Toxicity of three halogenated flame retardants to nitrifying bacteria, red clover (Trifolium pratense), and a soil invertebrate (Enchytraeus crypticus)

Chemosphere 64 (2006) 96–103 www.elsevier.com/locate/chemosphere

Toxicity of three halogenated flame retardants to nitrifying bacteria, red clover (Trifolium pratense), and a soil invertebrate (Enchytraeus crypticus) Line Emilie Sverdrup b

a,b,*

, Thomas Hartnik a, Espen Mariussen c, John Jensen

d

a Jordforsk—Norwegian Centre for Soil and Environmental Research, Frederik A. Dahlsvei 20, NO-1432 A˚s, Norway Department of Biology, University of Oslo, Program for Toxicology and Ecophysiology, P.O. Box 1050 Blindern, NO-0316 Oslo, Norway c Norwegian Institute for Air Research, P.O. Box 100, NO-2027 Kjeller, Norway d National Environmental Research Institute, Vejlsøvej 25, DK-8600 Silkeborg, Denmark

Received 13 August 2005; received in revised form 1 November 2005; accepted 9 November 2005 Available online 10 January 2006

Abstract Halogenated flame retardants have a high sorption affinity to particles, making soils and sediments important sinks. Here, three of the most commonly used flame retardants have been tested for sub-lethal toxicity towards soil nitrifying bacteria, a terrestrial plant (seed emergence and growth of the red clover, Trifolium pratense), and a soil invertebrate (survival and reproduction of Enchytraeus crypticus). Tetrabromobisphenol A (TBBPA) was quite toxic to enchytraeids, with significant effects on reproduction detected already at the 10 mg kg 1 exposure level (EC10 = 2.7 mg kg 1). In contrast, decabromodiphenyl ether (DeBDE) was not toxic at all, and short-chain chloroparaffins (CP10–13) only affected soil nitrifying bacteria at the highest test concentration (EC10 = 570 mg kg 1). Exposure concentrations were verified by chemical analysis for TBBPA and DeBDE, but not for CP10–13, as a reliable method was not available. Based on the generated data, a PNEC for soil organisms can be estimated at 0.3 mg kg 1 for TBBPA and 57 mg kg 1 for short-chain chloroparaffins. No PNEC could be estimated for DeBDE. Measurements of TBBPA in soil are not available, but measured concentrations in Swedish sludge are all lower than the estimated threshold value for biological effects in soil. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Ecological effects; Environment; Nitrification; PNEC; Soil

1. Introduction Flame retardants are substances used in plastics, textiles, electronic circuitry and other materials to prevent fires. Tetrabromobisphenol A (TBBPA) is one of the most extensively used flame retardants, with a worldwide usage of 130 000 metric tons in 2002 (BSEF, 2004a). For decabromodiphenyl ether (DeBDE), belonging to the group of * Corresponding author. Address: Department of Biology, University of Oslo, Program for Toxicology and Ecophysiology, P.O. Box 1050 Blindern, NO-0316 Oslo, Norway. Tel.: +47 22 85 46 47; fax: +47 22 85 46 05. E-mail addresses: [email protected], [email protected] (L.E. Sverdrup).

0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.11.056

polybrominated diphenyl ethers (PBDEs), the 2001 world-wide usage was 56 100 tons (BSEF, 2004b). Shortchain chloroparaffins (CP10–13, also called polychlorinated n-alkanes) are also an important group of flame retardants with an annual estimated consumption of 25 500 tons in the United States in 2001, and 4000 tons in Europe in 1998 (United Nations, 2003). The technical grade products in this group are mixtures of substances with chain lengths of C10–13 and a chlorine content of about 55–60%. In recent years, there has been an increasing concern about the presence of brominated flame retardants (BFR) and chloroparaffins (CPs) in the environment. Whereas the release and environmental concentrations of other persistent organic pollutants such as DDT and polychlorinated biphenyls are stable or decreasing, concentrations

L.E. Sverdrup et al. / Chemosphere 64 (2006) 96–103

of e.g. PBDEs in sediments (e.g. Nylund et al., 1992), blubber from ringed seals (Ikonomou et al., 1999), and lake trout (e.g. Luross et al., 2000) show a dramatic increase during the last 20 years. Flame retardants may reach the terrestrial environment by e.g. sewage sludge amendment or diffuse pollution. Various brominated flame retardants have been found in precipitation (e.g. Ter Schure and Larsson, 2002), soils (e.g. Matscheko et al., 2002), sewage sludge (e.g. Sellstro¨m and Jansson, 1995; Sellstro¨m, 1999; Sellstro¨m et al., 1999; ¨ berg et al., 2002; Amundsen and Hartnik, 2003), sediO ments (e.g. Luross et al., 2000) and biota (e.g. Schlabach et al., 2002), often far from emission sources. Chloroparaffins have been found in sewage sludge and compost (e.g. Amundsen and Hartnik, 2003), sediments (Schlabach et al., 2002), and for biota, Jansson et al. (1993) found higher concentrations of CPs in terrestrial mammals than in aquatic organisms. Some BFR (e.g. TBBPA) can be classified as reactive materials, which denotes that they are chemically bound into plastics. Others, such as polybrominated diphenyl ethers and CPs are additives in a wide variety of fluids, polymers and resins. Additive flame retardants are generally believed to be more easily released to the environment than reactive flame retardants (Hutzinger and Thoma, 1987). Some studies have been published on the toxicity of chlorinated and brominated flame retardants to aquatic organisms (e.g. WHO/ICPS, 1995; Kierkegaard et al., 1999), but the toxicity to soil organisms is principally unknown. This is of concern as the low water solubility and high binding affinity to particles possibly makes soils and sediments important sinks for substances like DeBDE, TBBPA and CP10–13. In a risk assessment for CP10–13, which was carried out by the European Commission (2000), the overall conclusion was that further information on the soil compartment was needed, including ecotoxicity data for soil dwelling organisms. For an evaluation of ecological risk to the terrestrial environment, toxicity testing with plants (primary producers), soil invertebrates (herbivores or decomposers) and soil microorganisms is usually recommended (e.g. European Communities, 2003). In line with this, the present study reports on the chronic toxicity of three halogenated flame retardants (TBBPA, DeBDE and short-chained chloroparaffins, CP10–13) to red clover (Trifolium pratense), enchytraeids (Enchytraeus crypticus) and soil nitrifying bacteria. 2. Materials and methods 2.1. Test substances Tetrabromobisphenol A (TBBPA; Cas no 79-94-7), decabromodiphenyl ether (DeBDE; Cas no 1163-19-5), and short-chained chloroparaffin (chain length C10–13—chlorine content of 60%, Cas No 85535-84-8) were purchased from Fluka (Germany).

97

2.2. Test soil The test soil was a Danish agricultural soil (Askov, Jutland), dried at 80 °C for 24 h. The soil was sieved through a 2 mm mesh before use. The Askov soil is a sandy loam, and it has the following particle size distribution: coarse sand (200–2000 lm) 38.4%, fine sand (63–200 lm) 23.6%, coarse silt (20–63 lm) 12.7%, fine silt (2–20 lm) 12.3%, clay (<2 lm) 13.0%. The humus content of the soil was 2.8% and the total organic carbon content was 1.6%. The soil pHH2 O and total cation exchange capacity (CEC) was 6.2 and 8.14 meq. * 100 g 1 (mmolc(+) kg 1), respectively. 2.3. Sample preparation A stock solution of the different substances was prepared by dissolving the test substances in acetone (J.T. Barker, Hayword, CA, USA; HPLC quality) in concentrations that are required to obtain the highest test dosage in soil. For each of the test concentrations used, dilutions from the stock solution were made using acetone. Acetone (amount corresponding to 20% of the soil dry wt) with test substance was added to each container and mixed thoroughly into the soil. We used 20 ± 0.05 g of soil (dry wt) per replicate for the enchytraeid and nitrification tests, and 250 g of soil/replicate for the plant tests. Pure acetone was used for the control samples. The solvent was evaporated under a fume head for 24 h. In addition to the solvent controls, each test included a set of controls without solvent. A range-finding study indicated some effects of chloroparaffin and TBBPA, and no effects of DeBDE at 1000 mg kg 1. It was therefore decided to run a smaller number of test concentrations for DeBDE than for the two other substances. 2.4. Enchytraeus crypticus tests The test species is permanently cultured at the Danish National Environmental Research Institute, where it is massbred in Petri dishes containing a Bacto agar. The worms were kept at 20 ± 1 °C, with a 12/12 h light–dark cycle, and fed oatmeal. Test individuals were transferred from the culture disks to a Petri dish with water for examination under a microscope. Sexually mature animals of approximately the same size were selected for the ex- periments. Tests were performed according to an ISO standardised procedure (ISO, 2002). Four replicates were used per concentration. Following the sample preparation, water content was adjusted to 65% of the water holding capacity. The soil was then transferred to the test vessels; plastic jars (5 cm high, inner diameter 3.5 cm) which were closed at the top with lids during exposure. Approximately 30 mg of cooked oatmeal was roughly mixed into the soil before adding 10 adult enchytraeids to each replicate. After addition of animals and food, the containers were weighed to the nearest 0.1 g. The tests were conducted at 20 ± 1 °C

98

L.E. Sverdrup et al. / Chemosphere 64 (2006) 96–103

with an illumination of about 400–800 lx and a 12/12 h light–dark cycle. The exposure period was 21 d, and the test containers were opened after 7 and 14 d for feeding and, in case of water loss, addition of water. At the end of the test period the organisms were extracted by dividing each sample into two plastic containers and adding water (approximately eight times the amount of soil). The plastic containers were closed with lids, shaken vigorously for 15 s, and then left to sediment for 24 h. Individuals of E. crypticus were then found on the surface of the settled soil particles. Enchytraeids were transferred from the plastic containers to Petri dishes using Pasteur pipettes. Samples were counted manually, using a microscope. Surviving adults could easily be distinguished from juveniles produced during the test period. 2.5. Trifolium pratense tests Seeds were purchased from DLF Trifolium (Randers, Denmark). Prior to addition to the soil, seeds were inspected and damaged/discoloured seeds were removed. Seeds of similar size were then selected for the experiments. Tests were performed according to OECD Guidelines 208: Terrestrial plants, Growth test (OECD, 1984). Four replicates were used per concentration. Following the sample preparation, water content was adjusted to 65% of the water holding capacity and the soil was transferred to test containers: transparent cylinders made of hard plastics (35 cm high, inner diameter 9.5 cm), closed at the top and bottom with lids. The upper lid was perforated with one hole, diameter 0.8 cm, to allow for transpiration and gaseous exchange. Five seeds were added to each replicate. Following the addition of seeds, the test tubes were weighed to the nearest 0.1 g and placed in the greenhouse. The exposure period was 21 d, which corresponded to 15–17 d of exposure after 50% of the seeds in the controls had emerged. Test containers were weighed twice a week, and lost weight was replenished with the appropriate amount of deionised water. All containers were placed in a randomised order, which was changed every week. The temperature in the greenhouse was continuously monitored. The minimum temperature was regulated, and set to 15 °C (night temperature). During daytime, the temperature usually increased, sometimes to more than 25 °C. A light–dark cycle of 16/8 h was provided. At the end of the test, seedlings were cut off and immediately weighed to the nearest 0.01 mg (fresh weight). For the effect estimations, the mean fresh weight of seedlings in each replicate was used. 2.6. Soil nitrification tests The test set-up was similar to a method proposed by ISO (1997). In this method, a nitrogen source is added to the soil samples at the beginning of the test, and the effect of different concentrations of a chemical on soil nitrification is determined by comparison with uncontaminated con-

trols after four weeks of exposure. In our tests, some deviations from this standard were considered necessary as we used dehydrated soil samples and a solvent (acetone) in the spiking process. Both the temperature treatment (to dehydrate the soil) and the solvent addition were likely to reduce the soil microbial diversity and biomass. An inoculum, prepared from fresh soil samples of the Askov soil type, was therefore added following solvent evaporation and addition of the nitrogen source. The nitrogen source used was horn meal (Solsikken, Lina˚, Denmark) at an amount of 1.00 ± 0.05 g kg 1 soil dry wt, as recommended in the standard (ISO, 1997). The nitrogen content of the horn meal batch was 9.1% w/w. Fresh Askov soil was sampled and stored at 5 °C prior to use. The inoculate was prepared according to a procedure described by Lindahl and Bakken (1995): 20 g soil + 180 ml deionised water was mixed using a kitchen blender for 1 min and cooled on ice for 5 min. This procedure was repeated once, then the slurry was mixed again for 1 min and cooled for 30 s. The slurry was then used for inoculation. The final water content corresponded to 57% of the water holding capacity of the soil. Test cylinders (diameter 3.5 cm, height 5.0 cm, sealed by plastic lids) were incubated for four weeks at 20 °C in the dark. Twice a week during the incubation period, test cylinders were weighed and water was added corresponding to the weight loss. At the end of the tests, the soil from each replicate was extracted with 1 M KCl and the nitrate/nitrite content was measured spectrophotometrically using a LaChat QuikChemÒ analyzer (LaChat Instruments, Milwaukee, WI, USA) after a procedure described in detail in Sverdrup et al. (2002). 2.7. Statistical analysis NOEC-values (no observable effect concentration) were assessed using ANOVA and Dunnett’s procedure (at a 5% significance level) by using the computer programme JMP ver. 5.0 (SAS Institute Inc., Cary, NC, USA) after testing for fulfillment of ANOVA requirements. Estimates of the concentrations that caused a 10% reduction (EC10-values) of the most sensitive endpoint (i.e. reproductive output (enchytraeids), seedling growth (red clover) and soil nitrification were calculated only for those tests where significant effects were detected (ANOVA, Dunnett’s p < 0.05 by comparison with controls). EC10-values were calculated by linear interpolation and the confidence intervals were calculated by the ICp method (Norberg-King, 1993). As this method is only suitable for monotonously decreasing response parameters, low test concentrations that were significantly higher than controls were excluded from calculations. 2.8. Chemical analysis Aliquots of the soils from the different bioassays were pooled with respect to the compound, the concentration

L.E. Sverdrup et al. / Chemosphere 64 (2006) 96–103

and the sampling date (start or end of the experiments). That means that for each concentration level measured, average concentrations for the three bioassays were obtained. Between 5 and 11 g of these pooled soils were extracted with 60 ml acetone and 60 ml dichloromethane for 3 h, using a Soxtec Avati 2050 Auto system (1.5 h boiling, 1.5 h extracting) following addition of internal standards (13C-TBBPA and 13C-DeBDE) to a portion of the extracts. The extracts were then cleaned by treatment with concentrated sulphuric acid. The purified extracts with a final volume of 100 ll in iso-octane were then added recovery standard (1,2,3,4-tetrachloronaphthalene, analytical grade (>99%), from Promochem, Sweden) and subjected to GC/HRMS-EI analysis. The solvent in the extracts for TBBPA analysis was shifted to methanol after sulphuric acid treatment and prior to analysis by LC/MS. 2.9. Analysis An HP5890 GC coupled to a VG AutoSpec, high resolution mass spectrometer operated in EI mode was used for the GC/MS analyses of DeBDE. The DeBDE was separated by a fused silica capillary column from Agilent (DB5-MS column, 20 m, 0.25 mm i.d., 0.10 lm film thickness). An Agilent 100 HPLC was coupled to a Micromass LC/TOF in electrospray mode, negative ions for the TBBPA analysis. The TBBPA was separated by a reversed phase C18-column from Xterra (150 mm, 2.1 mm id, 3.5 lm particle size) with a gradient of methanol and water as eluent. DeBDE and TBBPA were monitored at m/z of the molecular ions with 13C-TBBPA and 13C-DeBDE as internal standards respectively. 3. Results The chemical analysis of test soils showed that measured concentrations of DeBDE was about two times higher than added amounts in the 100 and 1000 mg kg 1 treatments. For the initial concentrations of TBBPA, somewhat lower values between 59% and 87% of the nominal concentrations were observed (Table 1). The concentrations at the end of the experiment did not differ more than 15% from the initial concentrations whereas for 10 mg kg 1 of DeBDE and TBBPA the initial concentration was two times higher than the concentrations at the end of the experiment. The concentrations of the controls were below 0.1 mg kg 1 for both TBBPA and DeBDE. In spite of some deviations between measured and nominal concentrations, the measured values largely confirm the nominal concentration levels of the test substances, with an approximate 10-fold increase between each test concentration. Therefore, dose– response curves for the tests, as well as estimated effect levels for toxicity, have been based on nominal concentrations. There was no significant difference between solvent controls and water controls for any of the tests performed. The control replicates were therefore pooled for each test. All tests could be considered valid according to the validity cri-

99

Table 1 Results of the chemical analysis of tetrabromobisphenol A (TBBPA) and decabromodiphenyl ether (DeBDE) in toxicity tests with E. crypticus, soil nitrifying bacteria and T. pratense Substance/sampling

Concentrations (mg kg

1

dry wt)

Nominal concentration

Measured concentration

TBBPA Start End Start End Start End Start End

1 1 10 10 100 100 1000 1000

0.59 0.59 6.33 3.68 64.3 74.8 866 839

DeBDE Start End Start End Start

10 10 100 100 1000

15.1 8.12 230 223 2274

Analyses of the short-chain chloroparaffins were not performed.

teria defined in the test standards. Test results for TBBPA, DeBDE, and chloroparaffins are presented in Table 2. We also recorded seedling emergence for plants and survival for enchytraeids, but no effect was observed for these endpoints. TBBPA showed a high toxicity to enchytraeid reproduction, with small, but significant effects already at the 10 mg kg 1 exposure level (ANOVA, Dunnett’s p < 0.05). The EC10-value was estimated to 2.7 mg kg 1. At 1000 mg TBBPA kg 1, there was also a significant effect on soil nitrification (ANOVA, Dunnett’s p < 0.05), and the estimated corresponding EC10-value was 295 mg kg 1. No phytotoxicity was observed for TBBPA. DeBDE was not toxic to any of the organisms tested, but the short-chained chloroparaffins had a significant effect on nitrification in soil at the 1000 mg kg 1 exposure level; the EC10-value was estimated to 570 mg kg 1. Figs. 1–3 shows the dose–response relationships for each of the individual tests. 4. Discussion The limited knowledge about effects of chlorinated and brominated flame retardants in the soil environment has made the risk assessment of these substances difficult. Although the main concern associated with these substances is food-chain effects through secondary poisoning, flame retardants may also cause direct effect through soil exposure. The data presented here will therefore be an important input to the ongoing work on risk assessment of TBBPA, CP10–13, and DeBDE. TBBPA is potentially an endrocrine disruptor as it has a chemical structure similar to the thyroid hormone thyroxine, and in vitro studies showed that the potency of TBBPA for its competitive binding to human transthyretin is the highest of all brominated and chlorinated substances tested

100

L.E. Sverdrup et al. / Chemosphere 64 (2006) 96–103

Table 2 No observable effect concentration (NOEC), lowest observable effect concentration (LOEC), and the 10% effect concentration (EC10) for the most sensitive endpoint in terrestrial toxicity tests with tetrabromobisphenol A (TBBPA), decabromodiphenyl ether (DeBDE), and short-chain chloroparaffin (Chl-P) Test organism

E. crypticus T. pratense Soil nitrifying bacteria E. crypticus T. pratense Soil nitrifying bacteria E. crypticus T. pratense Soil nitrifying bacteria

Substance

TBBPA TBBPA TBBPA DeBDE DeBDE DeBDE Chl-P Chl-P Chl-P

Effective concentrations (mg kg

1

soil dry wt)a

NOEC

LOEC

EC10 (95% c.i.)

3 P1000 300 P1000 P1000 P1000 P1000 P1000 300

10 >1000 1000 >1000 >1000 >1000 >1000 >1000 1000

2.7 (0.7–5.4) 295 (210–390)

570 (ne)

EC10-values were not calculated for tests where LOEC was larger than the highest tested concentration. ne: confidence interval could not be estimated. a Most sensitive endpoint was reproduction for E. crypticus and nitrate production for soil nitrifying bacteria.

so far (Meerts et al., 2000). However, this effect was not seen in in vivo studies with pregnant mice (Meerts et al., 1999), and it is therefore concluded that TBBPA does not bind to transthyretin in vivo. For fish, a study with fathead minnow gave a NOEC-value of 0.16 mg l 1 due to reduced survival of young at hatch and reduced survival of juveniles after 35 d of exposure to TBBPA (WHO/ICPS, 1995). A similar no-effect level was found in 21 d reproduction tests with the invertebrate Daphina magna (NOEC-value of 0.3 mg l 1 (WHO/ICPS, 1995)). In a 96 h test with the marine algae Skeletonema costatum an EC50-value was estimated in the range of 0.09–0.89 mg l 1 (WHO/ICPS, 1995). As TBBPA is metabolised and rapidly excreted in mammals (WHO/ICPS, 1995), secondary poisoning is not likely to occur. Soil organisms may therefore be the critical target for effects of this substance in the terrestrial environment. According to the Technical Guidance Document on risk assessment of chemicals (European Communities, 2003), a predicted no effect concentration (PNEC) for the soil compartment can be calculated by the use of toxicity data on plants, soil invertebrates and soil microorganisms. When chronic toxicity data are available for all three groups of organisms, and the chemicals are not expected to act by a specific mode of toxic action, the PNEC can be calculated by dividing the lowest NOEC-value by a factor of 10. Thus, our ecotoxicity data for TBBPA (Table 2) supports a PNEC value of 0.3 mg kg 1 soil dry weight for this substance. Soil environmental concentrations of TBBPA are currently not available, so whether or not TBBPA in the soil compartment may pose a threat to soil dwelling organisms remains uncertain. However, addition of sewage sludge to grassland and agricultural soils is assumed to be one of the major dispersal routes for flame retardants into the terrestrial environment, and reported TBBPA concentrations in sewage sludge are all lower than the suggested PNEC and in the range of 0.0036–0.22 mg kg 1 dry wt for five Swedish sewage treatment plants (Sellstro¨m and Jansson, ¨ berg et al., 1995; Sellstro¨m, 1999; Sellstro¨m et al., 1999; O 2002).

DeBDE was not toxic to any of the organisms tested in this study, and CP10–13 showed only a slight effect on soil nitrifying bacteria at very high concentrations. As many soil organisms are exposed through the soil solution (e.g. Van Gestel and Ma, 1988; Jager et al., 2000), the low toxicity of these two substances could be due to their very low water solubility. Ko¨nemann and Musch (1981) have suggested that for aquatic organisms the absence of toxicity for highly hydrophobic contaminants may be due to their low water solubility, in combination with a limited potential for bioaccumulation. However, it should be noted that highly hydrophobic contaminants may have a potential for biomagnification, and therefore may pose a risk to i.e. mammals and birds by secondary poisoning. The risk of secondary poisoning has been emphasised by the recent risk assessment of DeBDE performed by the European Commission (2002). DeBDE has a very high molecular weight (959.17) and a very low water solubility (<0.1 lg l 1) (Stenzel and Markley, 1997). It has been shown that DeBDE has a low accumulation in rats (Hardy, 2004), and with the exception of one study (Viberg et al., 2003), no carcinogenicity or adverse effects on development or reproduction have been detected in mammals (Hardy, 2002). Thus, absence of toxicity in terrestrial organisms is well in line with test results on higher organisms. For the short-chain chloroparaffins (CP10–13) a large range of aquatic toxicity data are available (European Commission, 2000), and they show a rather high toxicity to aquatic organisms. The lowest observed chronic toxicity level has been reported for reproduction of the crustacean D. magna, with a NOEC of 0.005 mg l 1 (Thompson and Madeley, 1983). From the dataset available, a PNEC for the aquatic compartment has been calculated at 0.5 lg l 1, and as no data were available for soil dwelling organisms, PNEC for the terrestrial compartment was estimated by the equilibrium partitioning method, and by applying an extra assessment factor of 10 for the possible ingestion of soil particles (European Commission, 2000). From this, a

L.E. Sverdrup et al. / Chemosphere 64 (2006) 96–103 0.1

14

Seedling weight (g)

Nitrate in extracts (mg l-1)

16

12 10 8 6 4

0.08 0.06 0.04 0.02

2 0

0 0

1

3

10

30

100

300

1000

0

Tetrabrombisphenol A (mg kg-1)

10

30

100

300

1000

0.1

14

Seedling weight (g)

Nitrate in extracts (mg l-1 )

3

Tetrabromobisphenol A (mg kg-1)

16

12 10 8 6 4

0.08 0.06 0.04 0.02

2 0

0 0

1

3

10

30

100

300

1000

0

Chloroparaffin (mg kg-1)

3

10

30

100

300

1000

Chloroparaffin (mg kg-1)

14

0.1

12

Seedling weight (g)

Nitrate in extracts (mg l-1)

101

10 8 6 4 2

0.08 0.06 0.04 0.02

0 0

10

30

100

Decabromodiphenyl ether (mg

300

1000

kg-1)

Fig. 1. Effects of TBBPA, short-chain chloroparaffin and DeBDE on the nitrifying ability of soil bacteria. The figure shows average nitrate production ± STD (n = 4, except from controls, where n = 8).

temporary PNEC of 0.8 mg kg 1 was suggested for the soil compartment, but with the recommendation that the PNEC should be revised through toxicity testing with soil-dwelling organisms (European Commission, 2000). Based on the results from the present study, a PNEC of 57 mg kg 1 can be calculated, using an assessment factor of 10 as suggested in the current guidelines from the European Communities (2003), which is more than 70 times higher than the one currently used. The results of the chemical analysis showed deviations from nominal values somewhat higher than usual for chemical analysis where recoveries between 70% and 130% can be accepted. A reason for the low recoveries of TBBPA might

0 0

100

1000

Decabromodiphenyl ether (mg kg-1) Fig. 2. Effects of TBBPA, short-chain chloroparaffin and DeBDE on the early life stage growth of the red clover (T. pratense). The figure shows average seedling weight ± STD (n = 4, except from controls, where n = 8).

be that a portion of the substance is bound so strongly to the soil that it cannot be extracted under the chosen conditions. It is known that hydrophobic compounds like DeBDPE and TBBPA form non-extractable residues in soil that can only be extracted by repetitive extraction or at especially harsh conditions (Northcott and Jones, 2000). Another reason for the observed deviations might be that internal standard was first added after extraction of the soil and to different portions of the extract. Possible losses of compound during extraction and pipetting errors can therefore not be compensated for. The analysis of DeBDE

102

L.E. Sverdrup et al. / Chemosphere 64 (2006) 96–103

icity of DeBDE was not at all detected, and short-chain chloroparaffins only affected one of the test organisms (soil nitrifying bacteria) at the highest test concentration (1000 mg kg 1). Based on the generated data, a PNEC for soil organisms can be estimated at 0.3 mg kg 1 for TBBPA and 57 mg kg 1 for short-chain chloroparaffins. Due to lack of toxicity, no PNEC could be estimated for DeBDE. Measurements of TBBPA in soil are not available, but concentrations in sludge are all lower than the estimated threshold value for biological effects in soil.

# of juveniles per replicate

800

600

400

200

0 0

1

3

10

30

100

300

1000

Tetrabromobisphenol A (mg kg-1)

This study was funded by the Norwegian Ministry of the Environment (Nasjonale oppgaver) and by LIBERATION.

# of juveniles per replicate

1000 800

References

600 400 200 0 0

1

3

10

30

100

Chloroparaffin (mg

300

1000

kg-1)

1000

# of juveniles per replicate

Acknowledgment

800 600 400 200 0 0

10

100

1000

Decabromodiphenyl ether (mg kg-1) Fig. 3. Effects of TBBPA, short-chain chloroparaffin and DeBDE on the reproduction of E. crypticus. The figure shows average no. of juveniles ± STD (n = 4, except from controls, where n = 8).

indicated higher concentrations than expected. This may be due to the extensive dilution procedure which was performed before analysis, making an apparent up-concentration of the substance. DeBDE has also in general an extremely low solubility, which may result in a not completely homogeneous material in the soil samples. 5. Conclusion TBBPA has a high toxicity to soil organisms, with significant effects on enchytraeid reproduction detected already at the 10 mg kg 1 exposure level. In contrast, tox-

Amundsen, C.E., Hartnik, T., 2003. Bromerte Flammehemmere Og Klorerte Parafiner I Avløpsslam, Slamkompost, Sedimenter Og Avlø psvann. Undersøkelser For Lindum Ressurs Og Gjenvinning AS Og ˚ s, Norway. Drammen Kommune 2003. Report 61/03, Jordforsk, A BSEF, 2004a. Fact sheet. Brominated Flame Retardant Deca-BDE, Decabromodiphenyl Ether. Bromine Science and Environmental Forum, Secretariat, Brussels, Belgium. BSEF, 2004b. Fact sheet. Brominated Flame Retardant TBBPA, Tetrabromobisphenol A. Bromine Science and Environmental Forum, Secretariat, Brussels, Belgium. European Commission, 2000. Alkanes C10–13, Chloro, Cas No 85535-84-8, Risk Assessment. European Union Risk Assessment Report. European Chemicals Bureau, Ispra, Italy. European Commission, 2002. Bis(pentabromophenyl), Cas no 1163-19-5, Risk Assessment. European Union Risk Assessment Report. European Chemicals Bureau, Ispra, Italy. European Communities, 2003. Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for New Notified Substances, Commission Regulation (EC) No 1488/94 on Risk Assessment for Existing Substances and Directive 98/8/EC of the European Parliament and of the Council Concerning the Placing of Biocidal Products on the Market. Document from the European Commission, 2003. Hardy, M.L., 2002. The toxicology of the three commercial polybrominated diphenyl oxide(ether) flame retardants. Chemosphere 46, 757– 777. Hardy, M.L., 2004. A comparison of the fish bioconcentration factors for brominated flame retardants with their nonbrominated analogues. Environ. Toxicol. Chem. 23, 656–661. Hutzinger, O., Thoma, H., 1987. Polybrominated dibenzo-p-dioxins and dibenzofurans: the flame retardant issue. Chemosphere 16, 1877–1880. Ikonomou, M.G., Crewe, N., He, T., Fischer, M., 1999. Polybrominateddiphenyl-ethers in biota samples from coastal British Columbia, Canada. Organohalogen Compds. 40, 341–345. ISO, 1997. Soil Quality—Biological Methods—Determination of Nitrogen Mineralization and Nitrification in Soils and the Influence of Chemicals on these Processes. ISO/DIS 14238. International Standardization Organization, Geneva, Switzerland. ISO, 2002. Soil Quality—Effects of Pollutants on Enchytraeidae (Enchytraeus sp.)—Determination of Effects on Reproduction and Survival. ISO/DIS 16387. International Standardization Organization, Geneva, Switzerland. Jager, T., Sa´nchez, F.A.A., Muijs, B., van der Velde, E.G., Posthuma, L., 2000. Toxicokinetics of polycyclic aromatic hydrocarbons in Eisenia andrei (oligochaeta) using spiked soil. Environ. Toxicol. Chem. 19, 953–961.

L.E. Sverdrup et al. / Chemosphere 64 (2006) 96–103 Jansson, B., Andersson, R., Asplund, L., Litze´n, K., Nylund, K., Sellstro¨m, U., Uvemo, U.B., Wahlberg, C., Widequist, U., Odsjo¨, T., Olsson, M., 1993. Chlorinated and brominated persistent organic compounds in biological samples from the environment. Environ. Toxicol. Chem. 12, 1163–1174. Kierkegaard, A., Balk, L., Tja¨rnlund, U., de Wit, C.A., Jansson, B., 1999. Dietary uptake and effects of decabromodiphenyl ether in the rainbow trout. Environ. Sci. Technol. 33, 1613–1617. Ko¨nemann, H., Musch, A., 1981. Quantitative structure-activity relationships in fish toxicity studies. Part 1. Relationship for 50 industrial pollutants. Toxicology 19, 209–221. Lindahl, V., Bakken, L.R., 1995. Evaluation of methods for extraction of bacteria from soil. FEMS Microbiol. Ecol. 16, 135–142. Luross, J.M., Alaee, M., Sergeant, D.B., Whittle, D.M., Solomon, K.R., 2000. Spatial and temporal distribution of polybrominated diphenyl ethers in lake trout from the Great Lakes. Organohalogen Compds. 47, 73–76. Matscheko, N., Tysklind, M., De Wit, C., Bergek, S., Andersson, R., Sellstrom, U., 2002. Application of sewage sludge to arable land-soil concentrations of polybrominated diphenyl ethers and polychorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls, and their accumulation in earthworms. Environ. Toxicol. Chem. 21, 2515–2525. Meerts, I.A.T.M., Assink, Y., Cenijn, P.H., Weijers, B.M., van den Berg, ˚ ., Koeman, J.H., Brouwer, A., 1999. Distribution H.H.J., Bergman, A of the flame retardant tetrabromobisphenol A in pregnant and fetal rats and effect on thyroid hormone homeostasis. Organohalogen Compds. 40, 375–378. Meerts, I.A.T.M., van Zanden, J.J., Luijks, E.A.C., van Leewen-Bol, I., ˚ ., Brouwer, A., 2000. Potent Marsh, G., Jakobsson, E., Bergman, A competitive interactions of some brominated flame retardants and related compounds with human transthyretin in vitro. Toxicol. Sci. 56, 95–104. Norberg-King, T.J., 1993. A Linear Interpolation Method for Sublethal Toxicity: The Inhibition Concentration (ICp) Approach (Version 2.0), July 1993. National Effluent Toxicity Assessment Center, Duluth, Minnesota, USA. Northcott, G.L., Jones, K.C., 2000. Experimental approaches and analytical techniques for determining organic compound bound residues in soil and sediment. Environ. Pollut. 108, 19–43. Nylund, K., Asplund, L., Jansson, B., Jonsson, P., Litze`n, K., Sellstro¨m, U., 1992. Analysis of some polyhalogenated organic pollutants in sediment and sewage sludge. Chemosphere 24, 1721–1730.

103

¨ berg, K., Warman, K., O ¨ berg, T., 2002. Distribution and levels of O brominated flame retardants in sewage sludge. Chemosphere 48, 805– 809. OECD, 1984. Terrestrial Plants, Growth Test. OECD Guidelines (208). Organization for Economic Cooperation and Development, Paris, France. Schlabach, M., Mariussen, E., Borgen, A., Dye, C., Enge, E.K., Steinnes, E., Green, N., Mohn, H., 2002. Screening of brominated flame retardants and chlorinated paraffins. Report TA-1924/2002. SFT (Statens Forurensningstilsyn), Oslo, Norway. Sellstro¨m, U., 1999. Determination of some polybrominated flame retardants in biota, sediment and sewage sludge. Ph.D. Dissertation, Stockholm University, Stockholm, Sweden. Sellstro¨m, U., Jansson, B., 1995. Analysis of tetrabromobisphenol A in a product and environmental samples. Chemosphere 31, 3085–3092. Sellstro¨m, U., Kierkegaard, A., Alsberg, T., Jonsson, P., Wahlberg, C., de Wit, C., 1999. Polybrominated flame retardants in sediments from European estuaries. Organohalogen Compds. 40, 383–386. Stenzel, J., Markley, B., 1997. Decabromodiphenyl Oxide (DBDPO): Determination of the Water Solubility. Wildlife international Ltd. Sverdrup, L.E., Ekelund, F., Krogh, P.H., Nielsen, T., Johnsen, K., 2002. Soil microbial toxicity of eight polycyclic aromatic compounds: effects on nitrification, the genetic diversity of bacteria and the total number of protozoans. Environ. Toxicol. Chem. 21, 1644–1650. Ter Schure, A.F.H., Larsson, P., 2002. Polybrominated diphenyl ethers in precipitation in Southern Sweden. Atmos. Environ. (Ska˚ne, Lund) 36, 4015–4022. Thompson, R.S., Madeley, J.R., 1983. The Acute and Chronic Toxicity of a Chlorinated Paraffin to Daphnia magna. ICI report no. BL/B/2358. United Nations, 2003. Further Assessment of Persistent Organic Pollutants (POPs). Report from the UN Expert Group on POPs. Report no EB.AIR/WG.5/2003/3, May 15 2003. Van Gestel, C.A.M., Ma, W.C., 1988. Toxicity and bioaccumulation of chlorophenols in earthworms, in relation to bioavailability in soil. Ecotox. Environ. Saf. 15, 289–297. Viberg, H., Fredriksson, A., Jakobsson, E., Orn, U., Eriksson, P., 2003. Neurobehavioral derangements in adult mice receiving decabrominated diphenyl ether (PBDE 209) during a defined period of neonatal brain development. Toxicol. Sci. 76, 112–120. WHO/ICPS, 1995. Environmental Health Criteria 172: Tetrabromobisphenol A and Derivatives. World Health Organisation, Geneva.