Enchytraeus crypticus as model species in soil ecotoxicology

Chemosphere 87 (2012) 1222–1227

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Enchytraeus crypticus as model species in soil ecotoxicology Marta P. Castro-Ferreira a,b,⇑, Dick Roelofs b, Cornelis A.M. van Gestel b, Rudo A. Verweij b, Amadeu M.V.M. Soares a, Mónica J.B. Amorim a a b

Department of Biology and CESAM, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal Department of Ecological Science, Faculty of Earth and Life Sciences, VU University, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands

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Article history: Received 15 July 2011 Received in revised form 23 December 2011 Accepted 13 January 2012 Available online 24 February 2012 Keywords: Enchytraeid reproduction test (ERT) Enchytraeus crypticus Cadmium Carbendazim Phenanthrene Chloroanilines

a b s t r a c t Enchytraeids are ecologically relevant soil organisms, due to their activity in decomposition and bioturbation in many soil types worldwide. The enchytraeid reproduction test (ERT) guidelines ISO 16387 and OECD 220 are exclusive to the genus Enchytraeus and recommend using the species E. albidus with a 6-week test period. The suggested alternative, E. crypticus has a shorter generation time which may enable the ERT to be twice as fast. To confirm the suitability of a 3-week test period for E. crypticus, the toxicity of five chemicals, with distinct properties and modes of action, was assessed in LUFA 2.2 soil. In all controls the validity criteria were met, as survival of E. crypticus was above 92% and more than 772 juveniles were produced. The good performance supports its appropriateness as model species. Reproduction was more sensitive than survival, with only cadmium and 3,5-dichloroaniline causing significant lethal effects in the tested concentration ranges. The effect concentration causing 50% reduction in the number of juveniles (EC50) was 35 mg kg1 for cadmium, 0.05), thus one control group (n = 10) was considered for further statistical analyses. 3.2. Ecotoxicological effects of the test chemicals As shown in Fig. 1, after 3 weeks of exposure to cadmium, adult survival was significantly different (p < 0.001) from the control only at the highest concentration tested and amounted 52% of the control; LOEC was 320 mg kg1. The number of juveniles showed a steep decrease with increasing cadmium concentration in a dose-related manner; EC50 was 35 mg kg1 (Table 3). No significant effect (p > 0.05) on adult survival was observed after 3 weeks of exposure to carbendazim at concentrations up to 32 mg kg1 (Fig. 1). The number of juveniles was significantly reduced (p < 0.001) to 20% of the control at the lowest test concentration and further decreased with increasing carbendazim concentration (Fig. 1); EC50 was 32 >400 >1000 51 (42–60)

Fig. 2. Survival of Enchytraeus crypticus after 3 weeks of exposure to 3,5-dichloroaniline in LUFA 2.2 soil. The x-axis shows the nominal concentration of chemical (mg kg1 DW soil); y-axis, mean ± standard error (SE) for the percentage of adult survival (j). The dose–response curve (solid line) and 95% confidence interval (dashed lines) for the survival dataset was fitted using the logistic model (Eq. (a)).

After 3 weeks of exposure to phenanthrene, no significant effect (p > 0.05) on adult survival was observed in the tested concentration range (Fig. 1). The number of juveniles decreased with increasing phenanthrene concentration in a dose-related manner. In comparison to the control it was significantly different (p < 0.001) at 100 mg kg1 and further reduced to 23% at 400 mg kg1; EC50 was 145 mg kg1 (Table 3). No significant effect (p > 0.05) on adult survival was observed after 3 weeks of exposure to pentachloroaniline up to 1000 mg kg1; survival was reduced to 85% of the control at the highest test concentration (Fig. 1). In comparison to the control the number of juveniles decreased significantly (p < 0.05) at the lowest concentration (62.5 mg kg1), and it was 30% of the control at the highest test concentration; EC50 was 275 mg kg1 (Table 3). After 3 weeks of exposure to 3,5-dichloroaniline, adult survival was significantly different (p < 0.001) from the control at 50 mg kg1 and further reduced in a dose-related manner (Fig. 2). LC10 was 33 mg kg1 and LC50 was 56 mg kg1 (Table 3). The numbers of juveniles at 25 and 50 mg kg1 were higher than the control, and significantly decreased (p < 0.05) at 100 mg kg1 and higher concentrations; this was indicative of hormesis. This reproduction dataset was fitted with the hormetic model (Eq. (b)); EC50 was 102 mg kg1 (Table 3). Comparing to the remaining test chemicals, after exposure to 3,5-dichloroaniline the overall juveniles were relatively smaller (1 mm versus 2–5 mm long). 4. Discussion 4.1. Control performance of the toxicity tests The guidelines (ISO, 2004; OECD, 2004) recommend that the criteria to assess the validity of the ERT should be adapted when using Enchytraeus species other than E. albidus. From ten initial

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adults, at the end of the test not less than 25 juveniles per control replicate should have been produced for E. albidus and twice as much for other suggested species (ISO, 2004; OECD, 2004). Considering the present and literature data regarding control performance using OECD and LUFA 2.2 soils, the reproductive output of E. crypticus was found to be 8–15-fold higher than for E. albidus (Collado et al., 1999; Amorim et al., 2005; Droge et al., 2006; Kuperman et al., 2006d; Römbke and Moser, 2002; Sverdrup et al., 2002b; van Gestel et al., 2011). Consequently the validity criterion regarding the number of juveniles for E. crypticus should be reviewed. The international ring test using E. albidus included 92 tests, from which nearly half passed the validity criteria. In 9 tests adult survival did not reach 80%, 26 tests had less than 25 juveniles and 21 tests had a CV > 50% (Römbke and Moser, 2002). In the literature are reported many tests using E. albidus in which the control performance did not pass the validity criteria (Collado et al., 1999; Kuperman et al., 2006d). On the other hand, previous tests using E. crypticus commonly met all the validity criteria; in both 4-week tests using LUFA 2.2 soil, OECD soil and eleven field soils (Droge et al., 2006; Kuperman et al., 2006d; van Gestel et al., 2011) and 3-week tests using two field soils (Sverdrup et al., 2002b; Menezes-Oliveira et al., 2011). In short, E. crypticus has demonstrated very good performance as soil ecotoxicological model. The 3-week tests with E. crypticus in LUFA 2.2 soil were successful with some advantages discussed here. For every replicate the control performance fulfilled the validity criteria set by the current guidelines (ISO, 2004; OECD, 2004), as both adult survival and juvenile production were high with small coefficient of variation (Table 2). Second, 3 weeks of exposure are sufficient to obtain generation F1 and also avoid excessive numbers of juveniles and occurrence of generation F2. Third, the size distinction between adults and juveniles after 3 weeks is more evident than after longer periods. Fourth, there was no need for intermediate removal of adults, a time-consuming invasive process which alters the test system and may destroy cocoons or juveniles. The fact that, between water and acetone controls, survival and reproduction did not significantly differ (p > 0.05) indicated that the acetone prespiking did not affect the enchytraeid endpoints and the methodological procedure used to evaporate the solvent was efficient. To conclude the 3-week period is suitable for ERT using E. crypticus and this test duration may be considered in future ERT guidelines. 4.2. Ecotoxicological effects of the test chemicals 4.2.1. Cadmium After exposure in LUFA 2.2 soil, the toxicity of cadmium for E. crypticus was compared to literature data for E. albidus (Novais et al., 2011). As shown in Table 4, the EC50 and LC50 values obtained for E. albidus were lower than for E. crypticus. Moreover survival of E. albidus was significantly different from the control at 3.2 mg kg1 and higher cadmium concentrations (Novais et al., 2011), whereas for E. crypticus significant decrease in survival was observed only at 320 mg kg1. This suggested E. crypticus is less susceptible than E. albidus to cadmium-induced toxicity. 4.2.2. Carbendazim Carbendazim was the reference substance recommended by the ERT guidelines (ISO, 2004; OECD, 2004) and the results from the ring test showed an EC50 of 1.2 mg kg1 for E. albidus (Römbke and Moser, 2002). Besides Novais et al. (2010) tested E. albidus and observed an EC50 of 0.1 mg kg1 (Table 4). On the other hand, E. crypticus was 4-week tested in OECD artificial soil and EC50 of 44 mg kg1 was obtained (Kuperman et al., 2006d). A remarkable difference was observed in the soil tested; due to lower organic matter content in LUFA 2.2 soil (compared to OECD soil), relatively more carbendazim might be available, thus higher toxicity to the

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M.P. Castro-Ferreira et al. / Chemosphere 87 (2012) 1222–1227 Table 4 Effect concentrations (with 95% confidence intervals) for the toxicity of three tested chemicals to the reproduction (EC50) and survival (LC50) of Enchytraeus crypticus and E. albidus after, respectively, 3 and 6 weeks of exposure in LUFA 2.2 soil. Effect concentrations are expressed as milligrams of chemical per kilogram of dry weight soil (mg kg1 DW soil). Test chemical Cadmium Carbendazim Phenanthrene

Species

EC50

LC50

References

E. E. E. E. E. E.

35 (31–38) 6.2 (n.d.) 320 14 (4–123) >32 1 (0–2) >400 135 (84–216)

Present study Novais et al., 2011 Present study Novais et al., 2010 Present study Amorim et al., 2011

crypticus albidus crypticus albidus crypticus albidus

enchytraeids may occur (Kuperman et al., 2006d). The previous results on E. crypticus were considered for the concentration range tested. It revealed to be high to study effects on reproduction and low to observe mortality of E. crypticus; since 80% of diminution in the number of juveniles was observed at 1 mg kg1 and no effect on survival was observed up to 32 mg kg1. Comparing E. crypticus to E. albidus the effect of carbendazim on reproduction may be similar for both species. Regarding survival, E. crypticus indicated a distinct response, without effect at higher concentration of carbendazim. This indicated the survival of E. crypticus has less susceptibility to carbendazim-induced toxicity, comparing to E. albidus.

4.2.3. Phenanthrene In the previous 4-week test with E. crypticus in LUFA 2.2 soil, EC50 for phenanthrene was 100 mg kg1 and LC50 was 376 mg kg1 (Droge et al., 2006). In the present 3-week test, EC50 was 145 mg kg1 and LC50 was >400 mg kg1, so results are accordingly considering more 1-week in test duration increase toxic effects. Comparing to literature on E. albidus (Amorim et al., 2011), less toxicity was observed for E. crypticus in the present study; the effect concentrations for E. crypticus were approximately five times higher than the ones obtained for E. albidus (Table 4). This indicated E. crypticus is less susceptible to phenanthrene-induced toxicity, in comparison to E. albidus.

4.2.4. Chloroanilines No previous data was available for enchytraeids exposed to chloroanilines. After 3-week tests with E. crypticus, the EC50 value for pentachloroaniline was higher than for 3,5-dichloroaniline (Table 3). A shallow dose–response curve was obtained for the effects of pentachloroaniline on the reproduction of E. crypticus and no effects observed on survival. For 3,5-dichloroaniline the EC50 was 2fold higher than the LC50 (Table 3) and effects on adult survival caused diminution in reproduction. The reproduction of E. crypticus was differently affected by 3,5-dichloroaniline concentration below versus above 100 mg kg1; as hormesis followed by a sharp diminution in the number of juveniles was observed. 3,5-Dichloroaniline also affected the development (hatching and growth) of juveniles, indicated by the smaller juveniles. Previous exposures to PAHs indicated a linear increase of toxicity with increasing lipophilicity, when lipophilicity is expressed as log octanol–water partition coefficient (log Kow, Table 1) and toxicity as porewater concentration (Di Toro et al., 2000; Sverdrup et al., 2002a; Droge et al., 2006; León Paumen et al., 2009). Comparing to pentachloroaniline, the log Kow is lower for 3,5-dichloroaniline, thus lower toxicity was expected for 3,5-dichloroaniline. The EC50 as porewater concentration for 3,5-dichloroaniline (67 lM) was higher than for pentachloroaniline (5 lM), confirming the assumption.

4.2.5. Chronic lethal/sublethal ratios Numerous acute to chronic ratios (ACR) have been previously analysed for several species and chemicals, and a relationship was found between the mode of action of the chemical and its ACR. In general, more specific modes of action were indicated by higher ACR, while narcotic chemicals presented lower ACR. As acute toxicity data is usually more variable than chronic toxicity data, ACR were rather variable too. In order to employ a less variable ratio, chronic toxicity data was used to establish a relationship between lethal and sublethal effects (Marinkovic´ et al., 2011). Chronic lethal/sublethal ratios for E. crypticus were estimated calculating the ratio LC50/EC10 using the results in Table 3. The lowest ratio was 1 for 3,5-dichloroaniline, intermediate ratios were estimated for cadmium (>21) and phenanthrene (>11), and the highest ratios were estimated for pentachloroaniline (>333) and carbendazim (>320, considering EC10 equal to 0.1 mg kg1). The very low LC50/EC10 ratio for 3,5-dichloroaniline suggested that the high lethality may be associated with narcotic toxicity. On the other hand, the very high LC50/EC10 ratios indicated the sublethal effects were considerably higher than the lethal effects, thus a more specific mode of action was assumed for pentachloroaniline and carbendazim. 5. Conclusions It was confirmed that the 3-week test period is reliable to assess the toxic effects on survival and reproduction of E. crypticus. In comparison to E. albidus, it was concluded that E. crypticus had relatively lower susceptibility to toxic effects caused by cadmium, carbendazim and phenanthrene. Advantages as good control performance and faster ERT support the appropriateness of E. crypticus as model species in soil ecotoxicology. Acknowledgements Thanks are due to Nico M. van Straalen, Matty P. Berg, Maria Diez Ortiz and Dalila Costa for advice and support at different stages of the work. This study was supported by FCT (Fundação para a Ciência e a Tecnologia) through a PhD grant to M.P. CastroFerreira (SFRH/BD/46759/2008). References Achazi, R.K., Flenner, C., Livingstone, D.R., Peters, L.D., Schaub, K., Scheiwe, E., 1998. Cytochrome P450 and dependent activities in unexposed and PAH-exposed terrestrial annelids. Comp. Biochem. Physiol. C 121, 339–350. Amorim, M.J.B., Römbke, J., Schallnass, H.J., Soares, A.M.V.M., 2005. Effect of soil properties and aging on the toxicity of copper for Enchytraeus albidus, Enchytraeus luxuriosus, and Folsomia candida. Environ. Toxicol. Chem. 24, 1875–1885. Amorim, M.J.B., Oliveira, E., Teixeira, A.S., Gravato, C.S., Loureiro, S., Guilhermino, L.C., van Gestel, C.A.M., Soares, A.M.V.M., 2011. Toxicity and bioaccumulation of phenanthrene in Enchytraeus albidus (Oligochaeta: Enchytraeidae). Environ. Toxicol. Chem. 30, 967–972.

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