Chemosphere 88 (2012) 501–506 Contents lists available at SciVerse ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Enchytraeidae Fridericia bulbosa as a new test species for soil ecotoxicity assessment ⇑ Daoli Yang a, Jiang Zhu b, , Rongbing Fu b, Wenhua Wang a, Xiaopin Guo b, Zhenqi Wang b, Hongwei Yao b a School of Environmental Science and Engineering, Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai 200240, PR China b Shanghai Academy of Environmental Sciences, 508 Qin-Zhou Road, Shanghai 200233, PR China article info abstract Article history: Fridericia bulbosa (Rosa, 1887) was proposed as a new test species to assess soil ecotoxicity. The acute tox- Received 28 September 2011 icity of mercury (Hg) and bromoxynil (BX) on the mortality of Fridericia bulbosa (F. bulbosa) in OECD soil Received in revised form 28 February 2012 was investigated. The results suggested there were statistically significant differences (p < 0.05) between Accepted 5 March 2012 controls and treatments except lower concentration of exposure to single pollutant. BX was more toxic to Available online 2 April 2012 À1 F. bulbosa than Hg. The 14 d LC50 values for F. bulbosa exposed to Hg and BX were 3.87 and 2.41 mg kg , respectively. The effects of mixtures with single pollutants on earthworms were observed differently. Keywords: Toxicity of Hg on earthworms was significantly influenced by the concentration of BX. BX was the main Enchytraeidae Fridericia bulbosa contributive factor of the combined toxic effects. It can be proved that F. bulbosa is a suitable bio-indicator Co-exposure Mixtures to measure the acute toxicity of mixed pollutants in soil and the mortality of earthworms may be con- Mercury sidered as a valuable and sensitive biomarker to diagnose adverse effect of Hg or BX in soil environment. Bromoxynil Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction the first use of F. bulbosa to evaluate the risk of mixture with a heavy metal and an herbicide. Enchytraeids (Oligochaeta) are important organisms of the soil Pesticides and heavy metals (HMs), as extraneous component fauna and are often present in soil where earthworms are not added to soils, will affect the quality of soils (Yang and Sun, found (An and Yang, 2009). They can be collected and cultivated 2007). Pesticides may not only affect the activities of enzymes easier than those common species used in tests (Eisenia fetida (Sannino and Gianfreda, 2001), but also the microbial activity and Eisenia andrei) under the stress of pollutants in field or labora- and biomass in soils (Haney et al., 2000). The negative impact of tory test and therefore, have been used in ecotoxicology diagnosis HMs results from their toxicity to biological processes (Kelly and (OECD, 2004; Zhu and Lu, 2008). Enchytraeus albidus is the recom- Tate, 1998). The combined effects of HM and herbicide are less ex- mended test species by OECD (2004), and has been widely used in plored and have received much attention in recent years (Barata ecotoxicological studies in soil (Lock and Janssen, 2001; Amorim et al., 2006; Wang and Zhou, 2006; Zhou et al., 2008). et al., 2005; Gomes et al., 2011). The other species of the genus Hg is a nonessential metal but quite contrary is one of the most Enchytraeus used in toxicity tests include Enchytraeus buchholzi toxic elements (Lock and Janssen, 2001). Despite growing concerns (Willuhn et al., 1996), Enchytraeus crypticus (Kuperman et al., 2006; about the potential adverse effect of elevated mercury concentra- Menezes-Oliveira et al., 2011), Enchytraeus luxuriosus (Amorim tions in the environment, only few toxicity data were available et al., 2005)andEnchytraeus doerjesi (Kramarz et al., 2005). Although for soil invertebrates (Sauve and Fournier, 2005; Bindesbol and Fridericia is regarded as the genus with most species in the Enchyt- Mark, 2009). The secondary standard of Hg in soils was 0.3 raeidae family (Oligochaete: Annelida: Clitellata: Enchytraeidae) to1.0 mg kgÀ1 (GB 15618, 1995). Abbasi and Soni (1983) found that (Schmelz, 2003), only a few researches have used Fridericia to eval- mercury (II) induced significant mortality of Octochaetus pattoni at uate the toxicity of environmental pollutants. Fridericia bulbosa all levels of exposure and it also caused an enhancement in repro- (Zhu and Lu, 2008) and Fridericia peregrinabunda (An and Yang, duction. Bindesbol and Mark (2009) reported the 4-week LC50 for 2009) were previously used in the toxicity test. F. bulbosa is Dendrobaena octaedra was 38 mg kgÀ1 exposed to mercury chlo- 9–10 mm long, and has 49–54 segments. It is found in Denmark, ride. Beyer et al. (1985) demonstrates that under laboratory condi- Armenia, American, Ireland, Germany, Italy, Tunisia, Algeria, and tions Eisenia fetida may accumulate concentrations of mercury; China (Schmelz, 2003). This paper describes what appears to be concentrations ranging from 0.50 mg kgÀ1 to 0.66 mg kgÀ1 Hg (dry wt). Fischer and Koszorus (1992) found 100% mortality in Eisenia fetida at 500 mg kgÀ1. Bromoxynil (BX, 3,5-dibromo-4-hydroxybenzonitrile) is a post- ⇑ Corresponding author. Tel.: +86 21 6408 5119 2324; fax: +86 21 6483 8991 6143. emergent herbicide registered for use on corn, wheat, and other E-mail address: [email protected] (J. Zhu). small grains to control certain broadleaf weeds and widely used 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2012.03.007 502 D. Yang et al. / Chemosphere 88 (2012) 501–506 in the world (Buhl et al., 1993a). After 28 d of continuous exposure 3. Results to bromoxynil octanoate (BO), survival of Daphnia magna was re- duced at 80 ug LÀ1 (Buhl et al., 1993a,b). Iglesias et al. (2002) re- 3.1. Toxic effect of Hg on F. bulbosa ported that bromoxynil killed all the eggs of D. reticulatum at doses <0.05 mg cmÀ2 after 24 h exposure. To date no report has fo- As shown in Fig. 1, with the time of exposure to Hg prolonged, cused on the toxicity of BX in earthworms, not mention in Enchyt- the mortality of F. bulbosa went up significantly (p < 0.05) in most raeidae family. In this study, Hg and BX were chosen to investigate treatment groups. No mortality was observed in earthworms even the acute toxicity on the mortality of F. bulbosa. The aim was to test on 6 d of exposure to Hg (Hg 6 1mgkgÀ1). After 14 d of exposure F. bulbosa as a potential species for soil ecotoxicity assessment. The to Hg, mortality of earthworms was observed in most of treatment results obtained in this work should lead to a better understanding groups (Hg P 0.5 mg kgÀ1). The mortality rate reached 100% at of the combined toxicity of mixtures and provide a technical basis 16 mg kgÀ1 after 12 d of exposure. for eco-toxicological soil risk assessment on combination. After the same time of exposure, the dose–response relation- ship was well found between the mortality rate of F. bulbosa and the Hg concentration (except at the lower concentration). Statisti- 2. Materials and methods cal differences were observed between treatment and control groups (p < 0.05). Regression linear equation of 14 d exposure to 2.1. Test chemicals Hg on earthworms is expressed as follows: Mercury chloride (99% purity) was supplied by Shanghai Acad- Y ¼0:71X2 þ 18:18X À 9:68ðR2 ¼ 0:98Þð1Þ emy of Environmental Sciences, China. Bromoxynil (99% purity) À1 was purchased from Shanghai NingYin Business Development where X is the concentration (mg kg )ofHg, and Y is the relative Co., Ltd., China. mortality of earthworms in artificial soils. The (NOEC) of 14 d À1 groups was 0.54 mg kg . The 14 d LC50 was calculated as 3.87 mg kgÀ1. The results from the present study indicated that 2.2. Test species Hg has toxic effect on F. bulbosa. F. bulbosa was collected and propagated by Shanghai Academy 3.2. Toxic effect of BX on F. bulbosa of Environmental Sciences, China. The culture was kept in a tem- perature-controlled incubator (20 ± 1 °C) in the darkness. After a As shown in Fig. 2A and B, the mortality of earthworms in- small adaptive phase, healthy adult earthworms with similar creased significantly with the time of exposure to BX prolonged. weight (1.5 mg) were selected for testing. For all test worms, con- There was no evident mortality of earthworms treated with BX less trol mortality was less than 10% (OECD, 1984). than 0.25 mg kgÀ1. No death was observed at lower concentration (BX 6 2mgkgÀ1) until after 4 d exposure time. The 10 d exposed 2.3. Artificial soil test groups were observed mortality at 8 mg kgÀ1. 14 d of exposure to BX, the mortality went up with increased concentration of BX The artificial soil was prepared according to OECD guideline 207 (BX P 0.5 mg kgÀ1). In other words, a long term exposure may (1984), which comprised (by dry weight) of 10% finely ground have toxic effect on earthworms even in very low concentration sphagnum peat, 20% kaolin clay, 70% industrial fine sand, with of pollutant. pH adjusted to 6.5 by addition of calcium carbonate. According While at the same exposure time, there was a dose–response to the results of preliminary experiments, a series of test concen- relationship between the mortality and the concentration of BX trations were determined. Test duration was set as 2, 4, 6, 8, 10, (Fig. 2B). Statistical differences were observed between all treat- 12 and 14 d mercury chloride was dissolved in deionized water ments and control groups (p < 0.05) except 0.25 mg kgÀ1 to obtain a range of exposure concentration (0.5, 1, 2, 4, 8 and (sig = 0.54, p > 0.05).