Chemosphere 88 (2012) 501–506
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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 1 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 kg 1 (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 kg 1 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 kg 1 to 0.66 mg kg 1 Hg (dry wt). Fischer and Koszorus (1992) found 100% mortality in Eisenia fetida at 500 mg kg 1. 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 L 1 (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 cm 2 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 1mgkg 1). 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 kg 1). The mortality rate reached 100% at of the combined toxicity of mixtures and provide a technical basis 16 mg kg 1 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 ¼ 0:71X2 þ 18:18X 9:68ðR2 ¼ 0:98Þð1Þ emy of Environmental Sciences, China. Bromoxynil (99% purity) 1 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 1 groups was 0.54 mg kg . The 14 d LC50 was calculated as 3.87 mg kg 1. 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 kg 1. No death was observed at lower concentration (BX 6 2mgkg 1) until after 4 d exposure time. The 10 d exposed 2.3. Artificial soil test groups were observed mortality at 8 mg kg 1. 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 kg 1). 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 kg 1 to obtain a range of exposure concentration (0.5, 1, 2, 4, 8 and (sig = 0.54, p > 0.05). The regression equation of 14 d exposure to 16 mg kg 1). BX was added to the soil by adding acetone stock BX on earthworm is expressed as follows: solutions. The concentrations of BX were prepared to 0.25, 0.5, 1, 2 2 2, 4 and 8 mg kg 1. According to the concentration–response rela- Y ¼ 2:23X þ 32:37X 15:01ðR ¼ 0:98Þð2Þ tionship of single Hg and BX on earthworms, the solution concen- where X is the concentration (mg kg 1)ofBX and Y is the relative 1 , trations of the co-exposure treatments were 0.5, 2, 8 mg kg of Hg mortality of earthworms in artificial soils. The 14 d NOEC for earth- 1 and 0.25, 1, 4 mg kg of BX. Fifty gram of dry soil was placed in a 1 worm was 0.48 mg kg . The 14 d LC50 for F. bulbosa was test unit and moisture content of the test soil was adjusted to 35% 2.41 mg kg 1. Earthworms were more sensitive than those treated (OECD, 1984). Controls without any artificial contaminant received with Hg. BX is more toxic than Hg on F. bulbosa. the same amounts of deionized water and acetone. Each experi- ment was repeated three times. 3.3. Toxic effect of co-exposure to Hg and BX on F. bulbosa
2.4. Statistics Figs. 3 and 4 shows the combined effect of Hg and BX on F. bulb- osa. Variance analysis and multi-comparison on single and mix- The data of this experiment were statistically processed using tures shows there were statistical differences between any two SPSS16.0 software (Standard Version 16.0, SPSS Inc.). All the values groups (p < 0.05). The concentration of pollutants significantly are presented as mean ± standard deviation (M ± SD). Differences influenced the mortality of earthworms (p < 0.01). At the same of treatments were evaluated by one-way analysis of variance (AN- concentration of mixtures, mortality increased significantly with OVA) followed by a Dunnett’s test for multiple pairwise compari- the exposure time of treatments prolonged (p < 0.01). Death hap- son (p < 0.05). Regression analysis was used to examine the pened on various dose of mixtures when the concentration of Hg relationship between the mortality and the concentration of reached 2 mg kg 1 after 2 d exposure. After 14 d of exposure to pollutants. mixtures, the mortality appeared even in the lowest concentration D. Yang et al. / Chemosphere 88 (2012) 501–506 503
(A) 120 0.5 1 100 2 4 80 ( % control ) 8 16 60
F. bulbosa 40
20
Mortality of 0 2 4 6 8 10 12 14 Time (d)
(B) 120 2 100 4 6
( % control ) 80 8 10 60 12 14 F. bulbosa 40
20
Mortality of 0 0.5 1 2 4 8 16 Concentration (mg kg-1)
Fig. 1. Mortality (%) of F. bulbosa exposed to Hg over (A) time and (B) concentration (data presented as M ± SD).
(Hg 0.5 mg kg 1 + BX 0.25 mg kg 1). LC50 values of exposure to 3.4. Main toxic effect of Hg or BX on F. bulbosa mixtures were determined at a given time by using the Weibull model (OECD, 2006). Again, as recommended in the OECD guide- The analysis of variance using SPSS16.0 software was done to line, the model was parameterized with the LC50. Model is written explore the main toxic effect of mixtures on earthworm F. bulbosa. as follows: The dose–response between mortality (Y) and the concentration
of Hg (XHg) and BX (XBX) fit the regression linear equation as – Log-probit: follows:
hi Y ¼ 4:01 þ 9:35XHg þ 11:08XBX ð5Þ c MðcÞ¼1 Fb log ð3Þ 10 LC50 For each of the variables, the beta BX (11.08) was higher than Hg (9.35), indicating BX was the main factor of contribution of – Weibull: mortality. The result was similar to those shows in Fig. 4. c b MðcÞ¼exp lnð3Þ ð4Þ LC50 4. Discussion with M(c) the mortality at concentration c; b regression coefficients The toxic data of heavy metals in Enchytraeidae are very scarce. and F the normal cumulative distribution function. The model was Comparing the data from other studies, F. bulbosa was more sensi- fitted to data expressed as numbers of mortality at 14 d and concen- tive to Hg than other soil organisms. Lock and Janssen (2001) re- tration c. ported mortality in Eisenia fetida (Annelida: Oligochaeta) was Moreover, the interaction of Hg with BX drastically affected observed even exposure to 100 mg kg 1of Hg after 3 weeks and, mortality (Fig. 3). The toxic effect depended on the constituents all Folsomia cadida Willem (Insecta: Collembola) died at 10 mg of the mixtures and may vary significantly (Zhu and Lu, 2008). Ef- kg 1, whereas the mortality at 5.6 mg kg 1 was less than 10% fect of co-exposure of pollutants may be similar (additive) to ex- (21 d exposure). Bindesbol (2009) found an LC50 for Dendrobaena pected effect from separate exposures, or stronger (synergistic, octaedra Savigny (Annelida: Oligochaeta) was 38 mg kg 1. The 14 1 more than additive), or weaker (antagonistic, less than additive) dLC50 of Hg (3.87 mg kg ) from the present study was lower than than those from separate exposures. The results suggested Hg those previous reports. Our previous research found that F. bulbosa and BX have synergistic or antagonistic effect on each other t was also more sensitive to Cd than other earthworms (Zhou et al., (Fig. 3). The concentrations of BX were affected the toxic effect of 2008). F. bulbosa as a microdrile earthworm may take up and accu- Hg on F. bulbosa. mulate pollutants in their body tissues through skin and digestive 504 D. Yang et al. / Chemosphere 88 (2012) 501–506
120 (A) 0.25 0.5 100 1 2 80 4 (% control) 8 60 F.bulbosa 40
20 Mortality of
0 2 4 6 8 10 12 14 Time (d)
(B) 120 2 100 4 6 80 8 (% control) 10 60 12 14 F. bulbosa F. 40
20 Mortality of
0 0.25 0.5 1 2 4 8 Cncentration (mg kg -1)
Fig. 2. Mortality (%) of F. bulbosa exposed to Hg over (A) time and (B) concentration (data presented as M ± SD). system. Changes in bioavailability are associated with different (Hg) > cooper (Cu) > cadmium (Cd) > chromium (Cr). BX was one exposure time and different constituents of soil. F. bulbosa with of the halogenated aromatic compounds as similar as Phe. 14 d more surface areas tend to take up and accumulate pollutants in LC50 of BX was lower than Phe in F. bulbosa. It revealed that the their bodies. The results show that F. bulbosa appears to be an structure of chemicals may influence the bioavailability of appropriate test potworm for assessment of heavy metals. In addi- pollutants. tion, the test method that was usually applied to earthworm assay Comparing the data in Fig. 3, it is observed that the combined appears to be suitable for the potworm assay. We propose the F. effect was similar with the single effect of BX. Low concentrations bulbosa as a new test species of soil ecotoxicity assessment. of BX can enhance the toxicity of Hg on earthworms. On the con- It was the first report of the acute toxicity of BX in earthworms. trary, when BX was kept at higher concentrations, the interaction No comparable LC50 data for BX were available for F. bulbosa. The effect was antagonistic. As we known, heavy metals are able to toxicity may be related to the natural and biological mechanisms change the lipid composition of membranes because of their ability of contaminants. Treatment with heavy metal and pesticide had to induce lipid peroxidation (Zhou et al., 2008; Bindesbol and an influence on the morphology of worms. The abnormalities such Mark, 2009). Compounds such as polycyclic aromatic hydrocar- as thinning, fragmentation and coiling were observed before the bons (PHAs) are likely to accumulate in membranes because of death of worms. The 14 d LC50 for F. bulbosa exposed to Hg and their lipophilic characteristics and their structure with no func- BX were 3.87 and 2.41 mg kg 1, respectively. It was found that tional groups, which may affect the fluidity and function of mem- BX was more toxic than Hg on F. bulbosa. In our previous work, branes (Belfroid and Sijm, 1996). It can be supposed that the the 14 d LC50 for F. bulbosa exposed to Cadimum (Cd) and phenan- accumulation of BX occurred before lipid peroxidation of Hg in threne (Phe) were 4.55 and 4.48 mg kg 1 (Zhu and Lu, 2008). Com- membranes of earthworms’ cells. The toxicity of mixtures on paring with those results, the toxic order for F. bulbosa may was as earthworms by affecting the membrane function was depended follows: Hg > Cd. This is agreement with Giller’ s research (1998), on BX concentration. It suggested BX is a main factor to decide that metal cation toxicity decreased as sliver (Ag) > mercury the co-effects of mixtures. D. Yang et al. / Chemosphere 88 (2012) 501–506 505
Hg 0.5 Hg 0.5+BX 0.25 BX 0.25 BX 0.25+Hg 0.5 120 Hg 0.5+BX 1 Hg 0.5+BX 4 120 BX 0.25+Hg 2 BX 0.25+Hg 8
100 100 ** ** ** 80 80 ** 60 60 **
40 40 **
Mortality (%) ** Mortality (%) ** ** ** 20 ** ** 20 ** * * * *** 0 0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 -20 time (day) -20 time (day)
BX 1 BX 1+Hg 0.5 Hg 2 Hg 2+BX 0.25 BX 1+Hg 2 BX 1+Hg 8 120 120 Hg 2+BX 1 Hg 2+BX 4 ** ** ** ** 100 100 ** ** ** ** ** 80 ** ** ** 80 ** ** ** ** 60 ** 60 ** ** ** 40 ** 40 ** ** Mortality (%) Mortality (%) ** ** 20 ** 20 ** 0 0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 -20 time (day) -20 time (day)
Hg 8 Hg 8+BX 0.25 BX 4 BX 4+Hg 0.5 Hg 8+BX 1 Hg 8+BX 4 BX 4+Hg 2 BX 4+Hg 8 120 120 ** ** ** ** ** 100 ** ** 100 ** * ** ** ** ** ** ** 80 80 ** * ** ** 60 60 ** ** ** 40 40 ** ** Mortality (%)
Mortality (%) ** ** 20 20 ** ** 0 0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 -20 -20 time (day) time (day)
Fig. 3. Mortality (%) of F. bulbosa co-exposed to mixtures (mg kg 1) over time. Data presented as M ± SD.
and 2.41 mg kg 1, respectively. BX was more toxic to F. bulbosa than Hg. There was a positive correlation between mortality and the concentration. Also, the morality increased with the exposure time. So morality of F. bulbosa may be considered as a valuable and sensitive biomarker to diagnose adverse effect of Hg and BX in soil environment. Different responses of earthworms were observed when they were exposed to single pollutant and mixtures. Moreover, BX mag- nificently affected the toxicity of Hg (p < 0.05). This is the first re- port using the Enchytraeidae F. bulbosa as a test species to evaluate the mixtures by heavy metal and herbicide. It can be con- cluded that F. bulbosa is a suitable test species to measure the acute Fig. 4. LC50 concentration (mg BX kg 1)ofF. bulbosa to mixtures over exposure time, as LC50 and 95% CI estimated from the Weibull model. toxicity of combination.
5. Conclusion Acknowledgements
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