone doses (McLaughlin et al. 1964). In contrast, Kitchin and Ebron (1984) used an in vitro rat whole-embryo culture system and found that acetone concentrations C0.5% caused structurally abnormal embryos and a high rate of embryo mortality when added to a culture medium. Moreover, acetone interacted with developmental toxicants in a FETAX test (frog embryo teratogenesis assay-xenopus) and increased the incidence of malformations induced by methylmercury (Rayburn et al. 1991b). Hallare et al. (2006) found no developmental defects with acetone at a 0.1% v/v concentration in a zebrafish embryo assay. Conclusions As expected no significant effects were observed on fecundity of Daphnia magna when exposed for 21 days to acetone at concentrations lower than the allowed threshold. However, daphnids born from mothers exposed to 79 mg acetone l -1, which is the threshold recommended by the international organizations (OCDE 2000) to vehiculate chemicals difficult to dissolve in water, showed a number of abnormalities, indicating the occurrence of some metabolic disorders in the parent generation. These results suggest that acetone addition should not exceed 7.9 mg l -1 in chronic assays on Daphnia magna in order to avoid misleading results when testing chemicals carried by a solvent. Moreover, greater attention should be paid to small, water-soluble molecules usually considered of low concern for chronic toxicity. 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