Effects of decabromodiphenyl ether (BDE-209) on the avoidance response, survival, growth and reproduction of earthworms (Eisenia fetida)

Effects of decabromodiphenyl ether (BDE-209) on the avoidance response, survival, growth and reproduction of earthworms (Eisenia fetida)
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  Effects of decabromodiphenyl ether (BDE-209) on the avoidance response,survival, growth and reproduction of earthworms ( Eisenia fetida ) Xianchuan Xie a,b, n , Yan Qian a , Yingxin Wu c , Jun Yin d , Jianping Zhai a, n a State Key Laboratory of Pollution Control and Resource Reuse, Center for Hydrosciences Research, School of the Environment, Nanjing University, Nanjing, PR China b Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, PR China c School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou, PR China d Department of Civil and Environmental Engineering, University of Missouri, Columbia, USA a r t i c l e i n f o  Article history: Received 25 June 2012Received in revised form26 November 2012Accepted 10 December 2012Available online 9 January 2013 Keywords: Decabromodiphenyl etherBDE-209ToxicityAvoidanceGrowthReproductionEarthworms a b s t r a c t The effects of decabromodiphenyl ether (BDE-209) on avoidance response, survival, growth, andreproduction of earthworms ( Eisenia fetida ) were investigated under laboratory conditions usingnatural and artificial soils as substrate. Results showed that no significant avoidance response wasobserved when earthworms were exposed to 0.1–1000 mg/kg of BDE-209 for 48 h. After 28-daysexposure, no significant effects on survival and growth of adult earthworms was induced by 0.1–1000 mg/kg of BDE-209 indicating the Lowest Observed Effect Level (LOEL) of BDE-209 on their survivaland body weight was more than 1000 mg/kg. Except for a significant decrease in the number of  juveniles per hatched cocoon in artificial soils at 1000 mg/kg of BDE-209, no significant effects onreproductive parameters (e.g. cocoon production per earthworms, weight per cocoon and cocoonhatchability) were observed. These results suggest that adult earthworms have a strong tolerance forBDE-209 exposure in soils, but a potential toxicity does exist for earthworm embryos or juveniles. &  2012 Elsevier Inc. All rights reserved. 1. Introduction Decabromodiphenyl ether (also known as decaBDE, deca-BDE,DBDPO, or BDE-209) which belongs to the group of polybromi-nated diphenyl ethers (PBDEs) is a group of additive brominatedfire retardants. Deca-BDE or BDE-209 is a fully brominateddiphenyl ether compound (i.e., ten bromine atoms) used as aflame retardant in electronic appliances, car seats, carpets, mat-tresses, furniture, and building materials (Darnerud et al., 2001;Talsness, 2008). The commercial deca-BDE, which accounts forover 80% of the total PBDE consumption, has been widelyproduced and currently used all over the world (De Wit, 2002;Rahman et al., 2001; Ikonomou et al., 2002). In China, the production of commercial deca-BDE was 13,500 t in 2001 andup to 30,000 t in 2005 (Zou et al., 2007). The commercial deca-BDE is composed primarily of the BDE-209 congener (above 97%).For combined with manufactured products through non-covalentbonds, BDE-209 may leach from products and find their way intoenvironment (Ikonomou et al., 2002).Owing to the high octanol–water partition coefficient, negligiblewater solubility and low vapor pressure, BDE-209 is easily adsorbedby soil particles and tends to stay in soils when released to theenvironment (WHO, 2004). Reports have shown the ubiquitousdistribution of BDE-209 in soils, especially in electronic-waste con-taminated soils (Covaci et al., 2003; Hassanin et al., 2004; Law et al., 2008). The concentrations of   S PBDEs in electronic-waste contami-nated soils from Guiyu of China ranged between 2.9 and 9156ng/gdw, and BDE-209 was the most dominant congener of PBDEs insoils, accounting for 35–82% of   S PBDEs (Luo et al., 2009). Anextremely high concentration of PBDEs (97400ng/g) was detectedin soils from an e-waste dismantling site in Guiyu, China and BDE-209 accounted for 82% of   S PBDEs in soils (Leung et al., 2007).Consequently, the BDE-209 pollution is becoming a major environ-mental problem in electronic-waste contaminated soils.Earthworms represent about 80% of the total soil biomass andplay a crucial role in health and fertility of soil ecosystems. Theyare also considered as a model organism for standard toxicitytests of terrestrial ecosystems according to the American Societyfor Testing and Materials (ASTM, 1995), the Organisation forEconomic Cooperation and Development (OECD, 2004) for thetesting of chemicals, and International Standards Organization(ISO, 2006).  E. fetida , which is an epigeic (litter-dwelling) earth-worm species is used frequently as standard toxicology testorganisms because they can be cultured easily in the laboratory Contents lists available at SciVerse ScienceDirectjournal homepage: Ecotoxicology and Environmental Safety 0147-6513/$-see front matter  &  2012 Elsevier Inc. All rights reserved. n Corresponding authors at: State Key Laboratory of Pollution Control andResource Reuse, Center for Hydrosciences Research, School of the Environment,Nanjing University, Nanjing, PR China. Fax:  þ 86 25 83594492. E-mail addresses:, (X. Xie). Ecotoxicology and Environmental Safety 90 (2013) 21–27  and are sensitive to a wide range of toxicants (OECD, 2004; ISO, 2006). Generally, acute toxicity tests emphasize lethal endpoints.However, toxicity tests designed to measure sub-lethal endpoints,such as body weight, cocoon viability and behavioral traits alsoprovide sensitive and ecologically relevant data allowing assess-ment of a contaminant at population and community levels(Bhattacharjee and Chaudhuri, 2002; Lukkari et al., 2005). Among all PBDEs congers, BDE-209 has the most extensivetoxicological database, and many standard toxicology studies havestudied to test the acute, sub-chronic, chronic, reproductive/devel-opmental toxicity of deca-BDE. It is demonstrated that deca-BDEappears to have very low toxicity (including acute, sub-chronic,chronic, reproductive/developmental toxicity) on mammals, suchas rats or mice when given by the oral, inhalation and dermal route(USEPA, 2008; Hardy, 2002; Hardy et al., 2002, 2009; Illinois-EPA, 2006; WHO, 2006). However, there are only a limited number of  published studies investigating the toxicity of BDE-209 on inverte-brates. European Commission (EC, 2002) investigated the effect of deca-BDE on earthworm according to the proposed OECD 207 testguideline. Sverdrup et al. (2006) observed that there was noadverse effect of deca-BDE on nitrifying bacteria, red clover( Trifolium pratense ), and a soil invertebrate ( Enchytraeus crypticus )at doses up to 1000 mg/kg dry wt. Breitholtz et al. (2008)investigated the toxicity of deca-BDE to the harpacticoid copepod( Nitocra spinipes ). Riva et al. (2007) studied the potential for deca-BDE to induce DNA damage in zebra mussels ( Dreissena polymor- pha ). Porch and Krueger (2001) studied the toxicity of decabromo-diphenyl ether to six species of plants has been determined usingOECD Guideline 208. In the present study, the main objective wasto evaluate the effects of decabromodiphenyl ether (BDE-209) onavoidance response, survival, growth and reproduction of earth-worms ( E. fetida ) under laboratory conditions using artificial andnatural soils as substrate. 2. Materials and methods  2.1. Reagents and apparatus Decabromodiphenyl ether (BDE-209) of analytical grade (purity  4 98%) waspurchased from Sigma-Aldrich Company (Shanghai, China), and hexane of HPLCgrade was purchased from Merck Company (Shanghai, China). All other chemicalsand reagents were of analytical grade and purchased from Sinopharm ChemicalReagent Co, Ltd. (SCRC) (Shanghai, China) and Nanjing Chemical Reagent Co., Ltd(China). Ultra-pure water was prepared with a Milli-Q system (Millipore, USA).The artificial climate chamber (PRX-350B) was supplied by Ningbo Saifu Labora-tory Apparatus Factory (Zhejiang, China) and was kept at the temperature of 20 7 2  1 C and the moisture of 75 7 5% for all experiments in this study.  2.2. Test organisms Adult  E. fetida  with well-developed clitellum each weighed 0.3–0.4 g and werepurchased from Chan-min of the earthworm culture farm in Nanjing, China. Theywere acclimatized in artificial soil for two weeks with cattle feces as food. The cattlefeces, which were collected from Wei-gan of cattle ranch in Nanjing, China, were air-dried, finely ground and pasteurized before use. The ambient temperature was keptat 20 7 2  1 C, the light–dark cycle was controlled at 16:8 h, and the air moisturecontent was set at 75 7 5%. The mortality of earthworms was lower than 5%. Prior toexposure experiments, earthworms were washed with deionized water and put onclean damp filter paper for 24 h to clear out the gut content.  2.3. Test soils Two test soils were used in this experiment: artificial soils and natural soils.The artificial soils were prepared according to OECD guidelines (OECD, 2004),which was comprised (by dry weight) of 70% quartz sand, 20% kaolin clay and 10%sphagnum peat. The pH was adjusted to 6.0 7 0.5 by adding a small amount of calcium carbonate. The moisture content was adjusted to about 50% of themaximum water holding capacity.The natural soils, which were collected in paddy fields (0–20 cm) from JiangsuAcademy of Agricultural Sciences, China, were used in the experiments. The air-dried soils were sieved (40 meshes; 0.45 mm) prior to the experiments. Theparticle size distributions of soils were 86 g/kg sand, 408 g/kg silt, and 354 g/kgclay. The organic carbon content was 1.80%, the pH was 7.73, and the cationexchange capacity (CEC) was 20.60 cmol/kg.  2.4. Preparation of the contaminated soils Spiked with  n -hexane as carrier, BDE-209 was mixed with quartz sands andthen mixed thoroughly with soils through tumbling for two days in dark. Toevaporate  n -hexane completely, the spiked soils were placed in a dark ventilationcabinet for two days.  2.5. Avoidance response test  The avoidance response test was performed as described by the ISO method(ISO, 2005), and the two-chamber plastic containers (approximately 8.5 cmdiameter; 6 cm height) were used as test vessels (Fig. 1). The spiked BDE-209concentrations in soils were set as 0, 0.1, 1, 5, 10, 50, 100, 500 and 1000 mg/kg dryweigh, and eight replicates (each container representing a replicate) were used foreach treatment. Each test container was divided in two equal sections by aremovable plastic divider. One section was filled with 300 g dry weight of controlsoil and the other with the same amount of contaminated soil. After the dividerwas removed, 10 adult earthworms were placed carefully at the midline of eachtest container. To reduce the water evaporation and the earthworm escape, thetest containers were covered with a transparent plastic lid in which pinholes weremade to facilitate air exchange, and then stored under constant conditions(20 7 2  1 C, 16:8 h light–dark photoperiod) for 48 h. Eight replicates for each testconcentration plus eight controls were conducted. On completion of the testperiod, the plastic divider was replaced at the midline of container, and thenumbers of earthworms in each half were recorded. The avoidance results wereexpressed as net response (NR) in percentage, which corresponds to avoidancefrom treated soil to control soil calculated according to the following equation:NR  ¼ ½  C   T  ð Þ = N    100%  ð 1 Þ where  C   is the number of earthworms observed in the control soils,  T   is thenumber of earthworms observed in the treated soils, and  N   is the number of earthworms per replicate. A positive ( þ ) net response indicates avoidance, and anegative (  ) net response indicates a non-response (or attraction).  2.6. Toxicity tests Toxicity tests were performed according to the OECD methods (OECD, 2004).The spiked BDE-209 concentrations in soils were set as 0, 0.1, 1, 5, 10, 50, 100, 500and 1000 mg/kg dry weight, and eight replicates (each container representing areplicate) for each test concentration and control. There were ten adult earth-worms and 500 g (dry weight) of soil in each container. Approximately 5 g of cattle dung was spread on the soil surface as food for worms. The container lidswere perforated to allow aeration and prevent the worms from escaping. Themoisture contents of artificial soils and earthworm food were maintained byreplenishing deionized water and 5 g cattle feces every week. After four weeks(i.e., 28 days) exposure, the living adult earthworms were hand collected, Fig. 1.  Scheme of the Avoidance Response test.  X. Xie et al. / Ecotoxicology and Environmental Safety 90 (2013) 21–27  22  observed and counted. Earthworms were classified as dead when they did notrespond to a gentle mechanical stimulus at the anterior end. After 24-h clearingout of gut content on moist filter paper, earthworms were weighed and checkedfor any morphological symptoms. All earthworms in each container were weighedas a group and recorded as a datum. The relative growth rate of earthworms wascalculated according to the following equation:Relative growth rate ¼  W  t   W  0 ð Þ =  W  0 ð Þ   100%  ð 2 Þ where  W  0  is the initial average weight of earthworms, and  W  t   is the averageweight at checking day. A positive ( þ ) relative rate indicates the growthstimulation, and a negative (  ) relative rate indicates the growth inhibition. T  ( d ) was the duration of experimental period.To assess the reproductive toxicity of BDE-209 on earthworms, the soils,cocoons, and any juvenile earthworms were put back into each container afteradult earthworms were removed, and the containers were incubated for anadditional four weeks under the same test conditions except that food only wasadded at the start of test and was not added during the second four-week period.On day 56, the cocoons and the juvenile earthworms hatched from cocoons werecounted using procedures described using OECD method (OECD, 2004). Thenumbers and weights of cocoons and juveniles per container were recorded asan individual replicate.  2.7. Determination of BDE-209 concentrations in soils and adult earthworms After the 28-day and 56-day exposure in toxicity tests, the soils and adultearthworms were collected separately, freeze-dried and then stored in glasscontainers at   20  1 C prior to chemical analysis. The BDE-209 concentrations insoils and adult earthworms were determined by the methods of  Mai et al. (2005)and Matscheko et al. (2002) with minor modifications, respectively. PCB-209 wasadded as surrogate standards to the samples prior to extraction.Chemical analysis was performed with a Thermo DSQII gas chromatographcoupled with a mass spectrometer (GC-MS) (Thermo, American) using negativechemical ionization (NCI) in the selected ion monitoring (SIM) mode. A ThermoTR-5msSQC column (15 m  0.25 mm, 0.25 m m film thickness) capillary columnwas used for the determination of BDE-209. Helium ( 4 99.999%) was used as thecarrier gas and methane ( 4 99.99%) as a chemical ionization moderating gas at aflow rate of 1.5 mL/min. The column temperature was programmed from 110 to300  1 C at a rate of 8  1 C/min (held for 20 min). Automatic injection of the 2 m lsamples was conducted at the splitless mode and the split mode was turned onafter 1 min.Other detailed procedures were conducted as the reported methods (Mai et al.,2005; Matscheko et al., 2002).  2.8. Statistical analysis All data obtained from different treatments were analyzed statistically usingone-way ANOVA and Dunnett’s test with the assistance of SPSS 13.0. Allexperimental data were expressed as mean 7 SD (standard deviation). Statisticaldifference was set to be significant when  p o 0.05 (denoted by  * ). Graphs weredrawn using Origin 7.0. 3. Results  3.1. Effect of BDE-209 on avoidance response of earthworms No worm died or escaped during the exposure period, which isconsistent with the results of the EU risk assessment (EC, 2002).The net response (NR) of showing avoidance of   E. fetida  to BDE-209 in artificial and natural soils is presented in Fig. 2. Althoughthe NR value of earthworms exposed to nominal 1000 mg/kg of BDE209 in artificial soils was significantly more than that of thecontrol group, all NR values were less than the trigger value of 80%, indicating no significant avoidance response of earthwormoccurring after 48-h exposure to BDE-209.  3.2. Effect of BDE-209 on survival and growth of earthworms Table 1 shows the survival and relative growth rate of   E. fetida in artificial and natural soils after 28 days exposure to BDE-209. Itwas observed an insignificant earthworm mortality rate in bothtested soils, indicating the tested levels of BDE-209 in this study(i.e. nominal 0–1000 mg/kg dry soils) was sub-lethal to  E. fetida .Table 1 also shows no significant difference in the relative growthrate of earthworms between the treated groups and the controlgroup, indicating no significant effect of BDE-209 on survival andgrowth of   E. fetida  after 28-day exposure. These results are inaccordance with that reported in the EU risk assessment (EC,2002).  3.3. Effect of BDE-209 on reproduction of earthworms Table 2 shows the effects of BDE-209 on reproductive para-meters of   E. fetida , including cocoon production per earthworms,weight per cocoon, cocoon hatchability and juvenile numbers perhatched cocoon in artificial and natural soils after 56-days expo-sure. In natural soils, it shows no significant difference in all testedreproductive parameters, including cocoons per worms, weight percocoon, cocoon hatchability, and juveniles per hatched cocoonbetween the treated groups and the control group, which indicatesno significant effect of BDE-209 on reproduction of earthworms innatural soils after 56-day exposure. In OECD artificial soils, nosignificant differences were observed in the number of cocoons perworms, weight per cocoon and cocoon hatchability between thetreated groups with the control group, while the juvenile numbers Fig. 2.  The avoidance behavior response of earthworms ( E. fetida ) after 48 h exposure to BDE-209 in artificial soils (A) and natural soils (B). Values are denoted asmean 7 SD ( n ¼ 8). Significant differences from the control are indicated ( n  p o 0.05). Dashed line indicates the trigger value of 80% for effect on the individuals.  X. Xie et al. / Ecotoxicology and Environmental Safety 90 (2013) 21–27   23  per hatched cocoon of   E. fetida  exposed to 1000 mg/kg of BDE-209was significantly decreased by about 15% (Table 2).  3.4. The BDE-209 concentrations in soils and adult earthworms The BDE-209 concentrations in soils and adult earthwormsafter 28-days and 56-days exposure were displayed in Table 3. Itshowed that the BDE-209 concentrations in adult earthwormsincreased as the spiked BDE-209 concentrations increased, and allthe measured BDE-209 concentrations in article and natural soilshave decreased in varying degrees compared with the initialspiked concentrations after 28-day and 56-day exposure. 4. Discussion For having chemical receptors in their prostomium and highlocomotory capacities, earthworms can sense and avoid pollutedor harmful soils. To assess soil quality and risk assessment,therefore, avoidance test of earthworms is increasingly regardedas a valuable tool for assessing the potential toxicity of contami-nated soils (Stephenson et al., 1998). Avoidance response of earthworms to various chemicals, including metal ions (Lukkariand Haimi, 2005), organic pollutants (Natal-da-Luz et al., 2008), and pesticides (Garcı´a-Santos and Keller-Forrer, 2011) has beenreported. In the present study, although the NR value in artificialsoils at 1000 mg/kg of BDE-209 was statistically more than that of the control group, the NR values for all tested levels of BDE-209were less than the 80% effect criterion of  ISO (2006), whichindicates that there was no statistical avoidance (Fig. 2). EuropeanCommission (EC, 2002) also observed that no abnormal burrow-ing or avoidance behaviors occurred when  E. fetida  was exposedto BDE-209. No avoidance of earthworms to certain chemicals(organophosphate pesticides and lead nitrate) has also beenreported (Hodge et al., 2000; Reinecke et al., 2002). BDE-209 levels in naturally occurring soils are far less than 1000 mg/kgmaking the avoidance test of earthworms unsuitable for assessingpotential toxicity. Numerous studies have reported that BDE-209appears to have very low acute toxicity on mammals when givenby oral, inhalation and dermal routes (USEPA, 2008). It wasreported that the no-effect level (NOEL) values of BDE-209 formammals in oral toxicity studies ranged from 100 mg/kg (Zhouet al., 2001) to 23,000 mg/kg (USEPA, 2008). In a short-term acute toxicity study, no adverse effects were observed in rats fed withapproximate 10,000 mg/kg/day of BDE-209 (99% pure) and inmice fed with 20,000 mg/kg/day for two weeks (USEPA, 2008). Nosigns of toxicity were observed in rabbits upon dermal exposureof BDE-209 at doses up to 8000 mg/kg and in rats that inhaled  Table 1 Effect of BDE-209 on survival and growth of adult earthworm ( Eisenia fetida ) in artificial and natural soils after 28-day exposure.Test soils Test parameters BDE-209 concentrations in soils (mg/kg dry weight)0 0.1 1 5 10 50 100 500 1000ArtificialsoilsSurvival of adult worms( n )80 78 80 79 80 80 80 80 78Relative growth rate (%) 9.18 7 2.21 8.83 7 1.82 9.79 7 1.36 10.45 7 2.13 10.03 7 1.92 9.35 7 1.83 9.16 7 1.45 9.05 7 2.12 8.67 7 2.14NaturalsoilsSurvival of adult worms( n )80 80 80 78 80 80 79 79 80Relative growth rate (%) 21.22 7 2.65 19.56 7 2.46 23.07 7 3.22 22.08 7 2.16 19.47 7 3.27 20.06 7 2.25 21.82 7 1.47 19.28 7 2.63 18.84 7 3.01Note: Values of survival are the sum of eight replicates for each concentration, and values of survival are denoted as mean 7 SD ( n ¼ 8).  Table 2 Effect of BDE-209 on reproduction of adult earthworm ( Eisenia fetida ) in artificial and natural soils after 56-day exposure.Test soils ReproductiveparametersBDE-209 concentrations in soils (mg/kg dry weight)0 0.1 1 5 10 50 100 500 1000ArtificialsoilsCocoons perworms ( n )2.18 7 0.54 2.35 7 0.53 2.36 7 0.39 2.08 7 0.46 2.33 7 0.45 2.13 7 0.56 2.20 7 0.47 2.05 7 0.44 2.19 7 0.41Weight percocoon (mg)8.85 7 2.14 10.13 7 3.13 9.26 7 2.25 8.38 7 2.30 11.01 7 3.14 9.25 7 2.62 9.12 7 1.35 8.71 7 2.43 8.43 7 2.08Cocoonhatchability (%)63.34 7 9.15 56.77 7 6.17 61.36 7 11.22 68.14 7 6.19 58.79 7 5.39 58.85 7 6.69 60.28 7 5.07 65.89 7 5.89 57.38 7 6.32 Juveniles perhatchedcocoon( n )3.58 7 0.40 4.10 7 0.30 3.62 7 0.39 3.47 7 0.57 3.55 7 0.35 3.32 7 0.47 3.38 7 0.40 3.25 7 0.21 3.02 7 0.35 n  Juvenilesperreplicate ( n )49.50 7 5.76 54.75 7 4.06 52.38 7 5.83 49.25 7 8.03 48.63 7 4.53 41.63 7 5.85 44.88 7 5.44 43.88 7 2.90 38.25 7 5.82NaturalsoilsCocoons perworms ( n )2.93 7 0.43 2.85 7 0.33 3.00 7 0.34 3.05 7 0.51 2.73 7 0.43 2.70 7 0.51 2.78 7 0.52 2.65 7 0.66 2.68 7 0.67Weight percocoon (mg)9.22 7 1.85 9.72 7 2.46 8.86 7 1.30 9.76 7 2.12 10.42 7 2.07 8.79 7 1.91 9.18 7 2.17 8.65 7 2.36 8.84 7 1.86Cocoonhatchability (%)72.18 7 7.29 76.04 7 8.23 68.47 7 9.06 71.01 7 6.54 75.24 7 8.50 70.70 7 8.11 68.84 7 7.15 71.12 7 5.05 69.24 7 5.56 Juveniles perhatched cocoon( n )4.84 7 0.51 4.68 7 0.26 4.74 7 0.38 5.07 7 0.42 4.86 7 0.36 4.78 7 0.40 4.80 7 0.42 4.63 7 0.55 4.80 7 0.39 Juveniles perreplicate ( n )102.38 7 12.45 101.25 7 7.30 97.38 7 7.50 109.75 7 12.28 99.88 7 12.17 91.25 7 8.38 91.88 7 7.64 87.25 7 10.78 89.13 7 7.08 Note : Values are denoted as mean 7 SD ( n ¼ 8). Significant differences from the control are indicated. n  p o 0.05.  X. Xie et al. / Ecotoxicology and Environmental Safety 90 (2013) 21–27  24  commercial BDE-209 products at concentrations of 48 mg/L or200 mg/L for 1–2 h exposure (Hardy et al., 2009). In toxicity tests,survival (or mortality) and body weight are important indicatorsof animals capacity for toxicant exposure. Zhou et al. (2001)reported that the rats, which were fed BDE-209 at the dose of 100 mg/kg-day for four days, did not show any visible toxicity orany effects on survival, body weight and liver-to-body-weightratios. It was also observed that mice, which were exposed toBDE-209 at the dose of 20,994 mg/kg-day in males and23,077 mg/kg-day in females for fourteen days, showed no effectson survival or body weight and no clinical signs of toxicity(USEPA, 2008). However, Kim et al. (2009) observed the signifi- cant decrease in body weight gain in F1 male offspring exposed tohigh-dose (320 mg/kg body weight/d) BDE-209. In the presentstudy, the survival and body weight of all tested earthwormswere not significantly different from the control group (Table 1),indicating the Lowest Observed Effect Level (LOEL) of BDE-209 onthe survival and body weight of   E. fetida  was more than 1000 mg/kg. This result is in agreement with the well-known results (NOEC Z 4910 mg/kg dry weight ) of the EU risk assessment (EC, 2002).Decabromodiphenyl ethane (DBDP-Ethane; CASRN84852-53-9) isalso a brominated flame retardant used frequently in products, of which the chemical structure with ten bromine atoms is similarto BDE-209. Hardy et al. (2011) reported that exposure to313–5000 mg/kg of DBDP-Ethane did not cause abnormal bur-rowing or avoidance behaviors of adult earthworms ( E. fetida ),and no effect on the mortality rate was observed after 28-dexposures to DBDP-Ethane.Generally, the reproductive and developmental toxicity valuesare much lower than the acute or chronic toxicity values. Tsenget al. (2008) reported on CD-1 mice which were fed doses of 10–5100 mg/kg BDE-209 (98% dissolved in corn oil) statingobserving no changes on reproductive parameters (e.g. gesta-tional length, litter size etc.), or on developmental landmarks (e.g.pinna detachment, incisor eruption, eyes opening etc.) except fordecreasing of the serum T3 levels and the minor histologicalchanges in thyroid at 1500 mg/kg of BDE-209. Kim, et al. (2009)also reported on Sprague-Dawley rats exposed to 320 mg/kg of BDE-209 stating that no effects on reproductive parameters wasobserved except for body weights decreasing and thyroid weightincreasing in offsprings. Hardy et al. (2002) found no adverseeffect of treatment at doses up to 1000 mg/kg/d in maternal orfetal rats administered DecaBDE by gavage over gestation days0–19. Qin et al. (2010) studied the effect of technical decabromo-diphenyl ether mixture DE-83R (containing 96–99% BDE-209) on  Xenopus laevis  amphibians (tadpoles). Although no malformationor abnormal behavior was found, the histological alterations of follicular epithelial cells and a decrease in thyroid receptor (TRbA)mRNA levels were observed at 100 and 1000 ng/L. For birds, theLowest Observed Effect Level (LOEL) of BDE-209 on avianembryos was 1800 ng/g (Sifleet, 2009). It was reported by Hardy et al. (2011) that no observed effect concentration (NOEC) andlowest-observed-effect concentration (LOEC) values of DBDP-Ethane on the earthworm’s 56-day reproduction cycle were1910 and 3720 mg/kg respectively. In the present study, nostatistically adverse effects on the reproductive parameters (e.g.cocoon production per earthworms, weight per cocoon, cocoonhatchability and juveniles per replicate) were observed (Table 2)at doses up to 1000 mg/kg, which indicates that adult earth-worms have a strong tolerance for BDE-209 exposure in soils.These results are consistent with that reported in the EU riskassessment at higher doses (EC, 2002). Nevertheless, the juvenilenumber per hatched cocoon in artificial soils was significantlydecreased when earthworms were exposed to 1000 mg/kg of BDE-209 (Table 2), indicating higher doses of BDE-209 exposuremight cause a potential toxicity for embryos of earthworm.Although the mean numbers of juveniles/replicate in artificialsoils of this study meet the requirement of test guideline (i.e. 30 juveniles/replicate), it is much lower than that reported in the EUrisk assessment (i.e. 60–120 juveniles/replicate) (EC, 2002). Thismay be one of reasons why the result of the juvenile number perhatched cocoon is not consistent with that of the prior study (EC,2002).Many studies have reported that BDE-209 could affect thedeveloping central nervous system and induce developmentalneurotoxicity including decreased learning and memory, changesin spontaneous behavior and reduced habituation (Viberg et al.,2003, 2007, 2008; Viberg, 2009; Rice et al., 2007). Several studies have demonstrated that PBDEs including BDE-209 could alsocause oxidative stress in neurons and lead to apoptotic neuronaldeath (He et al., 2008; Giordano et al., 2008; Huang et al., 2010). In our previous experiments, it was demonstrated that BDE-209could stimulate ROS levels and MDA content of   E. fetida  and havesignificant effects on its antioxidant enzyme activities. Thesepossibilities indicated that BDE-209 could cause oxidative stressin earthworms (Xie et al., 2011) which might be one of themechanisms leading to the adverse effect of BDE-209 on earth-worm embryos or juveniles.For the large molecules, low water solubility and extremelyhydrophobic, BDE-209 was thought to not readily cross mem-branes, and thus do not accumulate in organisms (Sellstr¨om et al., 2005). However, recent studies demonstrated BDE-209 had accu-mulative potential from soils to earthworms with Biota-soilaccumulation factors (BSAF) of 0.3–1.0 (Sellstr¨om et al., 2005;Matscheko et al., 2002). In the present study, BDE-209 wasdetected in adult earthworms, indicating that BDE-209 spiked inarticle and natural soils was bioavailable for  E. fetida . Basis on thisstudy’s analytical results for BDE-209 detected in soils and adult  Table 3 The BDE-209 concentrations in soils and adult earthworms ( Eisenia fetida ) after 28-days and 56-days exposure.Samples ExposuretimeBDE-209 concentrations in soils (mg/kg dry weight)0 0.1 1 5 10 50 100 500 1000Artificial soils (mg/kg dryweight)28 days ND 0.06 7 0.01 0.46 7 0.12 3.22 7 0.32 5.16 7 0.85 28.48 7 4.04 47.85 7 6.52 266.70 7 36.22 450.23 7 82.6256 days ND 0.05 7 0.02 0.38 7 0.09 2.84 7 0.40 4.08 7 1.03 25.77 7 2.75 42.90 7 7.20 248.36 7 28.48 435.82 7 62.90Natural soils (mg/kg dryweight)28 days ND 0.03 7 0.01 0.32 7 0.06 2.05 7 0.27 3.27 7 0.63 20.50 7 3.28 38.19 7 7.43 222.25 7 30.12 384.23 7 60.3356 days ND 0.02 7 0.02 0.28 7 0.04 2.04 7 0.19 3.18 7 0.54 18.27 7 3.52 36.28 7 8.20 208.49 7 19.54 360.43 7 42.74Adult earthworms inartificial soils (u˛g/g lipid wetweight)28 days ND 1.34 7 0.32 9.85 7 3.07 40.40 7 5.13 62.29 7 6.80 214.78 7 7.26 385.03 7 28.24 537.806 7 42.33 1025.60 7 76.58Adult earthworms inartificial soils (u˛g/g lipid wetweight)28 days ND 1.01 7 0.25 7.60 7 2.64 29.77 7 4.48 43.62 7 5.53 189.41 7 8.30 356.42 7 22.15 496.23 7 35.71 865.60 7 49.44 Note : Values are denoted as mean 7 SD ( n ¼ 4), ND means that PBDE was not detected.  X. Xie et al. / Ecotoxicology and Environmental Safety 90 (2013) 21–27   25
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